Obdulio López-Mayorga

Cooperative propagation of local stability changes from low-stability and high-stability regions in a SH3 domain

Site‐directed mutagenesis has been used to produce local stability changes at two regions of the binding site surface of the α‐spectrin SH3 domain (Spc‐SH3) differing in their intrinsic stability. Mutations were made at residue 56, located at the solvent‐exposed side of the short 310 helix, and at residue 21 in the tip of the flexible RT‐loop. NMR chemical‐shift analysis and X‐ray crystallography indicated negligible changes produced by the mutations in the native structure limited to subtle rearrangements near the mutated residue and at flexible loops. Additionally, mutations do not alter importantly the SH3 binding site structure, although produce significant changes in its affinity for a proline‐rich decapeptide. The changes in global stability measured by differential scanning calorimetry are consistent the local energy changes predicted by theoretical models, with the most significant effects observed for the Ala‐Gly mutations. Propagation of the local stability changes throughout the domain structure has been studied at a per‐residue level of resolution by NMR‐detected amide hydrogen–deuterium exchange (HX). Stability propagation is remarkably efficient in this small domain, apparently due to its intrinsically low stability. Nevertheless, the HX‐core of the domain is not fully cooperative, indicating the existence of co‐operative subunits within the core, which is markedly polarized. An equilibrium ϕ‐analysis of the changes in the apparent Gibbs energies of HX per residue produced by the mutations has allowed us to characterize structurally the conformational states leading to HX. Some of these states resemble notably the folding transition state of the Spc‐SH3 domain, suggesting a great potential of this approach to explore the folding energy landscape of proteins. An energy perturbation propagates more effectively from a flexible region to the core than in the opposite direction, because the former affects a broader region of the energy landscape than the latter. This might be of importance in understanding the special thermodynamic signature of the SH3‐peptide interaction and the relevance of the dual character of SH3 binding sites. Proteins 2007.

The high-resolution NMR structure of the R21A Spc-SH3:P41 complex: Understanding the determinants of binding affinity by comparison with Abl-SH3

BackgroundSH3 domains are small protein modules of 60–85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS) and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41) binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3), while it has a 100 times lower affinity for the α-spectrin SH3 domain (Spc-SH3).ResultsHere we present the high-resolution structure of the complex between the R21A mutant of Spc-SH3 and p41 derived from NMR data. Thermodynamic parameters of binding of p41 to both WT and R21A Spc-SH3 were measured by a combination of isothermal titration and differential scanning calorimetry. Mutation of arginine 21 to alanine in Spc-SH3 increases 3- to 4-fold the binding affinity for p41 due to elimination at the binding-site interface of the steric clash produced by the longer arginine side chain. Amide hydrogen-deuterium experiments on the free and p41-bound R21A Spc-SH3 domain indicate that binding elicits a strong reduction in the conformational flexibility of the domain. Despite the great differences in the thermodynamic magnitudes of binding, the structure of the R21A Spc-SH3:P41 complex is remarkably similar to that of the Abl-SH3:P41 complex, with only few differences in protein-ligand contacts at the specificity pocket. Using empirical methods for the prediction of binding energetics based on solvent-accessible surface area calculations, the differences in experimental energetics of binding between the two complexes could not be properly explained only on the basis of the structural differences observed between the complexes. We suggest that the experimental differences in binding energetics can be at least partially ascribed to the absence in the R21A Spc-SH3:P41 complex of several buried water molecules, which have been proposed previously to contribute largely to the highly negative enthalpy and entropy of binding in the Abl-SH3:P41 complex.ConclusionBased on a deep structural and thermodynamic analysis of a low and high affinity complex of two different SH3 domains with the same ligand p41, we underline the importance of taking into account in any effective strategy of rational design of ligands, factors different from the direct protein-ligand interactions, such as the mediation of interactions by water molecules or the existence of cooperative conformational effects induced by binding.

Detection and Characterization of Partially Unfolded Oligomers of the SH3 Domain of α-Spectrin

For the purpose of equilibrium and kinetic folding-unfolding studies, the SH3 domain of α-spectrin (spc-SH3) has long been considered a classic two-state folding protein. In this work we have indeed observed that the thermal unfolding curves of spc-SH3 measured at pH 3.0 by differential scanning calorimetry, circular dichroism, and NMR follow apparently the two-state model when each unfolding profile is considered individually. Nevertheless, we have found that protein concentration has a marked effect upon the thermal unfolding profiles. This effect cannot be properly explained in terms of the two-state unfolding model and can only be interpreted in terms of the accumulation of intermediate associated states in equilibrium with the monomeric native and unfolded states. By chemical cross-linking and pulsed-field gradient NMR diffusion experiments we have been able to confirm the existence of associated states formed during spc-SH3 unfolding. A three-state model, in which a dimeric intermediate state is assumed to be significantly populated, provides the simplest interpretation of the whole set of thermal unfolding data and affords a satisfactory explanation for the concentration effects observed. Whereas at low concentrations the population of the associated intermediate state is negligible and the unfolding process consequently takes place in a two-state fashion, at concentrations above ∼0.5 mM the population of the intermediate state becomes significant at temperatures between 45°C and 80°C and reaches up to 50% at the largest concentration investigated. The thermodynamic properties of the intermediate state implied by this analysis fall in between those of the unfolded state and the native ones, indicating a considerably disordered conformation, which appears to be stabilized by oligomerization.

pH dependence of the hydrogen exchange in the SH3 domain of α-spectrin

Using nuclear magnetic resonance we have measured the hydrogen exchange (HX) in the Src homology region 3 (SH3) domain of α‐spectrin as a function of pH*. At very acidic pH* values the exchange of most residues appears to occur via global unfolding, although several residues show abnormally large Gibbs energies of exchange, suggesting the presence of some residual structure in the unfolded state. At higher pH* HX occurs mainly via local or partial unfoldings. We have been able to characterize the coupling between the electrostatic interactions in this domain and the conformational fluctuations occurring under native conditions by analyzing the dependence upon pH* of the Gibbs energy of exchange. The SH3 domain seems to be composed of a central core, which requires large structural disruptions to become exposed to the solvent, surrounded by smaller subdomains, which fluctuate independently.

Structural cooperativity in the SH3 domain studied by site-directed mutagenesis and amide hydrogen exchange.

We have studied the effects produced by site‐directed mutagenesis upon energetic and structural cooperativity in the Src homology region 3 domain of α‐spectrin. The mutation of Asn47 to Gly or Ala in the distal loop brings about significant changes to the global stability of the domain in spite of not affecting its structure to any great extent. The binding affinity for a proline‐rich peptide is also largely diminished in both mutant domains. We have compared the apparent Gibbs energies of the amide hydrogen–deuterium exchange (HX) between the wild‐type and the Gly47 mutant. The observed changes in the Gibbs energy of HX indicate a remarkable energetic cooperativity in this small domain. Regions of the domains core have a high cooperativity with the position of the mutation, indicating that their HX occurs mainly in states in which the distal loop is unstructured. More flexible regions, which undergo HX mainly by local motions, show a lower but still considerable cooperativity with the distal loop. We conclude that there is an important correlation between regional stability and cooperativity in this small domain.

Thermodynamic Characterization of 5′-AMP Binding to Bovine Liver Glycogen Phosphorylase a

The binding of adenosine 5′-monophosphate to liver glycogen phosphorylase a (EC 2.4.1.1) has been studied by size exclusion high performance liquid chromatography and isothermal titration microcalorimetry at pH 6.9 over a temperature range of 25 to 35°C. The results are compared with those of the binding of the same nucleotide to the muscle isozyme and to liver phosphorylase b. Calorimetric measurements in various buffer systems with different ionization heats suggest that protons are released during the binding of the nucleotide. The dimer of liver glycogen phosphorylase a has been shown to have two equal and independent sites for 5′-AMP, which would correspond to the activator sites identified in the muscle isozyme. The binding constants as well as the changes in Gibbs energy, enthalpy, and entropy per site for 5′-AMP binding were calculated at each temperature. The results show that the major contribution to the negative value of ΔG0 stems from the value of ΔH in the range of 25 to 35°C. The enthalpy change of binding is strongly temperature-dependent, arising from a large negative ΔCp of binding equal to −1.45 ± 0.02 kJ K−1 (mol of 5′-AMP bound)−1, which suggests significant changes in the polar and apolar surfaces accessible to the solvent.

The temperature dependence of the hydrogen exchange in the SH3 domain of α-spectrin

The amide hydrogen–deuterium exchange (HX) in the Src homology region 3 (SH3) domain of α‐spectrin has been measured by nuclear magnetic resonance as a function of temperature between 8 and 46°C. The analysis of the temperature dependence of HX from a statistical thermodynamic point of view has allowed us to estimate the enthalpies and entropies of the conformational processes leading to HX. The results indicate that under native conditions the domain undergoes a wide variety of conformational fluctuations, ranging from local motions, mainly located in loops, turns and chain ends and involving only low enthalpy and entropy, to extensive structural disruptions affecting its core and involving enthalpies and entropies that come fairly close to those observed during global unfolding.