Acetylation of Surface Lysine Groups of a Protein Alters the Organization and Composition of Its Crystal Contacts.

Kyungtae Kang,Jeong-Mo Choi,George M Whitesides,Demetri T Moustakas,Phillip W Snyder,Jerome M Fox

doi:10.1021/acs.jpcb.6b01105

Abstract

This paper uses crystals of bovine carbonic anhydrase (CA) and its acetylated variant to examine (i) how a large negative formal charge can be accommodated in protein-protein interfaces, (ii) why lysine residues are often excluded from them, and (iii) how changes in the surface charge of a protein can alter the structure and organization of protein-protein interfaces. It demonstrates that acetylation of lysine residues on the surface of CA increases the participation of polar residues (particularly acetylated lysine) in protein-protein interfaces, and decreases the participation of nonpolar residues in those interfaces. Negatively charged residues are accommodated in protein-protein interfaces via (i) hydrogen bonds or van der Waals interactions with polar residues or (ii) salt bridges with other charged residues. The participation of acetylated lysine in protein-protein interfaces suggests that unacetylated lysine tends to be excluded from interfaces because of its positive charge, and not because of a loss in conformational entropy. Results also indicate that crystal contacts in acetylated CA become less constrained geometrically and, as a result, more closely packed (i.e., more tightly clustered spatially) than those of native CA. This study demonstrates a physical-organic approach-and a well-defined model system-for studying the role of charges in protein-protein interactions.

Highlights

This study uses the contact regions between molecules of bovine carbonic anhydrase (CA; E.C.4.2.1.1) in crystals of this protein as a physical-organic model system with which to study proteinprotein interfaces systematically
Four characteristics of the contact regions of crystals make them attractive as a subject for study: i) They are well defined structurally by X-ray and neutron crystallography. ii) They can be systematically modified through mutagenesis and/or chemical functionalization; these modifications often retain native protein structure. iii) Contact regions generate and define adjacent non-contact regions—regions that provide a set of examples of near-surface films or pools of water that might aid in understanding the role of water in protein-protein association. iv) A very large number of crystallographic data on protein-protein interfaces are available
This study addresses three questions pertaining to the influence of surface charge on the structure and organization of protein-protein interfaces: i) Can a protein with a large formal charge (i.e., Z = -19) be crystallized and, if so, how is that charge accommodated and/or compensated to enable stable protein-protein interfaces? ii) What attributes of lysine residues cause their often-observed exclusion from protein-protein interfaces? iii) How does the conversion of a surface of a protein from “polyzwitterionic” to “polyanionic” alter the structure and organization of interfaces that result when the protein is crystallized?

Summary

INTRODUCTION

This study uses the contact regions between molecules of bovine carbonic anhydrase (CA; E.C.4.2.1.1) in crystals of this protein as a physical-organic model system with which to study proteinprotein interfaces systematically. It has three characteristics that make it uniquely suited for carrying out a physical-organic examination of protein interfaces: i) It is structurally rigid; alterations to its surface, tend not to alter its conformation. ii) All 18 lysine residues on the surface of CA can be acetylated This acetylation results in a large increase in its negative charge (from Z = −3.4 for nonacetylated CA, to Z = −19 for peracetylated CA3; X-ray crystallography and circular dichroism spectroscopy indicate that this peracetylation does not alter the secondary or tertiary structure of CA). iii) Previous studies demonstrate that CA and many of its mutants can be crystallized.. We used X-ray crystallography to collect structures of CA with and without its positively charged, surface-exposed lysine residues acetylated; and using these structures, we examined how acetylation altered the organization and composition of crystal contacts

Background

RESULTS AND DUSCUSSION

CONCLUSIONS