Characterizing the Role of Ensemble Modulation in Mutation-Induced Changes in Binding Affinity

Abstract

Protein conformational fluctuations are key contributors to biological function, mediating important processes such as enzyme catalysis, molecular recognition and allosteric signaling. To better understand the role of conformational fluctuations in substrate:ligand recognition, we analyzed, experimentally and computationally, the binding reaction between an SH3 domain and the recognition peptide of its partner protein. The fluctuations in this SH3 domain were enumerated by using an algorithm based on the hard sphere collision model, and the binding energetics resulting from these fluctuations were calculated using a structure-based energy function parameterized to solvent accessible surface areas. Surprisingly, this simple model reproduced the effects of mutations on the experimentally determined SH3 binding energetics, within the uncertainties of the measurements, indicating that conformational fluctuations in SH3, and in particular the RT loop region, are structurally diverse and are well-approximated by the randomly configured states. The mutated positions in SH3 were distant to the binding site, and involved Ala and Gly substitutions of solvent exposed positions in the RT loop. To characterize these fluctuations, we applied principal coordinate analysis to the computed ensembles, uncovering the principal modes of conformational variation. It is shown that the observed differences in binding affinity between each mutant, and thus the apparent coupling between the mutated sites, can be described in terms of the changes in these principal modes. These results indicate that dynamic loops in proteins can populate a broad conformational ensemble, and that a quantitative understanding of molecular recognition requires consideration of the entire distribution of states.Protein conformational fluctuations are essential for biological function 1 , and perturbations to fluctuations brought about by mutation or environmental changes are known to affect proteins functionally 2 . Despite clear experimental evidence for a dependence of function on fluctuations 3,4,5 , the impact of conformational heterogeneity on important biological processes, such as substrate:ligand recognition, remains poorly understood 3,6 . Clearly, methods that can relate the functional and structural character of proteins are needed to understand mechanistically, and quantitatively, the role that fluctuations have in biological function.To investigate the impact of structural fluctuations on molecular recognition, we selected the C-terminal src homology 3 (SH3) domain of the C. Elegans protein SEM5 (SEM5 C-SH3). SH3 domains are a conserved structural motif found in many regulatory proteins 7 . The typical function of these domains is to mediate protein:protein interactions in signaling pathways by *Corresponding author: E-mail: vjhilser@utmb.edu. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript recognizing proline-rich sequences that adopt a left-handed polyproline II helical structure 8, 9,10,11 ...

Binding Mechanism of the N-Terminal SH3 Domain of CrkII and Proline-Rich Motifs in cAbl

“…For~10 amino-acid-long peptides used in this study, DH conf could be considerably larger than the reported values. In addition, the effect of conformational flexibility on the estimation of binding thermodynamic parameters was well highlighted in the study of the Sem-5 cSH3 domain (77,78).…”

Experimental and Computational Studies of Polyproline II Propensity

“…Employing an excluded -volume model from previous studies, Mini -Protein Modeler ( MPMOD ) [43,44] , the PII propensities of Ala and Gly are determined in the context [12] , an excluded -volume model (black dashed) [19] and in GGXGG/GXG from NMR (gray solid) [25] , an excluded -volume model (gray dashed) [19] , molecular dynamics simulation (gray dotted) [67] , and by a combination of spectroscopic techniques (gray dash -dotted) [27] . Positions marked by black arrows for Ala and Gly PII propensities have been experimentally measured [41] , and computationally validated [18,19] .…”

“…The impact of ligand PII formation on the thermodynamics of binding has been calorimetrically assessed using mutants of both the SH3 domain and Sos ligand [41,42] . Recent computational work has sought to elucidate the extent to which mutations in the SH3 domain modulate the native ensemble and infl uence Sos ligand binding [43] , as well as the impact of PII conformational bias on the binding of Sos peptides to SH3 [44] .…”

A Host–Guest Relationship in Bone Morphogenetic Protein Receptor-II Defines Specificity in Ligand–Receptor Recognition

“…This observation is consistent with the fact that the COREX/BEST algorithm has been employed successfully to capture many phenomena which require long range intra-molecular communications. 33,37–40 …”