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Ligand Binding Energy and Catalytic Efficiency from Improved Packing within Receptors and Enzymes
doi:10.1016/S0022-2836(03)00428-5
J. Mol. Biol. (2003) 329, 389–399
Ligand Binding Energy and Catalytic Efficiency from Improved Packing within Receptors and Enzymes Dudley H. Williams*, Elaine Stephens and Min Zhou Department of Chemistry University of Cambridge Lensfield Road, Cambridge CB2 1EW, UK
Some small molecules bind to their receptors, and transition states to enzymes, so strongly as to defy current understanding. We show that in the binding of biotin to streptavidin, the streptavidin structure becomes better packed. We conclude that this contraction of the streptavidin structure promotes biotin binding. The improved packing is associated with positively cooperative binding, occurring with a benefit in enthalpy and a cost in entropy. Evidence indicating that catalytic efficiency can also originate via improved packing in some enzyme transition states, derived from the work of others, is presented. Negatively cooperative ligand binding is concluded to induce converse effects (less efficient packing, a cost in enthalpy, and a benefit in entropy). It applies to the binding of O2 to haemoglobin, which indeed occurs with a hitherto unreported loosening of the amide backbones of the haemoglobin monomers. q 2003 Elsevier Science Ltd. All rights reserved
*Corresponding author
Keywords: positive cooperativity; negative cooperativity; streptavidin; haemoglobin; myoglobin
Introduction
The models for cooperativity and packing
The affinities with which some small molecules are bound by protein receptors and enzymes are so large as to be regarded as anomalous, or illunderstood. For example, the binding of biotin to streptavidin (K ¼ 1013.4 M21) is typically found to be an “outlier” in comparison with other affinities.1,2 Equally, where the association constants for the binding of transition states to enzymes have been estimated, the values are amazingly high (where examined, lying in the range 1016^4 M21).3 – 7 Here, we show that the binding of biotin to streptavidin can be increased via biotin-induced improvements in the packing of streptavidin. Analogously, in enzyme reactions, substrate transition states can improve the packing within the enzyme, and so increase catalytic efficiency. Evidence to support this concept utilising the data of others (either from very large enthalpies of transition state binding, or reduced dynamic behaviour of the enzyme in the transition state for reaction) is presented for nine enzymes.
The terms positive and negative cooperativity are most commonly used to understand binding involving multiple binding sites.8 In this usage, positively cooperative binding is said to occur when ligands that bind successively are bound with increasing affinities. Negatively cooperative binding is said to occur when successively binding ligands are bound with decreasing affinities. Classic papers by Monod, Wyman, and Changeux (MWC model),8 and by Koshland, Nemethy, and Filmer (KNF theory)9,10 have, respectively, considered the consequences of positively and negatively cooperative binding when there are multiple ligand binding sites in the receptor. Binding is described as positively cooperative where the binding curve for ligand binding is sigmoid in shape (i.e. subsequently bound ligands are bound more strongly than the first-bound ligand). This is the case for O2 binding to haemoglobin.11 Here, we adopt a different definition of cooperative binding. It is more general insofar as it is not confined to proteins with multiple ligand binding sites. A binding event is defined as positively cooperative with respect to a second interaction (or set of interactions) when its affinity is increased in the presence of that second interaction (or set of interactions). Conversely, a binding event is defined as negatively cooperative when its affinity
Abbreviations used: MWC model, Monod, Wyman, and Changeux model; HGPRT, hypoxanthine-guanine phosphoribosyl transferase. E-mail address of the corresponding author: [email protected]
0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved
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is decreased in the presence of that second interaction (or set of interactions). The structural basis of the positively cooperative binding of a ligand to a peptide receptor (as defined in the preceding paragraph) has been investigated using glycopeptide antibiotics of the vancomycin group.12 Positively cooperative binding of the natural ligand to the antibiotic dimers causes greater downfield shifts of a resonance due to a proton at the dimer interface. This observation indicates more efficient packing at the dimer interface due to the positively cooperative binding. Although the isolated dimer has only one noncovalently bonded interface, the dimerisation constants (Kdim) of several antibiotics can be increased up to 100-fold in the presence of two molecules of the natural ligand (e.g. Kdim increases from 105 M21 to 107 M21). Importantly, these antibiotic data show that ligand binding to a receptor system can be enhanced through ligand-induced structural tightening of the receptor system.13 The effect is understandable in the general case if the positively cooperative binding of a ligand to a receptor (analogous to the dimer in the above example) is likened to a cooling of portions of the receptor structure. The binding of the ligand locally reduces the dynamic motions of the residues with which it directly interacts (Figure 1(a)). Since non-covalent bonding is opposed by kinetic energy of motion, these residues are now in turn able to bond better to, and to organise, adjacent residues, and so on, until the effect has spread through the whole of the receptor where positive cooperativity can occur. The enhancement of ligand binding is specifically associated with better packing of the receptor system,12 and the benefits in enthalpy and costs in entropy of positively cooperative binding have been documented.14 The arguments above for effects analogous to cooling parts of a receptor as a model for positive cooperativity can be extended to propose the analogy of warming parts of a receptor as a model for negative cooperativity. In this case, the binding of a ligand is incompatible with the geometry existing in the isolated receptor at the potential ligand binding site (Figure 1(b)) and the making of these ligand/receptor bonds decreases the bonding efficiency of the non-covalent bonds within the receptor. Therefore, the general expectations for negatively cooperative binding of a ligand to a receptor are less efficient packing in the parts of the receptor where the negative cooperativity is expressed, with an associated cost in enthalpy and a benefit in entropy.14 We were encouraged that this model might have general applicability by a study of changes in the properties of tetrameric recombinant human tyrosine hydroxylase isoform 1 upon binding the natural cofactor (6R)-L -erythro-5,6,7,8tetrahydrobiopterin.15 The binding of the cofactor occurs with negative cooperativity, and this cofactor-bound form of the enzyme then shows a decreased resistance to limited tryptic proteolysis,
Improved Protein Packing Aids Binding, Catalysis
Figure 1. Structural models for positive and negative cooperativity, where the lower pair of hydrogen bonds is within a protein receptor and the upper pair of hydrogen bonds is formed upon binding a peptide ligand. (a) Positive cooperativity arises when the dynamic motion of the central chain is reduced by forming hydrogen bonds to the peptide ligand. The central chain then forms a better template for hydrogen bonding to the lower chain and the two sets of hydrogen bonds are strengthened and stabilized. (b) For negative cooperativity, the ligand – receptor interaction demands a distortion of the structure of the receptor. The decrease in bonding will increase the lengths of some of the hydrogen bonds and result in an increase in its dynamic behaviour.
as would be expected from a loosening of the enzyme structure. Although enthalpy/entropy compensation has previously been associated with protein/ligand binding,16 the circumstances under which it will promote, or oppose, ligand binding have not been defined. The work on the glycopeptide antibiotics,12,13 and the models described above, suggest structural consequences of both positively and negatively cooperative binding of a ligand. Here, we have examined the strong binding (K ¼ 1013.4 M21) of biotin to streptavidin to see if the proposed structural consequences of positively cooperative ligand binding are observed. In addition, the properties proposed for negatively cooperative ligand binding are here demonstrated for the binding of O2 to haemoglobin. Evidence is also presented (using the data of others) to show that nine enzymes derive catalytic efficiency through improved packing when they bind to substrate transition states.
Positively Cooperative Ligand Binding: Structural Tightening of Streptavidin upon Biotin Binding Four molecules of biotin bind to a streptavidin tetramer. It seemed that biotin-induced tightening of the streptavidin structure might be occurring, since the binding is remarkably exothermic and adverse in entropy at 25 8C (DH ¼ 2 134 kJ mol21 and TDS ¼ 2 57 kJ mol21,17 or DH ¼ 2 102 kJ mol21 and TDS ¼ 2 26 kJ mol21,18). That is, these
Improved Protein Packing Aids Binding, Catalysis
Figure 2. Time-course showing the extent of backbone amide NH to N2H exchange at pH 8.0 in streptavidin both in the absence (squares) and presence (diamonds) of biotin.
thermodynamic parameters might reflect a promotion of biotin binding because the streptavidin structure is simultaneously stabilised by an improvement in its internal bonding with an accompanying restriction in its dynamic behaviour. The dynamic behaviour of streptavidin upon biotin binding was monitored by well-established methods of hydrogen/deuterium (H/2H) exchange and mass spectrometry.19 The extent of NH to N2H exchange of the intact peptide backbone of streptavidin in the absence, and presence, of biotin was determined by electrospray mass spectrometry (ESI-MS). It was found that in the absence of biotin, each streptavidin (apo-streptavidin, residues 13 –139, from Streptomyces avidinii) underwent exchange of 72 – 74 backbone amide NHs in the time-scale of one to three hours. In the presence of biotin, only 48 – 50 of these NH groups underwent exchange on the same time-scale. Thus, through the binding of biotin, , 24 exchangable amide hydrogen atoms per sub-unit are protected from solvent exchange (Figure 2). It can be considered that the results of this experiment could be interpreted in terms of partial protection of larger numbers of amide NH groups. However, the relative invariance of the degree of exchange after 30 minutes (i.e. the exchange is essentially biphasic, Figure 2) suggests that an “all-or-none” interpretation is a useful approximation. The degree of protection is much larger than previously inferred from FT-IR studies, which indicated the protection of only about ten amide protons.20 A comparison of crystal structures of apostreptavidin and the streptavidin/biotin complex shows some structural ordering in certain regions of the protein when biotin is bound,21 that might account in principle for a portion of the amide NH protection observed in the current study. These regions include two surface loops incorporating residues 45– 50 and 63– 69, that lacked defined density in apo-streptavidin but became ordered upon biotin binding. The former flexible loop (45 – 50) formed hydrogen bonds to biotin and underwent an “open” to “closed” confor-
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mational change upon biotin binding. Therefore, a reduction in deuterium incorporation could occur in this region when biotin is bound. However, in the X-ray crystal structure of apo-streptavidin obtained by Freitag et al.22 the 45 –50 flexible loop found by Weber et al.21 is structured in a 310 helix and is stabilized in the open conformation. In this case, the number of hydrogen bonds within the loop involving backbone amide NH groups of apo-streptavidin is three in the biotin-free form, and only two in the biotin-bound form.22 Thus, the binding of biotin actually involves the formal removal of one such hydrogen bond in this loop. Furthermore, although biotin forms several hydrogen bonds to streptavidin, these involve only one backbone amide NH of streptavidin.21 Consequently, the protection of 24 backbone amide NH groups upon biotin binding is unexpected on the basis of the X-ray data regarding both hydrogen bonds made to biotin, and within a streptavidin subunit. We next consider whether the X-ray studies can throw any light on the mass spectrometric exchange data in terms of changed interactions between subunits. The extended b-strand that constitutes the C-terminal part of the streptavidin sequence (around Trp120) has a free edge. This free edge is indicated by the X-ray structure of apo-streptavidin to form two hydrogen bonds to the corresponding region of an adjacent subunit, and thus to promote van der Waals interactions between the subunit surfaces.21 Although the X-ray studies by Weber et al. indicated a small adjustment of the inter-subunit geometry upon biotin binding,21 the crystals obtained by Freitag et al. revealed no systematic change in the quaternary structure that can be associated with biotin binding.22 Both studies noted that there was no evidence for subunit cooperativity, e.g. through the formation of extra hydrogen bonds between subunits upon biotin binding. Thus, the protection of 24 backbone amide NH groups upon biotin binding is also unexpected on the basis of the X-ray data for interactions between streptavidin subunits. It is clear that the mass spectrometry experiments, carried out in solution, pick up extensive reductions in the dynamic behaviour of streptavidin upon biotin binding. These changes in dynamic behaviour are not apparent from the X-ray studies, presumably because they are masked by crystal packing forces, and/or they involve distance changes that are not discernible at the resolution of the structure determinations. However, a reduction in the dynamic behaviour of streptavidin upon biotin binding is supported by another solution study,23 where it is shown that the midpoint temperature for thermal denaturation of streptavidin changes from 75 8C to 112 8C upon binding of biotin. This increased stability was attributed to an enhanced inter-subunit association and an increase in structural order due to the organisation of the two disordered loops (45 –50
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Improved Protein Packing Aids Binding, Catalysis
Table 1. Deuterium content of streptavidin pepsin digest fragments Residues
Number of NHs in peptide
Apo-streptavidina
Streptavidin/biotin complexa
Deuterium changeb
14–21 22–29 30–42 43–56 59–72 71–78 79–95 102–119 120–124 125–138
7 7 12 13 12 7 16 17 4 12
3.9 3.9 6.4 8.6 11.9 1.3 3.7 6.8 1.9 8.0
2.1 1.5 3.8 3.6 10.7 1.0 1.4 2.4 1.1 7.0
21.8 22.4 22.6 25.0 21.2 20.3 22.3 24.4 20.8 21.0 (Total ¼ 21.8)
a Number of deuterium atoms incorporated after 60 minutes of deuterium exchange. Deuterium levels, determined from centroids of envelopes of isotope peaks, were corrected for deuterium loss during digestion and LC–MS.45 b Changes in deuterium content, relative to apo-streptavidin, when biotin is bound.
and 63 – 69), in the presence of biotin. Importantly, both the current, and the prior solution studies indicate reductions in dynamic behaviour upon ligand binding that are not evident from studies of crystals. To identify the regions of streptavidin that are protected from deuterium exchange by the binding of biotin, pepsin digestion and LC –MS were performed according to established protocols.24,25 Data covering almost the whole of the streptavidin backbone are summarised in Table 1. The peptides that show the greatest reduction in exchange of their backbone amide NH groups in the presence of biotin are 43 –56 (Figure 3(a)) and 22 –29. The
former includes the flexible loop (residues 45 – 50) and, depending upon the time allowed for exchange, the presence of biotin decreases the extent of exchange within it by five to six deuterium atoms (after correction for backexchange during the quenching, pepsin digestion and LC – MS steps, Figure 3(a)). The difference in deuterium content of apo-streptavidin and streptavidin/biotin was calculated for each peptide and expressed as a percentage of the total number of amide backbone NH groups (Figure 3(b)). The data show that the binding of biotin reduces the solvent accessibility of streptavidin backbone NH groups to different degrees throughout essentially
Figure 3. Deuterium incorporation into peptides obtained from pepsin digestion of streptavidin. (a) The level of deuterium incorporation in two peptides from pepsin digestion is shown in the absence (squares) and the presence (diamonds) of biotin as a function of time. (b) Position of peptides in a ribbon model of a streptavidin subunit with bound biotin (Figure prepared with the program MOLMOL46). The degree of NH protection in the presence of biotin is indicated by colour. Reduction in exchange . 30% (red), 20 – 30% (yellow), 10 – 20% (light blue), and ,10% (dark blue). Two regions not represented in peptic peptides are indicated in grey.
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Improved Protein Packing Aids Binding, Catalysis
the whole structure. The number of backbone amide NH groups protected from exchange by the presence of biotin, as assessed from analysis of the whole protein, is 24. This is in remarkably good agreement with that (21.8, Table 1) assessed from analysis of the available peptides (which cover 94% of the backbone). Relationships can be seen between the extent of mobility within a region of an apo-streptavidin subunit before biotin binding, the distance of this region from the biotin binding site, and the degree of tightening upon biotin binding. (i) The region of apo-streptavidin that undergoes least exchange (residues 71– 95, Table 1) shows little further protection from exchange upon biotin binding, despite being only moderately distant from the biotin binding site (Figure 3(b)). This region is found at the subunit interface and is stabilised by van der Waals interactions between the subunit barrel surfaces.21 According to our data, these tight binding regions of apo-streptavidin cannot further restrict their dynamic behaviour to give additional benefits in bonding upon biotin binding. (ii) The region of apo-streptavidin (residues 59– 72, Table 1) that is not only distant from the binding site of biotin, but also encompasses a loop that is outside the barrel structure (Figure 3(b)), undergoes the greatest exchange but shows little further protection from exchange upon biotin binding. Interestingly, this region incorporates the loop spanning residues 63 –69 that lacks defined density in the X-ray structure of apo-streptavidin, but becomes more ordered upon biotin binding.21 Our data suggest that this loop remains relatively mobile in the bound state in solution. This is presumably because of the lack of an efficient cooperative pathway from biotin to restrict its motion. (iii) All other parts of the protein that we have been able to examine (which constitute 63% of the residues) undergo extensive exchange (40 –67%) of their backbone NH groups prior to biotin binding, which is considerably reduced after binding (to the range 21 –58%). All these dynamic regions therefore have their mobility restricted by biotin binding. Biotin binding is particularly enhanced by improvement in the bonding within the four strands of the b-sheet and its attendant loops indicated in red (Figure 3(b)), and within the extensions of this sheet (Figure 3(b), yellow). The improved packing of the protein in the 102 – 124 region is also marked. The major part of the improved packing is within the subunits rather than between them. A useful analogy is to think of the apo-streptavidin tetramer as behaving like an impure crystal. When an impurity is removed from a crystal, the noncovalent bonding within it typically becomes more
positively cooperative and the melting transition of the crystal becomes higher and sharper. Biotin apparently acts as the antithesis of an impurity when it binds to apo-streptavidin. It causes the cooperative improvement of bonding, to varying degrees, throughout the streptavidin system, and the increase in the tm from 75 8C to 112 8C upon biotin binding23 is naturally accommodated. Biotin stabilises the structure of streptavidin, and this extensive structural stabilisation contributes to the affinity between biotin and streptavidin. We note that the binding of ligands to proteins often increases the thermal stability of the protein,26 – 28 and therefore the enhancement of ligand binding in this way is not uncommon. Jones & Kurzban have provided persuasive evidence that the binding affinity for biotin to the streptavidin tetramer, containing zero, one, two and three molecules of biotin is essentially identical.29 Therefore, the binding is not positively cooperative in the MWC sense. However, since biotin binding clearly tightens (the present work), and stabilises,23 the streptavidin structure, it is positively cooperative in terms of the definition used here (Figure 1(a)). Our data, taken in conjunction with those of Jones & Kurzban,29 indicate that upon biotin binding into each subunit, this part of the 4-fold symmetrical structure is tightened to essentially the same extent. That is, the degree of positive cooperativity is essentially the same for all four binding events. Previously, other solution studies have also noted structural stabilisation of protein receptors upon ligand binding that were not detected within the crystallographic resolution of their X-ray structures. A reduction in amide exchange rates of a protein antigen (lysozyme) upon binding any one of three antibodies has been observed.30 Theoretical considerations indicate that the binding of the antibodies stabilises the lysozyme structure.31 Also, NMR studies show that agonist binding to the peroxisome proliferator-activated receptor (PPAR)g causes a marked reduction in the conformational mobility of the receptor.32 The reductions in the dynamic behaviour of these proteins must enhance the ligand/receptor binding through positive cooperativity.12,13
Negatively Cooperative Ligand Binding: Structural Loosening of Haemoglobin upon O2 Binding We note that in the MWC model8 for O2 binding to haemoglobin (Figure 4), the O2 binding is positively cooperative because much of the work required for the T ! R conversion is effected by the first O2 to bind. Therefore, subsequently binding O2 molecules have the advantage to access a relatively high population of the R state, and bind with greater affinity (hence the binding is considered to be positively cooperative). However, in
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terms of the definition used here, negatively cooperative ligand binding occurs when O2 binds to the T state. The optimal binding of O2 is incompatible with the geometry existing in the T state. In analogy to Figure 1(b), the ligand therefore distorts the structure of the receptor. This distorted O2-bound structure is the R state. The negatively cooperative ligand binding does indeed force a loosening of the haemoglobin tetramer, through the breaking of inter-subunit salt bridges (as established by the classic work of Perutz and co-workers),33 to give the R state. However, and more importantly, widespread structural changes in the T to R transition seem possible. All noncovalent interactions within a receptor system that are coupled with negative cooperativity to ligand binding should loosen. It appeared possible from prior thermodynamic data that the properties proposed for negatively cooperative binding of a ligand might be displayed by the haemoglobin/O2 system. Thus, in the case of trout haemoglobin,34 binding of the first O2 ligand (when presented with the T state) occurs with an exothermicity very near zero, and a favourable TDS term of þ21 kJ mol21. However, subsequent O2 binding (to the R state) occurs with a large exothermicity (DH ¼ 232 kJ mol21) and slightly unfavourable entropy (TDS ¼ 23 kJ mol21). These thermodynamic data suggest that in the binding of the first ligand molecule, work must be done to drive the T to R state conversion, so that there is no significant net enthalpic benefit, despite the bonding of O2 to haemoglobin. Instead, the initial binding is driven by the fact that the R state is favourable in entropy (less well packed, i.e. looser) than the T state from which it is formed. Once the R state tetramer is more highly populated (as a result of the initial binding O2 binding (Figure 4)), binding to the R state is enthalpy driven (data above). To test the above inferences, we determined the change in dynamic behaviour of the horse haemoglobin tetramer peptide backbone when it binds O2. In experiments analogous to those described above for streptavidin, the extent of NH to N2H exchange of the peptide backbone of the a and b-chains of horse haemoglobin in the absence, and presence, of bound oxygen was determined by ESI-MS (Figure 5). In the absence of bound oxygen,
Figure 4. The MWC model for the binding of a ligand (L, here O2) to a tetrameric protein (haemoglobin) able to equilibrate between tense (T) and relaxed (R) forms.
Improved Protein Packing Aids Binding, Catalysis
Figure 5. Time-course showing the extent of backbone amide NH to N2H exchange in the a (triangles) and b (squares) subunits of oxygenated (filled) and deoxygenated (empty) haemoglobin.
the a-chains underwent exchange of 25 –27 backbone amide NH groups (out of a total of 134), and the b-chains underwent exchange of 24 –27 backbone amide NH groups (out of a total of 140) in the time-scale of half to two hours. In the presence of bound oxygen, the a-chains underwent exchange of 33 – 34, and the b-chains underwent exchange of 40– 43 in the same time-scale. Therefore, through the binding of oxygen, a further seven to eight exchangeable amide hydrogen atoms per a-chain (5.2 –6%), and a further 16 per b-chain (11.4%) are exposed to solvent exchange. Thus, complete saturation of the haemoglobin tetramer by O2 binding results in an increase of 46 –48 in the number of backbone NH groups undergoing exchange. X-ray structures of the haemoglobin tetramer were examined for the presence of any inter-subunit hydrogen bonds involving peptide backbone NH groups. Such hydrogen bonds are not evident, and therefore are not available to be broken in the T to R conversion. We conclude that the increase of 46– 48 in the number of backbone NH groups undergoing exchange upon O2 binding must reflect an increase in the internal dynamic behaviour of the subunits. These changes have not been described in prior work presumably, in the case of X-ray studies, because changes in dynamic behaviour can be masked by crystal packing forces. The conclusion is reinforced by the similar findings for myoglobin (which does not form a tetrameric structure) upon O2 binding (see later). The increase in amide NH exchange upon O2 binding to haemoglobin is opposite to the effect induced by biotin binding to streptavidin, and is in agreement with our predictions regarding the changes associated with negatively cooperative ligand binding. Thus, the relatively poor binding of O2 to the T form of haemoglobin is not simply due to the free energy cost of loosening the noncovalent bonding between its subunits.33 It is also due to extensive loosening of the non-covalent interactions within the haemoglobin subunits. We note that the structural loosening involving the
395
Improved Protein Packing Aids Binding, Catalysis
b-chains is much greater than of the a-chains. In this connection it is interesting to note that although all four subunits are distorted by the displacement of iron and its proximal histidine upon O2 binding, only in the b-subunits is the distal valine displaced.35 This observation is consistent with the greater expression of negative cooperativity within these b-subunits. Analogous experiments on myoglobin were clearly of interest, since it exists as a monomeric species. In the original MWC paper, it was assumed that “the subunits of the R form are closer to the conformation of the (haemoglobin) monomer” (cf. myoglobin) and that “myoglobin may be thought of as a relaxed subunit of haemoglobin”.8 According to this assumption, oxygen binds strongly to myoglobin, at least in part, because it does not have to pay the price of the T to R conversion (Figure 4). However, it is important to recall that the R structure has been shown above to be internally loosened relative to the T state, and this internal loosening must be driven by O2 binding. Thus, the myoglobin structure might also be loosened internally by O2 binding and, in its isolated form, may not be analogous to “a relaxed subunit of haemoglobin”.8 The extent of NH to N2H exchange of the peptide backbone of horse heart myoglobin in the absence, and presence, of bound oxygen was determined by ESI-MS. In the absence of bound oxygen, myoglobin underwent exchange of 27 –30 backbone amide NH groups (out of a total of 148) in the time-scale of half to two hours. In the presence of bound oxygen, it underwent exchange of 40 –45 in the same timescale. Thus, through the binding of oxygen, a further 13 – 15 exchangeable amide hydrogen atoms are exposed to solvent exchange. Myoglobin thus possesses a relatively tight (or “tense”) internal structure, and not a relaxed one as originally seemed likely.8 This conclusion makes physical sense for, as Perutz commented,35 it is the binding of O2 that perturbs the protein structure. Using the model described here (Figure 1(b)), both myoglobin and the T form of haemoglobin bind O2 with negative cooperativity, and the reduction in binding energy of the O2 to these proteins is spread among the network of non-covalent interactions.
exchange into backbone amide bonds in hypoxanthine-guanine phosphoribosyl transferase (HGPRT) 37 and purine nucleoside phosphorylase25 was used to compare the dynamic properties of the enzymes alone, in forms with bound reactant/ product, and in forms with bound transition-state analogues. In both cases, it was found that the rate and extent of deuterium incorporation decreased when the reactant/product was bound, and decreased to an even greater extent when the transition state analogue was bound. Thus, the greatest reduction in dynamic motion is caused by the transition state analogue. The effects are large: the binding of the transition state analogue protects 34 peptide backbone NH groups from exchange in the case of HGPRT, and 27 peptide backbone NH groups are similarly protected in the case of purine nucleoside phosphorylase. Our proposals indicate that both enzymes will provide binding energy for the reactant/product through becoming better packed. Crucially, even greater binding energy will be provided for the transition state analogue when the packing is further improved. Structural tightening of an enzyme when it binds a substrate transition state should have a further testable consequence. The benefit in entropy of enzyme catalysis (originating in the ordering of the catalytic groups on the enzyme structure) should be offset by the cost in entropy of reducing the dynamic behaviour of the enzyme in the transition state. Moreover, this proposed reduction in dynamic behaviour of the enzyme should provide a large benefit in improved non-covalent bonding (enthalpy) within the enzyme. Strikingly, where data are available, enzyme-catalysed reactions are, relative to the corresponding reaction in solution, greatly favoured in enthalpy (Table 2).4,38 In the case of cytidine deaminase, both the enthalpic and entropic contributions have been derived. Enzyme catalysis increases the reaction rate by 1016 M21, due to a benefit in enthalpy (DDH –) of 2 84 kJ mol21, and a benefit in entropy (TDDS –) of only 7 kJ mol21.39 Thus, where the second consequence of our model for the enhancement of catalysis through structural tightening of the enzyme can be tested, the available data are also in excellent accord with its requirements.
Implications for Enzyme Catalysis Our findings appear likely to have implications for the understanding of enzyme catalysis. Enzymes bind the transition states (S–) of substrates more strongly than they bind the substrates.36 According to the positive cooperativity model (Figure 1(a)), one way to effect this is through a greater degree of structural tightening of the enzyme upon binding the substrate transition state than upon binding the substrate. Two recent studies, as interpreted here, give convincing support to this idea. Hydrogen/deuterium (H/2H)
Table 2. Benefit in enthalpy (DDH –) of some enzymecatalysed reactions relative to the reactions in free solution4,38 Enzyme Chorismate dismutase Chymotrypsin Staphylococcal nuclease Bacterial a-glucosidase Urease Yeast OMP decarboxylase
DDH – (kJ mol21)
Rate accel. (s21) due to DDH –
233 266 263 280 293 2143
106 1012 1011 1014 1016 1025
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Conclusion Ligand binding causes a tightening of a glycopeptide receptor structure, and there is an associated increase in the free energy of binding of the ligand.12,13 Structural arguments suggest that this effect should be general. Thus, non-covalent interactions within protein receptors can be improved upon ligand binding to provide ligand binding energy. Evidence has been provided to show that the streptavidin structure tightens extensively upon biotin binding, and that this tightening will enhance biotin binding. In contrast, the binding of O2 to the T form of haemoglobin, and to myoglobin, is (using the definitions employed here, and their operational consequences) negatively cooperative. The internal structures of haemoglobin and myoglobin are loosened upon O2 binding, adding to our knowledge of why O2 binding to the T state of haemoglobin is less favourable than to the R state. Data for nine enzymes support the idea that the efficiency of enzyme catalysis is improved through the tightening of enzyme structures. Ligand binding to some receptors may of course occur with positive cooperativity to some portions of the receptor, and with negative cooperativity to other portions. Thus, it is a large net effect of ligand binding on receptor dynamics (as in the examples cited here) that will be a sign of a relatively large net positively or negatively cooperative effect. An intrinsic relationship between cooperative binding and enthalpy/entropy compensation accounts naturally for the occurrence of “tense” and “relaxed” systems.8 Negatively cooperative binding to a tense form of a receptor can drive it to a more relaxed state with a cost in enthalpy and a benefit in entropy. Since positively cooperative binding can occur with a benefit in enthalpy and a cost in entropy, and Nature utilises both positively and negatively cooperative binding, large variations in the values of these thermodynamic parameters for ligand – receptor interactions can be expected. The thermodynamics of binding of 136 drugs to biological receptors has been summarised in a graph of DH and DS.40 Some associations are endothermic by quantities in the region of 125 kJ mol21 (and therefore are likely to be negatively cooperative). At the other extreme, some are exothermic by ca 80 kJ mol21. In comparing the extremes, the differences are indeed remarkably large (ca 205 kJ mol21) as required for opposing forms of cooperativity. We note that the structural changes described here for positively and negatively cooperative binding may well be a general property of aggregated states, and therefore widespread. Additional examples are found among proteins. For example, addition of a seventh Notch ankyrin domain increases the folding equilibrium by 1000-fold (positive cooperativity), and increases the enthalpy of unfolding by ca 50%.41 The F0F1-type ATP synthase from the thermophilic bacillus PS3 binds
Improved Protein Packing Aids Binding, Catalysis
Mg-ADP with an increase in the thermal stability of the protein and a concomitant decrease in its flexibility, whereas the binding of Mg-ATP reduces its thermal stability and simultaneously increases its flexibility.42 Therefore the structural changes within the protein should enhance the binding of Mg-ADP and reduce the affinity for Mg-ATP at the binding sites probed in this work. Although these particular binding sites are reported to have a regulatory, rather than a direct catalytic function, the association of increased protein stability with decreased flexibility, and of decreased protein stability with increased flexibility, are in accord with the conclusions reached here. The characteristics of negative cooperativity are observed when “non-matching” substances are introduced into lipid bilayers. Thus, the incorporation of increasing quantities of cholesterol progressively reduces the positive cooperativity, the temperature, and enthalpy of the gel-to-liquid-crystalline phase transition of lipid bilayers constituted from phosphatidylserine.43 When pure, the lipid bilayer forms a positively cooperative aggregate. The binding of cholesterol into this bilayer is negatively cooperative because the cholesterol disrupts the large domains of positively cooperative interactions that previously existed within the bilayer. The changes in the property of the bilayer upon introduction of cholesterol therefore follow the changes posited here for negative cooperativity. Can the structural tightening of receptors and enzymes discussed here plausibly give rise to large increases in binding constants of ligands, or large increases in rates of catalysis? The glycopeptide dimeric receptors are stabilised by a factor of 100 when the system is elaborated by ligand binding.12 The elaborated system contains only 16 hydrogen bonds, plus a limited number of hydrophobic interactions, at its binding interfaces. Even simple receptors contain in the region of 100 or more hydrogen bonds, and simple enzymes in the region of 200 or more. Thus, aids to binding and catalysis of many orders of magnitude are expected in the systems discussed here. Indeed, the model for positively cooperative binding (Figure 1(a)) may suggest a reason why enzymes are large. Large systems involve large numbers of non-covalent interactions, and can thereby give rise to large benefits in free energy if portions of the enzyme system undergo a “pseudo-crystallisation” in the transition state for reaction.
Methods Preparation of oxy and deoxy-haemoglobin A 50 mg ml21 solution of horse ferric haemoglobin (Sigma) and 500 mg ml21 of sodium dithionite (Sigma) in elution buffer (100 mM ammonium acetate, 1 mM EDTA, pH 7.6) were prepared. For on-column reduction of ferric haemoglobin, 1 ml of the sodium dithionite solution was injected onto a Pharmacia XK16 column packed with Sephadex G-25 gel (bed volume 60 ml),
397
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followed by a 3 ml wash of elution buffer and 1 ml of ferric haemoglobin solution. The oxygenated protein ¨ KTA explorer FPLC system was eluted using A (Pharmacia Biotech), at a flow rate of 1 ml min21, and collected. For preparation of deoxygenated haemoglobin, the oxy-haemoglobin was first degassed under vacuum to remove most of the soluble oxygen, then 0.8 mg of sodium dithionite was added to 1 ml of degassed haemoglobin column eluent under a nitrogen atmosphere. The oxy and deoxy forms of haemoglobin were both confirmed by spectrophotometric analysis and were used for H/2H exchange studies within 30 hours. Experiments with horse heart myoglobin were carried out in an analogous manner, using 15 mg of the protein (Sigma M-1882). H/2H exchange protocols Streptavidin H/2H exchange was initiated by dilution of 10 ml of a 3 mM solution of streptavidin (Streptomyces avidinii) (Sigma) in 100 mM ammonium acetate buffer (pH 8.0) into 90 ml of 99.9 atom% excess 2H2O, to give a final protein concentration of 300 mM. The complex of streptavidin and D-biotin was formed by incubation of 3 mM streptavidin with a 10 – 20% molar excess of biotin at room temperature for more than one hour prior to dilution into 2H2O. Solutions were maintained at room temperature for H/2H exchange and allowed to exchange for the desired times. At appropriate intervals, 5 ml aliquots of the streptavidin solution were adjusted to pH 2.5 by the addition of 30 ml of chilled acidic quench solution and immediately cooled to 0 8C. A 10 ml aliquot was either loop-injected for ESI-MS, or digested with pepsin for LC-MS, to determine the deuterium content of streptavidin, or its peptic peptides, respectively. Haemoglobin H/2H exchange of haemoglobin was initiated by dilution of 20 ml of ca 20 mM oxygenated/deoxygenated haemoglobin into 180 ml of buffered 2H2O (100 mM ammonium acetate, 1 mM EDTA, p2H 7.6). The solutions were maintained at room temperature and allowed to exchange for the required times. At appropriate time intervals, 16 ml aliquots were removed from the labelling solution and adjusted to pH 2.7 by addition of an equal volume of chilled acidic solution. Quenched samples were immediately frozen in liquid nitrogen and stored at 2 80 8C until analysis by mass spectrometry. The H/2H exchange of myoglobin was achieved in an analogous manner. Mass spectrometry and LC –MS All mass spectra were acquired on a Q-TOF 1 mass spectrometer (Micromass, Manchester, UK) fitted with a Z-spray ion source. For the intact streptavidin experiments, a Harvard model syringe pump was used to infuse a solution of 90:9:1 water/methanol/formic acid (by vol.) to the ESI probe at a rate of 20 ml min21. Protein samples incubated for various time periods were introduced into the solution via a 10 ml loop Rheodyne 7125 injection valve. For deuterated haemoglobin, a 0.3 mm I.D. £ 1 mm C4 guard cartridge (Dionex) was used for desalting and mounted within the injection loop of the Rheodyne injector. The cartridge was equi-
librated with 20 mM ammonium acetate (pH 2.7) prior to 20 ml of sample injection, followed by a 200 ml wash of ice cold 20 mM ammonium acetate (pH 2.7). Haemoglobin was eluted from the C4 cartridge with ice cold 70% acetonitrile, 1% formic acid at a flow rate of 20 ml min21 and delivered to the Q-TOF 1 mass spectrometer. Pepsin digestions were performed on-line by linking a digestion cartridge made by packing a microbore guard column (1 mm £ 20 mm) (Upchurch Scientific) with pepsin Porozyme media (Applied Biosystems) to a Rheodyne 7010 injector coupled with LC – MS. The quenched protein solution was injected onto the pepsin cartridge and left to digest for three minutes at 0 8C. With the injector in the load position, the peptide mixture was then infused at 100 ml min21 through a C18 reversed phase peptide trap (0.3 mm I.D. £ 1 mm C18 guard cartridge (Dionex)) for two minutes using ice cold buffer (10 mM ammonium acetate, 2% acetic acid, pH 2.9). When the injector was switched to inject mode, the peptide trap was subsequently placed in-line with the LC column (PepMap C18, 300 mm £ 5 cm; LC-Packings, Dionex) and peptides were eluted with increasing organic concentration. An LC-Packings Ultimate capillary HPLC (Dionex) was used to generate the gradient, which flowed at 4 ml min21. Solvent A was 0.1% formic acid in H2O and solvent B was 90% acetonitrile containing 9.95% H2O and 0.05% formic acid. The peptic peptides eluted between 3.5 and nine minutes with a five minute 20% –50% B gradient, at which time the gradient was held at 50% B for ten minutes. The column effluent was delivered directly to a nanoflow ESI probe held at 3 kV. For all experiments, the solvents, Rheodyne injectors, peptide trap and HPLC column were all immersed in an ice bath (0 8C) to minimise back-exchange with solvents. To account for deuterium gain or loss under quenched conditions, two control samples were prepared. A “zero deuteration” control was prepared by diluting the protein solution directly into a 1:1 (v/v) mixture of deuterated buffer and quench buffer. A “full-deuteration” control was prepared by incubating streptavidin in 8 M urea-d4 in 2H2O at 55 8C for two hours. The extent of deuterium loss in the peptic peptides was ca 30 – 50%, consistent with previous reports.25,44 The deuterium content of each peptide was calculated after correction for back-exchange, as described.45 Identification of peptides from pepsin digestion Peptides were sequenced by LC – MS/MS following pepsin proteolysis under conditions identical with those used for the deuterium exchange experiments, except that 2H2O was omitted. Switching between MS and MS/ MS was done with automatic switching triggered by the detection of specific peptide ions entered into the Masslynx software as a peak list. Argon was used as the collision gas and collision energies from 32 eV to 35 eV were applied.
Acknowledgements Financial support was provided by Churchill College (to M.Z.), the EPSRC and BBSRC. We thank Dr S. Salisbury and Dr J. Goodman for
398
generous help in analysing the X-ray structures of the horse haemoglobin tetramer.
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18.
References 1. Bo¨hm, H.-J. (1994). The development of a simple scoring function to estimate the binding constant for a protein ligand complex of known 3-dimensional structure. J. Comput. Aided Mol. Des. 8, 243– 256. 2. Kuntz, I. D., Chen, K., Sharp, K. A. & Kollman, P. A. (1999). The maximal affinity of ligands. Proc. Natl Acad. Sci. USA, 96, 9997– 10002. 3. Bryant, R. A. R. & Hansen, D. E. (1996). Direct measurement of the uncatalyzed rate of hydrolysis of a peptide bond. J. Am. Chem. Soc. 118, 5498– 5499. 4. Radzicka, A. & Wolfenden, R. (1995). A proficient enzyme. Science, 267, 90 – 93. 5. Radzicka, A. & Wolfenden, R. (1996). Rates of uncatalyzed peptide bond hydrolysis in neutral solution and the transition state affinities of proteases. J. Am. Chem. Soc. 118, 6105– 6109. 6. Taylor, E. A., Palmer, D. R. J. & Gerlt, J. A. (2001). The lesser “burden borne” by o-succinylbenzoate synthase: an “easy” reaction involving a carboxylate carbon acid. J. Am. Chem. Soc. 123, 5824– 5825. 7. Wolfenden, R., Lu, X. D. & Young, G. (1998). Spontaneous hydrolysis of glycosides. J. Am. Chem. Soc. 120, 6814– 6815. 8. Monod, J., Wyman, J. & Changeux, J-P. (1965). On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 12, 88– 118. 9. Koshland, D. E., Jr, Ne´methy, G. & Filmer, D. (1966). Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry, 5, 365– 385. 10. Biemann, H.-P. & Koshland, D. E., Jr (1994). Aspartate receptors of Escherichia coli and Salmonella typhimurium bind ligand with negative and half-ofthe-sites cooperativity. Biochemistry, 33, 629– 634. 11. Fersht, A. R. (1999). Structure and Mechanism in Protein Science, W.H. Freeman, New York. 12. Williams, D. H., Maguire, A. J., Tsuzuki, W. & Westwell, M. S. (1998). An analysis of the origins of a cooperative binding energy of dimerization. Science, 280, 711 – 714. 13. Bardsley, B. & Williams, D. H. (1998). An illustration of the expression of cooperative binding energy in arrays of non-covalent interactions. Chem. Commun., 2305– 2306. 14. Williams, D. H., Calderone, C. T., O’Brien, D. P. & Zerella, R. (2002). Changes in motion vs. bonding in positively vs. negatively cooperative interactions. Chem. Commun., 1266– 1267. 15. Flatmark, T., Alma˚s, B., Knappskog, P. M., Berge, S. V., Svebak, R. M., Chehin, R. et al. (1999). Tyrosine hydroxylase binds tetrahydrobiopterin cofactor with negative cooperativity, as shown by kinetic analyses and surface plasmon resonance detection. Eur. J. Biochem. 262, 840– 849. 16. Eftink, M. R., Anusiem, A. C. & Biltonen, R. L. (1983). Enthalpy– entropy compensation and heat capacity changes for protein– ligand interactions: general thermodynamic models and data for the binding of nucleotides to ribonuclease A. Biochemistry, 22, 3884– 3896. 17. Weber, P. C., Wendoloski, J. J., Pantoliano, M. W. & Salemme, F. R. (1992). Crystallographic and thermodynamic comparison of natural and synthetic
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ligands bound to streptavidin. J. Am. Chem. Soc. 114, 3197– 3200. Hyre, D. E., Le Trong, I., Freitag, S., Stenkamp, R. E. & Stayton, P. S. (2000). Ser45 plays an important role in managing both the equilibrium and transition state energetics of the streptavidin – biotin system. Protein Sci. 9, 878– 885. Katta, V. & Chait, B. T. (1993). Mass spectrometry – a method for probing protein conformational changes in solution. J. Am. Chem. Soc. 115, 6317– 6321. Meskers, S., Ruysscgaert, J.-M. & Goormaghtigh, E. (1999). Hydrogen– deuterium exchange of streptavidin and its complex with biotin studied by 2Dattentuated total reflection Fourier transform infrared spectroscopy. J. Am. Chem. Soc. 121, 5115– 5122. Weber, P. C., Ohlendorf, D. H., Wendoloski, J. J. & Salemme, F. R. (1989). Structural origins of highaffinity biotin binding to streptavidin. Science, 243, 85 –88. Freitag, S., Le Trong, I., Klumb, L., Stayton, P. S. & Stenkamp, R. E. (1997). Structural studies of the streptavidin binding loop. Protein Sci. 6, 1157–1166. Gonzalez, M., Bagatolli, L. A., Echabe, I., Arrondo, J. L. R., Argarana, C. E., Cantor, C. R. & Fidelio, G. D. (1997). Interaction of biotin with streptavidin—thermostability and conformational changes upon binding. J. Biol. Chem. 272, 11288– 11294. Smith, D. L., Deng, Y. & Zhang, Z. (1997). Probing the non-covalent structure of proteins by amide hydrogen exchange and mass spectrometry. J. Mass Spectrom. 32, 135– 146. Wang, F., Miles, R. W., Kicsa, G., Nieves, E., Schramm, V. L. & Angeletti, R. H. (2000). Immucillin-H binding to purine nucleoside phosphorylase reduces dynamic solvent exchange. Protein Sci. 9, 1660– 1668. Brandts, J. F. & Lin, L. N. (1990). Study of strong to ultra-tight protein interaction using differential scanning calorimetry. Biochemistry, 29, 6927– 6940. Fukuda, H., Sturtevant, J. & Quiocho, F. (1983). Thermodynamics of the binding of L -arabinose and of D -galactose to the L -arabinose-binding protein of Escherichia coli. J. Biol. Chem. 258, 13193– 13198. Shrake, A. & Ross, P. D. (1990). Ligand-induced biphasic protein denaturation. J. Biol. Chem. 265, 5055– 5059. Jones, M. L. & Kurzban, G. P. (1995). Noncooperativity of biotin binding to tetrameric streptavidin. Biochemistry, 34, 11750– 11756. Williams, D. C., Benjamin, D. C., Poljak, R. J. & Rule, G. S. (1996). Global changes in amide hydrogen exchange rates for a protein antigen complex with three different antibiodies. J. Mol. Biol. 257, 866– 876. Freire, E. (1999). The propagation of binding interactions to remote sites in proteins: analysis of the binding of the monoclonal antibiody D1.3 to lysozyme. Proc. Natl Acad. Sci. USA, 96, 10118 – 10122. Johnson, B. A., Wilson, E. M., Li, Y., Moller, D. E., Smith, R. G. & Zhou, G. (2000). Ligand-induced stabilization of PPARg monitored by NMR spectroscopy: implications for nuclear receptor activation. J. Mol. Biol. 298, 187– 194. Perutz, M. F., Wilkinson, A. J., Paoli, M. & Dodson, G. G. (1998). The stereochemical mechanism of the cooperative effects in hemoglobin revisited. Annu. Rev. Biophys. Biomol. Struct. 27, 1 – 34. Colosimo, A., Coletta, M., Falconi, G., Giardina, B., Gill, S. J. & Brunori, M. (1982). Thermodynamics of
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oxygen binding to trout hemoglobin-I and its oxidation intermediates. J. Mol. Biol. 160, 531– 543. Perutz, M. F. (1979). Regulation of oxygen affinity of hemoglobin. Annu. Rev. Biochem. 48, 327– 386. Fersht, A. R. (1999). Structure and Mechanism in Protein Science, W.H. Freeman, New York. Wang, F., Shi, W., Nieves, E., Angeletti, R. H., Schramm, V. L. & Grubmeyer, C. (2001). A transition-state analogue reduces protein dynamics in hypoxanthine-guanine phosphoribosyltransferase. Biochemistry, 40, 8043– 8054. Wolfenden, R., Snider, M., Ridgway, C. & Miller, B. (1999). The temperature dependence of enzyme rate enhancement. J. Am. Chem. Soc. 121, 7419– 7420. Snider, M. J., Gaunitz, S., Ridgway, C., Short, S. A. & Wolfenden, R. (2000). Temperature effects on the catalytic efficiency, rate enhancement, and transition state affinity of cytidine deaminase, and the thermodynamic consequences for catalysis of removing a substrate “anchor”. Biochemistry, 39(32), 9746– 9753. Gilli, P., Ferretti, V., Gilli, G. & Borea, P. A. (1994). Enthalpy– entropy compensation in drug-receptor binding. J. Phys. Chem. 98, 1515– 1518. Zweifel, M. E. & Barrick, D. (2001). Studies of the ankyrin repeats of the Drosophila melanogaster Notch
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receptor. 2. Solution stability and cooperativity of unfolding. Biochemistry, 40, 14357– 14367. Villaverde, J., Cladera, J., Padros, E., Rigaud, J. L. & Dunach, J. L. (1997). Effect of nucleotides on the thermal stability and on the deuteration kinetics of the thermophilic F0F1 ATP synthase. Eur. J. Biochem. 244, 441– 448. McMullen, T. P. W., Lewis, R. N. A. H. & McElhaney, R. N. (2000). Differential scanning calorimetric and Fourier transform infrared spectroscopic studies of the effects of cholesterol on the thermotropic phase behavior and organization of a homologous series of linear saturated phosphatidylserine bilayer membranes. Biophys. J. 79, 2056– 2065. Johnson, R. S. & Walsh, K. A. (1994). Mass spectrometric measurement of protein amide hydrogen– deuterium exchange rates of apo-myoglobin and holo-myoglobin. Protein Sci. 3, 2411 –2418. Zhang, Z. & Smith, D. L. (1993). Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation. Protein Sci. 2, 522–531. Koradi, R., Billeter, M. & Wu¨thrich, K. (1996). MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51 – 55.
Edited by J. Karn (Received 30 January 2003; received in revised form 25 March 2003; accepted 27 March 2003)
J. Mol. Biol. (2003) 329, 389–399
Ligand Binding Energy and Catalytic Efficiency from Improved Packing within Receptors and Enzymes Dudley H. Williams*, Elaine Stephens and Min Zhou Department of Chemistry University of Cambridge Lensfield Road, Cambridge CB2 1EW, UK
Some small molecules bind to their receptors, and transition states to enzymes, so strongly as to defy current understanding. We show that in the binding of biotin to streptavidin, the streptavidin structure becomes better packed. We conclude that this contraction of the streptavidin structure promotes biotin binding. The improved packing is associated with positively cooperative binding, occurring with a benefit in enthalpy and a cost in entropy. Evidence indicating that catalytic efficiency can also originate via improved packing in some enzyme transition states, derived from the work of others, is presented. Negatively cooperative ligand binding is concluded to induce converse effects (less efficient packing, a cost in enthalpy, and a benefit in entropy). It applies to the binding of O2 to haemoglobin, which indeed occurs with a hitherto unreported loosening of the amide backbones of the haemoglobin monomers. q 2003 Elsevier Science Ltd. All rights reserved
*Corresponding author
Keywords: positive cooperativity; negative cooperativity; streptavidin; haemoglobin; myoglobin
Introduction
The models for cooperativity and packing
The affinities with which some small molecules are bound by protein receptors and enzymes are so large as to be regarded as anomalous, or illunderstood. For example, the binding of biotin to streptavidin (K ¼ 1013.4 M21) is typically found to be an “outlier” in comparison with other affinities.1,2 Equally, where the association constants for the binding of transition states to enzymes have been estimated, the values are amazingly high (where examined, lying in the range 1016^4 M21).3 – 7 Here, we show that the binding of biotin to streptavidin can be increased via biotin-induced improvements in the packing of streptavidin. Analogously, in enzyme reactions, substrate transition states can improve the packing within the enzyme, and so increase catalytic efficiency. Evidence to support this concept utilising the data of others (either from very large enthalpies of transition state binding, or reduced dynamic behaviour of the enzyme in the transition state for reaction) is presented for nine enzymes.
The terms positive and negative cooperativity are most commonly used to understand binding involving multiple binding sites.8 In this usage, positively cooperative binding is said to occur when ligands that bind successively are bound with increasing affinities. Negatively cooperative binding is said to occur when successively binding ligands are bound with decreasing affinities. Classic papers by Monod, Wyman, and Changeux (MWC model),8 and by Koshland, Nemethy, and Filmer (KNF theory)9,10 have, respectively, considered the consequences of positively and negatively cooperative binding when there are multiple ligand binding sites in the receptor. Binding is described as positively cooperative where the binding curve for ligand binding is sigmoid in shape (i.e. subsequently bound ligands are bound more strongly than the first-bound ligand). This is the case for O2 binding to haemoglobin.11 Here, we adopt a different definition of cooperative binding. It is more general insofar as it is not confined to proteins with multiple ligand binding sites. A binding event is defined as positively cooperative with respect to a second interaction (or set of interactions) when its affinity is increased in the presence of that second interaction (or set of interactions). Conversely, a binding event is defined as negatively cooperative when its affinity
Abbreviations used: MWC model, Monod, Wyman, and Changeux model; HGPRT, hypoxanthine-guanine phosphoribosyl transferase. E-mail address of the corresponding author: [email protected]
0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved
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is decreased in the presence of that second interaction (or set of interactions). The structural basis of the positively cooperative binding of a ligand to a peptide receptor (as defined in the preceding paragraph) has been investigated using glycopeptide antibiotics of the vancomycin group.12 Positively cooperative binding of the natural ligand to the antibiotic dimers causes greater downfield shifts of a resonance due to a proton at the dimer interface. This observation indicates more efficient packing at the dimer interface due to the positively cooperative binding. Although the isolated dimer has only one noncovalently bonded interface, the dimerisation constants (Kdim) of several antibiotics can be increased up to 100-fold in the presence of two molecules of the natural ligand (e.g. Kdim increases from 105 M21 to 107 M21). Importantly, these antibiotic data show that ligand binding to a receptor system can be enhanced through ligand-induced structural tightening of the receptor system.13 The effect is understandable in the general case if the positively cooperative binding of a ligand to a receptor (analogous to the dimer in the above example) is likened to a cooling of portions of the receptor structure. The binding of the ligand locally reduces the dynamic motions of the residues with which it directly interacts (Figure 1(a)). Since non-covalent bonding is opposed by kinetic energy of motion, these residues are now in turn able to bond better to, and to organise, adjacent residues, and so on, until the effect has spread through the whole of the receptor where positive cooperativity can occur. The enhancement of ligand binding is specifically associated with better packing of the receptor system,12 and the benefits in enthalpy and costs in entropy of positively cooperative binding have been documented.14 The arguments above for effects analogous to cooling parts of a receptor as a model for positive cooperativity can be extended to propose the analogy of warming parts of a receptor as a model for negative cooperativity. In this case, the binding of a ligand is incompatible with the geometry existing in the isolated receptor at the potential ligand binding site (Figure 1(b)) and the making of these ligand/receptor bonds decreases the bonding efficiency of the non-covalent bonds within the receptor. Therefore, the general expectations for negatively cooperative binding of a ligand to a receptor are less efficient packing in the parts of the receptor where the negative cooperativity is expressed, with an associated cost in enthalpy and a benefit in entropy.14 We were encouraged that this model might have general applicability by a study of changes in the properties of tetrameric recombinant human tyrosine hydroxylase isoform 1 upon binding the natural cofactor (6R)-L -erythro-5,6,7,8tetrahydrobiopterin.15 The binding of the cofactor occurs with negative cooperativity, and this cofactor-bound form of the enzyme then shows a decreased resistance to limited tryptic proteolysis,
Improved Protein Packing Aids Binding, Catalysis
Figure 1. Structural models for positive and negative cooperativity, where the lower pair of hydrogen bonds is within a protein receptor and the upper pair of hydrogen bonds is formed upon binding a peptide ligand. (a) Positive cooperativity arises when the dynamic motion of the central chain is reduced by forming hydrogen bonds to the peptide ligand. The central chain then forms a better template for hydrogen bonding to the lower chain and the two sets of hydrogen bonds are strengthened and stabilized. (b) For negative cooperativity, the ligand – receptor interaction demands a distortion of the structure of the receptor. The decrease in bonding will increase the lengths of some of the hydrogen bonds and result in an increase in its dynamic behaviour.
as would be expected from a loosening of the enzyme structure. Although enthalpy/entropy compensation has previously been associated with protein/ligand binding,16 the circumstances under which it will promote, or oppose, ligand binding have not been defined. The work on the glycopeptide antibiotics,12,13 and the models described above, suggest structural consequences of both positively and negatively cooperative binding of a ligand. Here, we have examined the strong binding (K ¼ 1013.4 M21) of biotin to streptavidin to see if the proposed structural consequences of positively cooperative ligand binding are observed. In addition, the properties proposed for negatively cooperative ligand binding are here demonstrated for the binding of O2 to haemoglobin. Evidence is also presented (using the data of others) to show that nine enzymes derive catalytic efficiency through improved packing when they bind to substrate transition states.
Positively Cooperative Ligand Binding: Structural Tightening of Streptavidin upon Biotin Binding Four molecules of biotin bind to a streptavidin tetramer. It seemed that biotin-induced tightening of the streptavidin structure might be occurring, since the binding is remarkably exothermic and adverse in entropy at 25 8C (DH ¼ 2 134 kJ mol21 and TDS ¼ 2 57 kJ mol21,17 or DH ¼ 2 102 kJ mol21 and TDS ¼ 2 26 kJ mol21,18). That is, these
Improved Protein Packing Aids Binding, Catalysis
Figure 2. Time-course showing the extent of backbone amide NH to N2H exchange at pH 8.0 in streptavidin both in the absence (squares) and presence (diamonds) of biotin.
thermodynamic parameters might reflect a promotion of biotin binding because the streptavidin structure is simultaneously stabilised by an improvement in its internal bonding with an accompanying restriction in its dynamic behaviour. The dynamic behaviour of streptavidin upon biotin binding was monitored by well-established methods of hydrogen/deuterium (H/2H) exchange and mass spectrometry.19 The extent of NH to N2H exchange of the intact peptide backbone of streptavidin in the absence, and presence, of biotin was determined by electrospray mass spectrometry (ESI-MS). It was found that in the absence of biotin, each streptavidin (apo-streptavidin, residues 13 –139, from Streptomyces avidinii) underwent exchange of 72 – 74 backbone amide NHs in the time-scale of one to three hours. In the presence of biotin, only 48 – 50 of these NH groups underwent exchange on the same time-scale. Thus, through the binding of biotin, , 24 exchangable amide hydrogen atoms per sub-unit are protected from solvent exchange (Figure 2). It can be considered that the results of this experiment could be interpreted in terms of partial protection of larger numbers of amide NH groups. However, the relative invariance of the degree of exchange after 30 minutes (i.e. the exchange is essentially biphasic, Figure 2) suggests that an “all-or-none” interpretation is a useful approximation. The degree of protection is much larger than previously inferred from FT-IR studies, which indicated the protection of only about ten amide protons.20 A comparison of crystal structures of apostreptavidin and the streptavidin/biotin complex shows some structural ordering in certain regions of the protein when biotin is bound,21 that might account in principle for a portion of the amide NH protection observed in the current study. These regions include two surface loops incorporating residues 45– 50 and 63– 69, that lacked defined density in apo-streptavidin but became ordered upon biotin binding. The former flexible loop (45 – 50) formed hydrogen bonds to biotin and underwent an “open” to “closed” confor-
391
mational change upon biotin binding. Therefore, a reduction in deuterium incorporation could occur in this region when biotin is bound. However, in the X-ray crystal structure of apo-streptavidin obtained by Freitag et al.22 the 45 –50 flexible loop found by Weber et al.21 is structured in a 310 helix and is stabilized in the open conformation. In this case, the number of hydrogen bonds within the loop involving backbone amide NH groups of apo-streptavidin is three in the biotin-free form, and only two in the biotin-bound form.22 Thus, the binding of biotin actually involves the formal removal of one such hydrogen bond in this loop. Furthermore, although biotin forms several hydrogen bonds to streptavidin, these involve only one backbone amide NH of streptavidin.21 Consequently, the protection of 24 backbone amide NH groups upon biotin binding is unexpected on the basis of the X-ray data regarding both hydrogen bonds made to biotin, and within a streptavidin subunit. We next consider whether the X-ray studies can throw any light on the mass spectrometric exchange data in terms of changed interactions between subunits. The extended b-strand that constitutes the C-terminal part of the streptavidin sequence (around Trp120) has a free edge. This free edge is indicated by the X-ray structure of apo-streptavidin to form two hydrogen bonds to the corresponding region of an adjacent subunit, and thus to promote van der Waals interactions between the subunit surfaces.21 Although the X-ray studies by Weber et al. indicated a small adjustment of the inter-subunit geometry upon biotin binding,21 the crystals obtained by Freitag et al. revealed no systematic change in the quaternary structure that can be associated with biotin binding.22 Both studies noted that there was no evidence for subunit cooperativity, e.g. through the formation of extra hydrogen bonds between subunits upon biotin binding. Thus, the protection of 24 backbone amide NH groups upon biotin binding is also unexpected on the basis of the X-ray data for interactions between streptavidin subunits. It is clear that the mass spectrometry experiments, carried out in solution, pick up extensive reductions in the dynamic behaviour of streptavidin upon biotin binding. These changes in dynamic behaviour are not apparent from the X-ray studies, presumably because they are masked by crystal packing forces, and/or they involve distance changes that are not discernible at the resolution of the structure determinations. However, a reduction in the dynamic behaviour of streptavidin upon biotin binding is supported by another solution study,23 where it is shown that the midpoint temperature for thermal denaturation of streptavidin changes from 75 8C to 112 8C upon binding of biotin. This increased stability was attributed to an enhanced inter-subunit association and an increase in structural order due to the organisation of the two disordered loops (45 –50
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Table 1. Deuterium content of streptavidin pepsin digest fragments Residues
Number of NHs in peptide
Apo-streptavidina
Streptavidin/biotin complexa
Deuterium changeb
14–21 22–29 30–42 43–56 59–72 71–78 79–95 102–119 120–124 125–138
7 7 12 13 12 7 16 17 4 12
3.9 3.9 6.4 8.6 11.9 1.3 3.7 6.8 1.9 8.0
2.1 1.5 3.8 3.6 10.7 1.0 1.4 2.4 1.1 7.0
21.8 22.4 22.6 25.0 21.2 20.3 22.3 24.4 20.8 21.0 (Total ¼ 21.8)
a Number of deuterium atoms incorporated after 60 minutes of deuterium exchange. Deuterium levels, determined from centroids of envelopes of isotope peaks, were corrected for deuterium loss during digestion and LC–MS.45 b Changes in deuterium content, relative to apo-streptavidin, when biotin is bound.
and 63 – 69), in the presence of biotin. Importantly, both the current, and the prior solution studies indicate reductions in dynamic behaviour upon ligand binding that are not evident from studies of crystals. To identify the regions of streptavidin that are protected from deuterium exchange by the binding of biotin, pepsin digestion and LC –MS were performed according to established protocols.24,25 Data covering almost the whole of the streptavidin backbone are summarised in Table 1. The peptides that show the greatest reduction in exchange of their backbone amide NH groups in the presence of biotin are 43 –56 (Figure 3(a)) and 22 –29. The
former includes the flexible loop (residues 45 – 50) and, depending upon the time allowed for exchange, the presence of biotin decreases the extent of exchange within it by five to six deuterium atoms (after correction for backexchange during the quenching, pepsin digestion and LC – MS steps, Figure 3(a)). The difference in deuterium content of apo-streptavidin and streptavidin/biotin was calculated for each peptide and expressed as a percentage of the total number of amide backbone NH groups (Figure 3(b)). The data show that the binding of biotin reduces the solvent accessibility of streptavidin backbone NH groups to different degrees throughout essentially
Figure 3. Deuterium incorporation into peptides obtained from pepsin digestion of streptavidin. (a) The level of deuterium incorporation in two peptides from pepsin digestion is shown in the absence (squares) and the presence (diamonds) of biotin as a function of time. (b) Position of peptides in a ribbon model of a streptavidin subunit with bound biotin (Figure prepared with the program MOLMOL46). The degree of NH protection in the presence of biotin is indicated by colour. Reduction in exchange . 30% (red), 20 – 30% (yellow), 10 – 20% (light blue), and ,10% (dark blue). Two regions not represented in peptic peptides are indicated in grey.
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the whole structure. The number of backbone amide NH groups protected from exchange by the presence of biotin, as assessed from analysis of the whole protein, is 24. This is in remarkably good agreement with that (21.8, Table 1) assessed from analysis of the available peptides (which cover 94% of the backbone). Relationships can be seen between the extent of mobility within a region of an apo-streptavidin subunit before biotin binding, the distance of this region from the biotin binding site, and the degree of tightening upon biotin binding. (i) The region of apo-streptavidin that undergoes least exchange (residues 71– 95, Table 1) shows little further protection from exchange upon biotin binding, despite being only moderately distant from the biotin binding site (Figure 3(b)). This region is found at the subunit interface and is stabilised by van der Waals interactions between the subunit barrel surfaces.21 According to our data, these tight binding regions of apo-streptavidin cannot further restrict their dynamic behaviour to give additional benefits in bonding upon biotin binding. (ii) The region of apo-streptavidin (residues 59– 72, Table 1) that is not only distant from the binding site of biotin, but also encompasses a loop that is outside the barrel structure (Figure 3(b)), undergoes the greatest exchange but shows little further protection from exchange upon biotin binding. Interestingly, this region incorporates the loop spanning residues 63 –69 that lacks defined density in the X-ray structure of apo-streptavidin, but becomes more ordered upon biotin binding.21 Our data suggest that this loop remains relatively mobile in the bound state in solution. This is presumably because of the lack of an efficient cooperative pathway from biotin to restrict its motion. (iii) All other parts of the protein that we have been able to examine (which constitute 63% of the residues) undergo extensive exchange (40 –67%) of their backbone NH groups prior to biotin binding, which is considerably reduced after binding (to the range 21 –58%). All these dynamic regions therefore have their mobility restricted by biotin binding. Biotin binding is particularly enhanced by improvement in the bonding within the four strands of the b-sheet and its attendant loops indicated in red (Figure 3(b)), and within the extensions of this sheet (Figure 3(b), yellow). The improved packing of the protein in the 102 – 124 region is also marked. The major part of the improved packing is within the subunits rather than between them. A useful analogy is to think of the apo-streptavidin tetramer as behaving like an impure crystal. When an impurity is removed from a crystal, the noncovalent bonding within it typically becomes more
positively cooperative and the melting transition of the crystal becomes higher and sharper. Biotin apparently acts as the antithesis of an impurity when it binds to apo-streptavidin. It causes the cooperative improvement of bonding, to varying degrees, throughout the streptavidin system, and the increase in the tm from 75 8C to 112 8C upon biotin binding23 is naturally accommodated. Biotin stabilises the structure of streptavidin, and this extensive structural stabilisation contributes to the affinity between biotin and streptavidin. We note that the binding of ligands to proteins often increases the thermal stability of the protein,26 – 28 and therefore the enhancement of ligand binding in this way is not uncommon. Jones & Kurzban have provided persuasive evidence that the binding affinity for biotin to the streptavidin tetramer, containing zero, one, two and three molecules of biotin is essentially identical.29 Therefore, the binding is not positively cooperative in the MWC sense. However, since biotin binding clearly tightens (the present work), and stabilises,23 the streptavidin structure, it is positively cooperative in terms of the definition used here (Figure 1(a)). Our data, taken in conjunction with those of Jones & Kurzban,29 indicate that upon biotin binding into each subunit, this part of the 4-fold symmetrical structure is tightened to essentially the same extent. That is, the degree of positive cooperativity is essentially the same for all four binding events. Previously, other solution studies have also noted structural stabilisation of protein receptors upon ligand binding that were not detected within the crystallographic resolution of their X-ray structures. A reduction in amide exchange rates of a protein antigen (lysozyme) upon binding any one of three antibodies has been observed.30 Theoretical considerations indicate that the binding of the antibodies stabilises the lysozyme structure.31 Also, NMR studies show that agonist binding to the peroxisome proliferator-activated receptor (PPAR)g causes a marked reduction in the conformational mobility of the receptor.32 The reductions in the dynamic behaviour of these proteins must enhance the ligand/receptor binding through positive cooperativity.12,13
Negatively Cooperative Ligand Binding: Structural Loosening of Haemoglobin upon O2 Binding We note that in the MWC model8 for O2 binding to haemoglobin (Figure 4), the O2 binding is positively cooperative because much of the work required for the T ! R conversion is effected by the first O2 to bind. Therefore, subsequently binding O2 molecules have the advantage to access a relatively high population of the R state, and bind with greater affinity (hence the binding is considered to be positively cooperative). However, in
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terms of the definition used here, negatively cooperative ligand binding occurs when O2 binds to the T state. The optimal binding of O2 is incompatible with the geometry existing in the T state. In analogy to Figure 1(b), the ligand therefore distorts the structure of the receptor. This distorted O2-bound structure is the R state. The negatively cooperative ligand binding does indeed force a loosening of the haemoglobin tetramer, through the breaking of inter-subunit salt bridges (as established by the classic work of Perutz and co-workers),33 to give the R state. However, and more importantly, widespread structural changes in the T to R transition seem possible. All noncovalent interactions within a receptor system that are coupled with negative cooperativity to ligand binding should loosen. It appeared possible from prior thermodynamic data that the properties proposed for negatively cooperative binding of a ligand might be displayed by the haemoglobin/O2 system. Thus, in the case of trout haemoglobin,34 binding of the first O2 ligand (when presented with the T state) occurs with an exothermicity very near zero, and a favourable TDS term of þ21 kJ mol21. However, subsequent O2 binding (to the R state) occurs with a large exothermicity (DH ¼ 232 kJ mol21) and slightly unfavourable entropy (TDS ¼ 23 kJ mol21). These thermodynamic data suggest that in the binding of the first ligand molecule, work must be done to drive the T to R state conversion, so that there is no significant net enthalpic benefit, despite the bonding of O2 to haemoglobin. Instead, the initial binding is driven by the fact that the R state is favourable in entropy (less well packed, i.e. looser) than the T state from which it is formed. Once the R state tetramer is more highly populated (as a result of the initial binding O2 binding (Figure 4)), binding to the R state is enthalpy driven (data above). To test the above inferences, we determined the change in dynamic behaviour of the horse haemoglobin tetramer peptide backbone when it binds O2. In experiments analogous to those described above for streptavidin, the extent of NH to N2H exchange of the peptide backbone of the a and b-chains of horse haemoglobin in the absence, and presence, of bound oxygen was determined by ESI-MS (Figure 5). In the absence of bound oxygen,
Figure 4. The MWC model for the binding of a ligand (L, here O2) to a tetrameric protein (haemoglobin) able to equilibrate between tense (T) and relaxed (R) forms.
Improved Protein Packing Aids Binding, Catalysis
Figure 5. Time-course showing the extent of backbone amide NH to N2H exchange in the a (triangles) and b (squares) subunits of oxygenated (filled) and deoxygenated (empty) haemoglobin.
the a-chains underwent exchange of 25 –27 backbone amide NH groups (out of a total of 134), and the b-chains underwent exchange of 24 –27 backbone amide NH groups (out of a total of 140) in the time-scale of half to two hours. In the presence of bound oxygen, the a-chains underwent exchange of 33 – 34, and the b-chains underwent exchange of 40– 43 in the same time-scale. Therefore, through the binding of oxygen, a further seven to eight exchangeable amide hydrogen atoms per a-chain (5.2 –6%), and a further 16 per b-chain (11.4%) are exposed to solvent exchange. Thus, complete saturation of the haemoglobin tetramer by O2 binding results in an increase of 46 –48 in the number of backbone NH groups undergoing exchange. X-ray structures of the haemoglobin tetramer were examined for the presence of any inter-subunit hydrogen bonds involving peptide backbone NH groups. Such hydrogen bonds are not evident, and therefore are not available to be broken in the T to R conversion. We conclude that the increase of 46– 48 in the number of backbone NH groups undergoing exchange upon O2 binding must reflect an increase in the internal dynamic behaviour of the subunits. These changes have not been described in prior work presumably, in the case of X-ray studies, because changes in dynamic behaviour can be masked by crystal packing forces. The conclusion is reinforced by the similar findings for myoglobin (which does not form a tetrameric structure) upon O2 binding (see later). The increase in amide NH exchange upon O2 binding to haemoglobin is opposite to the effect induced by biotin binding to streptavidin, and is in agreement with our predictions regarding the changes associated with negatively cooperative ligand binding. Thus, the relatively poor binding of O2 to the T form of haemoglobin is not simply due to the free energy cost of loosening the noncovalent bonding between its subunits.33 It is also due to extensive loosening of the non-covalent interactions within the haemoglobin subunits. We note that the structural loosening involving the
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b-chains is much greater than of the a-chains. In this connection it is interesting to note that although all four subunits are distorted by the displacement of iron and its proximal histidine upon O2 binding, only in the b-subunits is the distal valine displaced.35 This observation is consistent with the greater expression of negative cooperativity within these b-subunits. Analogous experiments on myoglobin were clearly of interest, since it exists as a monomeric species. In the original MWC paper, it was assumed that “the subunits of the R form are closer to the conformation of the (haemoglobin) monomer” (cf. myoglobin) and that “myoglobin may be thought of as a relaxed subunit of haemoglobin”.8 According to this assumption, oxygen binds strongly to myoglobin, at least in part, because it does not have to pay the price of the T to R conversion (Figure 4). However, it is important to recall that the R structure has been shown above to be internally loosened relative to the T state, and this internal loosening must be driven by O2 binding. Thus, the myoglobin structure might also be loosened internally by O2 binding and, in its isolated form, may not be analogous to “a relaxed subunit of haemoglobin”.8 The extent of NH to N2H exchange of the peptide backbone of horse heart myoglobin in the absence, and presence, of bound oxygen was determined by ESI-MS. In the absence of bound oxygen, myoglobin underwent exchange of 27 –30 backbone amide NH groups (out of a total of 148) in the time-scale of half to two hours. In the presence of bound oxygen, it underwent exchange of 40 –45 in the same timescale. Thus, through the binding of oxygen, a further 13 – 15 exchangeable amide hydrogen atoms are exposed to solvent exchange. Myoglobin thus possesses a relatively tight (or “tense”) internal structure, and not a relaxed one as originally seemed likely.8 This conclusion makes physical sense for, as Perutz commented,35 it is the binding of O2 that perturbs the protein structure. Using the model described here (Figure 1(b)), both myoglobin and the T form of haemoglobin bind O2 with negative cooperativity, and the reduction in binding energy of the O2 to these proteins is spread among the network of non-covalent interactions.
exchange into backbone amide bonds in hypoxanthine-guanine phosphoribosyl transferase (HGPRT) 37 and purine nucleoside phosphorylase25 was used to compare the dynamic properties of the enzymes alone, in forms with bound reactant/ product, and in forms with bound transition-state analogues. In both cases, it was found that the rate and extent of deuterium incorporation decreased when the reactant/product was bound, and decreased to an even greater extent when the transition state analogue was bound. Thus, the greatest reduction in dynamic motion is caused by the transition state analogue. The effects are large: the binding of the transition state analogue protects 34 peptide backbone NH groups from exchange in the case of HGPRT, and 27 peptide backbone NH groups are similarly protected in the case of purine nucleoside phosphorylase. Our proposals indicate that both enzymes will provide binding energy for the reactant/product through becoming better packed. Crucially, even greater binding energy will be provided for the transition state analogue when the packing is further improved. Structural tightening of an enzyme when it binds a substrate transition state should have a further testable consequence. The benefit in entropy of enzyme catalysis (originating in the ordering of the catalytic groups on the enzyme structure) should be offset by the cost in entropy of reducing the dynamic behaviour of the enzyme in the transition state. Moreover, this proposed reduction in dynamic behaviour of the enzyme should provide a large benefit in improved non-covalent bonding (enthalpy) within the enzyme. Strikingly, where data are available, enzyme-catalysed reactions are, relative to the corresponding reaction in solution, greatly favoured in enthalpy (Table 2).4,38 In the case of cytidine deaminase, both the enthalpic and entropic contributions have been derived. Enzyme catalysis increases the reaction rate by 1016 M21, due to a benefit in enthalpy (DDH –) of 2 84 kJ mol21, and a benefit in entropy (TDDS –) of only 7 kJ mol21.39 Thus, where the second consequence of our model for the enhancement of catalysis through structural tightening of the enzyme can be tested, the available data are also in excellent accord with its requirements.
Implications for Enzyme Catalysis Our findings appear likely to have implications for the understanding of enzyme catalysis. Enzymes bind the transition states (S–) of substrates more strongly than they bind the substrates.36 According to the positive cooperativity model (Figure 1(a)), one way to effect this is through a greater degree of structural tightening of the enzyme upon binding the substrate transition state than upon binding the substrate. Two recent studies, as interpreted here, give convincing support to this idea. Hydrogen/deuterium (H/2H)
Table 2. Benefit in enthalpy (DDH –) of some enzymecatalysed reactions relative to the reactions in free solution4,38 Enzyme Chorismate dismutase Chymotrypsin Staphylococcal nuclease Bacterial a-glucosidase Urease Yeast OMP decarboxylase
DDH – (kJ mol21)
Rate accel. (s21) due to DDH –
233 266 263 280 293 2143
106 1012 1011 1014 1016 1025
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Conclusion Ligand binding causes a tightening of a glycopeptide receptor structure, and there is an associated increase in the free energy of binding of the ligand.12,13 Structural arguments suggest that this effect should be general. Thus, non-covalent interactions within protein receptors can be improved upon ligand binding to provide ligand binding energy. Evidence has been provided to show that the streptavidin structure tightens extensively upon biotin binding, and that this tightening will enhance biotin binding. In contrast, the binding of O2 to the T form of haemoglobin, and to myoglobin, is (using the definitions employed here, and their operational consequences) negatively cooperative. The internal structures of haemoglobin and myoglobin are loosened upon O2 binding, adding to our knowledge of why O2 binding to the T state of haemoglobin is less favourable than to the R state. Data for nine enzymes support the idea that the efficiency of enzyme catalysis is improved through the tightening of enzyme structures. Ligand binding to some receptors may of course occur with positive cooperativity to some portions of the receptor, and with negative cooperativity to other portions. Thus, it is a large net effect of ligand binding on receptor dynamics (as in the examples cited here) that will be a sign of a relatively large net positively or negatively cooperative effect. An intrinsic relationship between cooperative binding and enthalpy/entropy compensation accounts naturally for the occurrence of “tense” and “relaxed” systems.8 Negatively cooperative binding to a tense form of a receptor can drive it to a more relaxed state with a cost in enthalpy and a benefit in entropy. Since positively cooperative binding can occur with a benefit in enthalpy and a cost in entropy, and Nature utilises both positively and negatively cooperative binding, large variations in the values of these thermodynamic parameters for ligand – receptor interactions can be expected. The thermodynamics of binding of 136 drugs to biological receptors has been summarised in a graph of DH and DS.40 Some associations are endothermic by quantities in the region of 125 kJ mol21 (and therefore are likely to be negatively cooperative). At the other extreme, some are exothermic by ca 80 kJ mol21. In comparing the extremes, the differences are indeed remarkably large (ca 205 kJ mol21) as required for opposing forms of cooperativity. We note that the structural changes described here for positively and negatively cooperative binding may well be a general property of aggregated states, and therefore widespread. Additional examples are found among proteins. For example, addition of a seventh Notch ankyrin domain increases the folding equilibrium by 1000-fold (positive cooperativity), and increases the enthalpy of unfolding by ca 50%.41 The F0F1-type ATP synthase from the thermophilic bacillus PS3 binds
Improved Protein Packing Aids Binding, Catalysis
Mg-ADP with an increase in the thermal stability of the protein and a concomitant decrease in its flexibility, whereas the binding of Mg-ATP reduces its thermal stability and simultaneously increases its flexibility.42 Therefore the structural changes within the protein should enhance the binding of Mg-ADP and reduce the affinity for Mg-ATP at the binding sites probed in this work. Although these particular binding sites are reported to have a regulatory, rather than a direct catalytic function, the association of increased protein stability with decreased flexibility, and of decreased protein stability with increased flexibility, are in accord with the conclusions reached here. The characteristics of negative cooperativity are observed when “non-matching” substances are introduced into lipid bilayers. Thus, the incorporation of increasing quantities of cholesterol progressively reduces the positive cooperativity, the temperature, and enthalpy of the gel-to-liquid-crystalline phase transition of lipid bilayers constituted from phosphatidylserine.43 When pure, the lipid bilayer forms a positively cooperative aggregate. The binding of cholesterol into this bilayer is negatively cooperative because the cholesterol disrupts the large domains of positively cooperative interactions that previously existed within the bilayer. The changes in the property of the bilayer upon introduction of cholesterol therefore follow the changes posited here for negative cooperativity. Can the structural tightening of receptors and enzymes discussed here plausibly give rise to large increases in binding constants of ligands, or large increases in rates of catalysis? The glycopeptide dimeric receptors are stabilised by a factor of 100 when the system is elaborated by ligand binding.12 The elaborated system contains only 16 hydrogen bonds, plus a limited number of hydrophobic interactions, at its binding interfaces. Even simple receptors contain in the region of 100 or more hydrogen bonds, and simple enzymes in the region of 200 or more. Thus, aids to binding and catalysis of many orders of magnitude are expected in the systems discussed here. Indeed, the model for positively cooperative binding (Figure 1(a)) may suggest a reason why enzymes are large. Large systems involve large numbers of non-covalent interactions, and can thereby give rise to large benefits in free energy if portions of the enzyme system undergo a “pseudo-crystallisation” in the transition state for reaction.
Methods Preparation of oxy and deoxy-haemoglobin A 50 mg ml21 solution of horse ferric haemoglobin (Sigma) and 500 mg ml21 of sodium dithionite (Sigma) in elution buffer (100 mM ammonium acetate, 1 mM EDTA, pH 7.6) were prepared. For on-column reduction of ferric haemoglobin, 1 ml of the sodium dithionite solution was injected onto a Pharmacia XK16 column packed with Sephadex G-25 gel (bed volume 60 ml),
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followed by a 3 ml wash of elution buffer and 1 ml of ferric haemoglobin solution. The oxygenated protein ¨ KTA explorer FPLC system was eluted using A (Pharmacia Biotech), at a flow rate of 1 ml min21, and collected. For preparation of deoxygenated haemoglobin, the oxy-haemoglobin was first degassed under vacuum to remove most of the soluble oxygen, then 0.8 mg of sodium dithionite was added to 1 ml of degassed haemoglobin column eluent under a nitrogen atmosphere. The oxy and deoxy forms of haemoglobin were both confirmed by spectrophotometric analysis and were used for H/2H exchange studies within 30 hours. Experiments with horse heart myoglobin were carried out in an analogous manner, using 15 mg of the protein (Sigma M-1882). H/2H exchange protocols Streptavidin H/2H exchange was initiated by dilution of 10 ml of a 3 mM solution of streptavidin (Streptomyces avidinii) (Sigma) in 100 mM ammonium acetate buffer (pH 8.0) into 90 ml of 99.9 atom% excess 2H2O, to give a final protein concentration of 300 mM. The complex of streptavidin and D-biotin was formed by incubation of 3 mM streptavidin with a 10 – 20% molar excess of biotin at room temperature for more than one hour prior to dilution into 2H2O. Solutions were maintained at room temperature for H/2H exchange and allowed to exchange for the desired times. At appropriate intervals, 5 ml aliquots of the streptavidin solution were adjusted to pH 2.5 by the addition of 30 ml of chilled acidic quench solution and immediately cooled to 0 8C. A 10 ml aliquot was either loop-injected for ESI-MS, or digested with pepsin for LC-MS, to determine the deuterium content of streptavidin, or its peptic peptides, respectively. Haemoglobin H/2H exchange of haemoglobin was initiated by dilution of 20 ml of ca 20 mM oxygenated/deoxygenated haemoglobin into 180 ml of buffered 2H2O (100 mM ammonium acetate, 1 mM EDTA, p2H 7.6). The solutions were maintained at room temperature and allowed to exchange for the required times. At appropriate time intervals, 16 ml aliquots were removed from the labelling solution and adjusted to pH 2.7 by addition of an equal volume of chilled acidic solution. Quenched samples were immediately frozen in liquid nitrogen and stored at 2 80 8C until analysis by mass spectrometry. The H/2H exchange of myoglobin was achieved in an analogous manner. Mass spectrometry and LC –MS All mass spectra were acquired on a Q-TOF 1 mass spectrometer (Micromass, Manchester, UK) fitted with a Z-spray ion source. For the intact streptavidin experiments, a Harvard model syringe pump was used to infuse a solution of 90:9:1 water/methanol/formic acid (by vol.) to the ESI probe at a rate of 20 ml min21. Protein samples incubated for various time periods were introduced into the solution via a 10 ml loop Rheodyne 7125 injection valve. For deuterated haemoglobin, a 0.3 mm I.D. £ 1 mm C4 guard cartridge (Dionex) was used for desalting and mounted within the injection loop of the Rheodyne injector. The cartridge was equi-
librated with 20 mM ammonium acetate (pH 2.7) prior to 20 ml of sample injection, followed by a 200 ml wash of ice cold 20 mM ammonium acetate (pH 2.7). Haemoglobin was eluted from the C4 cartridge with ice cold 70% acetonitrile, 1% formic acid at a flow rate of 20 ml min21 and delivered to the Q-TOF 1 mass spectrometer. Pepsin digestions were performed on-line by linking a digestion cartridge made by packing a microbore guard column (1 mm £ 20 mm) (Upchurch Scientific) with pepsin Porozyme media (Applied Biosystems) to a Rheodyne 7010 injector coupled with LC – MS. The quenched protein solution was injected onto the pepsin cartridge and left to digest for three minutes at 0 8C. With the injector in the load position, the peptide mixture was then infused at 100 ml min21 through a C18 reversed phase peptide trap (0.3 mm I.D. £ 1 mm C18 guard cartridge (Dionex)) for two minutes using ice cold buffer (10 mM ammonium acetate, 2% acetic acid, pH 2.9). When the injector was switched to inject mode, the peptide trap was subsequently placed in-line with the LC column (PepMap C18, 300 mm £ 5 cm; LC-Packings, Dionex) and peptides were eluted with increasing organic concentration. An LC-Packings Ultimate capillary HPLC (Dionex) was used to generate the gradient, which flowed at 4 ml min21. Solvent A was 0.1% formic acid in H2O and solvent B was 90% acetonitrile containing 9.95% H2O and 0.05% formic acid. The peptic peptides eluted between 3.5 and nine minutes with a five minute 20% –50% B gradient, at which time the gradient was held at 50% B for ten minutes. The column effluent was delivered directly to a nanoflow ESI probe held at 3 kV. For all experiments, the solvents, Rheodyne injectors, peptide trap and HPLC column were all immersed in an ice bath (0 8C) to minimise back-exchange with solvents. To account for deuterium gain or loss under quenched conditions, two control samples were prepared. A “zero deuteration” control was prepared by diluting the protein solution directly into a 1:1 (v/v) mixture of deuterated buffer and quench buffer. A “full-deuteration” control was prepared by incubating streptavidin in 8 M urea-d4 in 2H2O at 55 8C for two hours. The extent of deuterium loss in the peptic peptides was ca 30 – 50%, consistent with previous reports.25,44 The deuterium content of each peptide was calculated after correction for back-exchange, as described.45 Identification of peptides from pepsin digestion Peptides were sequenced by LC – MS/MS following pepsin proteolysis under conditions identical with those used for the deuterium exchange experiments, except that 2H2O was omitted. Switching between MS and MS/ MS was done with automatic switching triggered by the detection of specific peptide ions entered into the Masslynx software as a peak list. Argon was used as the collision gas and collision energies from 32 eV to 35 eV were applied.
Acknowledgements Financial support was provided by Churchill College (to M.Z.), the EPSRC and BBSRC. We thank Dr S. Salisbury and Dr J. Goodman for
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generous help in analysing the X-ray structures of the horse haemoglobin tetramer.
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18.
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Edited by J. Karn (Received 30 January 2003; received in revised form 25 March 2003; accepted 27 March 2003)