Eukaryotic protein homeostasis (proteostasis) is largely dependent on the action of highly conserved Hsp70 molecular chaperones. Recent evidence indicates that, apart from conserved molecular allostery, Hsp70 proteins have retained and adapted the ability to assemble as functionally relevant ATP-bound dimers throughout evolution. Here, we have compared the ATP-dependent dimerization of DnaK, human stress-inducible Hsp70, Hsc70 and BiP Hsp70 proteins, showing that their dimerization propensities differ, with stress-inducible Hsp70 being predominantly dimeric in the presence of ATP. Structural analyses using hydrogen/deuterium exchange mass spectrometry, native electrospray ionization mass spectrometry and small-angle X-ray scattering revealed that stress-inducible Hsp70 assembles in solution as an antiparallel dimer with the intermolecular interface closely resembling the ATP-bound dimer interfaces captured in DnaK and BiP crystal structures. ATP-dependent dimerization of stress-inducible Hsp70 is necessary for its efficient interaction with Hsp40, as shown by experiments with dimerization-deficient mutants. Moreover, dimerization of ATP-bound Hsp70 is required for its participation in high molecular weight protein complexes detected ex vivo, supporting its functional role in vivo As human cytosolic Hsp70 can interact with tetratricopeptide repeat (TPR) domain containing cochaperones, we tested the interaction of Hsp70 ATP-dependent dimers with Chip and Tomm34 cochaperones. Although Chip associates with intact Hsp70 dimers to form a larger complex, binding of Tomm34 disrupts the Hsp70 dimer and this event plays an important role in Hsp70 activity regulation. In summary, this study provides structural evidence of robust ATP-dependent antiparallel dimerization of human inducible Hsp70 protein and suggests a novel role of TPR domain cochaperones in multichaperone complexes involving Hsp70 ATP-bound dimers.

• Klíčová slova
• Allostery, Chaperone, Cochaperone, Mass Spectrometry, Protein Conformation, Protein Structure, Protein-Protein Interactions, Structural Biology,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• fyziologický stres MeSH
• HEK293 buňky MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• maloúhlový rozptyl MeSH
• mitochondriální importní komplex MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• proteiny tepelného šoku HSP70 chemie   metabolismus   MeSH
• transportní proteiny mitochondriální membrány metabolismus   MeSH
• ubikvitinligasy metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• mitochondriální importní komplex MeSH
• proteiny tepelného šoku HSP70 MeSH
• STUB1 protein, human MeSH Prohlížeč
• TOMM34 protein, human MeSH Prohlížeč
• transportní proteiny mitochondriální membrány MeSH
• ubikvitinligasy MeSH

All eukaryotic genomes encode multiple members of the heat shock protein 70 (HSP70) family, which evolved distinctive structural and functional features in response to specific environmental constraints. Phylogenetic analysis of this protein family thus can inform on genetic and molecular mechanisms that drive species-specific environmental adaptation. Here we use the eukaryotic pathogen Leishmania spp. as a model system to investigate the evolution of the HSP70 protein family in an early-branching eukaryote that is prone to gene amplification and adapts to cytotoxic host environments by stress-induced and chaperone-dependent stage differentiation. Combining phylogenetic and comparative analyses of trypanosomatid genomes, draft genome of Paratrypanosoma and recently published genome sequences of 204 L. donovani field isolates, we gained unique insight into the evolutionary dynamics of the Leishmania HSP70 protein family. We provide evidence for (i) significant evolutionary expansion of this protein family in Leishmania through gene amplification and functional specialization of highly conserved canonical HSP70 members, (ii) evolution of trypanosomatid-specific, non-canonical family members that likely gained ATPase-independent functions, and (iii) loss of one atypical HSP70 member in the Trypanosoma genus. Finally, we reveal considerable copy number variation of canonical cytoplasmic HSP70 in highly related L. donovani field isolates, thus identifying this locus as a potential hot spot of environment-genotype interaction. Our data draw a complex picture of the genetic history of HSP70 in trypanosomatids that is driven by the remarkable plasticity of the Leishmania genome to undergo massive intra-chromosomal gene amplification to compensate for the absence of regulated transcriptional control in these parasites.

• Klíčová slova
• HSP70, Leishmania, copy number variation, evolution, gene loss, heat shock protein, phylogeny, synteny,
• MeSH
• amplifikace genu genetika   MeSH
• druhová specificita MeSH
• fylogeneze MeSH
• genom MeSH
• Leishmania genetika   patogenita   MeSH
• leishmanióza genetika   parazitologie   MeSH
• lidé MeSH
• molekulární evoluce * MeSH
• proteiny tepelného šoku HSP70 genetika   MeSH
• variabilita počtu kopií segmentů DNA genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny tepelného šoku HSP70 MeSH

Co-chaperones containing tetratricopeptide repeat (TPR) domains enable cooperation between Hsp70 and Hsp90 to maintain cellular proteostasis. Although the details of the molecular interactions between some TPR domains and heat shock proteins are known, we describe a novel mechanism by which Tomm34 interacts with and coordinates Hsp70 activities. In contrast to the previously defined Hsp70/Hsp90-organizing protein (Hop), Tomm34 interaction is dependent on the Hsp70 chaperone cycle. Tomm34 binds Hsp70 in a complex process; anchorage of the Hsp70 C terminus by the TPR1 domain is accompanied by additional contacts formed exclusively in the ATP-bound state of Hsp70 resulting in a high affinity entropically driven interaction. Tomm34 induces structural changes in determinants within the Hsp70-lid subdomain and modulates Hsp70/Hsp40-mediated refolding and Hsp40-stimulated Hsp70 ATPase activity. Because Tomm34 recruits Hsp90 through its TPR2 domain, we propose a model in which Tomm34 enables Hsp70/Hsp90 scaffolding and influences the Hsp70 chaperone cycle, providing an additional role for co-chaperones that contain multiple TPR domains in regulating protein homeostasis.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• mitochondriální importní komplex MeSH
• molekulární modely MeSH
• mutace MeSH
• proteiny tepelného šoku HSP70 chemie   metabolismus   MeSH
• sbalování proteinů MeSH
• simulace molekulového dockingu MeSH
• terciární struktura proteinů MeSH
• transportní proteiny mitochondriální membrány chemie   genetika   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát MeSH
• mitochondriální importní komplex MeSH
• proteiny tepelného šoku HSP70 MeSH
• TOMM34 protein, human MeSH Prohlížeč
• transportní proteiny mitochondriální membrány MeSH