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J Biol Chem. Author manuscript; available in PMC 2009 November 2.
Published in final edited form as:
PMCID: PMC2771598



CD2 is a T cell surface molecule that enhances T and Natural Killer cell function by binding its ligands CD58 (humans) and CD48 (rodents) on antigen presenting or target cells. Here we show that the CD2/CD58 interaction is enthalpically-driven and accompanied by unfavourable entropic changes. Taken together with structural studies, this indicates that binding is accompanied by energetically-significant conformational adjustments. Despite having a highly charged binding interface neither the affinity nor rate constants of the CD2/CD58 interaction were affected by changes in ionic strength, indicating that long-range electrostatic forces make no net contribution to binding.

The CD2 family of cell-surface glycoproteins are structurally-related members of the immunoglobulin (Ig1) superfamily (1,2). Expressed mainly on haematopoietic cells, they modulate immune responses by homotypic or heterotypic interactions with other members of the CD2 family. CD2 binds CD58 (in humans) or CD48 (in rodents), and these interactions enhance T cell recognition of antigen on antigen-presenting or target cells. This enhancement is thought to involve both adhesion and signalling mechanisms (1,3-7).

CD2 and its interaction with its ligands have been intensively studied and have emerged as an important paradigm for understanding the molecular basis of cell-cell recognition (1,8,9). CD2 and its ligands have structurally-related ectodomains comprised of two Ig domains, with the membrane-distal domains involved in ligand binding (1,8). The interaction of human CD2 with CD58 is characterised by a low affinity (Kd ~10 μM at 37°C), which is the result of a very fast dissociation rate constant (koff > 4 s−1) (10). Structural studies of the individual proteins and site-directed mutagenesis have located the binding sites on the equivalent GFCC′C″ β-sheets of CD2 and CD58, and revealed them to be highly charged (11-13). Solution of the crystal structure of the complex between the human CD2 and CD58 ligand binding domains has provided a detailed view of the binding interface (14). This is relatively small (buried surface area ~1160 Å2) and has poor surface-shape complementarity, consistent with the low affinity (14). Comparison of the structure of the complex with the structure of unbound CD2 (15-17) and CD58 (12,13) revealed significant differences, particularly in the case of CD2. The most prominent differences were in the C′C″ and FG loops of both molecules (14,16). In addition NMR analysis has shown that the CD58 binding site on unbound CD2 is highly flexible, with most of the movement occurring in the C′C″ and FG loops (16,17). Taken together, these data suggest CD2 binding to CD58 is accompanied by conformational adjustment and stabilization of a flexible interface. While these conformational changes provide an explanation for the low affinity of the CD2/CD58 interaction they appear inconsistent with its relatively fast kon (10).

In order to further investigate these putative conformational changes and the discrepancy between the structural and kinetic data we undertook a detailed thermodynamic and kinetic analysis of the CD2/CD58 interaction. We show that the interaction is enthalpically-driven and accompanied by unfavourable entropic changes, consistent with stabilisation of a flexible binding interface. We also show that, despite having a highly charged binding interface, long-range electrostatic interactions have no net effect on the CD2/CD58 interaction.



Soluble forms of CD2 and CD58 were prepared and purified as previously described (18). These comprised the full ectodomains with C-terminal oligohistidine tags. The C-termini of the encoded CD2 and CD58 were SCPEKHHHHHH and TCIPSSHHHHHH respectively.

Surface Plasmon Resonance

These studies were performed on a BIAcore 2000 (BIAcore AB) (19). Unless otherwise stated experiments were performed at 25°C using HBS buffer [10mM HEPES (pH 7.4), 150 mM NaCl, 1mM CaCl2, and 1mM MgCl2] at a flow rate of 10 μL.min−1. Human CD2 was directly coupled to Research Grade CM5 sensor chips (BIAcore AB) using the Amine Coupling Kit (BIAcore) as previously described (10). Kinetics measurements were performed at a flow rate of 50 μL.min−1 and confirmed at three different immobilization levels of CD2, in order to rule out mass transport artefacts.

Affinity, kinetic and thermodynamic properties were determined as described (20). Equilibrium thermodynamic parameters were obtained by measuring the affinity over a range of temperatures (5 to 37°C), and fitting the non-linear form of the van't Hoff equation to these data (21)


where T is the temperature (in K); T0 is an arbitrary reference temperature (e.g. 298.15K); ΔG is the free energy of binding at the standard state (all components at 1 mol.L−1); ΔHTo is the enthalpy change at T0 (kcal.mol−1); ΔCp is the heat capacity change (kcal.mol−1K−1 at constant pressure; and ΔS is the entropy change at the standard state .

ΔG was calculated from the affinity constant (KD) using the equation


where R is 1.987 cal.mol−1K−1; KD is expressed in mol/L; and C is the standard state concentration (1 M).

The activation enthalpy of dissociation (ΔHdiss) was determined by measuring the koff over a range of temperature (10-30°C) and plotting ln(koff/T) against 1/T, the slope of which equals −ΔHdiss/R (20). The ΔHass was calculated from the relationship ΔHassHdiss +ΔH.

In experiments varying the ionic strength a series of HBS stocks were prepared with the indicated NaCl or KF concentration. CD58 samples were diluted in the appropriate HBS buffer and the same HBS buffer was used as the running buffer.

Isothermal Titration Calorimetry (ITC)

These measurements were performed at 25°C using a MicroCal VP-ITC unit (MicroCal Inc.) as described previously (22). Samples were dialysed extensively into HBS. CD58 at 245 μM was injected in 10 μL aliquots into a cell containing 1345 μL of CD2 (at 35 μM), and the heat release was measured. The heat of dilution was obtained by injection of CD58 into HBS and subtracted prior to data analysis. The titration data were fitted by non-linear curve fitting using the Origin software supplied with the instrument to obtain the KD, stoichiometry, and ΔH. TΔS was determined by the relationship TΔS = ΔHRTln(KD/C).


Thermodynamics of human CD2 binding to CD58

In order to investigate whether CD2 binding to CD58 is accompanied by energetically significant conformational changes we undertook a detailed thermodynamic analysis. The binding of CD58 to CD2 was analysed by surface plasmon resonance. The affinity constant (KD) was measured by equilibrium binding analysis (10). The binding free energy of an interaction (ΔG), which can be calculated from the affinity constant (see Experimental Procedures), is comprised of enthalpic (ΔH) and entropic (−TΔS) components (ΔG = ΔH - TΔS).

The relative contribution of enthalpic and entropic components can be determined by measuring the dependence of ΔG on temperature, a procedure termed van't Hoff analysis (see Experimental Procedures). For protein/protein interactions ΔH and TΔS typically vary with temperature, and this variation is measured as the change in heat capacity (at constant pressure) or ΔCp. The binding energy of the CD2/CD58 interaction was measured over the temperature range of 5 to 37°C and ΔH, −TΔS, and ΔCp were determined by fitting the non-linear form of the van't Hoff equation to the data (Fig. 1A). From several different determinations the ΔH at 25°C was determined to be −11.5 ± 0.2 kcal.mol−1 (mean ± SEM, n = 3), which is highly favourable. The corresponding entropic component (−TΔS) was +4.4 ± 0.2 kcal.mol−1, which is unfavourable (Table I).

FIG. 1FIG. 1
Thermodynamic analysis of the CD2/CD58 interaction by SPR
Thermodynamic properties of the CD2/CD58 interaction

The ΔCp determined by van't Hoff analysis was −118 cal.mol−1K−1 (Table I). This falls at the high end of values typically reported for protein/protein interactions, which range from −1000 to 0 cal−1mol.K−1, with an average value of ~−300 cal−1mol.K−1 (23). A negative ΔCp, which is typical of protein/protein interactions, is thought to be the result of the tendency of water to form an ordered ‘shell’ adjacent to non-polar surfaces which ‘melts’ at higher temperatures (24). Upon binding, the burial of non-polar surfaces disrupts this shell, ejecting the water into free solution, with favourable entropic and unfavourable enthalpic effects. Increasing the temperature ‘melts’ the shell so that these effects are gradually lost. The relatively high ΔCp measured for the CD2/CD58 interaction is consistent with the fact that the binding interfaces are highly polar and that, as a result, a comparatively small amount of non-polar surface is buried upon binding.

It is possible to estimate ΔCp from structural data using the empirically-determined relationship


where ΔAnp is the change in the buried non-polar surface area and ΔAp is the change in the buried polar surface area. Using an implementation of the Lee and Richards algorithm developed by Hubbard (25-27), the ΔAnp and ΔAp were estimated from the crystal structure of the CD2/CD58 complex to be 660 and 690 Å2, respectively. Using these values the calculated ΔCp is −116 cal.mol−1, which is in good agreement with the experimental value obtained from van't Hoff analysis (Table II).

Dissection of binding entropy (ΔS)

Several studies have reported discrepancies between ΔH measured indirectly by van't Hoff analysis (ΔHvdH) and ΔH measured directly by calorimetry (ΔHcal) (28-30). We therefore measured ΔH directly using ITC (Fig. 2). The ΔH and −TΔS thus measured were similar to those determined by van't Hoff analysis (Table I). It has been suggested that differences between ΔHcal and ΔHvdH indicate the presence of linked equilibria that contribute to ΔHcal but not ΔHvdH. Recent studies dispute this, however, arguing that ΔHcal should equal ΔHvdH and that differences are more likely to be the result of experimental artefact (31-33).

FIG. 2
Thermodynamic analysis or the CD2/CD58 interaction by ITC

When conformational changes are required for binding this may result in a high activation enthalpy of association (ΔHass), which is a measure of the net number of bonds that need to be broken in order to form the transition state complex (34). Because the binding kinetics were too fast to measure the ΔHass directly, we determined the ΔHdiss and calculated the ΔHass from the relationship ΔHass = ΔH+TΔHdiss. We determined ΔHdiss by measuring the koff over a range of temperatures (10-30°C) and plotting ln(koff/T) against 1/T (Fig. 1B), the slope of which equals −ΔHdiss/R (20). Using this approach the ΔHass thus estimated was 5.3 ± 0.8 kcal.mol−1, which is relatively small (35). While this does not support conformational change it does not rule it out either, since it is possible that new bonds, such as long-range electrostatic interactions, are formed in the transition state which compensate for the bonds that are broken (36). The preponderance of charged residues in the binding interface supports this possibility, leading us to test below whether long-range electrostatic interactions accelerate binding.

Our results show that, at physiological temperatures, the CD2/CD58 interaction is enthalpically driven and accompanied by unfavourable entropic changes. This contrasts with most protein/protein interactions (23), which are accompanied by favourable entropic and enthalpic changes (Fig. 3). It is similar to what has been observed with protein/protein interactions that are known to be accompanied by conformational adjustments and a reduction in conformational flexibility, such T cell receptor/ligand (Fig. 3), and gp120/CD4 (37) interactions. However the unfavourable entropic change is relatively modest in comparison with these examples, and it is possible that it arises from sources other than changes in conformational flexibility. For example it may arise purely from solvent effects, such as the trapping of water molecules in the binding interface. We therefore investigated the source of the unfavourable entropic change.

FIG. 3
Comparison of thermodynamics of protein/protein interactions

Entropic changes accompanying protein/protein interactions are the sum of favourable changes in solvent (water) entropy and unfavourable changes in protein entropy. Changes in solvent entropy arise mainly from the burial of non-polar surfaces (i.e. the hydrophobic effect). This arises from the movement of water adjacent to hydrophobic surfaces, where it adopts an organized, shell-like structure, into free solution, where it is more disorganised. Changes in protein entropy arise from loss of rotational and translational freedom and conformational flexibility. In order to investigate the extent to which unfavourable entropic changes arise from changes in conformational flexibility we used the approach suggested by Spolar and Record (38) to dissect total entropy change (ΔS) into three main components.


where ΔSHE is the entropy change associated with the hydrophobic effect, ΔSRT is the change associated with loss of translational and rotational freedom of the interacting proteins, and ΔSOTHER is the change arising from other sources, including a reduction in conformational flexibility upon binding. ΔSHE arises from the burial of non-polar surface area; it can be calculated from structural data using the empirically-determined relationship:


where Anp is the buried non-polar surface area (in Å2). As noted previously, Anp is estimated to be 660 Å2 for the CD2/CD58 interaction.

Based on empirical measurements Spolar and Record estimated ΔSRT to be −50 cal.mol−1K−1 for a 1:1 protein/protein interaction.

Since for protein/protein interactions the ΔS is temperature dependent, there exists a temperature (Ts) where ΔS = 0. This can be calculated using the relationship


where T is an arbitrary temperature (e.g. 298.15 K) and ΔST is the entropy change at that T. Using the ΔST and ΔCp values determined above the Ts was calculated to be 261 K for the CD2/CD58 interaction. The ΔSHE at Ts was calculated using equation 3 to be 83 cal.mol−lK−1.

ΔSOTHER at Ts can thus be calculated from the relationship


If one assumes that ΔSRT is temperature independent over this temperature range, as suggested by Spolar and Record (38), then ΔSOTHER is calculated to be −33 cal−1mol−1K−1 (Table II). This negative ΔSOTHER value indicates that binding is accompanied by a reduction in conformational entropy. Spolar and Record (38) show that the number of residues (R) undergoing the transition from a flexible to a folded or rigid state can be estimated from the empirical relationship


suggesting that ~6 or more residues are converted from a flexible to a stable state upon CD2 binding to CD58.

Recently it has been argued that a better estimate of ΔSRT for protein/protein association would be the cratic entropy (41), which is −8 cal mol−1 K−1 (Rln1/55.5) for a bimolecular interaction in water in the standard state. Substituting this value into equation 5 would give −74.6 cal mol−1K−1 for ΔSOTHER and substituting this in equation 6 gives a value of 13 for R (Table II). Thus dissection of the binding entropy suggests that CD2 binding to CD58 is accompanied by a reduction in conformational flexibility involving 6-13 residues. While these changes might occur anywhere in the protein complex, structural studies support the notion that this reduction in conformational flexibility involves the BC, C′C″ and FG loops of CD2 (17,42) and the C′C″ and FG loops of CD58 (14). As noted above, CD2 binding to CD58 is accompanied by and driven by a large favourable enthalpy change. Such favourable enthalpy changes may arise from an increased number of contacts forming at the interface and/or from the trapping of solvent within the binding interface. The binding interface of the CD2/CD58 complex is approximately 1150 Å2 (14). While this is at the low end of the range for protein/protein interactions (23,43), there appear to be numerous contacts; ten salt bridges and five hydrogen bonds were proposed based on analysis of the crystal structure (14). Site-directed mutagenesis studies of the CD2/CD58 interaction suggest that a centrally positioned CD2 tyrosine and surrounding charged residues make major contributions to the binding energy (44-47). Interestingly, the CD2/CD58 binding interface exhibits poor surface shape complementarity (14), suggesting that water molecules bridge the binding surfaces and raising the possibility that trapped water contributes to the favourable enthalpy changes.

Effect of electrostatic interactions on CD2 binding to CD58

Proteins typically associate in solution with a rate constant of 105-106 M−1s−1 (36,48). This rate may decrease if binding requires conformational adjustments (37,49). Given the evidence from structural and thermodynamic studies that CD2 binding to CD58 is accompanied by conformational change, it is notable that the association rate constant of the CD2/CD58 interaction is not particularly slow (kon ≥ 4 × 105 M−1s−1) (10). The association may be accelerated, sometimes dramatically (50), by favourable long-range electrostatic interactions. The mechanism is believed to involve accelerated collisions and/or steering proteins into the correct orientation for binding (36,48,51). Given that the CD2/CD58 binding interface is highly charged (14), we investigated the contribution of long-range electrostatic interactions to binding by examining the effect of varying solution ionic strength.

We initially varied the ionic strength by changing the NaCl concentration. Increasing the NaCl concentration from 150 mM to 1500 mM resulted in a ~6 fold decrease in affinity (Fig. 4A), consistent with a previous report (45). This effect was primarily a result of an effect on the kon (Fig. 4B), consistent with screening of long range electrostatic interactions. Recent studies have shown that electrostatic screening results in a linear relationship between lnkon and 1/(1 + κa), where the latter is proportional to the ionic strength (36). In the case of the CD2/CD58 interaction this relationship was clearly not linear (Fig. 4C), suggesting that the effect of NaCl was not solely the result of electrostatic screening. One possible explanation was that the Na and/or Cl ions were disrupting the structure of CD2 and/or CD58 either by direct interactions with the proteins or through effects on solvent structure. Since such effects are dependent on the salt we used a different salt (KF) to vary the ionic strength. In contrast to the effect of NaCl, the affinity and kinetics of CD2 binding to CD58 showed no significant change when the concentration of KF was varied from 150 mM to 1500 mM (Fig. 4A-C). It follows that the effect of varying NaCl on binding is not the result of changes in ionic strength and must result from another mechanism. Interestingly, the rat CD2/CD48 interaction was unaffected by varying the NaCl concentration (52), implying that rat CD2 and/or CD48 are resistant to this effect. Given that the surface charge distributions on CD2/ligand binding surfaces are not conserved across species, and that the ligand binding surfaces of the human CD2/ligand pair are more highly charged, it is not surprising that they exhibit differential sensitivity to NaCl (12,14,53).

FIG. 4
The dependence of binding on ionic strength

The absence of an effect when varying KF concentration demonstrates that long-range electrostatic interactions have no net effect on the CD2/CD58 interaction. This is somewhat unexpected given that the binding interfaces in the complex are highly charged with excellent charge complementarity (14). However, it remains possible that there are some favourable electrostatic interactions that are balanced by unfavourable electrostatic interactions. To investigate this further we used site-directed mutagenesis to disrupt a subset of electrostatic interactions. We have recently generated a number of human CD2 mutants in which single charged residues in the binding interface were mutated to alanine (18). Since it was not possible directly to measure kon we calculated it from the KD and koff (Table III). While mutation of CD2 residues K41, K51 and K91 to alanine had no significant effect on the kon, mutation of D31 to alanine led to a five fold decrease in the kon from 4 × 105 to 0.8 × 105 M−1s−1 (Table III). Interestingly, D31 forms a salt bridge with the CD58 residue R44 in the bound complex (14). Taken together, these data suggest that a favourable electrostatic interaction involving D31 and R44 accelerates CD2 binding to CD58 but that other, unfavourable interactions inhibit association. Interestingly, there is also evidence that the rat CD2/CD48 interaction is also characterised by balancing unfavourable and favourable electrostatic interactions (52); whereas this interaction is unaffected by changes in salt concentration, mutation to alanine of individual charged residues in the binding interface resulted in both decreases and increases in affinity (52).

Effect of CD2 mutations on association rate constant

Given the highly-charged nature of the CD2/CD58 binding interface, our finding that long-range electrostatic interactions do not accelerate binding is unexpected, and in striking contrast to the barnase/barstar interaction, where electrostatic attractions between highly charged surfaces accelerate binding by several orders of magnitude (50). One difference between the CD2/CD58 and barnase/barnstar interfaces is that the latter is less heterogenous; barnase is predominantly positively charged and barstar is uniformly negatively charged. In contrast, the CD2 and CD58 binding surfaces each carry an intricate mixture of negative and positive charges (12,14,53). We suggest that the presence of heterogenous charges on each surface results in a complex electrostatic potential energy landscape with balancing favourable and unfavourable long-range electrostatic interactions. Given that it has no effect on binding kinetics, what is the functional significance of charged nature of the CD2/CD58 binding interface? We have previously shown, in an analysis of the rat CD2/CD48 interaction, that charged residues that contribute little to the binding affinity may nevertheless contribute to binding specificity, by imposing a requirement for charge complementarity on the other surface (52). The reason proposed for their small contribution to affinity is that, in order for them to form favourable interactions (e.g. salt bridges) with the ligand, existing favourable electrostatic interactions (e.g. with water, salt or adjacent residues) need to be broken (54). The molecular interactions that mediate transient cell-cell interactions need to be low affinity to facilitate detachment (55). We suggest that CD2/CD58 binding interface is charged because electrostatic complementarity enables the interaction to be both weak and specific.


In this report we show that the CD2/CD58 interaction is accompanied by an unfavourable entropy change. Dissection of the entropy change using the approach of Spolar and Record (38) suggests that the unfavourable entropy change results in part from a reduction in conformational flexibility. Taken together with previous structural studies, these findings suggest that energetically significant structural rearrangements accompany binding. Despite the highly-charged binding interface, we show that long-range electrostatic interactions have no net favourable effect on CD2 binding to CD58, probably because of the presence of balancing favourable and unfavourable electrostatic interactions.


*We thank Neil Barclay for valuable advice, Jennifer Byrne for assistance with tissue culture, and David Mahoney and Anthony Day for assistance with ITC measurements. P.A. vdM. and A.K. are supported by the Medical Research Council, S.J.D is supported by the Wellcome Trust, and M.A.A.C. and A.C. are supported by FEDER and Fundação para a Ciência e a Tecnologia.

1The abbreviations used are: APC, antigen presenting cell; CD, cluster of differentiation; CHO, Chinese hamster ovary; Cp, heat capacity at constant pressure; HBS, hepes buffered saline; Ig, immunoglobulin; ITC, isothermal titration calorimetry; NK, natural killer; NMR, nuclear magnetic resonance; PCR, polymerase chain reaction; pep-MHC, peptide loaded major histocompatibility complex; SPR, surface plasmon resonance; TCR, T cell receptor; WT, wild type.


1. Davis SJ, van der Merwe PA. Immunol Today. 1996;17(4):177–187. [PubMed]
2. Engel P, Eck MJ, Terhorst C. Nat Rev Immunol. 2003;3(10):813–821. [PubMed]
3. Bachmann MF, Barner M, Kopf M. J Exp Med. 1999;190(10):1383–1392. [PMC free article] [PubMed]
4. Bierer BE, Burakoff SJ. Immunol Rev. 1989;111(267):267–294. [PubMed]
5. Green JM, Karpitskiy V, Kimzey SL, Shaw AS. J Immunol. 2000;164(7):3591–3595. [PubMed]
6. Moingeon P, Chang HC, Sayre PH, Clayton LK, Alcover A, Gardner P, Reinherz EL. Immunol Rev. 1989;111(111):111–144. [PubMed]
7. van der Merwe PA. J Exp Med. 1999;190(10):1371–1374. [PMC free article] [PubMed]
8. Davis SJ, Ikemizu S, Wild MK, van der Merwe PA. Immunol Rev. 1998;163:217–236. [PubMed]
9. van der Merwe PA, Davis SJ. Annu Rev Immunol. 2003;21:659–684. [PubMed]
10. van der Merwe PA, Barclay AN, Mason DW, Davies EA, Morgan BP, Tone M, Krishnam AK, Ianelli C, Davis SJ. Biochemistry. 1994;33(33):10149–10160. [PubMed]
11. Bodian DL, Jones EY, Stuart DI, Harlos KH, Davies EA, Davis SJ. Structure. 1994;2:755–766. [PubMed]
12. Ikemizu S, Sparks LM, van der Merwe PA, Harlos K, Stuart DI, Jones EY, Davis SJ. Proc Natl Acad Sci U S A. 1999;96(8):4289–4294. [PubMed]
13. Sun ZY, Dötsch V, Kim M, Li J, Reinherz EL, Wagner G. EMBO J. 1999;18(11):2941–2949. [PubMed]
14. Wang JH, Smolyar A, Tan K, Liu JH, Kim M, Sun ZY, Wagner G, Reinherz EL. Cell. 1999;97(6):791–803. [PubMed]
15. Wyss DF, Choi JS, Jing L, Knoppers MH, Willis KJ, Arulanandam ARN, Reinherz EL, Wagner G. Science. 1995;269:1273–1278. [PubMed]
16. Kitao A, Wagner G. Proc Natl Acad Sci U S A. 2000;97(5):2064–2068. [PubMed]
17. Wyss DF, Dayie KT, Wagner G. Protein Sci. 1997;6(3):534–542. [PubMed]
18. Bayas MV, Kearney A, Avramovic A, van der Merwe PA, Leckband DE. J. Biol. Chem. 2007;282:5589–5596. [PubMed]
19. Karlsson R, Ståhlberg R. Anal Biochem. 1995;228:274–280. [PubMed]
20. Lee JK, Stewart-Jones G, Dong T, Harlos K, Di Gleria K, Dorrell L, Douek DC, van der Merwe PA, Jones EY, McMichael AJ. J Exp Med. 2004;200(11):1455–1466. [PMC free article] [PubMed]
21. Yoo SH, Lewis MS. Biochemistry. 1995;34:632–638. [PubMed]
22. Ladbury JE, Chowdhry BZ. Chem Biol. 1996;3(10):791–801. [PubMed]
23. Stites WE. Chem Rev. 1997;97:1233–1250. [PubMed]
24. Creighton T. Proteins. Structures and Molecular properties. 2nd Ed. WH Freeman; New York: 1993.
25. Lee B, Richards FM. J Mol Biol. 1971;55(3):379–400. [PubMed]
26. McDonald IK, Thornton JM. J Mol Biol. 1994;238(5):777–793. [PubMed]
27. Laskowski RA. J Mol Graph. 1995;13(5):323–330. 307-328. [PubMed]
28. Naghibi H, Tamura A, Sturtevant JM. Proc Natl Acad Sci U S A. 1995;92(12):5597–5599. [PubMed]
29. Sigurskjold BW, Bundle DR. J Biol Chem. 1992;267(12):8371–8376. [PubMed]
30. Liu Y, Sturtevant JM. Protein Sci. 1995;4(12):2559–2561. [PubMed]
31. Horn JR, Russell D, Lewis EA, Murphy KP. Biochemistry. 2001;40(6):1774–1778. [PubMed]
32. Horn JR, Brandts JF, Murphy KP. Biochemistry. 2002;41(23):7501–7507. [PubMed]
33. Chaires JB. Biophys Chem. 1997;64(1-3):15–23. [PubMed]
34. Atkins P. Physical Chemistry. Oxford University Press; Oxford: 1998.
35. Gutfreund H. Kinetics for the life sciences. Receptors, transmitters, and catalysis. Cambridge University Press; Cambridge: 1995.
36. Schreiber G. Curr Opin Struct Biol. 2002;12(1):41–47. [PubMed]
37. Myszka DG, Sweet RW, Hensley P, Brigham-Burke M, Kwong PD, Hendrickson WA, Wyatt R, Sodroski J, Doyle ML. Proc Natl Acad Sci U S A. 2000;97(16):9026–9031. [PubMed]
38. Spolar RS, Record MT., Jr. Science. 1994;263(5148):777–784. [PubMed]
39. Potter TA, Rajan TV, Dick R. F. d., Bluestone JA. Nature. 1989;337:73–75. [PubMed]
40. Ziegler SF, Levin SD, Johnson L, Copeland NG, Gilbert DJ, Jenkins NA, Baker E, Sutherland GR, Feldhaus AL, Ramsdell F. T. m. C. g. S. expression, and mapping to the NK. Journal of Immunology. 1994;152:1228–1236. [PubMed]
41. Murphy KP, Xie D, Thompson KS, Amzel LM, Freire E. Proteins. 1994;18(1):63–67. [PubMed]
42. Kitao A, Wagner G. Proc Natl Acad Sci U S A. 2000;97(5):2064–2068. [PubMed]
43. Lo Conte L, Chothia C, Janin J. J Mol Biol. 1999;285(5):2177–2198. [PubMed]
44. Arulanandam ARN, Withka JM, Wyss DF, Wagner G, Kister A, Pallai P, Recny MA, Reinherz EL. Proc Natl Acad Sci USA. 1993;90:11613–11617. [PubMed]
45. Kim M, Sun ZY, Byron O, Campbell G, Wagner G, Wang J, Reinherz EL. J Mol Biol. 2001;312(4):711–720. [PubMed]
46. Arulanandam ARN, Kister A, McGregor MJ, Wyss DF, Wagner G, Reinherz EL. J Exp Med. 1994;180:1861–1871. [PMC free article] [PubMed]
47. Somoza C, Driscoll PC, Cyster JG, Williams AF. J. Exp. Med. 1993;178:549–558. [PMC free article] [PubMed]
48. Janin J. Proteins. 1997;28(2):153–161. [PubMed]
49. Willcox BE, Gao GF, Wyer JR, Ladbury JE, Bell JI, Jakobsen BK, van der Merwe PA. Immunity. 1999;10(3):357–365. [PubMed]
50. Schreiber G, Fersht AR. Nat Struct Biol. 1996;3(5):427–431. [PubMed]
51. Sheinerman FB, Norel R, Honig B. Curr Opin Struct Biol. 2000;10(2):153–159. [PubMed]
52. Davis SJ, Davies EA, Tucknott MG, Jones EY, van der Merwe PA. Proc Natl Acad Sci U S A. 1998;95(10):5490–5494. [PubMed]
53. Evans EJ, Castro MA, O'Brien R, Kearney A, Walsh H, Sparks LM, Tucknott MG, Davies EA, Carmo AM, van der Merwe PA, Stuart DI, Jones EY, Ladbury JE, Ikemizu S, Davis SJ. J Biol Chem. 2006;281(39):29309–29320. [PubMed]
54. Honig B, Nicholls A. Science. 1995;268:1144–1149. [PubMed]
55. van der Merwe PA, Barclay AN. Trends Biochem Sci. 1994;19(9):354–358. [PubMed]
56. Anikeeva N, Lebedeva T, Krogsgaard M, Tetin SY, Martinez-Hackert E, Kalams SA, Davis MM, Sykulev Y. Biochemistry. 2003;42(16):4709–4716. [PubMed]
57. Davis-Harrison RL, Armstrong KM, Baker BM. J Mol Biol. 2005;346(2):533–550. [PubMed]
58. Garcia KC, Radu CG, Ho J, Ober RJ, Ward ES. Proc Natl Acad Sci U S A. 2001;98(12):6818–6823. [PubMed]
59. Krogsgaard M, Prado N, Adams EJ, He XL, Chow DC, Wilson DB, Garcia KC, Davis MM. Mol Cell. 2003;12(6):1367–1378. [PubMed]