Nucleation and the Transition State of the SH3 Domain

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We present a verified computational model of the SH3 domain transition state (TS) ensemble. This model was built for three separate SH3 domains using experimental ϕ-values as structural constraints in all-atom protein folding simulations. While averaging over all conformations incorrectly considers non-TS conformations as transition states, quantifying structures as pre-TS, TS, and post-TS by measurement of their transmission coefficient (“probability to fold”, or pfold) allows for rigorous conclusions regarding the structure of the folding nucleus and a full mechanistic analysis of the folding process. Through analysis of the TS, we observe a highly polarized nucleus in which many residues are solvent-exposed. Mechanistic analysis suggests the hydrophobic core forms largely after an early nucleation step. SH3 presents an ideal system for studying the nucleation-condensation mechanism and highlights the synergistic relationship between experiment and simulation in the study of protein folding.

Introduction

Current understanding of the protein folding process is largely based on the theory of nucleation1, 2 and energy landscape theory.3 For the majority of small proteins, which serve as model systems for studying protein folding, the process occurs via a two-state mechanism.4 In analogy to a first-order phase transition, the folded and unfolded states may be thought of as different phases (i.e. liquid and gas) and the transition state (TS) as the “critical nucleus” that seeds the transition5. Since the kinetics of the transformation between the thermodynamically stable folded and unfolded states is determined by the TS, understanding this state allows for a deeper understanding of the rate and mechanism of folding. The folding process may also be thought of statistically with the formation of a transition state ensemble (TSE) as the rate-limiting process in folding.5 Experiments, mainly based on the protein engineering method of “ϕ-value analysis”,6 have made significant progress in testing theories of folding. Concurrently, theory and simulation have continued to improve our understanding of the nature of nucleation in the TSE.7

A prototypical example of the interplay between theory and experiment is the structural interpretation of experimental ϕ-values through simulation.8, 9 This method for reconstructing a protein's TSE from experimental constraints has proven highly useful in understanding folding, especially as it was refined to test the assumption that a set of structures meeting experimental constraints is, in fact, a TSE.10, 11 A TS is characterized by a transmission coefficient of 1/2. In proteins, this corresponds to a probability of folding, or “pfold5 equal to one half. Verifying this condition prior to making statements regarding computational models of the TSE is absolutely essential as it has been shown that an ensemble of structures meeting a set of ϕ-value restraints is not composed entirely of TS conformations.11, 12 Once a model of the TSE is obtained, it is possible to conduct analyses that are currently impossible by experiment, such as exploring the role of residues for which mutation does not cause sufficient destabilization and building an atom-level structural model of the TSE.

SH3 is a widely studied protein that exhibits two-state folding, and is composed of two orthogonally packed three-stranded β-sheets that form a single hydrophobic core.13 SH3 domains have served as the experimental13, 14 and computational15, 16, 17, 18 model for numerous recent studies of nucleation in protein folding. Although there have been simulations that attempted to reconstruct the TSE using ϕ-values,17, 18 the essential step of testing the assumption that the resulting structures are transition states was not necessarily taken.18 As it has been shown that not all conformations consistent with experimental ϕ-values are members of the TSE, it is difficult to accurately interpret these conclusions regarding nucleation and the TSE. In the following study we use the src, fyn, and spectrin SH3 domain and reported experimental ϕ-values19 as model systems to study the nature of nucleation in the protein folding transition state. Importantly, pfold analysis is used to quantify the position of each structure along the folding pathway. We also examine the role and formation of the hydrophobic core and explore the order of folding events in this classic nucleation-condensation mechanism. In SH3, nucleation is early and separate from hydrophobic core formation. The early, polarized TS that we observe is largely solvent-exposed and is formed by a minimal number of contacts.

Section snippets

A model of the TSE

A side-by-side comparison of the native and transition states is presented in Figure 1. The general features of all observed SH3 TSEs include denaturation of the N and C termini, turns and loops, and a small amount of secondary structures located in the central β strands. The TSE for the three different SH3 domains exhibit average Cα root mean square deviation (RMSD) ranging from 7.1 to 11.4 Å and radii of gyration (Rg) of 12.5 to 14.53 Å, which is largely expanded compared to the ∼10 Å Rg of the

Discussion

We have presented a pfold verified, all-atom model of the TSE in three different SH3 domains. This allows for rigorous conclusions regarding the nature and mechanism of nucleation. In constructing the TSE, residues were restrained, but the way in which the minimal set of restraints was met (contacts between residues) was not predetermined. Importantly, the true TSE was identified through (unconstrained) pfold analysis. It is noteworthy that the set of available ϕ-values for SH3, and other

Constructing and verifying the TSE

Simulations were initiated using coordinates from the crystal structures of the src-SH3 of tyrosine kinase37 (1FMK), fynSH322, 23 (1FYN), and α-spectrin SH324 domains (1BK2). The simulation was propagated by Monte Carlo dynamics using a move set that included rotations of backbone (ϕ-ψ) angles and side-chain (χ) torsion angles, while maintaining planar peptide bonds. All non-hydrogen atoms are represented as impenetrable hard spheres38, 39 and excluded volume is continually enforced. Energy was

Acknowledgements

We thank Dr Brian N. Dominy for assistance with CHARMM, and Eric J. Deeds for his comments on the manuscript. This work was supported by an HHMI pre-doctoral fellowship (I.A.H.).

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