Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly.
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.
Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland.
Tomasz
T
Kochańczyk
Biological and Chemical Research Center, Faculty of Chemistry, University of Warsaw, Żwirki i Wigury 101, 02-089 Warsaw, Poland.
Michał
M
Nowakowski
Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland.
Dominika
D
Wojewska
Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland.
Anna
A
Kocyła
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106, Warsaw, Poland.
Andrzej
A
Ejchart
Biological and Chemical Research Center, Faculty of Chemistry, University of Warsaw, Żwirki i Wigury 101, 02-089 Warsaw, Poland.
Wiktor
W
Koźmiński
Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland.
Artur
A
Krężel
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019277:Entropy; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D000072417:Protein Domains; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D020027:Pyrococcus furiosus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D015032:Zinc / Q000378:metabolism*
D019843:Archaeal Proteins; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C415984:Rad50 protein, archaeal; D015032:Zinc