Conservation of Flexible Residue Clusters among Structural and Functional Enzyme Homologues

Donald Gagné,Laurie-Anne Charest,Sébastien Morin,Evgenii L Kovrigin,Nicolas Doucet

doi:10.1074/jbc.m112.394866

Abstract

Conformational flexibility between structural ensembles is an essential component of enzyme function. Although the broad dynamical landscape of proteins is known to promote a number of functional events on multiple time scales, it is yet unknown whether structural and functional enzyme homologues rely on the same concerted residue motions to perform their catalytic function. It is hypothesized that networks of contiguous and flexible residue motions occurring on the biologically relevant millisecond time scale evolved to promote and/or preserve optimal enzyme catalysis. In this study, we use a combination of NMR relaxation dispersion, model-free analysis, and ligand titration experiments to successfully capture and compare the role of conformational flexibility between two structural homologues of the pancreatic ribonuclease family: RNase A and eosinophil cationic protein (or RNase 3). In addition to conserving the same catalytic residues and structural fold, both homologues show similar yet functionally distinct clusters of millisecond dynamics, suggesting that conformational flexibility can be conserved among analogous protein folds displaying low sequence identity. Our work shows that the reduced conformational flexibility of eosinophil cationic protein can be dynamically and functionally reproduced in the RNase A scaffold upon creation of a chimeric hybrid between the two proteins. These results support the hypothesis that conformational flexibility is partly required for catalytic function in homologous enzyme folds, further highlighting the importance of dynamic residue sectors in the structural organization of proteins.

Highlights

It remains unclear whether structural homologues rely on similar concerted motions to promote enzyme function
Our work shows that the reduced conformational flexibility of eosinophil cationic protein can be dynamically and functionally reproduced in the RNase A scaffold upon creation of a chimeric hybrid between the two proteins
These studies typically use coarse-grained models, such as normal mode analysis, to infer catalytic time scale motional similarities between protein folds [58]. These studies involve the direct comparison of simulated data with crystallographically resolved and/or NMR-resolved enzymes in apo and ligand-bound forms, inferring dynamic information through, but not limited to, residual dipolar coupling measurements and/or Debye-Waller factors (B-factors) [59]. The latter provides a rough approximation of residue flexibility in protein crystals, albeit with no clear definition of dynamic time scale

Summary

Background

It remains unclear whether structural homologues rely on similar concerted motions to promote enzyme function. The dynamically important residue pair His48-Thr is 86% conserved in the mammalian and vertebrate ribonuclease superfamily [23], ECP is one of only two human ribonucleases lacking this important interaction, coinciding with a steep decrease in ribonucleolytic activity [18] Based on these observations, it is tempting to verify whether evolutionarily conserved motional networks between homologous ribonucleases could partially account for their divergent catalytic activities. We aimed at validating whether highly homologous structural and functional ribonucleases catalyzing the same transphosphorylation reaction display conserved dynamic behaviors both on the fast ps-ns and the slower microsecond-to-millisecond (␮s-ms) time scales We challenge this hypothesis by investigating the conformational exchange and the conservation of dynamic clusters between ECP and RNase A, two structural homologues of the ribonuclease superfamily. The present work demonstrates the importance of controlling millisecond dynamics to modulate protein function, a central concept with broad implications in protein engineering and allosteric drug design [4, 5, 28, 29]

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION