- Email: [email protected]
Cooperative hemoglobins: conserved fold, diverse quaternary assemblies and allosteric mechanisms
Review
40 Ponting, C.P. and Phillips, C. (1997) Identification of homer as a homologue of the Wiskott–Aldrich syndrome protein suggests a receptor-binding function for WH1 domains. J. Mol. Med. 75, 769–771 41 Sun, J. et al. (1998) Isolation of PSD-Zip45, a novel Homer/vesl family protein containing leucine zipper motifs, from rat brain. FEBS Lett. 437, 304–308 42 Xiao, B. et al. (1998) Homer regulates the association of group 1 metabotropic glutamate receptors with multivalent complexes of homerrelated, synaptic proteins. Neuron 21, 707–716 43 Tu, J.C. et al. (1998) Homer binds a novel proline-rich motif and links group 1 metabotropic glutamate receptors with IP3 receptors. Neuron 21, 717–726 44 Tu, J.C. et al. (1999) Coupling of mGluR/Homer and PSD-95 complexes by the Shank family of postsynaptic density proteins. Neuron 23, 583–592 45 Naisbitt, S. et al. (1999) Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin. Neuron 23, 569–582
TRENDS in Biochemical Sciences Vol.26 No.5 May 2001
46 Weinman, E.J. et al. (1998) Structure-function of recombinant Na/H exchanger regulatory factor (NHE-RF). J. Clin. Invest. 101, 2199–2206 47 Hall, R.A. et al. (1998) A C-terminal motif found in the β2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins. Proc. Natl. Acad. Sci. U. S. A. 95, 8496–8501 48 Hall, R.A. et al. (1999) G protein-coupled receptor kinase 6A phosphorylates the Na(+)/H(+) exchanger regulatory factor via a PDZ domain-mediated interaction. J. Biol. Chem. 274, 24328–24334 49 Hall, R.A. et al. (1998) The β2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange. Nature 392, 626–630 50 Liu, X. et al. (1999) Activation of a frizzled-2/β-adrenergic receptor chimera promotes Wnt signaling and differentiation of mouse F9 teratocarcinoma cells via Gαo and Gαt. Proc. Natl. Acad. Sci. U. S. A. 96, 14383–14388
297
51 Liu, T. et al. (1999) Activation of rat frizzled-1 promotes Wnt signaling and differentiation of mouse F9 teratocarcinoma cells via pathways that require Galpha(q) and Galpha(o) function. J. Biol. Chem. 274, 33539–33544 52 Sheldahl, L.C. et al. (1999) Protein kinase C is differentially stimulated by Wnt and Frizzled homologs in a G-protein-dependent manner. Curr. Biol. 9, 695–698 53 Kuhl, M. et al. (2000) Ca(2+)/calmodulindependent protein kinase II is stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in Xenopus. J. Biol. Chem. 275, 12701–12711 54 Wu, C. et al. (2000) RGS proteins inhibit Xwnt-8 signaling in Xenopus embryonic development. Development 127, 2773–2784 55 Lefkowitz, R.J. (2000) The superfamily of heptahelical receptors. Nat. Cell Biol. 2, E133–E136 56 Luttrell, L.M. et al. (2001) Activation and targeting of extracellular signal-regulated kinases by β-arrestin scaffolds. Proc. Natl. Acad. Sci. U. S. A. 98, 2449–2453
Cooperative hemoglobins: conserved fold, diverse quaternary assemblies and allosteric mechanisms William E. Royer, Jr, James E. Knapp, Kristen Strand and Holly A. Heaslet Assembly of hemoglobin subunits into cooperative complexes produces a remarkable variety of architectures, ranging in oligomeric state from dimers to complexes containing 144 hemoglobin subunits. Diverse stereochemical mechanisms for modulating ligand affinity through intersubunit interactions have been revealed from studies of three distinct hemoglobin assemblages. This mechanistic diversity, which occurs between assemblies of subunits that have the same fold, provides insight into the range of regulatory strategies that are available to protein molecules.
William E. Royer, Jr* James E. Knapp Kristen Strand Holly A. Heaslet Dept of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA. *e-mail: William.Royer@ umassmed.edu
For nearly a century, mammalian tetrameric hemoglobin (composed of two α and two β subunits) has been a major paradigm for the allosteric regulation of protein function. Experiments in the early 1900s by Christian Bohr and others demonstrated that the oxygen binding curve of hemoglobin was sigmoidal rather than hyperbolic; thus, oxygen binding is cooperative, with a stepwise increase in oxygen affinity as the binding reaction proceeds. A structural explanation for this cooperativity was proposed in 1935 by Linus Pauling, who suggested that binding of oxygen triggers an interaction between adjacent heme groups1. A major breakthrough in our understanding of cooperativity came from the
pioneering studies of Max Perutz, whose structures of mammalian hemoglobin showed that the heme groups are actually too far apart for the type of direct interaction envisioned by Pauling2. Perutz also demonstrated that mammalian hemoglobins can exist in alternate quaternary structures depending on ligand state3. These structures provided the framework for our understanding of the cooperative ligand binding that arises from the transition between a low-affinity state and higher-affinity state(s) as ligand binding proceeds. In contrast to the conserved tetrameric hemoglobin structure found in most vertebrates, a remarkable diversity in quaternary assemblage is observed among invertebrate and primitive vertebrate hemoglobins, despite conserved tertiary structures of the subunits. To achieve intersubunit communication, the diverse assemblages use a variety of mechanisms, comparisons of which reveal several broad principles that define the regulation of ligand affinity. Specifically, ligand affinity is modulated by limiting access of the ligand to the heme pocket, by altering the chemical reactivity of the heme iron, or by stabilizing the bound ligand by
http://tibs.trends.com 0968-0004/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0968-0004(01)01811-4
Review
298
TRENDS in Biochemical Sciences Vol.26 No.5 May 2001
Diversity in binding affinity
(a)
(b)
Sperm whale myoglobin
Ascaris hemoglobin, domain 1
(c)
Paramecium hemoglobin
Fig. 1. Structures of three myoglobin-fold monomers. For each protein, a ribbon diagram is shown with the amino-terminal helices in light gray, E and F helices in cyan, and the carboxy terminal G and H helices in dark gray. Heme groups are shown in red, proximal His residues (F8) in cyan, and oxygen ligands in yellow. Residues at distal positions E7 (His or Gln), B10 (Leu or Tyr) and CD1 (Phe) are shown in cyan, magenta and purple, respectively. (By convention, residues are designated by their position within a helix or corner, based on the myoglobin structure. Therefore, the proximal His is always F8, even though, for instance, in Ascaris hemoglobin it is the 12th residue along the F helix.) Oxygen affinities differ dramatically among these hemoglobins, and can be designated by p50 values; that is, the partial pressure of oxygen at half-saturation. (a) Sperm whale myoglobin (PDB code 1mbo)34 (p50 = 0.5 Torr). (b) Ascaris hemoglobin domain (1ash)8. Ascaris hemoglobin has one of the highest known oxygen affinities (p50 = 0.004 Torr), which results primarily from ligand stabilization resulting from hydrogen bonds contributed by Tyr B10 and Gln E7. (c) Paramecium hemoglobin (1dlw)35. This ‘truncated hemoglobin’ (~75% the length of myoglobin) is missing part of the A helix, most of the CD corner and D helix, and much of the F helix, yet it still provides a proximal His in the single turn F helix and an environment for reversible oxygen binding (p50 = 0.5 Torr). This figure was produced using the program MIDAS (Ref. 36).
interaction with neighboring residues. This review focuses on how subunit assembly regulates ligand affinity and cooperativity using one or more of these strategies. Tertiary structures
The first two protein crystal structures to be determined were those of sperm whale myoglobin4 and horse hemoglobin2. The structural similarity between the hemoglobin α and β subunits and myoglobin provided the initial evidence of structural conservation in the globin family and suggested that this tertiary structure, the myoglobin fold, optimally uses a heme group for reversible oxygen binding. Since then, the myoglobin fold has been found in proteins widely distributed among bacteria, plants, invertebrates and vertebrates. The classic myoglobin fold consists of seven or eight α helices encapsulating a heme prosthetic group, with helices (A–H) being connected by corners (e.g. the AB corner connects helices A and B) (Fig. 1). The heme iron is coordinated to five ligands in hemoglobin – four pyrrole nitrogen atoms and a strictly conserved proximal His F8 (i.e. the eighth residue in the F helix of sperm whale myoglobin) – leaving the sixth site available to bind oxygen on the distal face of the heme. A second conserved residue is Phe CD1 (first residue in the CD corner), which helps to wedge the heme in the protein. Two other key residues are distal residues E7 and B10 (Fig. 1), both of which can play crucial roles in stabilizing the bound oxygen. Although the hemoglobins shown in Fig. 1 have significant structural differences, they all provide an environment for reversible binding of oxygen to the heme group. http://tibs.trends.com
The proteins shown in Fig. 1 bind oxygen with drastically different affinities. Reported oxygen dissociation rate constants of hemoglobins range over six orders of magnitude from approximately 10–3 to 103 sec–1, whereas association rate constants vary over three orders of magnitude from approximately 105 to 108 M–1sec–1 (Ref. 5). How are such large differences achieved? Contributions of ligand access and distal stabilization to ligand affinity were highlighted in studies using mutants of sperm whale myoglobin. Several groups have investigated ligand access in sperm whale myoglobin, one study involving the generation of 1500 mutants by random mutagenesis6. Recently, Olson and his colleagues have analyzed 90 mutations at 27 positions that affect ligand access and stability7. The oxygen affinities of these mutants vary over three orders of magnitude. Decreases in affinity are observed when the distal His (E7) is replaced with residues that do not form hydrogen bonds with bound oxygen (e.g. H64V) or when access to the ligand-binding site is hindered (e.g. V68I). Increases in the affinity are observed in L29F (B10) as the Phe ring stabilizes the bound ligand. A more dramatic illustration of the power of ligand stabilization is observed in the high-affinity hemoglobin from the nematode parasite, Ascaris suum. This hemoglobin is assembled as an octamer of 33-kDa chains, each of which consists of two hemoglobin domains. The high affinity (Kd = 4 × 10–9 M) results primarily from an extremely slow dissociation rate of 0.004 sec–1 (Ref. 5). How is oxygen held so tightly? Clues come from the structure of an isolated domain that binds oxygen with the same affinity as does the non-cooperative octamer (Fig. 1b). Major ligand stabilization appears to result from favorable hydrogen bonding of Tyr B10 and Gln E7 with bound oxygen in the distal pocket8. The proximal His, which is in a staggered orientation with respect to the pyrrole nitrogen atoms, also contributes to ligand affinity by increasing the reactivity of the heme iron8. This orientation allows optimal positioning of the iron atom in the heme plane such that the six iron ligands are almost in an octahedral arrangement. By contrast, an eclipsed proximal His orientation, as observed in sperm whale myoglobin, can restrict movement of the iron into the heme plane thereby decreasing reactivity. (A different orientation of the proximal His residues is evident when comparing Fig. 1a and 1b.) This analysis indicates that stereochemistry from both the proximal and distal regions can contribute to regulation of ligand affinity; the contribution of such stereochemical effects are discussed in more detail with respect to allosteric mechanisms below. Quaternary assemblies
The large variation in properties among monomeric hemoglobins illustrates the plasticity of the myoglobin fold as the heme binding site
Review
TRENDS in Biochemical Sciences Vol.26 No.5 May 2001
Fig. 2. Quaternary assemblages of eight oligomeric hemoglobins viewed along molecular (or quasi) two-folds. Structures are depicted with van der Waals spheres for main-chain and heme atoms. In each subunit, heme groups are shown in red, the heme-encapsulating E and F helices are shown in cyan and the rest of the main-chain atoms in gray. Hemoglobin assemblies (a)–(e) show cooperative oxygen binding. Note how these hemoglobins have contacts that involve the E and/or F helices or the FG corner. Cooperative assemblages with the EF-dimer are seen in both the molluscan and echinoderm hemoglobins and might be widespread among invertebrates. (In many cases, a given organism has multiple hemoglobins, which are designated by a letter; for example, HbA, for human adult hemoglobin, or HbV, the fifth and largest electrophoretic component in lamprey erythrocytes.) (a) Human hemoglobin (PDB code 2hhb)37. (b) Scapharca tetrameric hemoglobin (1sct). (c) Deoxy lamprey HbV (3lhb)28. (d) Scapharca dimeric hemoglobin (3sdh)21. (e) Caudina hemoglobin (1hlm)38. (f) Vitreoscilla hemoglobin (1vhb)39. (g) Rice hemoglobin (1d8u)40. (h) Urechis hemoglobin (1ith)41. This figure was produced using the program MIDAS (Ref. 36).
stereochemistry is molded to the functional needs of the organism. Assembly of hemoglobin subunits into larger oligomeric forms can endow the protein with important regulatory properties if the assembly impacts the active site stereochemistry. A remarkable diversity in molecular architecture is apparent from the eight known hemoglobin assemblages (Fig. 2), five of which (Fig. 2a–e) exhibit cooperative ligand binding. Among these diverse forms, only one oligomeric assemblage (which we will term the ‘EF-dimer’ because of extensive subunit interactions between the http://tibs.trends.com
299
E and F helices) has been found in more than one phylum. This EF-dimer is not only observed in the molluscan (Fig. 2d) and echinoderm (Fig. 2e) dimers, but is also a subassembly of both a molluscan tetramer (Fig. 2b) and an annelid multimer (see below). Based on the oxidation-linked subunit dissociation behavior, Riggs has suggested that the EF-dimer might be even more widely distributed among invertebrate hemoglobins9. The other two cooperative hemoglobins (Fig. 2a,c) link ligand binding to the subunit interface using the E helices (Fig. 2c) or the FG corner (Fig. 2a). By contrast, the assemblages shown in Fig. 2f–h either do not exhibit cooperative oxygen binding or their binding properties are still under investigation. In these hemoglobins, contacts are formed using portions of the D, E, F, G and H helices and AB, BC, EF and GH corners. Comparison of these assemblies suggest that cooperative interactions require intersubunit contacts involving a limited region of the E helix, F helix or FG corner, which can be sensitive to ligand state of the heme group. One of the most elaborate uses of the EF-dimer is seen in the large hemoglobin assemblage of the respiratory proteins found in annelids. These proteins have molecular masses of ~3.6 × 106 Da and exhibit strong cooperative oxygen binding. Both the allosteric properties and subunit assembly are dependent upon calcium10. A structure of one of these assemblages, from Lumbricus terrestris (the common earthworm), has recently been determined at a resolution of 5.5Å (Ref. 11). This structure reveals 144 hemoglobin subunits and 36 nonhemoglobin (‘linker’) subunits that are crucial for the formation of the complete complex (Fig. 3). The whole molecule is assembled as two hexagonal discs, with linker subunits comprising the central portion. A key component of the linker assemblage is the interdigitation of 12 triple-stranded coiled-coil elements near the molecular center that appear to dictate aspects of the molecular hierarchical symmetry. The hemoglobin subunits occupy the surface of the complex, arranged as 12 dodecamers (gray and magenta in Fig. 3a). Each dodecamer consists of three disulfide-linked trimers (chains a, b and c) along with three copies of a fourth subunit (chain d) (Fig. 3b). A key structural unit appears to be a tetramer (abc′d) of hemoglobin subunits, each half of which is assembled similarly to the EF-dimers discussed above (Fig. 3d). Thus, although used in very different ways, the EF-dimer assembly is conserved in at least three different invertebrate phyla. Allosteric mechanisms
Does the large variability of quaternary assemblages of cooperative hemoglobins translate into a large variety of cooperative mechanisms? Protein allostery requires that there are at least two alternate binding modes that exhibit significant differences in ligand affinity. Such differences can be made physiologically
300
Review
TRENDS in Biochemical Sciences Vol.26 No.5 May 2001
Fig. 3. The structure of the extracellular respiratory protein found in the annelid Lumbricus terrestris11 shown as a surface representation of the 5.5Å electron density map. The whole molecule, termed erythrocruorin, is composed of 144 hemoglobin subunits, arranged as 12 dodecamers and 36 linker subunits, which are required for assembly. (a) Representation of the whole molecule viewed along the molecular sixfold. Hemoglobin subunits are depicted in gray, but with one dodecamer in magenta, whereas half of the linker chains are shown in yellow and the other half in cyan. Note the interdigitation of 12 triple-stranded coiled-coils from the linker subunits near the molecular center. (b) Hemoglobin dodecamer viewed along its local threefold axis. The dodecamer is formed from three disulfide-linked trimers (abc), each of which is shown in a different color, and three ‘monomeric’ d-chains shown in gray. (Heme density is red.) (c) Tetrameric (abc’d) assemblage, viewed along a quasi twofold, with E and F helices in cyan, the heme groups in red and remaining tetramer density in gray. This tetramer is composed of a and b chains from one trimer, one c chain of a neighboring trimer and one d chain. Note the approximate similarity in subunit arrangement to the molluscan tetramer Scapharca HbII (Fig. 2b). (d) Assembly of the two halves of the tetramer. Note the similarity of these dimeric assemblages with those of the molluscan and echinoderm dimers (Fig. 2d,e). This figure was produced using the program RIBBONS (Ref. 42).
useful if the protein conformation switches between alternate structures in response to appropriate environmental signals. The functional properties measured from four allosteric hemoglobins are shown in Table 1. As we have seen, diverse binding affinities are found in different myoglobin-fold molecules. In an allosteric system, differences in affinity occur within a single molecule. A prerequisite for understanding the stereochemical basis for allosteric function is the structure of the molecule in alternate affinity states.
These states are termed the T (low affinity, ‘tense’) state and R (high affinity, ‘relaxed’) state, following the definitions in the classical MWC (Monod, Wyman and Changeux) model for cooperativity12. Structures of alternate states have now been determined for three cooperative hemoglobins (Fig. 4): the classic mammalian tetramer (e.g. human HbA), a molluscan dimer and a primitive vertebrate hemoglobin. Comparison of these hemoglobins reveals considerable diversity in regulation of binding affinity. Mammalian tetrameric hemoglobin
No allosteric system has been studied in greater detail than mammalian hemoglobin. Although impressive progress has been made in our understanding of this molecule, including the difficult task of a thermodynamic description of intermediate states, several differing viewpoints still exist13–15. Because of the vast literature on mammalian hemoglobins, we will only briefly discuss certain aspects of one proposed structural allosteric mechanism to form the basis for comparison with other hemoglobins. Crystallographic investigations have shown that ligand binding is coupled with a large quaternary change involving a rotation of ~15° between the two αβ dimers in the T and classical R states3. Whereas a single T state has always been observed
Table 1. Selected functional data on cooperative hemoglobins Hemoglobin
P50a (Torr)
nmaxb
Bohr effectc ∆logP50/∆ ∆pH) (∆
pH, temperature
Notes
Refs
Human HbA
1.6–18.8
2.5–3.0
–0.5
pH 6.0–9.0, 25°C
Dependent upon Cl–, organic phosphates, CO2
10,33
Lamprey Hb
5.0–68.0
1.0–1.2
–0.6 to –1.0
pH 6.3–7.7, 20°C
Dependent upon Hb concentration 26,29
Scapharca HbI
7.8
1.5
0.0
pH 5.5–9.5, 20°C
No known heterotropic effectors
20
Lumbricus erythrocruorin
1.6–17.3
2.5–7.9
–0.35 to –0.77
pH 6.2–9.0, 25°C
Ca2+-dependent Bohr effect
10
aP is the partial pressure of oxygen at half saturation. 50 bThe Hill coefficient, n, a measure of cooperativity, will
of interacting sites. c The Bohr effect is a measure of the sensitivity to pH.
http://tibs.trends.com
have a value of 1.0 for a non-cooperative system and will always be less than the number
Review
TRENDS in Biochemical Sciences Vol.26 No.5 May 2001
Fig. 4. Ligand-linked structural transitions in three cooperative hemoglobins. Three turns of the E and F helices are shown for each, along with heme groups (red), and four heme-contacting side chains. (a) Human hemoglobin HbA beta chains deoxygenated (unligated) (PDB code 2hhb)37 and oxygenated (1hho)43. The key residue in modulation of oxygen affinity in both alpha and beta chains of human hemoglobin is the proximal His (cyan), which is coordinated to the heme iron. In the low-affinity deoxy state, the proximal His orientation restricts movement of the iron into the heme plane by van der Waals contacts between the His and heme atoms. Transition to the high-affinity state, represented by the oxygenated structure, results from a movement of the F helix that orients the proximal His normal to the heme plane. This provides communication between subunits by altering the α1β2 contact involving the FG corner. [Note the transition of Cysβ93 (C93) and Aspβ94 (D94), two of the many residues at subunit interfaces whose conformations are altered upon ligand binding.] In the β subunits (but not the α subunits) Val E11 contributes to modulation of oxygen affinity by sterically restricting ligand access to the binding site in the deoxygenated state. (b) Scapharca dimeric hemoglobin (HbI)21 in the deoxygenated (4sdh) and oxygenated (1hbi) forms. Regulation of oxygen affinity in HbI is primarily determined by the disposition of Phe97 (cyan). In the deoxy state, Phe97 packs against the proximal face of the heme such that it sterically restricts movement of the proximal His and heme iron into the heme plane. In the oxygenated form, Phe97 is displaced from the heme pocket into the interface, where it disrupts a cluster of well-ordered interface water molecules and increases the oxygen affinity of the second subunit. (c) Lamprey hemoglobin in the deoxygenated (3lhb)28 and CN-met (2lhb)44 forms. [The CN-met form, in which CN binds to the heme iron in the oxidized (+3) state, is believed to have a similar structure to oxygenated hemoglobin.] Dimerization involves the E helix and key interface residues Trp72 (W72; E6) and Glu75 (E75; E9). The movement of the E helix results in a distal His orientation that sterically blocks ligand entry. Binding of ligand requires movement of the distal His and E helix, disrupting the dimeric interface leading to subunit dissociation and release of the Bohr protons. [The Bohr protons provide lamprey hemoglobin with strong pH sensitivity (Table 1) by binding at the dimeric interface between Glu75 and Glu31, in its partner subunit.] This figure was produced using the program MIDAS (Ref. 36).
in deoxyhemoglobin, regardless of crystal conditions, more variability has been found in the ligated state. A second quaternary arrangement, termed the Y or R2 state, has been found under conditions believed to be more physiological16, and recent results on bovine hemoglobin show additional quaternary arrangements of ligated hemoglobin that are between R and R2 (Ref. 17). Based on ligand-coupled structural transitions, Perutz http://tibs.trends.com
301
proposed that a major component of oxygen regulation derived from the orientation of the proximal His relative to the heme plane15. In the T state, the proximal His restricts motion of the iron into the heme plane, and thus lowers the iron reactivity. In ligated states, the F helix is shifted towards the FG corner, allowing movement of the proximal His and heme iron into the heme plane. Based on an analysis of the tertiary structural alterations that occur upon ligand binding, Gelin et al.18 proposed that an ‘allosteric core’, composed of the heme, proximal His, FG corner and a portion of the F helix, plays an essential role in cooperativity. The involvement of the FG corner in the subunit interface allows coupling between the active site and quaternary structure. Barrick et al.19 conducted an important test of the Perutz hypothesis by severing the covalent link between the F helix and the heme by mutation of His F8 to Gly, and simultaneously adding exogenous imidazole to coordinate the heme iron. These imidazole–hemoglobin complexes had increased ligand affinity and attenuated the cooperativity to about one-third of the wild-type value, thus directly supporting the Perutz mechanism. However, residual cooperativity exists, suggesting the role of alternate pathways of communication, which could involve distal residues, such as the distal Val (E11) and His (E7) in the β chains, that appear to sterically block ligand binding in the deoxy state. Thus, regulation in HbA appears to involve proximal regulation of iron reactivity, and also possibly ligand access to the distal pocket. However, it is important to note that the large quaternary changes that occur upon ligand binding make it difficult to define the stereochemical basis unambiguously for the difference in affinity between the T and R states, which is only a few kcal mol–1 (Ref. 14). Scapharca dimeric hemoglobin
Scapharca dimeric hemoglobin (HbI) represents one of the simplest possible allosteric systems. This molecule binds oxygen cooperatively at two chemically identical sites, with no affinity modulation by classical non-heme ligands such as organic phosphates, protons or chloride20. Structural analysis at high resolution (1.4–1.6Å) reveals that ligand binding is coupled with significant tertiary changes at the interface but only minor quaternary structural changes21. The independence of cooperativity from large quaternary structural changes is shown by the finding that cooperative ligand binding is exhibited within the crystalline state22. The localized nature of ligand-linked transitions considerably simplifies the task of elucidating a stereochemical basis for cooperativity. A key change is the movement of Phe97 (F4) from the heme pocket into the interface upon ligand binding (Fig. 4b). The low affinity of the deoxy state largely results from the packing of the Phe side chain in the heme pocket23. In this position, it
302
Review
TRENDS in Biochemical Sciences Vol.26 No.5 May 2001
restricts movement of the heme iron into the heme plane and lengthens a hydrogen bond involving the proximal His (Ref. 21). Thus, in HbI, proximal modulation of iron reactivity plays a central role in control of ligand affinity. How is the allosteric signal propagated between subunits in HbI? The deoxy HbI structure has a very well-ordered water cluster in the subunit interface that appears to contribute to the stabilization of the low-affinity form of each subunit21. The ligand-coupled movement of Phe97 disrupts this water cluster leaving a smaller and less well-ordered interface water network. Could disruption of the water cluster be the signal by which a subunit detects the ligand state of its partner subunit? To test this hypothesis, the water cluster was disrupted by mutagenesis and osmotic stress24. Both methods led to significantly increased oxygen affinity, and the mutation studies show that structural changes are limited to the interface water structure. These experiments indicate a direct link between the interface water structure and oxygen affinity that is exploited for cooperative function. The water effect explains the result that cooperativity is entropically driven in HbI (Ref. 25). Evidently, the large energetic contribution of water molecules can be crucial for stabilizing alternate states that are required for allostery. An intriguing aspect of Scapharca HbI is the participation of the heme groups in cooperative structural transitions at the subunit interface, similar to that envisioned by Pauling in 1935 for mammalian hemoglobin. In the absence of ligand, the heme is positioned such that the distal His sterically hinders ligation. Binding of oxygen results in the movement of the heme deeper into the subunit, which permits optimal hydrogen bonding and is coupled with a rearrangement of the heme propionates at the subunit interface. These transitions might be responsible for the residual cooperativity observed upon mutation of Phe97 (Ref. 23). Moreover, mutations that restrict the heme movement prevent extrusion of Phe97 from the heme pocket and severely diminish cooperativity (J.E. Knapp and W.E. Royer, unpublished). Thus, there is a tight coupling between the ligand-linked heme and Phe97 transitions that affect ligand affinity from both the proximal and distal regions.
experiments reveal that the lamprey dimer has the slowest known oxygen association rate constant (2.5 × 105 M–1sec–1) but, upon dissociation into monomers, this rate increases 40-fold27. This difference in association rate constants is far greater than that observed for HbA or HbI, indicating an allosteric mechanism based largely on modulating ligand access to the active site. How is this achieved? The recent structure of an assembled form of lamprey hemoglobin reveals that dimerization occurs through contacts between the E helices of each subunit (Fig. 2c), which directly affects the distal (ligand-binding) pocket, but produces no discernible changes in the proximal pocket28 (Fig. 4c). In the dimeric state, the distal His adopts a conformation that sterically blocks the ligand-binding site. In very recent studies, the binding of carbon monoxide to deoxy crystals of assembled lamprey hemoglobin has permitted visualization of ligand binding to the lowaffinity dimeric form of lamprey hemoglobin (H.A. Heaslet and W.E. Royer, unpublished). This work shows displacement of the distal His to several alternate conformations that preclude formation of the stabilizing hydrogen bond that probably exists in the monomeric form. Thus, in lamprey hemoglobin, regulation of oxygen affinity results entirely from distal effects by altering accessibility and distal stabilization of bound oxygen. The most physiologically important aspect of ligand-linked assembly of lamprey hemoglobin is the resultant strong Bohr effect in which binding of oxygen is linked with proton loss. How is the Bohr effect achieved? The interface of the lamprey dimer contains a cluster of four Glu residues, with each Glu75 (Fig. 4c) in a position to form a hydrogen bond with Glu31 from the adjacent subunit. One of these two Glu residues must be protonated in the dimer, requiring an increase in pK of about two pH units to reach the measured pK 6.5 for the Bohr group. Evidently the favorable interface interactions, driven by the burial of Trp72 (Fig. 4c) in the interface, are sufficient to bring these Glu residues into close proximity and raise their pK. Mutagenesis studies support the involvement of these residues in the Bohr effect29. Thus, the interface of lamprey hemoglobin has been exquisitely designed to balance favorable and unfavorable interactions such that binding of oxygen or protons can tip the balance.
Lamprey hemoglobin
Ligand-linked assembly
The hemoglobins found in the primitive vertebrate lampreys provide alternative model systems for allosteric regulation. Unlike HbA or HbI, lamprey hemoglobin manifests regulatory behavior as a result of an equilibrium between low-affinity dimers and higher-affinity monomers, which results in slight cooperativity but strong pH sensitivity known as the Bohr effect. The lamprey Bohr effect is nearly twice that of human hemoglobin26. Kinetic
Despite these mechanistic differences, there is one important aspect of similarity in the ligand-linked assembly of the HbA, HbI and lamprey hemoglobins. In all cases, the oligomeric assemblage is more tightly assembled in the unligated state than in the ligated state. Although most striking in lamprey hemoglobin, with ligated monomers, this is also true of the deoxy HbA interface13 and the highly hydrated deoxy HbI
http://tibs.trends.com
Review
Acknowledgements We thank Warner Love for first inspiring interest in these molecules, Austen Riggs, Quentin Gibson, Marvin Hackert, Mark Hargrove, Celia Schiffer and Kendall Knight for helpful comments on the manuscript. We apologize for the omission of many relevant citations imposed by space limitations. This work was supported by a grant from NIH (W.E.R.) and a postdoctoral award from the New England affiliate of the American Heart Association (J.E.K.).
TRENDS in Biochemical Sciences Vol.26 No.5 May 2001
interface30. Over 50 years ago, Wyman31 suggested that cooperative interaction energy could either be present as stabilizing energy between unligated subunits (quaternary constraint12) or ligated subunits (quaternary enhancement13). In the case of quaternary constraint, the unligated assembly would be assembled more tightly with interface interactions acting to lower intrinsic ligand affinity. Cooperativity would result from the release of intersubunit constraints as ligand binding proceeds. In the case of quaternary enhancement, the reverse occurs – the ligated structure is assembled more tightly and the intersubunit interactions enhance intrinsic binding affinity. Why is quaternary constraint much more commonly observed? A clue comes from the recent crystal structure of isolated, highly active, catalytic subunits of aspartate transcarbamoylase32. Surprisingly, this structure shows more similarity with catalytic subunits in the T state than with those in the presumed R-state structure. However, the isolated trimer also exhibits greater flexibility than catalytic subunits in the T-state structure. The authors suggest that the T-state quaternary interactions hinder local conformational changes required for catalysis and that allostery depends upon modulation of flexibility. This would appear to be impossible in a quaternary-enhancement approach in which the strongest interactions between subunits would occur between high-affinity subunits and tend to limit flexibility. Thus, flexibility required for optimal activity, in either an enzyme or a binding protein, might be the decisive factor that leads to quaternary constraint being the primary means used by nature for allosteric regulation.
References 1 Pauling, L. (1935) The oxygen equilibrium of hemoglobin and its structural interpretation. Proc. Natl. Acad. Sci. U. S. A. 21, 186–191 2 Perutz, M.F. et al. (1968) Three-dimensional Fourier synthesis of horse oxyhaemoglobin at 2.8Å resolution: II – the atomic model. Nature 219, 131–139 3 Perutz, M.F. (1970) Stereochemistry of cooperative effects in haemoglobin. Nature 228, 726–739 4 Kendrew, J.C. et al. (1960) Structure of myoglobin: a three-dimensional Fourier synthesis at 2Å resolution. Nature 185, 422–427 5 Bolognesi, M. et al. (1997) Nonvertebrate hemoglobins: structural bases for reactivity. Prog. Biophys. Mol. Biol. 68, 29–68 6 Huang, X. and Boxer, S.G. (1994) Discovery of new ligand binding pathways in myoglobin by random mutagenesis. Nat. Struct. Biol. 1, 226–229 7 Scott, E.E. et al. (2001) Mapping the pathways for O2 entry into and exit from myoglobin. J. Biol. Chem. 276, 5177–5188 8 Yang, J. et al. (1995) The structure of Ascaris hemoglobin domain I at 2.2Å resolution: molecular features of oxygen avidity. Proc. Natl. Acad. Sci. U. S. A. 92, 4224–4228 http://tibs.trends.com
303
Conclusion
Assembly of hemoglobin subunits into oligomeric complexes is often coupled with acquisition of allosteric function. Different cooperative assemblies have been observed and a striking variety of strategies are used to modulate binding affinity of individual subunits. Thus, although all hemoglobin subunits are evolutionarily related, allosteric mechanisms have clearly developed independently on multiple occasions. Future investigations will undoubtedly increase this variety, which might even extend to similar assemblages such as the EF-dimer assembly present in cooperative hemoglobins of mollusks, echinoderms and annelids. Surprisingly, the key residues that underlie cooperativity in the molluscan dimer are not conserved in the echinoderm and annelid hemoglobin sequences. Moreover, the localized transitions characteristic of the molluscan dimer are not compatible with the high cooperativity of the annelid erythrocruorins (Table 1). How are these similar assemblages used in different ways to obtain cooperativity? Other important questions also remain. Structural analysis of lamprey hemoglobin indicates that regulation results entirely from direct effects on the distal, ligand-binding pocket. There are hints that distal regulation might also be important in mammalian and molluscan hemoglobins. Does this imply that distal regulation is universal in cooperative hemoglobins? Finally, one of the key, but experimentally difficult, questions for any cooperative system is – what are the structural, thermodynamic and kinetic properties of the intermediate forms of ligation? By addressing such questions in these proteins, we hope to obtain a better understanding of general principles that can be applied to many other allosteric systems.
9 Riggs, A.F. (1998) Self-association, cooperativity and supercooperativity of oxygen binding by hemoglobins. J. Exp. Biol. 201, 1073–1084 10 Fushitani, K. et al. (1986) Oxygenation properties of hemoglobin from the earthworm, Lumbricus terrestris. Effects of pH, salts, and temperature. J. Biol. Chem. 261, 8414–8423 11 Royer, W.E., Jr et al. (2000) Structural hierarchy in erythrocruorin, the giant respiratory assemblage of annelids. Proc. Natl. Acad. Sci. U. S. A. 97, 7107–7111 12 Monod, J. et al. (1965) On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 12, 88–118 13 Ackers, G.K. (1998) Deciphering the molecular code of hemoglobin allostery. Adv. Protein Chem. 51, 185–253 14 Eaton, W.A. et al. (1999) Is cooperative oxygen binding by hemoglobin really understood? Nat. Struct. Biol. 6, 351–357 15 Perutz, M.F. et al. (1998) The stereochemical mechanism of the cooperative effects in hemoglobin revisited. Annu. Rev. Biophys. Biomol. Struct. 27, 1–34 16 Smith, F.R. and Simmons, K.C. (1994) Cyanomet human hemoglobin crystallized under physiological conditions exhibits the Y quaternary structure. Proteins 18, 295–300
17 Mueser, T.C. et al. (2000) Interface sliding as illustrated by the multiple quaternary structures of liganded hemoglobin. Biochemistry 39, 15353–15364 18 Gelin, B.R. et al. (1983) Hemoglobin tertiary structural change on ligand binding. Its role in the co-operative mechanism. J. Mol. Biol. 171, 489–559 19 Barrick, D. et al. (1997) A test of the role of the proximal histidines in the Perutz model for cooperativity in haemoglobin. Nat. Struct. Biol. 4, 78–83 20 Chiancone, E. et al. (1981) Dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis. Structural and functional properties. J. Mol. Biol. 152, 577–592 21 Royer, W.E., Jr (1994) High-resolution crystallographic analysis of a co-operative dimeric hemoglobin. J. Mol. Biol. 235, 657–681 22 Mozzarelli, A. et al. (1996) Cooperative oxygen binding to Scapharca inaequivalvis hemoglobin in the crystal. J. Biol. Chem. 271, 3627–3632 23 Pardanani, A. et al. (1997) Mutation of residue Phe97 to Leu disrupts the central allosteric pathway in Scapharca dimeric hemoglobin. J. Biol. Chem. 272, 13171–13179
304
Review
24 Royer, W.E., Jr et al. (1996) Ordered water molecules as key allosteric mediators in a cooperative dimeric hemoglobin. Proc. Natl. Acad. Sci. U. S. A. 93, 14526–14531 25 Chiancone, E. and Boffi, A. (2000) Structural and thermodynamic aspects of cooperativity in the homodimeric hemoglobin from Scapharca inaequivalvis. Biophys. Chem. 86, 173–178 26 Wald, G. and Riggs, A. (1951) The hemoglobin of the sea lamprey, Petromyzon marinus. J. Gen. Physiol. 35, 45–53 27 Andersen, M.E. and Gibson, Q.H. (1971) A kinetic analysis of the binding of oxygen and carbon monoxide to lamprey hemoglobin. J. Biol. Chem. 246, 4790–4799 28 Heaslet, H.A. and Royer, W.E., Jr (1999) The 2.7 Å crystal structure of deoxygenated hemoglobin from the sea lamprey (Petromyzon marinus): structural basis for a lowered oxygen affinity and Bohr effect. Struct. Fold. Des. 7, 517–526 29 Qiu, Y. et al. (2000) Lamprey hemoglobin. Structural basis for the Bohr effect. J. Biol. Chem. 275, 13517–13528
TRENDS in Biochemical Sciences Vol.26 No.5 May 2001
30 Royer, W.E., Jr et al. (1997) Ligand linked assembly of Scapharca dimeric hemoglobin. J. Biol. Chem. 272, 5689–5694 31 Wyman, J., Jr (1948) Heme proteins. Adv. Protein Chem. 4, 407–531 32 Beernink, P.T. et al. (1999) Assessment of the allosteric mechanism of aspartate transcarbamoylase based on the crystalline structure of the unregulated catalytic subunit. Proc. Natl. Acad. Sci. U. S. A. 96, 5388–5393 33 Imai, K. (1982) Allosteric Effects in Haemoglobin, Cambridge University Press 34 Phillips, S.E.V. (1980) Structure and refinement of oxymyoglobin at 1.6Å resolution. J. Mol. Biol. 142, 531–554 35 Pesce, A. et al. (2000) A novel two-over-two α-helical sandwich is characteristic of the truncated hemoglobin family. EMBO J. 19, 2424–2434 36 Ferrin, T.E. et al. (1988) The MIDAS display system. J. Mol. Graphics 6, 13–27 37 Fermi, G. et al. (1984) The crystal structure of human deoxy hemoglobin at 1.74Å resolution. J. Mol. Biol. 175, 159–174
38 Mitchell, D.T. et al. (1995) Structural analysis of monomeric hemichrome and dimeric cyanomet hemoglobins from Caudina arenicola. J. Mol. Biol. 251, 421–431 39 Tarricone, C. et al. (1997) Unusual structure of the oxygen-binding site in the dimeric bacterial hemoglobin from Vitreoscilla sp. Structure 45, 497–507 40 Hargrove, M.S. et al. (2000) Crystal structure of a nonsymbiotic plant hemoglobin. Struct. Fold. Des. 8, 1005–1014 41 Kolatkar, P.R. et al. (1994) Structural analysis of Urechis caupo hemoglobin. J. Mol. Biol. 237, 87–97 42 Carson, M. (1997) Ribbons. Methods Enzymol. 277, 493–505 43 Shaanan, B. (1983) Structure of human oxyhemoglobin at 2.1Å resolution. J. Mol. Biol. 171, 31–59 44 Honzatko, R.B. et al. (1985) Refinement of a molecular model for lamprey hemoglobin from Petromyzon marinus. J. Mol. Biol. 184, 147–164
Receptor clustering and transmembrane signaling in T cells Jennifer R. Cochran, Dikran Aivazian, Thomas O. Cameron and Lawrence J. Stern T cells are activated via engagement of their cell-surface receptors with molecules of the major histocompatibility complex (MHC) displayed on another cell surface. This process, which is a key step in the recognition of foreign antigens by the immune system, involves oligomerization of receptor components. Recent characterization of the T-cell response to soluble arrays of MHC–peptide complexes has provided insights into the triggering mechanism for T-cell activation.
Jennifer R. Cochran Thomas O. Cameron Lawrence J. Stern* Dept of Chemistry Dikran Aivazian Dept of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. *e-mail: [email protected]
Ligand binding to cell-surface receptors can activate intracellular signal transduction pathways through a variety of mechanisms (Fig. 1). In one common mechanism, typical of seven-transmembrane span receptors that activate G proteins1, ligand binding induces a structural change in the receptor that can be sensed by effector proteins within the cell. In another mechanism, binding of a multivalent ligand induces receptor colocalization. This mechanism is typical of receptor tyrosine kinases (RTKs), in which receptor clustering facilitates transphosphorylation by the cytoplasmic kinase domains2. Other mechanisms driven by mass action can be envisioned (see following text). In a third mechanism, ligand binding induces rearrangement of a receptor oligomer. One example of this mechanism is the bacterial aspartic acid receptor, in which ligand binding induces a helix reorganization that activates receptor-associated cytoplasmic signaling proteins3. For soluble ligands, several examples of each of these
mechanisms are known. When both the ligand and receptor are cell-surface proteins, the situation can be complicated; for example, by redistribution of membrane components or interaction of other proteins in the juxtaposed membranes. Cases of both of these situations have been observed in the interaction of T-cell antigen receptors (TCRs) with their ligands – major histocompatibility complex (MHC) proteins that are displayed on the surface of antigen-presenting cells. Recent investigation of this cell–cell signaling system, using soluble oligomeric arrays of MHC proteins as mimics of the antigen-presenting cell, have provided insights into the mechanism of T-cell triggering. However, more work is necessary for us to understand the roles of membrane-proximal signaling events in the overall T-cell activation process. MHC–TCR interaction
T cells play an important role in the initiation and control of immune responses by recognizing antigenic (foreign) peptides bound to MHC proteins on the surface of antigen-presenting cells4. MHC molecules bind an extensive variety of peptides from the local cellular environment and display these peptides at the cell surface, providing a diverse peptide library for interaction with T cells. TCRs are generated by clonotypic recombination of genomic constant and
http://tibs.trends.com 0968-0004/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0968-0004(01)01815-1
40 Ponting, C.P. and Phillips, C. (1997) Identification of homer as a homologue of the Wiskott–Aldrich syndrome protein suggests a receptor-binding function for WH1 domains. J. Mol. Med. 75, 769–771 41 Sun, J. et al. (1998) Isolation of PSD-Zip45, a novel Homer/vesl family protein containing leucine zipper motifs, from rat brain. FEBS Lett. 437, 304–308 42 Xiao, B. et al. (1998) Homer regulates the association of group 1 metabotropic glutamate receptors with multivalent complexes of homerrelated, synaptic proteins. Neuron 21, 707–716 43 Tu, J.C. et al. (1998) Homer binds a novel proline-rich motif and links group 1 metabotropic glutamate receptors with IP3 receptors. Neuron 21, 717–726 44 Tu, J.C. et al. (1999) Coupling of mGluR/Homer and PSD-95 complexes by the Shank family of postsynaptic density proteins. Neuron 23, 583–592 45 Naisbitt, S. et al. (1999) Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin. Neuron 23, 569–582
TRENDS in Biochemical Sciences Vol.26 No.5 May 2001
46 Weinman, E.J. et al. (1998) Structure-function of recombinant Na/H exchanger regulatory factor (NHE-RF). J. Clin. Invest. 101, 2199–2206 47 Hall, R.A. et al. (1998) A C-terminal motif found in the β2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins. Proc. Natl. Acad. Sci. U. S. A. 95, 8496–8501 48 Hall, R.A. et al. (1999) G protein-coupled receptor kinase 6A phosphorylates the Na(+)/H(+) exchanger regulatory factor via a PDZ domain-mediated interaction. J. Biol. Chem. 274, 24328–24334 49 Hall, R.A. et al. (1998) The β2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange. Nature 392, 626–630 50 Liu, X. et al. (1999) Activation of a frizzled-2/β-adrenergic receptor chimera promotes Wnt signaling and differentiation of mouse F9 teratocarcinoma cells via Gαo and Gαt. Proc. Natl. Acad. Sci. U. S. A. 96, 14383–14388
297
51 Liu, T. et al. (1999) Activation of rat frizzled-1 promotes Wnt signaling and differentiation of mouse F9 teratocarcinoma cells via pathways that require Galpha(q) and Galpha(o) function. J. Biol. Chem. 274, 33539–33544 52 Sheldahl, L.C. et al. (1999) Protein kinase C is differentially stimulated by Wnt and Frizzled homologs in a G-protein-dependent manner. Curr. Biol. 9, 695–698 53 Kuhl, M. et al. (2000) Ca(2+)/calmodulindependent protein kinase II is stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in Xenopus. J. Biol. Chem. 275, 12701–12711 54 Wu, C. et al. (2000) RGS proteins inhibit Xwnt-8 signaling in Xenopus embryonic development. Development 127, 2773–2784 55 Lefkowitz, R.J. (2000) The superfamily of heptahelical receptors. Nat. Cell Biol. 2, E133–E136 56 Luttrell, L.M. et al. (2001) Activation and targeting of extracellular signal-regulated kinases by β-arrestin scaffolds. Proc. Natl. Acad. Sci. U. S. A. 98, 2449–2453
Cooperative hemoglobins: conserved fold, diverse quaternary assemblies and allosteric mechanisms William E. Royer, Jr, James E. Knapp, Kristen Strand and Holly A. Heaslet Assembly of hemoglobin subunits into cooperative complexes produces a remarkable variety of architectures, ranging in oligomeric state from dimers to complexes containing 144 hemoglobin subunits. Diverse stereochemical mechanisms for modulating ligand affinity through intersubunit interactions have been revealed from studies of three distinct hemoglobin assemblages. This mechanistic diversity, which occurs between assemblies of subunits that have the same fold, provides insight into the range of regulatory strategies that are available to protein molecules.
William E. Royer, Jr* James E. Knapp Kristen Strand Holly A. Heaslet Dept of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA. *e-mail: William.Royer@ umassmed.edu
For nearly a century, mammalian tetrameric hemoglobin (composed of two α and two β subunits) has been a major paradigm for the allosteric regulation of protein function. Experiments in the early 1900s by Christian Bohr and others demonstrated that the oxygen binding curve of hemoglobin was sigmoidal rather than hyperbolic; thus, oxygen binding is cooperative, with a stepwise increase in oxygen affinity as the binding reaction proceeds. A structural explanation for this cooperativity was proposed in 1935 by Linus Pauling, who suggested that binding of oxygen triggers an interaction between adjacent heme groups1. A major breakthrough in our understanding of cooperativity came from the
pioneering studies of Max Perutz, whose structures of mammalian hemoglobin showed that the heme groups are actually too far apart for the type of direct interaction envisioned by Pauling2. Perutz also demonstrated that mammalian hemoglobins can exist in alternate quaternary structures depending on ligand state3. These structures provided the framework for our understanding of the cooperative ligand binding that arises from the transition between a low-affinity state and higher-affinity state(s) as ligand binding proceeds. In contrast to the conserved tetrameric hemoglobin structure found in most vertebrates, a remarkable diversity in quaternary assemblage is observed among invertebrate and primitive vertebrate hemoglobins, despite conserved tertiary structures of the subunits. To achieve intersubunit communication, the diverse assemblages use a variety of mechanisms, comparisons of which reveal several broad principles that define the regulation of ligand affinity. Specifically, ligand affinity is modulated by limiting access of the ligand to the heme pocket, by altering the chemical reactivity of the heme iron, or by stabilizing the bound ligand by
http://tibs.trends.com 0968-0004/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0968-0004(01)01811-4
Review
298
TRENDS in Biochemical Sciences Vol.26 No.5 May 2001
Diversity in binding affinity
(a)
(b)
Sperm whale myoglobin
Ascaris hemoglobin, domain 1
(c)
Paramecium hemoglobin
Fig. 1. Structures of three myoglobin-fold monomers. For each protein, a ribbon diagram is shown with the amino-terminal helices in light gray, E and F helices in cyan, and the carboxy terminal G and H helices in dark gray. Heme groups are shown in red, proximal His residues (F8) in cyan, and oxygen ligands in yellow. Residues at distal positions E7 (His or Gln), B10 (Leu or Tyr) and CD1 (Phe) are shown in cyan, magenta and purple, respectively. (By convention, residues are designated by their position within a helix or corner, based on the myoglobin structure. Therefore, the proximal His is always F8, even though, for instance, in Ascaris hemoglobin it is the 12th residue along the F helix.) Oxygen affinities differ dramatically among these hemoglobins, and can be designated by p50 values; that is, the partial pressure of oxygen at half-saturation. (a) Sperm whale myoglobin (PDB code 1mbo)34 (p50 = 0.5 Torr). (b) Ascaris hemoglobin domain (1ash)8. Ascaris hemoglobin has one of the highest known oxygen affinities (p50 = 0.004 Torr), which results primarily from ligand stabilization resulting from hydrogen bonds contributed by Tyr B10 and Gln E7. (c) Paramecium hemoglobin (1dlw)35. This ‘truncated hemoglobin’ (~75% the length of myoglobin) is missing part of the A helix, most of the CD corner and D helix, and much of the F helix, yet it still provides a proximal His in the single turn F helix and an environment for reversible oxygen binding (p50 = 0.5 Torr). This figure was produced using the program MIDAS (Ref. 36).
interaction with neighboring residues. This review focuses on how subunit assembly regulates ligand affinity and cooperativity using one or more of these strategies. Tertiary structures
The first two protein crystal structures to be determined were those of sperm whale myoglobin4 and horse hemoglobin2. The structural similarity between the hemoglobin α and β subunits and myoglobin provided the initial evidence of structural conservation in the globin family and suggested that this tertiary structure, the myoglobin fold, optimally uses a heme group for reversible oxygen binding. Since then, the myoglobin fold has been found in proteins widely distributed among bacteria, plants, invertebrates and vertebrates. The classic myoglobin fold consists of seven or eight α helices encapsulating a heme prosthetic group, with helices (A–H) being connected by corners (e.g. the AB corner connects helices A and B) (Fig. 1). The heme iron is coordinated to five ligands in hemoglobin – four pyrrole nitrogen atoms and a strictly conserved proximal His F8 (i.e. the eighth residue in the F helix of sperm whale myoglobin) – leaving the sixth site available to bind oxygen on the distal face of the heme. A second conserved residue is Phe CD1 (first residue in the CD corner), which helps to wedge the heme in the protein. Two other key residues are distal residues E7 and B10 (Fig. 1), both of which can play crucial roles in stabilizing the bound oxygen. Although the hemoglobins shown in Fig. 1 have significant structural differences, they all provide an environment for reversible binding of oxygen to the heme group. http://tibs.trends.com
The proteins shown in Fig. 1 bind oxygen with drastically different affinities. Reported oxygen dissociation rate constants of hemoglobins range over six orders of magnitude from approximately 10–3 to 103 sec–1, whereas association rate constants vary over three orders of magnitude from approximately 105 to 108 M–1sec–1 (Ref. 5). How are such large differences achieved? Contributions of ligand access and distal stabilization to ligand affinity were highlighted in studies using mutants of sperm whale myoglobin. Several groups have investigated ligand access in sperm whale myoglobin, one study involving the generation of 1500 mutants by random mutagenesis6. Recently, Olson and his colleagues have analyzed 90 mutations at 27 positions that affect ligand access and stability7. The oxygen affinities of these mutants vary over three orders of magnitude. Decreases in affinity are observed when the distal His (E7) is replaced with residues that do not form hydrogen bonds with bound oxygen (e.g. H64V) or when access to the ligand-binding site is hindered (e.g. V68I). Increases in the affinity are observed in L29F (B10) as the Phe ring stabilizes the bound ligand. A more dramatic illustration of the power of ligand stabilization is observed in the high-affinity hemoglobin from the nematode parasite, Ascaris suum. This hemoglobin is assembled as an octamer of 33-kDa chains, each of which consists of two hemoglobin domains. The high affinity (Kd = 4 × 10–9 M) results primarily from an extremely slow dissociation rate of 0.004 sec–1 (Ref. 5). How is oxygen held so tightly? Clues come from the structure of an isolated domain that binds oxygen with the same affinity as does the non-cooperative octamer (Fig. 1b). Major ligand stabilization appears to result from favorable hydrogen bonding of Tyr B10 and Gln E7 with bound oxygen in the distal pocket8. The proximal His, which is in a staggered orientation with respect to the pyrrole nitrogen atoms, also contributes to ligand affinity by increasing the reactivity of the heme iron8. This orientation allows optimal positioning of the iron atom in the heme plane such that the six iron ligands are almost in an octahedral arrangement. By contrast, an eclipsed proximal His orientation, as observed in sperm whale myoglobin, can restrict movement of the iron into the heme plane thereby decreasing reactivity. (A different orientation of the proximal His residues is evident when comparing Fig. 1a and 1b.) This analysis indicates that stereochemistry from both the proximal and distal regions can contribute to regulation of ligand affinity; the contribution of such stereochemical effects are discussed in more detail with respect to allosteric mechanisms below. Quaternary assemblies
The large variation in properties among monomeric hemoglobins illustrates the plasticity of the myoglobin fold as the heme binding site
Review
TRENDS in Biochemical Sciences Vol.26 No.5 May 2001
Fig. 2. Quaternary assemblages of eight oligomeric hemoglobins viewed along molecular (or quasi) two-folds. Structures are depicted with van der Waals spheres for main-chain and heme atoms. In each subunit, heme groups are shown in red, the heme-encapsulating E and F helices are shown in cyan and the rest of the main-chain atoms in gray. Hemoglobin assemblies (a)–(e) show cooperative oxygen binding. Note how these hemoglobins have contacts that involve the E and/or F helices or the FG corner. Cooperative assemblages with the EF-dimer are seen in both the molluscan and echinoderm hemoglobins and might be widespread among invertebrates. (In many cases, a given organism has multiple hemoglobins, which are designated by a letter; for example, HbA, for human adult hemoglobin, or HbV, the fifth and largest electrophoretic component in lamprey erythrocytes.) (a) Human hemoglobin (PDB code 2hhb)37. (b) Scapharca tetrameric hemoglobin (1sct). (c) Deoxy lamprey HbV (3lhb)28. (d) Scapharca dimeric hemoglobin (3sdh)21. (e) Caudina hemoglobin (1hlm)38. (f) Vitreoscilla hemoglobin (1vhb)39. (g) Rice hemoglobin (1d8u)40. (h) Urechis hemoglobin (1ith)41. This figure was produced using the program MIDAS (Ref. 36).
stereochemistry is molded to the functional needs of the organism. Assembly of hemoglobin subunits into larger oligomeric forms can endow the protein with important regulatory properties if the assembly impacts the active site stereochemistry. A remarkable diversity in molecular architecture is apparent from the eight known hemoglobin assemblages (Fig. 2), five of which (Fig. 2a–e) exhibit cooperative ligand binding. Among these diverse forms, only one oligomeric assemblage (which we will term the ‘EF-dimer’ because of extensive subunit interactions between the http://tibs.trends.com
299
E and F helices) has been found in more than one phylum. This EF-dimer is not only observed in the molluscan (Fig. 2d) and echinoderm (Fig. 2e) dimers, but is also a subassembly of both a molluscan tetramer (Fig. 2b) and an annelid multimer (see below). Based on the oxidation-linked subunit dissociation behavior, Riggs has suggested that the EF-dimer might be even more widely distributed among invertebrate hemoglobins9. The other two cooperative hemoglobins (Fig. 2a,c) link ligand binding to the subunit interface using the E helices (Fig. 2c) or the FG corner (Fig. 2a). By contrast, the assemblages shown in Fig. 2f–h either do not exhibit cooperative oxygen binding or their binding properties are still under investigation. In these hemoglobins, contacts are formed using portions of the D, E, F, G and H helices and AB, BC, EF and GH corners. Comparison of these assemblies suggest that cooperative interactions require intersubunit contacts involving a limited region of the E helix, F helix or FG corner, which can be sensitive to ligand state of the heme group. One of the most elaborate uses of the EF-dimer is seen in the large hemoglobin assemblage of the respiratory proteins found in annelids. These proteins have molecular masses of ~3.6 × 106 Da and exhibit strong cooperative oxygen binding. Both the allosteric properties and subunit assembly are dependent upon calcium10. A structure of one of these assemblages, from Lumbricus terrestris (the common earthworm), has recently been determined at a resolution of 5.5Å (Ref. 11). This structure reveals 144 hemoglobin subunits and 36 nonhemoglobin (‘linker’) subunits that are crucial for the formation of the complete complex (Fig. 3). The whole molecule is assembled as two hexagonal discs, with linker subunits comprising the central portion. A key component of the linker assemblage is the interdigitation of 12 triple-stranded coiled-coil elements near the molecular center that appear to dictate aspects of the molecular hierarchical symmetry. The hemoglobin subunits occupy the surface of the complex, arranged as 12 dodecamers (gray and magenta in Fig. 3a). Each dodecamer consists of three disulfide-linked trimers (chains a, b and c) along with three copies of a fourth subunit (chain d) (Fig. 3b). A key structural unit appears to be a tetramer (abc′d) of hemoglobin subunits, each half of which is assembled similarly to the EF-dimers discussed above (Fig. 3d). Thus, although used in very different ways, the EF-dimer assembly is conserved in at least three different invertebrate phyla. Allosteric mechanisms
Does the large variability of quaternary assemblages of cooperative hemoglobins translate into a large variety of cooperative mechanisms? Protein allostery requires that there are at least two alternate binding modes that exhibit significant differences in ligand affinity. Such differences can be made physiologically
300
Review
TRENDS in Biochemical Sciences Vol.26 No.5 May 2001
Fig. 3. The structure of the extracellular respiratory protein found in the annelid Lumbricus terrestris11 shown as a surface representation of the 5.5Å electron density map. The whole molecule, termed erythrocruorin, is composed of 144 hemoglobin subunits, arranged as 12 dodecamers and 36 linker subunits, which are required for assembly. (a) Representation of the whole molecule viewed along the molecular sixfold. Hemoglobin subunits are depicted in gray, but with one dodecamer in magenta, whereas half of the linker chains are shown in yellow and the other half in cyan. Note the interdigitation of 12 triple-stranded coiled-coils from the linker subunits near the molecular center. (b) Hemoglobin dodecamer viewed along its local threefold axis. The dodecamer is formed from three disulfide-linked trimers (abc), each of which is shown in a different color, and three ‘monomeric’ d-chains shown in gray. (Heme density is red.) (c) Tetrameric (abc’d) assemblage, viewed along a quasi twofold, with E and F helices in cyan, the heme groups in red and remaining tetramer density in gray. This tetramer is composed of a and b chains from one trimer, one c chain of a neighboring trimer and one d chain. Note the approximate similarity in subunit arrangement to the molluscan tetramer Scapharca HbII (Fig. 2b). (d) Assembly of the two halves of the tetramer. Note the similarity of these dimeric assemblages with those of the molluscan and echinoderm dimers (Fig. 2d,e). This figure was produced using the program RIBBONS (Ref. 42).
useful if the protein conformation switches between alternate structures in response to appropriate environmental signals. The functional properties measured from four allosteric hemoglobins are shown in Table 1. As we have seen, diverse binding affinities are found in different myoglobin-fold molecules. In an allosteric system, differences in affinity occur within a single molecule. A prerequisite for understanding the stereochemical basis for allosteric function is the structure of the molecule in alternate affinity states.
These states are termed the T (low affinity, ‘tense’) state and R (high affinity, ‘relaxed’) state, following the definitions in the classical MWC (Monod, Wyman and Changeux) model for cooperativity12. Structures of alternate states have now been determined for three cooperative hemoglobins (Fig. 4): the classic mammalian tetramer (e.g. human HbA), a molluscan dimer and a primitive vertebrate hemoglobin. Comparison of these hemoglobins reveals considerable diversity in regulation of binding affinity. Mammalian tetrameric hemoglobin
No allosteric system has been studied in greater detail than mammalian hemoglobin. Although impressive progress has been made in our understanding of this molecule, including the difficult task of a thermodynamic description of intermediate states, several differing viewpoints still exist13–15. Because of the vast literature on mammalian hemoglobins, we will only briefly discuss certain aspects of one proposed structural allosteric mechanism to form the basis for comparison with other hemoglobins. Crystallographic investigations have shown that ligand binding is coupled with a large quaternary change involving a rotation of ~15° between the two αβ dimers in the T and classical R states3. Whereas a single T state has always been observed
Table 1. Selected functional data on cooperative hemoglobins Hemoglobin
P50a (Torr)
nmaxb
Bohr effectc ∆logP50/∆ ∆pH) (∆
pH, temperature
Notes
Refs
Human HbA
1.6–18.8
2.5–3.0
–0.5
pH 6.0–9.0, 25°C
Dependent upon Cl–, organic phosphates, CO2
10,33
Lamprey Hb
5.0–68.0
1.0–1.2
–0.6 to –1.0
pH 6.3–7.7, 20°C
Dependent upon Hb concentration 26,29
Scapharca HbI
7.8
1.5
0.0
pH 5.5–9.5, 20°C
No known heterotropic effectors
20
Lumbricus erythrocruorin
1.6–17.3
2.5–7.9
–0.35 to –0.77
pH 6.2–9.0, 25°C
Ca2+-dependent Bohr effect
10
aP is the partial pressure of oxygen at half saturation. 50 bThe Hill coefficient, n, a measure of cooperativity, will
of interacting sites. c The Bohr effect is a measure of the sensitivity to pH.
http://tibs.trends.com
have a value of 1.0 for a non-cooperative system and will always be less than the number
Review
TRENDS in Biochemical Sciences Vol.26 No.5 May 2001
Fig. 4. Ligand-linked structural transitions in three cooperative hemoglobins. Three turns of the E and F helices are shown for each, along with heme groups (red), and four heme-contacting side chains. (a) Human hemoglobin HbA beta chains deoxygenated (unligated) (PDB code 2hhb)37 and oxygenated (1hho)43. The key residue in modulation of oxygen affinity in both alpha and beta chains of human hemoglobin is the proximal His (cyan), which is coordinated to the heme iron. In the low-affinity deoxy state, the proximal His orientation restricts movement of the iron into the heme plane by van der Waals contacts between the His and heme atoms. Transition to the high-affinity state, represented by the oxygenated structure, results from a movement of the F helix that orients the proximal His normal to the heme plane. This provides communication between subunits by altering the α1β2 contact involving the FG corner. [Note the transition of Cysβ93 (C93) and Aspβ94 (D94), two of the many residues at subunit interfaces whose conformations are altered upon ligand binding.] In the β subunits (but not the α subunits) Val E11 contributes to modulation of oxygen affinity by sterically restricting ligand access to the binding site in the deoxygenated state. (b) Scapharca dimeric hemoglobin (HbI)21 in the deoxygenated (4sdh) and oxygenated (1hbi) forms. Regulation of oxygen affinity in HbI is primarily determined by the disposition of Phe97 (cyan). In the deoxy state, Phe97 packs against the proximal face of the heme such that it sterically restricts movement of the proximal His and heme iron into the heme plane. In the oxygenated form, Phe97 is displaced from the heme pocket into the interface, where it disrupts a cluster of well-ordered interface water molecules and increases the oxygen affinity of the second subunit. (c) Lamprey hemoglobin in the deoxygenated (3lhb)28 and CN-met (2lhb)44 forms. [The CN-met form, in which CN binds to the heme iron in the oxidized (+3) state, is believed to have a similar structure to oxygenated hemoglobin.] Dimerization involves the E helix and key interface residues Trp72 (W72; E6) and Glu75 (E75; E9). The movement of the E helix results in a distal His orientation that sterically blocks ligand entry. Binding of ligand requires movement of the distal His and E helix, disrupting the dimeric interface leading to subunit dissociation and release of the Bohr protons. [The Bohr protons provide lamprey hemoglobin with strong pH sensitivity (Table 1) by binding at the dimeric interface between Glu75 and Glu31, in its partner subunit.] This figure was produced using the program MIDAS (Ref. 36).
in deoxyhemoglobin, regardless of crystal conditions, more variability has been found in the ligated state. A second quaternary arrangement, termed the Y or R2 state, has been found under conditions believed to be more physiological16, and recent results on bovine hemoglobin show additional quaternary arrangements of ligated hemoglobin that are between R and R2 (Ref. 17). Based on ligand-coupled structural transitions, Perutz http://tibs.trends.com
301
proposed that a major component of oxygen regulation derived from the orientation of the proximal His relative to the heme plane15. In the T state, the proximal His restricts motion of the iron into the heme plane, and thus lowers the iron reactivity. In ligated states, the F helix is shifted towards the FG corner, allowing movement of the proximal His and heme iron into the heme plane. Based on an analysis of the tertiary structural alterations that occur upon ligand binding, Gelin et al.18 proposed that an ‘allosteric core’, composed of the heme, proximal His, FG corner and a portion of the F helix, plays an essential role in cooperativity. The involvement of the FG corner in the subunit interface allows coupling between the active site and quaternary structure. Barrick et al.19 conducted an important test of the Perutz hypothesis by severing the covalent link between the F helix and the heme by mutation of His F8 to Gly, and simultaneously adding exogenous imidazole to coordinate the heme iron. These imidazole–hemoglobin complexes had increased ligand affinity and attenuated the cooperativity to about one-third of the wild-type value, thus directly supporting the Perutz mechanism. However, residual cooperativity exists, suggesting the role of alternate pathways of communication, which could involve distal residues, such as the distal Val (E11) and His (E7) in the β chains, that appear to sterically block ligand binding in the deoxy state. Thus, regulation in HbA appears to involve proximal regulation of iron reactivity, and also possibly ligand access to the distal pocket. However, it is important to note that the large quaternary changes that occur upon ligand binding make it difficult to define the stereochemical basis unambiguously for the difference in affinity between the T and R states, which is only a few kcal mol–1 (Ref. 14). Scapharca dimeric hemoglobin
Scapharca dimeric hemoglobin (HbI) represents one of the simplest possible allosteric systems. This molecule binds oxygen cooperatively at two chemically identical sites, with no affinity modulation by classical non-heme ligands such as organic phosphates, protons or chloride20. Structural analysis at high resolution (1.4–1.6Å) reveals that ligand binding is coupled with significant tertiary changes at the interface but only minor quaternary structural changes21. The independence of cooperativity from large quaternary structural changes is shown by the finding that cooperative ligand binding is exhibited within the crystalline state22. The localized nature of ligand-linked transitions considerably simplifies the task of elucidating a stereochemical basis for cooperativity. A key change is the movement of Phe97 (F4) from the heme pocket into the interface upon ligand binding (Fig. 4b). The low affinity of the deoxy state largely results from the packing of the Phe side chain in the heme pocket23. In this position, it
302
Review
TRENDS in Biochemical Sciences Vol.26 No.5 May 2001
restricts movement of the heme iron into the heme plane and lengthens a hydrogen bond involving the proximal His (Ref. 21). Thus, in HbI, proximal modulation of iron reactivity plays a central role in control of ligand affinity. How is the allosteric signal propagated between subunits in HbI? The deoxy HbI structure has a very well-ordered water cluster in the subunit interface that appears to contribute to the stabilization of the low-affinity form of each subunit21. The ligand-coupled movement of Phe97 disrupts this water cluster leaving a smaller and less well-ordered interface water network. Could disruption of the water cluster be the signal by which a subunit detects the ligand state of its partner subunit? To test this hypothesis, the water cluster was disrupted by mutagenesis and osmotic stress24. Both methods led to significantly increased oxygen affinity, and the mutation studies show that structural changes are limited to the interface water structure. These experiments indicate a direct link between the interface water structure and oxygen affinity that is exploited for cooperative function. The water effect explains the result that cooperativity is entropically driven in HbI (Ref. 25). Evidently, the large energetic contribution of water molecules can be crucial for stabilizing alternate states that are required for allostery. An intriguing aspect of Scapharca HbI is the participation of the heme groups in cooperative structural transitions at the subunit interface, similar to that envisioned by Pauling in 1935 for mammalian hemoglobin. In the absence of ligand, the heme is positioned such that the distal His sterically hinders ligation. Binding of oxygen results in the movement of the heme deeper into the subunit, which permits optimal hydrogen bonding and is coupled with a rearrangement of the heme propionates at the subunit interface. These transitions might be responsible for the residual cooperativity observed upon mutation of Phe97 (Ref. 23). Moreover, mutations that restrict the heme movement prevent extrusion of Phe97 from the heme pocket and severely diminish cooperativity (J.E. Knapp and W.E. Royer, unpublished). Thus, there is a tight coupling between the ligand-linked heme and Phe97 transitions that affect ligand affinity from both the proximal and distal regions.
experiments reveal that the lamprey dimer has the slowest known oxygen association rate constant (2.5 × 105 M–1sec–1) but, upon dissociation into monomers, this rate increases 40-fold27. This difference in association rate constants is far greater than that observed for HbA or HbI, indicating an allosteric mechanism based largely on modulating ligand access to the active site. How is this achieved? The recent structure of an assembled form of lamprey hemoglobin reveals that dimerization occurs through contacts between the E helices of each subunit (Fig. 2c), which directly affects the distal (ligand-binding) pocket, but produces no discernible changes in the proximal pocket28 (Fig. 4c). In the dimeric state, the distal His adopts a conformation that sterically blocks the ligand-binding site. In very recent studies, the binding of carbon monoxide to deoxy crystals of assembled lamprey hemoglobin has permitted visualization of ligand binding to the lowaffinity dimeric form of lamprey hemoglobin (H.A. Heaslet and W.E. Royer, unpublished). This work shows displacement of the distal His to several alternate conformations that preclude formation of the stabilizing hydrogen bond that probably exists in the monomeric form. Thus, in lamprey hemoglobin, regulation of oxygen affinity results entirely from distal effects by altering accessibility and distal stabilization of bound oxygen. The most physiologically important aspect of ligand-linked assembly of lamprey hemoglobin is the resultant strong Bohr effect in which binding of oxygen is linked with proton loss. How is the Bohr effect achieved? The interface of the lamprey dimer contains a cluster of four Glu residues, with each Glu75 (Fig. 4c) in a position to form a hydrogen bond with Glu31 from the adjacent subunit. One of these two Glu residues must be protonated in the dimer, requiring an increase in pK of about two pH units to reach the measured pK 6.5 for the Bohr group. Evidently the favorable interface interactions, driven by the burial of Trp72 (Fig. 4c) in the interface, are sufficient to bring these Glu residues into close proximity and raise their pK. Mutagenesis studies support the involvement of these residues in the Bohr effect29. Thus, the interface of lamprey hemoglobin has been exquisitely designed to balance favorable and unfavorable interactions such that binding of oxygen or protons can tip the balance.
Lamprey hemoglobin
Ligand-linked assembly
The hemoglobins found in the primitive vertebrate lampreys provide alternative model systems for allosteric regulation. Unlike HbA or HbI, lamprey hemoglobin manifests regulatory behavior as a result of an equilibrium between low-affinity dimers and higher-affinity monomers, which results in slight cooperativity but strong pH sensitivity known as the Bohr effect. The lamprey Bohr effect is nearly twice that of human hemoglobin26. Kinetic
Despite these mechanistic differences, there is one important aspect of similarity in the ligand-linked assembly of the HbA, HbI and lamprey hemoglobins. In all cases, the oligomeric assemblage is more tightly assembled in the unligated state than in the ligated state. Although most striking in lamprey hemoglobin, with ligated monomers, this is also true of the deoxy HbA interface13 and the highly hydrated deoxy HbI
http://tibs.trends.com
Review
Acknowledgements We thank Warner Love for first inspiring interest in these molecules, Austen Riggs, Quentin Gibson, Marvin Hackert, Mark Hargrove, Celia Schiffer and Kendall Knight for helpful comments on the manuscript. We apologize for the omission of many relevant citations imposed by space limitations. This work was supported by a grant from NIH (W.E.R.) and a postdoctoral award from the New England affiliate of the American Heart Association (J.E.K.).
TRENDS in Biochemical Sciences Vol.26 No.5 May 2001
interface30. Over 50 years ago, Wyman31 suggested that cooperative interaction energy could either be present as stabilizing energy between unligated subunits (quaternary constraint12) or ligated subunits (quaternary enhancement13). In the case of quaternary constraint, the unligated assembly would be assembled more tightly with interface interactions acting to lower intrinsic ligand affinity. Cooperativity would result from the release of intersubunit constraints as ligand binding proceeds. In the case of quaternary enhancement, the reverse occurs – the ligated structure is assembled more tightly and the intersubunit interactions enhance intrinsic binding affinity. Why is quaternary constraint much more commonly observed? A clue comes from the recent crystal structure of isolated, highly active, catalytic subunits of aspartate transcarbamoylase32. Surprisingly, this structure shows more similarity with catalytic subunits in the T state than with those in the presumed R-state structure. However, the isolated trimer also exhibits greater flexibility than catalytic subunits in the T-state structure. The authors suggest that the T-state quaternary interactions hinder local conformational changes required for catalysis and that allostery depends upon modulation of flexibility. This would appear to be impossible in a quaternary-enhancement approach in which the strongest interactions between subunits would occur between high-affinity subunits and tend to limit flexibility. Thus, flexibility required for optimal activity, in either an enzyme or a binding protein, might be the decisive factor that leads to quaternary constraint being the primary means used by nature for allosteric regulation.
References 1 Pauling, L. (1935) The oxygen equilibrium of hemoglobin and its structural interpretation. Proc. Natl. Acad. Sci. U. S. A. 21, 186–191 2 Perutz, M.F. et al. (1968) Three-dimensional Fourier synthesis of horse oxyhaemoglobin at 2.8Å resolution: II – the atomic model. Nature 219, 131–139 3 Perutz, M.F. (1970) Stereochemistry of cooperative effects in haemoglobin. Nature 228, 726–739 4 Kendrew, J.C. et al. (1960) Structure of myoglobin: a three-dimensional Fourier synthesis at 2Å resolution. Nature 185, 422–427 5 Bolognesi, M. et al. (1997) Nonvertebrate hemoglobins: structural bases for reactivity. Prog. Biophys. Mol. Biol. 68, 29–68 6 Huang, X. and Boxer, S.G. (1994) Discovery of new ligand binding pathways in myoglobin by random mutagenesis. Nat. Struct. Biol. 1, 226–229 7 Scott, E.E. et al. (2001) Mapping the pathways for O2 entry into and exit from myoglobin. J. Biol. Chem. 276, 5177–5188 8 Yang, J. et al. (1995) The structure of Ascaris hemoglobin domain I at 2.2Å resolution: molecular features of oxygen avidity. Proc. Natl. Acad. Sci. U. S. A. 92, 4224–4228 http://tibs.trends.com
303
Conclusion
Assembly of hemoglobin subunits into oligomeric complexes is often coupled with acquisition of allosteric function. Different cooperative assemblies have been observed and a striking variety of strategies are used to modulate binding affinity of individual subunits. Thus, although all hemoglobin subunits are evolutionarily related, allosteric mechanisms have clearly developed independently on multiple occasions. Future investigations will undoubtedly increase this variety, which might even extend to similar assemblages such as the EF-dimer assembly present in cooperative hemoglobins of mollusks, echinoderms and annelids. Surprisingly, the key residues that underlie cooperativity in the molluscan dimer are not conserved in the echinoderm and annelid hemoglobin sequences. Moreover, the localized transitions characteristic of the molluscan dimer are not compatible with the high cooperativity of the annelid erythrocruorins (Table 1). How are these similar assemblages used in different ways to obtain cooperativity? Other important questions also remain. Structural analysis of lamprey hemoglobin indicates that regulation results entirely from direct effects on the distal, ligand-binding pocket. There are hints that distal regulation might also be important in mammalian and molluscan hemoglobins. Does this imply that distal regulation is universal in cooperative hemoglobins? Finally, one of the key, but experimentally difficult, questions for any cooperative system is – what are the structural, thermodynamic and kinetic properties of the intermediate forms of ligation? By addressing such questions in these proteins, we hope to obtain a better understanding of general principles that can be applied to many other allosteric systems.
9 Riggs, A.F. (1998) Self-association, cooperativity and supercooperativity of oxygen binding by hemoglobins. J. Exp. Biol. 201, 1073–1084 10 Fushitani, K. et al. (1986) Oxygenation properties of hemoglobin from the earthworm, Lumbricus terrestris. Effects of pH, salts, and temperature. J. Biol. Chem. 261, 8414–8423 11 Royer, W.E., Jr et al. (2000) Structural hierarchy in erythrocruorin, the giant respiratory assemblage of annelids. Proc. Natl. Acad. Sci. U. S. A. 97, 7107–7111 12 Monod, J. et al. (1965) On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 12, 88–118 13 Ackers, G.K. (1998) Deciphering the molecular code of hemoglobin allostery. Adv. Protein Chem. 51, 185–253 14 Eaton, W.A. et al. (1999) Is cooperative oxygen binding by hemoglobin really understood? Nat. Struct. Biol. 6, 351–357 15 Perutz, M.F. et al. (1998) The stereochemical mechanism of the cooperative effects in hemoglobin revisited. Annu. Rev. Biophys. Biomol. Struct. 27, 1–34 16 Smith, F.R. and Simmons, K.C. (1994) Cyanomet human hemoglobin crystallized under physiological conditions exhibits the Y quaternary structure. Proteins 18, 295–300
17 Mueser, T.C. et al. (2000) Interface sliding as illustrated by the multiple quaternary structures of liganded hemoglobin. Biochemistry 39, 15353–15364 18 Gelin, B.R. et al. (1983) Hemoglobin tertiary structural change on ligand binding. Its role in the co-operative mechanism. J. Mol. Biol. 171, 489–559 19 Barrick, D. et al. (1997) A test of the role of the proximal histidines in the Perutz model for cooperativity in haemoglobin. Nat. Struct. Biol. 4, 78–83 20 Chiancone, E. et al. (1981) Dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis. Structural and functional properties. J. Mol. Biol. 152, 577–592 21 Royer, W.E., Jr (1994) High-resolution crystallographic analysis of a co-operative dimeric hemoglobin. J. Mol. Biol. 235, 657–681 22 Mozzarelli, A. et al. (1996) Cooperative oxygen binding to Scapharca inaequivalvis hemoglobin in the crystal. J. Biol. Chem. 271, 3627–3632 23 Pardanani, A. et al. (1997) Mutation of residue Phe97 to Leu disrupts the central allosteric pathway in Scapharca dimeric hemoglobin. J. Biol. Chem. 272, 13171–13179
304
Review
24 Royer, W.E., Jr et al. (1996) Ordered water molecules as key allosteric mediators in a cooperative dimeric hemoglobin. Proc. Natl. Acad. Sci. U. S. A. 93, 14526–14531 25 Chiancone, E. and Boffi, A. (2000) Structural and thermodynamic aspects of cooperativity in the homodimeric hemoglobin from Scapharca inaequivalvis. Biophys. Chem. 86, 173–178 26 Wald, G. and Riggs, A. (1951) The hemoglobin of the sea lamprey, Petromyzon marinus. J. Gen. Physiol. 35, 45–53 27 Andersen, M.E. and Gibson, Q.H. (1971) A kinetic analysis of the binding of oxygen and carbon monoxide to lamprey hemoglobin. J. Biol. Chem. 246, 4790–4799 28 Heaslet, H.A. and Royer, W.E., Jr (1999) The 2.7 Å crystal structure of deoxygenated hemoglobin from the sea lamprey (Petromyzon marinus): structural basis for a lowered oxygen affinity and Bohr effect. Struct. Fold. Des. 7, 517–526 29 Qiu, Y. et al. (2000) Lamprey hemoglobin. Structural basis for the Bohr effect. J. Biol. Chem. 275, 13517–13528
TRENDS in Biochemical Sciences Vol.26 No.5 May 2001
30 Royer, W.E., Jr et al. (1997) Ligand linked assembly of Scapharca dimeric hemoglobin. J. Biol. Chem. 272, 5689–5694 31 Wyman, J., Jr (1948) Heme proteins. Adv. Protein Chem. 4, 407–531 32 Beernink, P.T. et al. (1999) Assessment of the allosteric mechanism of aspartate transcarbamoylase based on the crystalline structure of the unregulated catalytic subunit. Proc. Natl. Acad. Sci. U. S. A. 96, 5388–5393 33 Imai, K. (1982) Allosteric Effects in Haemoglobin, Cambridge University Press 34 Phillips, S.E.V. (1980) Structure and refinement of oxymyoglobin at 1.6Å resolution. J. Mol. Biol. 142, 531–554 35 Pesce, A. et al. (2000) A novel two-over-two α-helical sandwich is characteristic of the truncated hemoglobin family. EMBO J. 19, 2424–2434 36 Ferrin, T.E. et al. (1988) The MIDAS display system. J. Mol. Graphics 6, 13–27 37 Fermi, G. et al. (1984) The crystal structure of human deoxy hemoglobin at 1.74Å resolution. J. Mol. Biol. 175, 159–174
38 Mitchell, D.T. et al. (1995) Structural analysis of monomeric hemichrome and dimeric cyanomet hemoglobins from Caudina arenicola. J. Mol. Biol. 251, 421–431 39 Tarricone, C. et al. (1997) Unusual structure of the oxygen-binding site in the dimeric bacterial hemoglobin from Vitreoscilla sp. Structure 45, 497–507 40 Hargrove, M.S. et al. (2000) Crystal structure of a nonsymbiotic plant hemoglobin. Struct. Fold. Des. 8, 1005–1014 41 Kolatkar, P.R. et al. (1994) Structural analysis of Urechis caupo hemoglobin. J. Mol. Biol. 237, 87–97 42 Carson, M. (1997) Ribbons. Methods Enzymol. 277, 493–505 43 Shaanan, B. (1983) Structure of human oxyhemoglobin at 2.1Å resolution. J. Mol. Biol. 171, 31–59 44 Honzatko, R.B. et al. (1985) Refinement of a molecular model for lamprey hemoglobin from Petromyzon marinus. J. Mol. Biol. 184, 147–164
Receptor clustering and transmembrane signaling in T cells Jennifer R. Cochran, Dikran Aivazian, Thomas O. Cameron and Lawrence J. Stern T cells are activated via engagement of their cell-surface receptors with molecules of the major histocompatibility complex (MHC) displayed on another cell surface. This process, which is a key step in the recognition of foreign antigens by the immune system, involves oligomerization of receptor components. Recent characterization of the T-cell response to soluble arrays of MHC–peptide complexes has provided insights into the triggering mechanism for T-cell activation.
Jennifer R. Cochran Thomas O. Cameron Lawrence J. Stern* Dept of Chemistry Dikran Aivazian Dept of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. *e-mail: [email protected]
Ligand binding to cell-surface receptors can activate intracellular signal transduction pathways through a variety of mechanisms (Fig. 1). In one common mechanism, typical of seven-transmembrane span receptors that activate G proteins1, ligand binding induces a structural change in the receptor that can be sensed by effector proteins within the cell. In another mechanism, binding of a multivalent ligand induces receptor colocalization. This mechanism is typical of receptor tyrosine kinases (RTKs), in which receptor clustering facilitates transphosphorylation by the cytoplasmic kinase domains2. Other mechanisms driven by mass action can be envisioned (see following text). In a third mechanism, ligand binding induces rearrangement of a receptor oligomer. One example of this mechanism is the bacterial aspartic acid receptor, in which ligand binding induces a helix reorganization that activates receptor-associated cytoplasmic signaling proteins3. For soluble ligands, several examples of each of these
mechanisms are known. When both the ligand and receptor are cell-surface proteins, the situation can be complicated; for example, by redistribution of membrane components or interaction of other proteins in the juxtaposed membranes. Cases of both of these situations have been observed in the interaction of T-cell antigen receptors (TCRs) with their ligands – major histocompatibility complex (MHC) proteins that are displayed on the surface of antigen-presenting cells. Recent investigation of this cell–cell signaling system, using soluble oligomeric arrays of MHC proteins as mimics of the antigen-presenting cell, have provided insights into the mechanism of T-cell triggering. However, more work is necessary for us to understand the roles of membrane-proximal signaling events in the overall T-cell activation process. MHC–TCR interaction
T cells play an important role in the initiation and control of immune responses by recognizing antigenic (foreign) peptides bound to MHC proteins on the surface of antigen-presenting cells4. MHC molecules bind an extensive variety of peptides from the local cellular environment and display these peptides at the cell surface, providing a diverse peptide library for interaction with T cells. TCRs are generated by clonotypic recombination of genomic constant and
http://tibs.trends.com 0968-0004/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0968-0004(01)01815-1