Differences in backbone dynamics of two homologous bacterial albumin-binding modules: implications for binding specificity and bacterial adaptation1

doi:10.1006/jmbi.2001.5398 available online at http://www.idealibrary.com on

J. Mol. Biol. (2002) 316, 1083±1099

Differences in Backbone Dynamics of Two Homologous Bacterial Albumin-binding Modules: Implications for Binding Specificity and Bacterial Adaptation Maria U. Johansson1*, Hanna Nilsson1, Johan EvenaÈs1, Sture ForseÂn 1 TorbjoÈrn Drakenberg1Lars BjoÈrck2 and Mats WikstroÈm3 1

Department of Biophysical Chemistry, Lund University P.O. Box 124, SE-221 00 Lund, Sweden 2

Department of Cell and Molecular Biology, Section for Molecular Pathogenesis, BMC B14 Lund University TornavaÈgen 10, SE-221 84 Lund, Sweden 3

Department of Structural Chemistry, Biovitrum AB Norden¯ychtsvaÈgen 62:5 SE-112 76 Stockholm, Sweden

Proteins G and PAB are bacterial albumin-binding proteins expressed at the surface of group C and G streptococci and Peptostreptococcus magnus, respectively. Repeated albumin-binding domains, known as GA modules, are found in both proteins. The third GA module of protein G from the group G streptococcal strain G148 (G148-GA3) and the second GA module of protein PAB from P. magnus strain ALB8 (ALB8-GA) exhibit 59 % sequence identity and both fold to form three-helix bundle structures that are very stable against thermal denaturation. ALB8-GA binds human serum albumin with higher af®nity than G148-GA3, but G148-GA3 shows substantially broader albumin-binding speci®city than ALB8-GA. The 15N nuclear magnetic resonance spin relaxation measurements reported here, show that the two GA modules exhibit mobility on the picosecond-nanosecond time scale in directly corresponding regions (loops and termini). Most residues in G148-GA3 were seen to be involved in conformational exchange processes on the microsecond-millisecond time scale, whereas for ALB8-GA such motions were only identi®ed for the beginning of helix 2 and its preceding loop. Furthermore, and more importantly, hydrogen-deuterium exchange and saturation transfer experiments reveal large differences between the two GA modules with respect to motions on the second-hour time scale. The high degree of similarity between the two GA modules with respect to sequence, structure and stability, and the observed differences in dynamics, binding af®nity and binding speci®city to different albumins, suggest a distinct correlation between dynamics, binding af®nity and binding speci®city. Finally, it is noteworthy in this context that the module G148-GA3, which has broad albumin-binding speci®city, is expressed by group C and G streptococci known to infect all mammalian species, whereas P. magnus with the ALB8-GA module has been isolated only from humans. # 2002 Elsevier Science Ltd.

Present address: Johan EvenaÈs, Medicinal Chemistry, AstraZeneca R&D Lund, SE-221 87 Lund. Abbreviations used: ALB8-GA, the second albumin-binding domain of protein PAB from Peptostreptococcus magnus strain ALB8; CPMG, Carr-Purcell-Meiboom-Gill; CSA, chemical shift anisotropy; DSS, 2,2-dimethylsilapentane-5sulphonic acid; Zxy, transverse 1H-15N dipolar/15N CSA cross-correlated cross-relaxation rate constant; Zz, longitudinal 1H-15N dipolar/15N CSA cross-correlated cross-relaxation rate constant; FID, free induction decay; G148GA3, the third albumin-binding domain of protein G from Streptococcus strain G148; GA, protein G-related albuminbinding; H-2H, hydrogen-deuterium; HPLC, high performance liquid chromatography; HSA, human serum albumin; HSQC, heteronuclear single quantum coherence; IgG, immunoglobulin G; kex, hydrogen exchange rate constant; NOE, steady state 1H-15N nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; ppm, parts per million; P, protection factor; R1, 1H-15N longitudinal auto-relaxation rate constant; R2, 1H-15N transverse autorelaxation rate constant; R02, conformational exchange-free R2; Rex, conformational exchange contribution to R2; rms, root mean squared; S2, generalised order parameter; S2f, order parameter for fast internal motions; S2s, order parameter for slow internal motions; sd, standard deviation; sNH, 1H-15N dipolar cross-relaxation rate constant; tc, global rotational correlation time; tc,i, local rotational correlation time; tf, effective correlation time for fast internal motions; ts, effective correlation time for slow internal motions; TOCSY, total correlation spectroscopy. E-mail address of the corresponding author: [email protected] 0022-2836/02/021083±17 $35.00/0

# 2002 Elsevier Science Ltd.

1084

*Corresponding author

Dynamics of Two Homologous GA Modules

Keywords: albumin-binding; backbone dynamics; bacterial surface proteins; evolution; NMR

Introduction Protein G, a surface protein of group C and G streptococci, was originally described as an immunoglobulin G (IgG)-binding protein.1,2 Apart from IgG-binding domains, protein G also contains separate domains with af®nity for human serum albumin (HSA)3,4 as well as albumin from various animal species.5 Some strains of the strictly anaerobic bacterial species Peptostreptococcus magnus also express an albumin-binding surface protein called protein PAB.6 Sequence analyses of the pab gene and its mosaic organisation revealed that the second of the two domains of pab encoding albumin-binding activity had been shuf¯ed from group C and G streptococci into P. magnus. This ®rst contemporary example of module shuf¯ing was probably promoted by inter-domain so-called recer sequences,7 and these albumin-binding modules were named GA, protein G-related albumin-binding modules.6 The biological function(s) of the GA module remains unclear, but available data suggest that this albumin-binding structural motif promotes bacterial growth, and helps the bacteria to evade detection by the immune system.8 The third albumin-binding domain of protein G from the group G streptococcal strain G148 (G148GA3) and the second albumin-binding domain of protein PAB from P. magnus strain ALB8 (ALB8GA) studied here exhibit 59 % sequence identity (Figure 1(a)), adopt super®cially indistinguishable global folds9 ± 11 (Figure 1(b)) and show similar (high) thermal stability.9,12 Despite all these similarities, which are due to common ancestry, the two GA modules differ noticeably with respect to speci®city for serum albumins of different species.12 In brief, ALB8-GA binds to HSA with higher af®nity than G148-GA3, whereas on the other hand G148-GA3 binds to serum albumins from a much wider variety of species. Believing that the observed differences in serum albumin speci®cities of the two GA modules may be a consequence of differences in local mobility, the backbone dynamics of the two GA modules have been characterised on a wide range of time scales using nuclear magnetic resonance (NMR) spectroscopy, fast internal motions on the picosecondnanosecond time scale, overall molecular rotational diffusion on the nanosecond time scale and conformational exchange processes on the microsecond-millisecond time scale have been probed by performing 15N spin relaxation experiments while hydrogen exchange rate constants (kex) on the second-hour time scale have been investigated using hydrogen-deuterium (H-2H) exchange and saturation transfer experiments. In summary, the two homologous GA modules exhibit substantial

differences in backbone dynamics exemplifying the power of bacterial evolution and adaptation in action.

Results and Discussion In the following, the backbone dynamics of the two GA modules will be described. Fast internal motions, overall molecular rotational diffusion and conformational exchange processes have been characterised by 15N spin relaxation measurements and kex values have been determined using H-2H exchange and saturation transfer experiments. Five different 15N spin relaxation parameters have been determined in this study: 15N longitudinal and transverse auto-relaxation rate constants (R1 and R2, respectively), steady state 1H-15N nuclear Overhauser effect (NOE), and longitudinal and transverse 1H-15N dipolar/15N chemical shift anisotropy (CSA) cross-correlated cross-relaxation rate constants (Zz and Zxy, respectively). The ALB8-GA construct used in this study contains residues 213-265 of intact protein PAB,6 whereas the ®rst 19 residues in the G148-GA3 sequence are remnants of the cloning procedure, followed by residues 141-186 of intact protein G.13 There are several reasons for missing data; severe overlap, the residue being a proline and therefore having no amide proton, too low signal-to-noise ratio of the peak for reliability or when the peak is not observed at all (possibly due to fast exchange with solvent or exchange broadening beyond detection). We will not occupy ourselves with presenting the exact subset of reasons for missing data in each particular case. For practical reasons, data for Trp01 Ne1H will be presented in the Figure legends. Assignment of ALB8-GA and G148-GA3 The 15N assignments of ALB8-GA were based on our previous 1H assignments9 of this GA module using an unlabelled sample in combination with two-dimensional 1H-15N HSQC-TOCSY14 experiments acquired on the 15N-labelled sample with a DIPSI-2 relaxation compensated isotropic mixing sequence15 of length 80 ms. Likewise, 15N assignments of G148-GA3 were made based on previously published assignments10 of a 13C-15Nlabelled sample in combination with two-dimensional 1H-15N HSQC-TOCSY experiments (50 ms) acquired on our 15N-labelled sample. R1, R2 and NOE data In Figure 2(a)-(c) and Table 1 the R1, R2 and NOE data are summarised. In Table 1 the statistical

1085

Dynamics of Two Homologous GA Modules

(a) 10

20

30

40

ALB8GA LKNAKEDA AIAELK KKAGITSDF FYFNAINK KAKTVEE EVNALKNEILK KAHA TIDQWLL G148 GA3 MKAIFVLNAQHDEAVDANSL LAEAKVLA ANRELD DKYGV SDY YYKNLINN NAKTVEG GVKALIDEILA AALP Consensus (of 16 GA modules)

L..AKE.AI.ELK..GI.SD.Y...INKAKTVEGV.ALK.EIL..

(b)

45 13 20

28 4 32

Figure 1. Organisation of the two homologous GA modules. (a) Alignment of ALB8-GA sequence6 and G148-GA3 sequence.13 Bold-face letters indicate residues that are identical in both GA modules. The GA module that has been shuf¯ed from streptococcal protein G (exempli®ed by G148-GA3) into protein PAB (denoted ALB8-GA) of P. magnus, comprises 45 residues6. This sequence (boxed) is conserved, whereas the G148-GA3 and ALB8-GA studied here contain 21 (19 ‡ 2) or 8 (6 ‡ 2) non-homologous additional residues, respectively. The consensus sequence resulting from sequence alignment of 16 GA modules11 is shown at the bottom. The residue numbers above the ALB8-GA sequence are used in the text to refer to individual residues of each construct. The numbering corresponds to a subtraction of six from every residue number in our previous papers regarding ALB8-GA.9,11 Segments 4-13, 20-28 and 32-45 in ALB8-GA, and segments (ÿ1)-15, 20-27 and 32-45 in G148-GA3 are determined to be helical12 and are indicated by black bars above each sequence. (b) Image of the solution structure of ALB8-GA11 (N terminus downwards) using the co-ordinate ®le 1GAB. The radius of the tube, representing the mean backbone conformation, is proportional to the rms deviation of the backbone co-ordinates of the 20 structures representing the solution structure (1GJS). The approximate locations for the ®rst and last residue of each helix are indicated by their corresponding residue numbers. It has previously been shown that the global fold of G148-GA3 is nearly identical to that of ALB8-GA.10 The Figure was generated using MOLMOL.65

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Dynamics of Two Homologous GA Modules

Table 1. Summary of relaxation data at 14.1 T and 18.8 T Parameter

Average  sd

R1 R2 NOE Zz Zxy

2.16  0.10 6.53  0.84 0.73  0.06 1.54  0.10 4.44  0.41

R1 R2 NOE Zz Zxy

1.93  0.08 9.41  0.77 0.73  0.05 1.41  0.06 5.95  0.45

R1 NOE Zz Zxy

1.64  0.07 0.79  0.05 1.33  0.07 5.48  0.49

Range Residues 1-45 of ALB8-GA at 14.1 T 1.73-2.31 4.09-8.83 0.51-0.80 1.20-1.69 2.76-4.86 Residues 1-45 of G148-GA3 at 14.1 T 1.66-2.04 7.48-12.16 0.55-0.78 1.26-1.58 4.63-6.86 Residues 1-45 of ALB8-GA at 18.8 T 1.45-1.78 0.64-0.85 1.13-1.53 3.59-6.05

Uncertainty (%) 2.5 3.0 2.9 1.5 1.2 8.7 6.1 3.5 2.0 2.6 4.0 2.6 2.1 1.8

All parameter values and ranges are given in sÿ1. The uncertainty is the average relative error.

data are only given for residues belonging to the de®ned GA module sequence (Figure 1(a)) in order to simplify comparison between the two GA modules. Clear differences between the two GA modules can be seen. The different relaxation behaviour of the two GA modules is particularly pronounced in the R2 values, and to a lesser extent in the R1 values. The average R2 value is much higher for G148-GA3 than for ALB8-GA, and the average R1 value is noticeably lower for G148-GA3 than for ALB8-GA. However, the average NOE value is the same for both GA modules when including only data for residues belonging to the de®ned GA module sequence. For ALB8-GA, several residues in the loop between helix 1 and 2, and also in the beginning of helix 2 display substantially larger than average R2 values. For G148GA3, especially Val17 in the loop between helices 1 and 2 has a substantially larger than average R2 value. For both GA modules, most residues in the termini have low R1, low R2 and low NOE values, indicating the presence of substantial motions on the picosecond-nanosecond time scale. Calculating the average R2/R1 ratio for residues with negligible motions on the picosecond-nanosecond time scale (NOE > 0.65 at 14.1 T, as is customary) after excluding residues with possible motions on the microsecond-millisecond time scale (according to the expression and criterion of Tjandra et al.16) gives a value of 2.9 for ALB8-GA and a value of 4.7 for G148-GA3. This yields estimates of the single global rotational correlation time for all spins (tc) according to equation (13) as 4.3 and 6.1 ns for ALB8-GA and G148-GA3, respectively. The value for G148-GA3 (7.1 kDa) is much larger than expected for an isotropically tumbling molecule according to the Stokes-Einstein relation. A high value of an individual R2/R1 ratio can be a consequence of anisotropic rotational diffusion and/or conformational exchange. To distinguish between these two factors, Zz and Zxy values have been determined and will be described in the following.

In general, high values of R2/R1 (and tc), such as observed for G148-GA3, can also be due to nonspeci®c aggregation. However, in the present case that is not the most plausible explanation (vide infra). Z z and Z xy data In Figure 2(d)-(e) and Table 1, the Zz and Zxy data are summarised. Again, clear differences between the two GA modules are evident. The average Zxy value is much higher for G148-GA3 than for ALB8-GA, and the average Zz value is lower for G148-GA3 than for ALB8-GA. The residues in ALB8-GA displaying substantially larger than average R2 values do not have larger than average Zxy values and therefore the reason for the elevated R2 values most likely is conformational exchange processes and not rotational anisotropic diffusion effects. For both GA modules, most residues in the termini have low Zz and low Zxy values, again indicating the presence of substantial motions on the picosecond-nanosecond time scale in these regions. By the same argument, it is also seen that the loop between helices 1 and 2 exhibits motions on the picosecond-nanosecond time scale, a fact indicated by low R1 and low NOE values but obscured in the R2 data most likely because of elevated R2 values due to conformational exchange processes. Calculating the average Zxy/Zz ratio for residues with negligible motions on the picosecond-nanosecond time scale gives a value of 2.9 for ALB8-GA and a value of 4.2 for G148-GA3. A bene®t of using Zxy/Zz ratios is that only residues with motions on the picosecond-nanosecond time scale (NOE 4 0.65 at 14.1 T) need to be excluded from consideration, since a large Zxy/Zz ratio cannot be a consequence of conformational exchange processes but instead must be due to rotational diffusion anisotropy. The value of tc obtained from equation (13) is 4.2 and 5.6 ns for ALB8-GA and G148-GA3, respectively. This value of tc of G148-

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Dynamics of Two Homologous GA Modules

Figure 2. Graphs of the 15N spin relaxation data at 14.1 T for ALB8GA (left) and G148-GA3 (right). (a) R1, (b) R2, (c) NOE, (d) Zz and (e) Zxy values versus residue number. R1 ˆ 1.467 (0.033) sÿ1, R2 ˆ 3.796 (0.168) sÿ1, NOE ˆ 0.39 (0.02), Zz ˆ 0.840 (0.032) sÿ1 and Zxy ˆ 1.697 (0.023) sÿ1 for Trp ÿ 01 Ne1H of ALB8-GA (not included in the Figure). Helices are indicated by black bars above each column of panels.

GA3 is still too high for a 7.1 kDa molecule tumbling isotropically in solution. It thus seems as if the high values of the R2/R1 ratios described above are mostly (but not entirely) due to rotational diffusion anisotropy as conformational exchange processes cannot contribute to high Zxy/Zz ratios. This makes Zxy/Zz ratios more valuable in determining rotational correlation times and de®ning rotational diffusion tensors. We have calculated the conformation exchange-free R2 (R02) from Zz and Zxy data according to equation (7) in order to obtain residue-speci®c conformational exchange contributions to R2 (Rex) as will be described in the next section.

Conformational exchange For ALB8-GA, seven residues (Gly16, Asp20, Phe21, Tyr22, Phe23, Lys28, Thr31) were excluded from the calculation of tc (see above) from R2/R1 ratios due to possible conformational exchange. Thr18 also has a high R2/R1 ratio but was excluded because of a NOE 4 0.65, indicative of motions on the picosecond-nanosecond time scale. Using Rex ˆ R2 ÿ R02 to calculate Rex values, ®ve residues (Thr18, Asp20, Phe21, Tyr22 and Phe23) in the beginning of helix 2 and the preceding loop, were found to have Rex values in the range 1.5-3.2 sÿ1 (Figure 3(a)). The remaining three residues with

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Dynamics of Two Homologous GA Modules

Figure 3. Graphs of Rex and spectral density values for ALB8-GA (left) and G148-GA3 (right). (a) Rex, (b) J(0), (c) J(oN) and (d) J(0.870oH) values versus residue number. Values obtained at 14.1 T are indicated in black whereas values obtained at 18.8 T (only ALB8-GA) are indicated in white. The J(0) values for ALB8-GA were formed by averaging J(0) values at 14.1 and 18.8 T and are therefore shown in grey. Only residues belonging to the de®ned GA module sequence are shown in order to simplify comparison between the two GA modules. Rex ˆ 0.963 (0.212) sÿ1, J(0) (14.1 T) ˆ 0.582 (0.035) ns, J(0) (18.8 T) ˆ 0.557 (0.024) ns, J(oN) (14.1 T) ˆ 0.255 (0.007) ns, J(oN) (18.8 T) ˆ 0.175 (0.004) ns, J(0.870oH) (14.1 T) ˆ 13.961 (0.556) ps and J(0.870oH) (18.8 T) ˆ 10.101 (0.430) ps for Trp ÿ 01 Ne1H of ALB8-GA (not included in the Figure). Helices are indicated by black bars above each column of panels.

high R2/R1 ratios (Gly16, Lys28 and Thr31) were, along with several other residues, also found to have elevated Rex values but less than 1 sÿ1 and therefore not analysed further here. No Rex value could be obtained for Leu11, Ile17 and Val35 due to overlap. As seen (Figure 3(a)) by the many residues having non-zero Rex, G148-GA3 is much more ¯exible on the microsecond-millisecond time scale than ALB8-GA. This could be connected to the faster hydrogen exchange (higher kexvalues) observed for G148-GA3 as will be described in detail below. Val17 has the largest value of Rex, 5.8 sÿ1 (Figure 3(a)), not surprisingly as it had a much larger than average R2 value without a concomitant large Zxy value. The corresponding residue in

ALB8-GA, Ile17, was unfortunately severely overlapped with Val35, preventing extraction of any data for this residue. However, the neighbouring residue, Thr18, has the highest value of Rex in ALB8-GA (3.2 sÿ1). Unfortunately, comparison of the two GA modules is again impossible since G148-GA3 has a deletion at this position. No Rex values could be obtained for Leu01, Asp20 and Lys30 due to unreliable ®ts of some of the relaxation data and Tyr21 could not be identi®ed in any spectra (possibly exchange broadened beyond detection). The two glycine residues (Gly16 and Gly34) in G148-GA3 have the lowest Rex values in G148-GA3. In Figure 3(a), Rex values are only shown for residues belonging to the de®ned GA module sequence (Figure 1(a)) in order to simplify

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Dynamics of Two Homologous GA Modules

comparison between the two GA modules. None of the small negative Rex values are statistically signi®cant, indicating the reliability of this method in separating conformational exchange and rotational anisotropic diffusion effects. The Rex term represents conformational exchange processes contributing to R2 occurring on the microsecond-millisecond time scale. The Rex values (for a two-site exchange) are dependent on the difference in chemical shift of the nucleus in the two conformational states, the populations of the two states, and the rate of exchange between the two states. As we do not know the chemical shift differences and the populations of the two states, the Rex values obtained here can only be interpreted qualitatively. Rotational diffusion anisotropy The advantage of using Zz and Zxy data when determining rotational correlation times and rotational diffusion tensors is that large ratios, which are particularly important in de®ning the diffusion tensor, do not have to be excluded. By contrast, using R2/R1 ratios, an elevated ratio may be excluded believing that it is caused by conformational exchange when in reality it is caused by rotational diffusion anisotropy, which in turn makes it important to include the value in a correct analysis. The principal moments of inertia of ALB8-GA have relative magnitudes of 1.00  0.92 (0.02)  0.41 (0.02) when calculated as an average (1 standard deviation (sd)) of the 20 structures (1GAB) representing its solution structure. The corresponding values for G148-GA3 are 1.00  0.97 (0.02)  0.20 (0.04) (average of 30 structures}12 representing its solution structure (1GJS)). This indicates that both protein domains should exhibit anisotropic rotational diffusion to some extent, with G148-GA3 being the more anisotropic molecule. By a visual inspection of the structure of ALB8-GA in Figure 1(b) it is suggested that the domain could be approximated by a prolate ellipsoid. The same holds for G148-GA3, which has an even larger ratio of long to short axes due to the presence of the 19 extra N-terminal residues (see Figure 1(a) for details). One simple approach to get a crude estimate of the rotational diffusion anisotropy is to calculate the rotational diffusion coef®cients from standard hydrodynamic equations.17 The ratios between long and short axes calculated from the principal moments of inertia are 1.53 and 2.22 for ALB8-GA and G148-GA3, respectively. The so-called Perrin shape factors are Fa ˆ 0.86 and Fb ˆ 1.21 for ALB8GA, and Fa ˆ 0.79 and Fb ˆ 1.67 for G148-GA3. These factors yield values of 1.20 and 1.56 for the ratio of rotational correlation times for NH bond vectors oriented along the long axis and along the short axis for ALB8-GA and G148-GA3, respectively. Furthermore, the rotational diffusion anisotropy, which is commonly expressed as Dk/D? (Dk

and D? are the diffusion constants parallel and perpendicular to the symmetry axis of an axially symmetric diffusion tensor) is 1.41 and 2.11 for ALB8-GA and G148-GA3, respectively. Naturally, the reliability of this method in estimating the anisotropy relies on the approximation of modelling the molecule as an axially symmetric ellipsoid with a smooth surface. The rotational diffusion anisotropy can also be determined by ®tting of R2/R1 ratios18,19 or through an analysis of local diffusion coef®cients20,21 and information about the angle between the NH bond vector and the axis of symmetry given by the structural co-ordinates. Since in our case most well-de®ned residues are located in one of the three parallel helices (Figure 1(b)) making more or less the same angle with the symmetry axis, the angle distribution is limited and therefore it becomes impossible to de®ne the anisotropy using such an approach in the present case. However, the fact that a majority of the NH bond vectors are oriented approximately along the long axis of the molecule explains the high tc value calculated for G148-GA3. In the reduced spectral density mapping approach, the assumption of isotropic tumbling is not required. Therefore, we have analysed our 15N spin relaxation data using this approach described immediately below. Reduced spectral density mapping We have determined R1, NOE, Zz and Zxy values at 14.1 and 18.8 T for ALB8-GA and at 14.1 T for G148-GA3. R2 values have only been determined at 14.1 T and are merely utilised for the determination of Rex values since we determine an exchange-free J(0) from Zz and Zxy data (equation (10)) rather than the more common approach of calculating J(0)eff (which can contain Rex) using R2 values (equation not shown). We therefore do not have the problem of opposing effects of motions on picosecond-nanosecond and microsecond-millisecond time scales. The reduced spectral density maps of the two GA modules, under the assumption that the reduced spectral density approach22 is valid, are shown in Figure 3(b)-(d). The relaxation behaviour of G148-GA3 as judged by reduced spectral density maps is somewhat obscured by the large error estimates, resulting from wider relaxation rate constant error estimates and successive error propagation. The J(0) values show the largest variation across the sequences (Figure 3(b)). As we have determined J(0) from Zz and Zxy values, we only have contributions from motions on the picosecond-nanosecond time scale and no contributions from conformational exchange processes occurring on the microsecond-millisecond time scale. Residues with low J(0), high J(0.870oH), and to a lesser extent, low J(oN) undergo motions on the picosecond-nanosecond time scale. For both GA modules this occurs primarily for residues in the loops between helices (loop 1 in particular) and the

1090

Dynamics of Two Homologous GA Modules

also ®t the relaxation data to the model-free functions as is described in detail below. Model-free calculations

Figure 4. Graphical summary of the ®ts of standard (equation 11) and extended (equation 12) model-free functions to 15N spin relaxation parameters of ALB8-GA using two different approaches (single tc for all spins or independent tc,i for each spin i). (a) Blue bars correspond to independent tc,i for each spin i, and the red line corresponds to the optimised single tc (ˆ4.24 ns) for all spins. The tc,i value was 2.893 (0.072) ns (extended model-free function) for Trpÿ01 Ne1H of ALB8-GA (not included in the Figure). (b) Generalised overall order parameters S2 obtained for an optimised single tc (ˆ4.24 ns) for all spins are shown in red whereas generalised overall order parameters S2 obtained for independent tc,i for each spin i are shown in blue, together with the rms deviation of the backbone co-ordinates (black line) of the 20 structures representing the solution structure of ALB8-GA. Filled (red or blue) circles represent S2 values obtained using the standard model-free function and open circles (red or blue) represent S2 values obtained using the extended model-free function. S2 ˆ 0.341 (0.021) and S2 ˆ 0.619 (0.027) (extended model-free function was chosen in both protocols) for Trpÿ01 Ne1H of ALB8-GA using a single tc for all spins or independent tc,i values for each spin i, respectively. Helices are indicated by black bars.

C terminus. The reason that the same effects are not seen for the residues with lowest numbers in each panel of Figure 3(b)-(d) is that we in this study have chosen to concentrate on residues that belong to the de®ned GA module sequence (Figure 1(a)) and therefore the rather long N terminus (and two residues in the C terminus) has been excluded to clarify the presentation and to facilitate comparison of the two GA modules. As indicated by low R1, low R2 and low NOE values described above, both termini experience ¯exibility on the picosecond-nanosecond time scale. In Figure 3(b)(d) it is also seen that J(oN) and J(0.870oH) decrease with increasing oN and oH and we have therefore

The high quality of the spectra recorded for ALB8-GA and its moderate degree of rotational diffusion anisotropy, makes it the more suitable candidate for model-free analysis. By contrast, the spectra recorded for G148-GA3 are poorer, resulting in wider error estimates for relaxation rate constants (Table 1), and the degree of anisotropy is of necessity greater. For these reasons, only ALB8-GA has been subject to model-free analysis, using R1, NOE, Zz and Zxy values at 14.1 and 18.8 T. Furthermore, the reduced spectral density mappings reveal no signi®cantly different trends when it comes to motions on the picosecond-nanosecond time scale, and also Rex values, indicative of motions on the microsecond-millisecond time scale, are estimated by other means. In this context, it is important to note that model-free calculations using the standard 15N spin relaxation data set (R1, R2 and NOE at one magnetic ®eld strength) do not enable simultaneous optimisation of Rex and fast motions on two time scales. By calculating R02 or using the Zxy/Zz directly (as implemented in Modelfree 4.10, kindly provided by A. G. Palmer) and thereby avoiding model-free models with Rex, an improvement in the description of fast motions will be achieved, as previously described.23 Model-free calculations for ALB8-GA were ®rst performed assuming isotropic tumbling, even though the molecule is anisotropic to some extent. Initially, the single tc for all spins was set to 4.18 ns (average of 4.23 ns (14.1 T) and 4.13 ns (18.8 T)). Model selection (equation (11) or (12)) was based on statistical considerations. A total of 36 residues were ®t with the standard Lipari-Szabo model-free function (S2 and tf, these variables are de®ned in Materials and Methods) according to equation (11), whereas the other 12 residues (Trpÿ01 Ne1H, Aspÿ03, Thr18, Ser19, Asp20, Phe21, Tyr22, Ala29, Lys30, Ala45, His46 and Ala47), in the termini, ends or beginnings of helices and the loops inbetween, were ®t with the so-called extended model-free function (S2f, S2s and ts, these variables are de®ned in Materials and Methods) according to equation (12) with the second term neglected, as is customary. This is quite expected, since termini, ends or beginnings of structural elements and loops often have additional motions on the nanosecond time scale as previously exempli®ed.24 ± 26 However, it has been shown that use of the extended model-free function can result in substantial amplitudes of non-existent internal motions on the nanosecond time scale if true rotational diffusion anisotropy is neglected.27 With the optimised tc value (4.24 ns, Figure 4(a)) it was found that all residues were ®t with the same model as in the ®rst cycle. Figure 4(b), shows the S2 values and their corresponding errors. One reason for errors in S2 values being larger than reported in most other

Dynamics of Two Homologous GA Modules 15

N spin relaxation studies is that CSA was treated as a random variable with Gaussian distribution. It is seen that the termini and the loops in-between apparently are more ¯exible on the picosecondnanosecond time scale than their adjacent residues, a fact also indicated by the reduced spectral density mappings described. Since the molecule is not isotropic the relaxation depends on the orientation of the NH bond vector with respect to the symmetry axis of the rotational diffusion tensor and more elaborate spectral density functions are needed to fully capture the motions of such molecules.28 However, for limited degrees of rotational diffusion anisotropy, the simple spectral density function can be used with an independent local rotational correlation time (tc,i) for each residue.27,29,30 Therefore, the model selection was also performed using an independent tc,i for each spin i (Figure 4(a)). As expected, more residues could now be ®t with the simpler standard modelfree function. Only residues Ser19, Phe21, His46 and Ala47 required the more complex extended model-free function to ®t the data. As seen in Figure 4(b), introducing an independent tc,i for each spin i does not alter the overall trend in S2, despite large variability in tc,i values (Figure 4(a)). For residues that were ®t with the standard LipariSzabo model-free function in both the global and local approach, the S2 values are nearly identical, as expected.27 The exact values of S2 may differ somewhat between the two approaches if different (standard or extended) model-free equations have been chosen. For the case when using a single tc for all spins the average S2 values obtained were 0.78 (0.13) for backbone residues, 0.81 (0.09) for GA module residues and 0.83 (0.06) for helical residues. For the case when using an independent tc,i for each spin i the average S2 values obtained were 0.80 (0.10) for backbone residues, 0.82 (0.06) for GA module residues and 0.83 (0.05) for helical residues. This implies that the three-helix bundle of ALB8-GA is a compact structure with low ¯exibility when it comes to motions on the nanosecond-picosecond time scale. In a previous study describing the solution structure of ALB8-GA, the N terminus, and to a lesser extent the C terminus and the loops between the helices, were less well-de®ned, mainly explained by a lack of inter-proton constraints possibly being a consequence of ¯exibility. Here it has been con®rmed that the higher root mean squared (rms) deviations in these regions result from ¯exibility as the S2 values are lower in the corresponding regions (Figure 4(b)) and not caused by a low density of inter-proton constraints. The values of tf, were small for most residues and were smaller than 70 ps for both protocols (single tc for all spins/independent tc,i for each spin i), and the values of ts were in the range 0.3-2.6 ns. For de®nitions of tf and ts, see Materials and Methods.

1091 The resulting tc,i values ranged from 3.50 to 4.61 ns (when excluding the C-terminal residue and Trpÿ01 Ne1H). This veri®es that the molecule is indeed anisotropic although the rotational diffusion anisotropy does not in¯uence the obtained S2 values noticeably if the same model is chosen in both protocols. The symmetry axis of the inertia tensor runs through the centre of the bundle of helices and consequently the three helices (Figure 1(b)) make approximately the same angle to the axis of symmetry. The tc value from the model-free calculations and also the tc value from Zxy/Zz ratios is to a large extent dominated by the high values of tc,i in the helices (the NH bond vectors within a given helix are more or less parallel). The smaller the angle between the helix and the symmetry axis of the inertia (or diffusion, to be correct) tensor the more the tc value will be overestimated (for a prolate ellipsoid, and underestimated for an oblate ellipsoid). In conclusion, the fact that most of the NH bond vectors are oriented approximately along the long axis of the molecule is a likely explanation for the slightly high tc value of ALB8-GA. In addition, in a previous study we observed a linear dependence between the ellipticity at 222 nm and protein concentration in the range 85 mM to 1.3 mM, indicative of the absence of signi®cant oligomerisation.9 Another example of the signi®cant in¯uence of limited angle distribution on the value of tc is the bacteriorhodopsin fragment (1-36)BR comprising a single helix (residues 9-31). In analogy to ALB8-GA (and G148-GA3), the structure is anisotropic and its NH bond vectors are nearly parallel to the long axis and, not surprisingly, 15N spin relaxation studies yielded a value of tc as high as 5.8 ns for this 36-residue protein fragment.31 G148-GA3 represents yet another case where the tc value is dominated by the high values of tc,i in the helices, resulting from the small angles between the helices and the symmetry axis of the inertia tensor, and for this reason the value of tc will be overestimated. It is illustrative to compare G148-GA3 with the Ig-light-chain-binding domain of protein L32 as both have a long, unstructured N terminus (17 residues) protruding out from the axis of symmetry, both have signi®cant rotational diffusion anisotropy (values of 1.4 versus 1.6 (G148-GA3) for the ratio of rotational correlation times for NH bond vectors oriented along the long axis and along the short axis) and are of approximately the same size (78 versus 65 residues (G148-GA3)). When calculating the tc value for helical residues in G148-GA3, a value of 5.71 ns was obtained from the Zxy/Zz ratios at 14.1 T. This value should then be compared with the tc value for the single helix, packed in a parallel fashion against the fourstranded b-sheet, in protein L. Indeed, a large value is found for the helix (6.0 ns) as compared to residues in the b-sheet (4.4 ns) of protein L. This again highlights the importance of separating conformational exchange effects from rotational diffu-

1092

Dynamics of Two Homologous GA Modules

Table 2. Summary of hydrogen exchange data Res. no.

ALB-GA kex, pH 6.0

ALB-GA kex, pH 7.2

ALB8-GA Log(P), pH 7.2

G148-GA3 kex, pH 7.2

G148-GA3 Log(P), pH 7.2

ÿ18 ÿ17 ÿ16 ÿ15 ÿ14 ÿ13 ÿ12 ÿ11 ÿ10 ÿ9 ÿ8 ÿ7 ÿ6 ÿ5 ÿ4 ÿ3 ÿ2 ÿ1 ÿ1 Ne1H 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.d. n.d. 1.5 2.0 0.7 1.2 >2  10ÿ3 2  10ÿ3-0.2 2  10ÿ3-0.2 2  10ÿ3-0.2 1.53(0.07)  10ÿ3 2.30(0.15)  10ÿ4 1.23(0.09)  10ÿ4 1.07(0.07)  10ÿ4 6.37(0.59)  10ÿ5 1.93(0.03)  10ÿ5 5.51(0.27)  10ÿ5 5.37(0.68)  10ÿ5 3.89(0.30)  10ÿ5 1.33(0.08)  10ÿ4 3.52(0.18)  10ÿ4 2.59(0.18)  10ÿ4 2  10ÿ3 5.59  10ÿ4 0.8 0.7 3.4 2.1 0.4 1.45(0.20)  10ÿ3 2  10ÿ3-0.2 2  10ÿ3-0.2 2.35(0.22)  10ÿ4 7.75(0.36)  10ÿ4 2  10ÿ3 6.27(0.24)  10ÿ4 1.9 2  10ÿ3 0.9 0.5 2  10ÿ3 1.40  10ÿ4 1.00(0.07)  10ÿ3 5.88(0.38)  10ÿ4 1.03(0.05)  10ÿ4 1.70(0.11)  10ÿ4 9.99(0.51)  10ÿ4 6.49(0.75)  10ÿ4 1.14(0.09)  10ÿ4 1.96(0.12)  10ÿ4 2  10ÿ3-0.2 2  10ÿ3-0.2 1.0 0.3

n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.d. n.d. >8 >8 6.3 >8 0.7 1.1 0.3 2  10ÿ3-0.2 2  10ÿ3 1.14(0.21)  10ÿ3 5.18(0.37)  10ÿ4 4.59(0.45)  10ÿ4 2.65(0.20)  10ÿ4 7.84(0.73)  10ÿ5 2.18(0.13)  10ÿ4 2.36(0.12)  10ÿ4 1.59(0.14)  10ÿ4 6.13(0.39)  10ÿ4 2.27(0.38)  10ÿ3 1.43(0.09)  10ÿ3 2  10ÿ3-0.2 2  10ÿ3 6.6 4.3 >8 >8 0.7 2  10ÿ3 0.8 0.5 1.39(0.21)  10ÿ3 2  10ÿ3 2  10ÿ3-0.2 2  10ÿ3 >8 2  10ÿ3-0.2 2.3 1.4 2  10ÿ3-0.2 6.71(0.53)  10ÿ4 2  10ÿ3 2  10ÿ3 4.97(0.36) 10ÿ4 6.71(0.68)  10ÿ4 2  10ÿ3 2  10ÿ3 5.14(0.31)  10ÿ4 9.74(0.55)  10ÿ4 2  10ÿ3-0.2 0.6 3.2 0.9

n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.d. n.d. <0.07 <0.48 0.49 n.a. 0.97 0.67 1.77 4.03-2.03 3.74 3.62 3.61 3.61 4.12 4.09 4.15 3.84 3.79 3.68 3.44 3.68 4.47-2.47 2,86 0.38 1.44 <0.60 <0.18 1.45 3.23 2.15 2.12 2.85 4.46 4.48-2.48 3.54 <0.56 4.25-2.25 0.64 0.71 3.54-1.54 3.04 3.77 3.74 3.44 3.64 4.04 3.23 3.13 2.92 3.95-1.95 1.84 1.13 0.07

n.d. n.d. >8 1.9 5.2 5.4 3.6 >8 >8 n.d. n.d. >8 3.2 3.1 1.3 5.6 >8 >8 n.a. >8 7.2 0.7 0.3 2  10ÿ3-0.2 2  10ÿ3-0.2 2  10ÿ3-0.2 2  10ÿ3-0.2 2  10ÿ3-0.2 2  10ÿ3-0.2 2  10ÿ3-0.2 2  10ÿ3-0.2 2  10ÿ3-0.2 0.4 n.d. 0.5 1.4 0.5 n.a. 1.4 >8 n.d. 0.8 0.6 1.7 0.4 2  10ÿ3 0.4 1.1 0.9 >8 1.5 2.3 1.1 0.9 2  10ÿ3 2  10ÿ3-0.2 2  10ÿ3 2  10ÿ3 1.21(0.19)  10ÿ3 2  10ÿ3 n.d. 9.09(1.33)  10ÿ4 1.67(0.26)  10ÿ3 2  10ÿ3-0.2 2  10ÿ3-0.2 2  10ÿ3-0.2 n.d.

n.d. n.d. <0.72 0.49 0.32 0.13 0.23 <0.88 <0.92 n.d. n.d. <0.62 0.31 0.86 0.69 0.32 <0.42 <1.09 n.a. <1.29 0.37 1.45 1.52 4.05-2.05 4.16-2.16 3.62-1.62 3.48-1.48 3.99-1.99 4.69-2.69 4.60-2.60 3.91-1.91 3.47-1.47 1.39 n.d. 1.65 1.68 1.27 n.a. 1.59 <0.60 n.d. 1.38 1.73 1.88 1.64 2.48 2.16 2.27 1.87 <0.56 1.38 0.64 0.81 1.67 2.89 4.02-2.02 3.54 2.84 2.70 2.89 n.d. 2.88 2.69 3.99-1.99 4.20-2.20 3.62-1.62 n.d.

Table 2 (legend opposite)

1093

log(protection factor)

Dynamics of Two Homologous GA Modules

5.0 4.0 3.0 2.0 1.0 0.0 1. 0

0

10

20

30

40

50

residue Figure 5. Logarithm of protection factors obtained at pH 7.2 from data in Table 2 versus residue number for ALB8-GA (open circles and dotted lines) and G148-GA3 (®lled circles and continuous line). For residues having hydrogen exchange rates in between those obtained from saturation transfer and H-2H exchange experiments the symbols were placed in the centres of the ranges of log(P). Helix extents are indicated by open (ALB8-GA) and ®lled (G148-GA3) bars.

sion anisotropic effects. The signi®cant in¯uence of extended tails on the tc value has also been observed for the dynamin pleckstrin homology domain33. Thus, the high value of tc for G148-GA3 is mostly due to anisotropic tumbling. However, we cannot rule out the possibility of minor oligomerisation for this GA module and this would also contribute to an elevated tc value, but anisotropic tumbling is likely the dominant cause of the high tc value. Model-free calculations are known to be sensitive to even small degrees of oligomerisation,27 but no such calculations were performed for this GA module. However, the differences in dynamics between the two GA modules actually observed would in no way be reduced by minor oligomerisation. In fact, this would add to the differences in behaviour between them. Hydrogen exchange In addition to 15N spin relaxation data we have performed measurements of hydrogen exchange rates using H-2H exchange and saturation transfer

methods to investigate motions on the second-hour time scale of the two GA modules. The largest kex value possible to determine from H-2H exchange experiments was approximately 2  10ÿ3 sÿ1 (Table 2). The range of hydrogen exchange rate constants possible to determine from saturation transfer methods is 0.2-8 sÿ1, given that the average R1 value is about 2 sÿ1 at 14.1 T (equation (6) and assuming that reductions of peak intensities in the spectrum with water presaturation in the range 20-90 % of corresponding intensities in the spectrum without presaturation can be reliably determined). As evident from Table 2, ALB8-GA at pH 6.0 displays slow hydrogen exchange (low kex values) in the three helices, a fact previously described9,11 but now established with higher accuracy and precision. Utilisation of rapidly acquired 1H-15N HSQC spectra after dissolution of each lyophilised 15 N-labelled GA module in 2H2O and saturation transfer methods have resulted in kex estimates for almost all residues. The present results are consistent with the previous studies9,11 of ALB8-GA except that Tyr22 (Tyr28 with the old numbering) in our previous study11 was seen in a NOESY spectrum 20 minutes after dissolution into 2H2O but is not visible even in the ®rst 1H-15N HSQC in this study and seems to have a kex value of 0.4 sÿ1 as measured in saturation transfer experiments. Even though this high kex value may be a consequence of not accounting for possible cross-relaxation (vide infra), the reason why we do not see the peak in the 1H-15N HSQC-series remains unclear. For ALB8-GA, the residues for which the kex values are measurable by H-2H exchange experiments are primarily present in helices while the amide hydrogens that exchange rapidly and are therefore measured by saturation transfer methods are located preferentially in the loops between the helices and in the termini. The residue in ALB8-GA with slowest hydrogen exchange is Ile09 in the centre of helix 1 with a kex value of 1.93 (0.03)  10ÿ5 sÿ1 at pH 6.0. In general, residues in helix 1 display the slowest hydrogen exchange, followed by residues in helix 3 with slightly faster hydrogen exchange. Residues in helix 2 display

All values are given in sÿ1. All values with an error estimate were determined from the ®tting of H-2H exchange experiments; values for Ile17 and Val35 of ALB8-GA at pH 6.0 were obtained simultaneously by biexponential ®tting of H-2H data and no error estimates were calculated; a few peaks were only seen in the ®rst H-2H exchange experiments preventing ®tting of the data and were therefore assigned the value of 2  10ÿ3 sÿ1 (as this is the approximate value of the fastest rate that can be determined from the ®tting of H-2H experiments (Lys14 of ALB8-GA at pH 7.2)); values in the range 2  10ÿ3-0.2 sÿ1 are too fast to be determined from H-2H experiments and too slow to be determined from saturation transfer experiments; values in the range 0.2-8 sÿ1 are determined from saturation transfer experiments and no error was calculated since possible cross-relaxation is not corrected for and therefore the values are regarded as approximate upper limits; values >8 sÿ1 indicate that the amide hydrogen exchange rates were too fast to be determined by saturation transfer experiments. For ALB8-GA Leu00 is normally overlapped with Glu11 at pH 6.0 but the decay in the H-2H experiments was clearly monoexponential and therefore originates solely from Glu11 (based on comparison with data at pH 7.2), in the saturation transfer experiments it is not possible to resolve the contribution of each residue and therefore Leu00 is assigned the value of >2  10ÿ3 sÿ1 at pH 6.0. n.a. indicates that the corresponding NH is not present in the ALB8-GA or G148-GA3 sequence; and ®nally n.d. indicates that no data could be determined.

1094

Figure 6. Tube representations of ALB8-GA (left) and G148-GA3 (right) colour-coded by the magnitude of (a) Rex and (b) log(P) values. The structures are oriented with the N terminus upwards. Residues with the largest Rex or log(P) values are coloured red and residues with the smallest Rex or log(P) values are coloured yellow. For the remaining characterised residues, the colour is interpolated between yellow and red to represent intermediate values. Residues for which no data are available are coloured grey. The radius of the tube is proportional to the Rex or log(P) values and for residues coloured in grey (not characterised) the radius represent an average of those calculated for neighbouring residues. Only residues belonging to the de®ned GA module sequence are shown in order to simplify comparison between the two GA modules. The Figure was generated using MOLMOL.65

much faster hydrogen exchange than the other two helices. In order to be able to compare hydrogen exchange properties of ALB8-GA with those of G148-GA3, we have performed H-2H exchange and saturation transfer experiments for both GA modules at pH 7.2, for which results and calculated protection factor (P) values are also shown in Table 2. Inspection of the data in Table 2 and Figure 5 reveals a large difference in behaviour of the two GA modules with respect to the rate of hydrogen exchange and degree of protection. It is clearly seen that ALB8-GA in general displays slower hydrogen exchange (lower kex values, Table 2) and higher degree of protection (higher P values, Figure 5), than G148-GA3. For G148-GA3, only the kex value of a few residues in the end of the third helix are measurable by H-2H exchange

Dynamics of Two Homologous GA Modules

experiments. The residue in G148-GA3 with slowest hydrogen exchange is Ile42 with a kex value of 9.09 (1.33)  10ÿ4 sÿ1 at pH 7.2. Since the differences in hydrogen exchange behaviour cannot at present be explained by structural differences this may be interpreted as being due to differences in mobility, which would give rise to transient accessibility. It is revealing to compare the dynamics data obtained from 15N spin relaxation experiments with the data obtained from hydrogen exchange experiments. Interestingly, in both cases G148-GA3 turns out to be the more ¯exible one. This GA module shows a high number of residues exhibiting motions on the millisecond-microsecond time scale, indicating that it is a quite ¯exible protein module as compared to ALB8-GA. An analogous trend was observed through the measurements of hydrogen exchange rates, indicative of motions on the second-hour time scale. In fact, the fast hydrogen exchange (low degree of protection) of G148GA3 is most likely a re¯ection of the high ¯exibility. Tube structures of ALB8-GA and G148-GA3 summarising the millisecond-microsecond and second-hour time-scale motions are shown in Figure 6(a) and (b). Biological implications There are interesting biological implications of the different dynamical and binding properties of the two GA modules. Although the detailed biological consequences of albumin binding to bacterial surfaces remain unclear, previous studies suggest that the binding adds selective advantages to the bacteria by promoting their growth.8 Infections with group C and G streptococci are known to occur in many mammalian species and virtually all C and G streptococcal strains bind albumin,34 underlining the signi®cance of this interaction. For these bacteria the expression of a ¯exible albuminbinding module like G148-GA3 with broad species speci®city therefore appears rational. In contrast, P. magnus has been isolated only from humans. This anaerobic bacterial species is part of the normal ¯ora on most body surfaces and mucous membranes in the mouth, the upper respiratory, and the alimentary and urogenital tracts. Most P. magnus strains do not bind albumin (35 Holst and L.B., unpublished results) but those that do are frequently isolated from patients with deep wound infections,8 suggesting that the acquisition of the GA module turns this commensal into a potential pathogen. Previous work has shown that the ALB8-GA module studied here, which is found in protein PAB of P. magnus strain ALB8,6 originates from a protein G gene of group C or G streptococci. The transfer of this GA module is probably the result of a selective pressure driven by broad spectrum antibiotics, which are often used in the treatment of patients with deep wound infections. The ALB8-GA module, present only in a few human isolates of P. magnus, has subsequently evolved

1095

Dynamics of Two Homologous GA Modules

to bind human albumin with higher af®nity than its predecessor G148-GA3. This evolutionary focusing on human albumin, the only albumin that P. magnus will come across, has resulted in a loss of molecular ¯exibility and thereby also a narrower albumin-binding speci®city. The differences in dynamics and albumin-binding properties of the two GA modules studied here, clearly and beautifully re¯ect the power of bacterial evolution and adaptation at the molecular level.

Materials and Methods Expression and purification The cloning work and production of 15N-labelled ALB8-GA was done using Escherichia coli strain SG20048. E. coli SG-20048 harbouring the expression vector pHD389 was used to inoculate 50 ml LB medium supplemented with ampicillin (50 mg lÿ1) and was left to grow overnight at 30  C with shaking. 2.5 ml of the overnight culture was used to inoculate 60 ml M9 medium (0.64 g lÿ1 15NH4Cl, 3.0 g lÿ1 D-glucose, 0.13 g lÿ1 MgSO4, 11.0 mg lÿ1 CaCl2, 1.0 g lÿ1 thiamine, 2.9 mg lÿ1 FeCl3, 6.0 g lÿ1 Na2HPO4, 3.0 g lÿ1 KH2PO4 and 0.5 g lÿ1 NaCl). The culture was grown at 30  C with shaking until the absorbance at 600 nm (A600) reached 0.5-0.6 and was subsequently used to inoculate 400 ml of the M9 medium which again was allowed to grow at 30  C with shaking until A600 reached 0.5-0.6 at which time expression of ALB8-GA was induced by adding 100 ml M9 medium (70  C) and raising the temperature to 41  C. The cultures were harvested after 4.5 hours (A600 ˆ 1.31.4) by centrifugation followed by resuspension of the cell pellet in phosphate buffer (10 mM, pH 6.5) prior to sonication. After clari®cation, the supernatant was applied to an HSA-Sepharose column. After extensive washing of the column with the phosphate buffer, the protein was eluted with HCl (pH 2.0), neutralised with Tris buffer (1 M) and lyophilised. Lyophilised HSA-binding protein was dissolved in water and loaded on a Sephadex G-50 column with a bed volume of 1830 ml. The running buffer was ammonium acetate (50 mM, pH 6.0) and the ¯ow rate was 0.8 ml minÿ1. The fractions containing HSA-binding protein of the correct size were collected and lyophilised. Lyophilised protein was further puri®ed using high performance liquid chromatography (HPLC). Fractions containing the puri®ed ALB8-GA were subsequently collected and lyophilised. The expression and puri®cation of G148-GA3 was carried out as previously described10 (see Figure 1(a) for details regarding the protein fragments in this study). Sample preparation Protein samples were dissolved in either 0.33 ml H2O (ALB8-GA, pH 6.0) or 0.33 ml 20 mM phosphate buffer (G148-GA3, pH 7.2) containing 0.02 % (w/v) NaN3, 8 mM 2,2-dimethylsilapentane-5-sulphonic acid (DSS) for reference and 10 % 2H2O for the lock signal at a protein concentration of 2 mM. The choices of solvent and pH were based on the conditions used in previous studies of the two domains.9,10 All pH values are electrode readings with no correction for the deuterium isotope effects. Samples for H-2H exchange experiments were prepared as described above but using pure (99.96 %) 2H2O instead of H2O (ALB8-GA, pH 6.0) or 2H2O-based

20 mM phosphate buffer (both GA modules at pH 7.2 to make comparison of kex values possible). Relaxation rate equations The relaxation rates of a protonated 15N nucleus spin are dominated by dipole-dipole relaxation of the directly attached proton and, to a smaller extent, CSA of the 15N nucleus and are related to the spectral density function of the NH bond vector as detailed below:36,37 R1 ˆ d2 fJ…oH ÿ oN † ‡ 3J…oN † ‡ 6J…oH ‡ oN †g ‡ 3c2 J…oN †

R2 ˆ

…1†

d2 f4J…0† ‡ J…oH ÿ oN † ‡ 3J…oN † 2 ‡ 6J…oH † ‡ 6J…oH ‡ oN †g ‡

…2†

c2 f4J…0† ‡ 3J…oN †g ‡ Rex 2

NOE ˆ 1 ‡

d2 gH f6J…oH ‡ oN †J…oH ÿ oN †g R1 gN

…3†

Zxy ˆ 6cdP2 …cos b†J…oN †

…4†

Zxy ˆ cdP2 …cos b†f4J…0† ‡ 3J…oN †g

…5†

where d ˆ {ÿm0hgHgN}/16p2}hrÿ3 c ˆ oN
s/3, m0 NHi, (4p  10ÿ7 kg m sÿ2 Aÿ2) is the permeability of vacuum, h (6.62618  10ÿ34 Js) is Planck's constant, gH (2.67519  108 rad Tÿ1 sÿ1) and gN (ÿ2.712  107 rad Tÿ1 sÿ1) are the gyromagnetic ratios of amide proton and Ê ) represents amide nitrogen, respectively, rNH (1.02 A the distance between amide proton and amide nitrogen, oH and oN are the Larmor frequencies of amide proton and amide nitrogen, respectively,
s is the CSA (often16,38 ± 40 taken to be ÿ170 parts per million (ppm)) and J(o) is the value of the spectral density function at frequency o. For
s, we used random values drawn from a Gaussian distribution with a sd of 15 ppm41 and centred at ÿ170 ppm16,38 ± 40 for backbone NH or ÿ90 ppm42 for Ne1H of Trpÿ01, as appropriate. The Rex term43 represents processes contributing to R2 during the Carr-Purcell-Meiboom-Gill44,45 (CPMG) pulse train, such as conformational exchange processes on the microsecond-millisecond time scale. Zz is the rate constant for cross-correlated cross-relaxation between Nz and 2HzNz. Correspondingly, Zz is the rate constant between Nx (or Ny) and 2HzNx (or 2HzNy). NMR spectroscopy Spectra were recorded using Varian Inova spectrometers operating at 14.1 or 18.8 T. All data sets were acquired with relaxation delays (when applicable) in random order at a temperature of 27  C. All 15N spin relaxation experiments and experiments for determining amide hydrogen exchange rates were performed with spectral widths of 6982.6 or 6896.6 (Zz for ALB8-GA only) and 1800 Hz in 1H and 15N dimensions, respectively at 14.1 T and with spectral widths of

1096 9210.2 and 2400 Hz in 1H and 15N dimensions, respectively, for ALB8-GA at 18.8 T. The R1, R2 and NOE spectra46 were acquired with 192 t1 increments of 4096 complex points. The total relaxation delays between scans were set to 1.5 (R1), 3.0 (R2) and 6.0 (NOE) seconds. Eight (R1 and R2) or 16 (NOE) scans were added for each free induction decay (FID). The relaxation delays used in the R1 measurements were 2  11, 90, 140, 200, 280, 370, 480, 2  640 and 920 ms for ALB8-GA and 2  11, 90, 140, 200, 280, 370, 480, 640 and 2  920 ms for G148-GA3. The relaxation delays used in R2 measurements were 2  10, 45, 71, 100, 140, 180, 240, 2  320 and 460 ms for ALB8-GA and 10, 2  42, 71, 100, 140, 180, 240, 320 and 2  460 ms for G148-GA3. The NOE values were determined by acquiring two different types of spectra, one with four seconds of proton presaturation and one without proton presaturation. Each type of spectra was recorded twice. Both Zz and Z16,47 were determined from pairs of xy cross-correlation and auto-correlation experiments (vide infra). All these experiments were acquired with 150 t1 increments of 4096 complex points, the total relaxation delay between scans was set to 2.0 seconds. Either 48 (for Zz) or 32 (for Zxy) scans were collected for each FID in the cross-correlation experiments, whereas 16 scans/ FID (both Zz and Zxy) were collected in the auto-correlation experiments. The cross-correlation experiment describes the cross-relaxation between 2HzNz (or 2HzNxy) and Nz (or Nxy) due to dipolar/CSA cross-correlation, whereas the auto-correlation experiment describes the auto-relaxation of the 2HzNz (or 2HzNxy) magnetisation. Relaxation delays for the Zz measurement were (for both GA modules) 2  100, 155, 210 and 265 ms. Relaxation delays for the Zxy measurements were 2  20, 40, 60 and 80 ms for ALB8-GA and 2  15, 30, 45 and 60 ms for G148-GA3. For H-2H exchange measurements several sets of 1 H-15N HSQC48 experiments were performed after a quick shimming. The ®rst such spectrum in each set was started less than three minutes after dissolution of the protonated protein in 2H2O, except for a set of 1H-15N HSQC experiments intended to measure slowly exchanging amide hydrogens (ALB8-GA at pH 6.0) where it was less critical to start the experiment quickly. This larger set consisted of a total of 46 1H-15N HSQC experiments whereas the other three (ALB8-GA at pH 6.0 and 7.2, and G148-GA3 at pH 7.2) sets consisted of 15 or 16 1 H-15N HSQC experiments. The spectra were acquired with 64 t1 increments of 4096 complex points. The relaxation delay was set to 1.3 seconds. The recording times were 7, 14, 28, 56, 112 or 225 minutes corresponding to 2, 4, 8, 16, 32 or 64 scans for each FID. To estimate kex values too large to be determined from H-2H exchange experiments the method of saturation transfer from water to amide protons was applied.49,50 The spectra were acquired with 256 t1 increments of 4096 complex points. The relaxation delay was set to 3.0 seconds. A total of 14 scans were added for each FID. The values of kex are determined from two spectra acquired with or without water presaturation for 1.0 second. Spectral data processing All data sets were processed using Felix 95.0/98.0 software (Biosym/Molecular Simulations). Two distinct processing protocols were applied to all data sets. Protocol 1 was utilised for signals that were well resolved in

Dynamics of Two Homologous GA Modules both dimensions of the spectra, whereas protocol 2 was applied when signals were slightly overlapped in either or both dimensions of the spectra. In all protocols data were convolved with a ten-point sinebell function in both o2 and o1, and the ®rst point was replaced by the average of the ®rst and last point immediately prior to Fourier transformation.51 Protocol 1 applies a ``matched'' 7 Hz exponential ®lter in o2 before Fourier transformation. An 80  shifted squared sinebell ®lter was applied to the data in o1, which was then zero-®lled to 1024 data points prior to Fourier transformation. Protocol 2 applies a 50  shifted sinebell ®lter in o2 before Fourier transformation. The t1 interferograms were extended to twice the number of original points using modi®ed ``forwardbackward'' linear prediction,52 a 50  shifted squared sinebell ®lter was applied followed by zero-®lling to 1024 data points before Fourier transformation.

Rate constant determination and error estimation Values for R1 and R2 were determined by non-linear least squares ®tting of two-parameter monoexponential functions to the measured peak volumes. Values for Zz and Zxy were determined by non-linear least squares ®tting of tanh(Zzt) and tanh(Zxyt) to the quotients Icross(t)/ Iauto(t) of peak volumes at relaxation delays t (t ˆ t1,t2,...) from the cross- and auto-correlation experiments, respectively. The NOE value was calculated as the ratio between the peak volume measured with and without proton presaturation and NOE values reported are formed as the average of two independent measurements. The kex values resulting from H-2H exchange experiments were determined by non-linear least squares ®tting of two-parameter monoexponential functions to the measured peak volumes. The kex values from saturation transfer experiments can be extracted from the following equation:49,50     kex T1 ÿt…kex T1 † I…t† ˆ I…0†  1 ÿ 1 ÿ exp T1 1 ‡ kex

…6†

where I(t) is the peak intensity (volume) after t seconds of water presaturation, I(0) is the peak intensity without water presaturation and T1 is the inverse of R1. We have not tried to distinguish resonance attenuation caused by hydrogen exchange from attenuation caused by crossrelaxation such as can be achieved by recording the experiments at several different pH values.53 Constant w2 boundaries were used to estimate 68.3 % con®dence limits54 for the R1, R2, Zz and Zxy values. Con®dence limits for the NOE values were estimated by application of the error propagation formula for ratios55 to the 68.3 % con®dence limits obtained from duplicate measurements with and without proton presaturation, respectively. Constant w2 boundaries were also used to estimate 68.3 % con®dence limits for the kex values determined from H-2H exchange experiments. Since we take the kex values derived from saturation transfer methods as upper limits only (kex values derived from saturation transfer methods tend to overestimate the corresponding rate when not accounting for possible cross-relaxation), no error estimates have been derived for these values.

1097

Dynamics of Two Homologous GA Modules Conformational exchange By inspection of equations (1), (2), (4) and (5) it is clear that R2/R1 and Zxy/Zz ratios differ only in high-frequency terms ((J(oH) ‡ J(oN)), J(oH) and (J(oH) ÿ J(oN))) of the spectral density functions. These terms can be approximated using the reduced spectral density mapping approach22 as will be described below. By setting Rex ˆ 0 in equation (2) and identifying corresponding components, one obtains an estimate of the value of R02 as outlined by:47 R02 ˆ …R1 ÿ 1:249sNH †

Zxy ‡ 1:079sNH Zz

…7†

where sNH is the 1H-15N dipolar cross-relaxation rate constant, which is calculated as sNH ˆ (gH/gN)(NOE ÿ 1)R1. Finally, Rex values are computed as Rex ˆ R2-R02.

Reduced spectral density mapping As an alternative to the model-free approach, which will described further below, the relaxation parameters can be analysed using the reduced spectral density mapping approach.22,56 ± 58 From R1 and NOE data we can calculate J(0.870oH) as follows: J…0:870oH † ˆ

1 gN …NOE ÿ 1†R1 5d2 gH

…8†

…9†

2

where the approximation J(eoH) ˆ (0.870/e) J(0.870oH) is used to obtain J(0.921oH) from J(0.870oH).22 Finally, J(0) can be determined from Zz and Zxy values:   3 Zxy J…0† ˆ 2 ÿ 1 J…oN † …10† 4 Zz Error estimates for J-values were obtained by propagating the error estimates for R1, NOE, Zz and Zxy values, through the expressions given in equations (8)-(10) by repeated use of the error propagation formulae for sums, differences, products and ratios,55 as appropriate, and assuming errors in corresponding R1, NOE, Zz and Zxy values to be independent.

The model-free formalism In the Lipari-Szabo model-free formalism59,60 the spectral density is described by: ( ) …1 ÿ S2 †t0f 2 S2 tc J…o† ˆ ‡ …11† 5 1 ‡ …otc †2 1 ‡ …ot0f †2 where S2 is the generalised order parameter which characterises the amplitude of the internal motions and 1/tf0 ˆ 1/tc ‡ 1/tf, where tf is the effective correlation time for the (fast) internal motions. In order to account for two internal motions on different time scales, Clore and co-workers24,61 have suggested the following so-called extended form of the spectral density function:

…12†

where S2 is de®ned as S2f
S2s and 1/ts0 ˆ 1/tc ‡ 1/ts, where S2f and S2s are the order parameters of the fast and slow internal motions, and ts is the effective correlation time for the slow internal motions. Customarily, tf is assumed to be small, in which case the second term of equation (12) can be neglected. Model-free parameters were determined by ®tting standard (equation (11)) and extended (equation (12)) model-free functions to the determined relaxation rate constants and measured NOE values using the program Modelfree 4.10 (kindly provided by A. G. Palmer).

Molecular rotational diffusion Initially, residues subject to conformational exchange processes on the microsecond-millisecond time scale were identi®ed by using an expression and a criterion given by Tjandra et al.16. The single tc for all spins was determined using R2/R1 ratios62 for residues with negligible motions on microsecond-millisecond and picosecond-nanosecond time scales (NOE > 0.65 at 14.1 T) using the equation one obtains from equation (11) by assuming that internal motions are limited and fast (large S2 and very short tf) as the ratios then are independent of both S2 and tf: J…o† ˆ

J(oN) can then be calculated from J(0.870oH) as: J…oN † ˆ fR1 ÿ 7d2 J…0:921oH †g=…c2 ‡ d2 †

( ) …1 ÿ S2 †t0f …S2f ÿ S2 †t0s 2 S2 tc ‡ ‡ J…o† ˆ 5 1 ‡ …otc †2 1 ‡ …ot0f †2 1 ‡ …ot0s †2

2 S2 tc 5 1 ‡ …otc †2

…13†

When using Zxy/Zz ratios only residues with non-negligible motions on picosecond-nanosecond time scales have to be excluded. Equation (11) (and its simpli®cation, equation (13)) and equation (12) are only valid for molecules that reorient isotropically in solution.

Calculation of protection factors Intrinsic peptide amide hydrogen exchange rates (kint) were calculated using the program SPHERE which uses correction factors and empirical rates.63,64 The protection factors (P) are then calculated as P ˆ kint/kex.

Acknowledgements This work was supported by the Swedish Research Council (projects 7480 (L.B.) and 31X-13088-03C (T.D.)). The 600 MHz Varian Unity NMR spectrometer (Lund University) was purchased with grants from the Knut and Alice Wallenberg foundation, and the Swedish Council for Planning and Coordination of Research. NMR studies at 18.8 T/800 MHz were carried out at the Swedish NMR Center. M.U.J. acknowledges travel grants from the Swedish Foundation of Strategic Research. We are indebted to Charlotta Damberg at the Swedish NMR Center for excellent technical assistance and stimulating discussions and Dr Per J. Kraulis (Stockholm Bioinformatics Center) for making the original resonance assignment list of G148-GA3 available to us.

1098

Dynamics of Two Homologous GA Modules

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