Changing the topology of protein backbone: the effect of backbone cyclization on the structure and dynamics of a SH3 domain.

Frank H Schumann,Praveen P Tayakuniyil,Julio A Camarero,Jennifer H Grossman,David Fushman,Ranjani Varadan

doi:10.3389/fchem.2015.00026

Abstract

Understanding of the effects of the backbone cyclization on the structure and dynamics of a protein is essential for using protein topology engineering to alter protein stability and function. Here we have determined, for the first time, the structure and dynamics of the linear and various circular constructs of the N-SH3 domain from protein c-Crk. These constructs differ in the length and amino acid composition of the cyclization region. The backbone cyclization was carried out using intein-mediated intramolecular chemical ligation between the juxtaposed N- and the C-termini. The structure and backbone dynamics studies were performed using solution NMR. Our data suggest that the backbone cyclization has little effect on the overall three-dimensional structure of the SH3 domain: besides the termini, only minor structural changes were found in the proximity of the cyclization region. In contrast to the structure, backbone dynamics are significantly affected by the cyclization. On the subnanosecond time scale, the backbone of all circular constructs on average appears more rigid than that of the linear SH3 domain; this effect is observed over the entire backbone and is not limited to the cyclization site. The backbone mobility of the circular constructs becomes less restricted with increasing length of the circularization loop. In addition, significant conformational exchange motions (on the sub-millisecond time scale) were found in the N-Src loop and in the adjacent β-strands in all circular constructs studied in this work. These effects of backbone cyclization on protein dynamics have potential implications for the stability of the protein fold and for ligand binding.

Highlights

Perturbation of protein structure by changing the polypeptide chain topology from a linear to a circular one is a promising tool for exploring the protein energy landscape in order to gain insights into mechanisms underlying protein stability and folding, and biological function
The current study provides a detailed, atomic-resolution analysis of the effect of the backbone circularization on the structure, backbone dynamics, and function of the SH3 domain
We determined the three-dimensional structure and backbone dynamics of a linear and several circular forms of the N-terminal SH3 domain of c-Crk in order to examine the effect of backbone circularization

Summary

Introduction

Perturbation of protein structure by changing the polypeptide chain topology from a linear to a circular one is a promising tool for exploring the protein energy landscape in order to gain insights into mechanisms underlying protein stability and folding, and biological function. The backbone cyclization of a polypeptide chain, i.e., the ligation of its N- and C- termini via a peptide bond (hereafter called circularization) is expected to reduce the backbone conformational entropy, Structure and dynamics of circularized proteins especially in the intermediate and unfolded state, potentially resulting in increased thermodynamic stability and backbone rigidity of the folded state (Iwai and Pluckthun, 1999) This fact is well known for peptides, and backbone cyclization has become a widely used strategy to control the structure and biological function of small peptides and to improve their in vivo stability (Hruby, 1982; Kessler, 1982; Hruby et al, 1990), the impact of backbone circularization in proteins has not been fully explored. Restricting the mobility of the termini by circularization can, to some extent, mimic the “natural” situation in multidomain proteins, and could be useful for understanding the effect of the “environmental” factors on the structure and function of individual domains in these systems

Methods

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Discussion

Conclusion