Hsp70 ATP-dependent Dimerization and Interactions

Filip Trcka¹, Michal Durech¹, Pavla Vankova^2,3 , Josef Chmelik^2,3, Veronika Martinkova¹, Jiri Hausner^2,.3, Alan Kadek^2,3, Julien Marcoux⁴, Tomas Klumpler⁵, Borivoj Vojtesek¹, Petr Muller¹, Petr Man^2,3

¹Regional Centre for Applied Molecular Oncology, Zluty kopec 7, 656 53 Brno.

²BioCeV - Institute of Microbiology of the Czech Academy of Sciences, Prumyslova 595, 252 50 Vestec.

³Dep. of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43 Prague.

⁴Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, Toulouse.

⁵CEITEC–Central European Institute of Technology, Masaryk University, 625 00 Brno.

pman@biomed.cas.cz

The 70kDa heat shock protein (Hsp70) is the central component of nanomachinery maintaining the cellular proteostasis [1]. Sequence and structural conservation of Hsp70 across all domains of life suggest the evolutionary importance and conserved, but protean mechanism of action [2]. Hsp70 system is able to bind nascent polypeptide chains at ribosomes or translocons[3,4]; misfolded, stress-denatured proteins or solubilize protein aggregates or amyloid fibrils [5]. Hsp70 proteins are composed an nucleotide-binding domain (NBD) and substrate-binding domain (SBD) connected via highly conserved linker [6]. SBD interacts with short degenerative sequence motifs present in virtually every globular protein, while NBD covers the SBD as a helical lid and stabilizes substrate binding [7]. Beside this allosteric opening of the polypeptide binding site upon ATP binding [8], Hsp70 proteins were reported to form antiparallel dimers facilitated by Hsp40, which is part of Hsp70 system [9].

In this study, we inspect the human Hsp70 by hydrogen/deuterium exchange mass spectroscopy, native electrospray ionization mass spectroscopy and small angle x-ray scattering, showing the Hsp70 to form an antiparallel dimer in solution in ATP-dependent manner. Dimerization of Hsp70 is essential for interaction with Hsp40 as was shown by dimerization-deficient mutatnts. Formation of multichaperon complexes of Hsp70 with two tetratricopeptide repeat domain co-chaperones Chip and Tomm34 was inspected by size exclusion chromatography in presence and absence of ATP. Dimeric co-chaperone Chip interacts with ATP-induced Hsp70 dimer and forms mutichaperon complex with stoichiometry 2:2, while monomeric co-chaperon disrupts the ATP-induced Hsp70 dimer and forms 1:1 complex with Hsp70.

This work was supported by Czech Science Foundation (16-20860S); Ministry of Education, Youth and Sports of the Czech Republic (MEYS CR, LO1413, LO1604, LM2015043, LM2011020 and LO1509); Ministry of Health of the Czech Republic – conceptual development of research organization (MMCI, 00209805) and EU (CZ.1.05/1.1.00/02.0109; OPPK CZ.2.16/3.1.00/24023).

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