Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly.

Tomasz Kochańczyk,Dominika Wojewska,Wiktor Koźmiński,Artur Krężel,Andrzej Ejchart,Anna Kocyła,Michał Nowakowski

doi:10.1038/srep36346

Abstract

The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed analysis of the structural and thermodynamic effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small β-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation constant of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is critical for the tight association of protein subunits.

Highlights

DNA damage response, DNA recombination, telomere integrity and meiosis
The binding of metal ions at the protein-protein interface significantly contributes to the complex formation thermodynamics, and may promote molecular recognition and induce changes in the conformation of the resulting complex
Based on the currently available structure of the zinc hook domain of Rad[50] from P. furiosus (PDB ID: 1L8D)[6] (Fig. 1a), we designed a series of peptides of various lengths ranging from the CXXC motif required for intermolecular Zn2+ binding to the full-length domain

Summary

Introduction

DNA damage response, DNA recombination, telomere integrity and meiosis. The precise role of the zinc hook complex in these processes and how the stability of this complex relates to its function are unknown[11,13]. The total contact area of the interface formed by the zinc hook domain of Rad[50] protein is only 640 Å2 6,20, suggesting a major effect from a low number of interactions and a significant contribution of intermolecular Zn2+ binding to the stability of the interaction. We used alanine substitutions and the replacement of the amide bond with an ester bond to address the influence of certain residues and hydrogen bond formation in the peptide backbone on zinc hook complex stability. Using these models, we dissected the structural and thermodynamic determinants governing the stability of the metal-mediated dimer assembly formed by the zinc hook domain

Methods

Results

Conclusion