Relations between Structure and Zn(II) Binding Affinity Shed Light on the Mechanisms of Rad50 Hook Domain Functioning and Its Phosphorylation

Abstract

The metal binding at protein–protein interfaces is still uncharted territory in intermolecular interactions. To date, only a few protein complexes binding Zn(II) in an intermolecular manner have been deeply investigated. The most notable example of such interfaces is located in the highly conserved Rad50 protein, part of the Mre11-Rad50-Nbs1 (MRN) complex, where Zn(II) is required for homodimerization (Zn(Rad50)2). The high stability of Zn(Rad50)2 is conserved not only for the protein derived from the thermophilic archaeon Pyrococcus furiosus (logK12 = 20.95 for 130-amino-acid-long fragment), which was the first one studied, but also for the human paralog studied here (logK12 = 19.52 for a 183-amino-acid-long fragment). As we reported previously, the extremely high stability results from the metal-coupled folding process where particular Rad50 protein fragments play a critical role. The sequence–structure–stability analysis based on human Rad50 presented here separates the individual structural components that increase the stability of the complex, pointing to amino acid residues far away from the Zn(II) binding site as being largely responsible for the complex stabilization. The influence of the individual components is very well reflected by the previously published crystal structure of the human Rad50 zinc hook (PDB: 5GOX). In addition, we hereby report the effect of phosphorylation of the zinc hook domain, which exerts a destabilizing effect on the domain. This study identifies factors governing the stability of metal-mediated protein–protein interactions and illuminates their molecular basis.

Dynamic Properties of the DNA Damage Response Mre11/Rad50 Complex

“…The Zn hook is necessary for the association of two coiled coils by sharing the metal ion among the four Cysteine residues of two Rad50 subunits belonging to the same complex (intra-complex bridge) [32], and it was proposed that it could be shared by two different complexes (inter-complex bridge) [33,34]. The Zn hook association is stabilized by the interactions through the distal coiled-coil rods [29] and is probably regulated via phosphorylation on Thr690 [35]. The transition from an oval-shaped ring to a rod was also observed when the complex bound to the DNA end; thus, the oval-shaped ring was proposed to be involved in genomic DNA scanning [8] (Figure 2C).…”