Defining a novel domain that provides an essential contribution to site-specific interaction of Rep protein with DNA

15 p.-6 fig.An essential feature of replication initiation proteins is their ability to bind to DNA. In this work, we describe a new domain that contributes to a replication initiator sequence-specific interaction with DNA. Applying biochemical assays and structure prediction methods coupled with DNA-protein crosslinking, mass spectrometry, and construction and analysis of mutant proteins, we identified that the replication initiator of the broad host range plasmid RK2, in addition to two winged helix domains, contains a third DNA-binding domain. The phylogenetic analysis revealed that the composition of this unique domain is typical within the described TrfA-like protein family. Both in vitro and in vivo experiments involving the constructed TrfA mutant proteins showed that the newly identified domain is essential for the formation of the protein complex with DNA, contributes to the avidity for interaction with DNA, and the replication activity of the initiator. The analysis of mutant proteins, each containing a single substitution, showed that each of the three domains composing TrfA is essential for the formation of the protein complex with DNA. Furthermore, the new domain, along with the winged helix domains, contributes to the sequence specificity of replication initiator interaction within the plasmid replication origin.National Science Centre [2012/04/A/NZ1/00048 to I.K.;2017/26/D/NZ1/00239 to K.W.]; Foundation for Polish Science [TEAM, POIR.04.04.00-00-5C75/17-00 to I.K.]; International Institute of Molecular and Cell Biology in Warsaw (to J.M.B.); Ministerio de Economía,Industria y Competitividad (MINECO/AEI) [BIO2012-30852, RTI2018-094549-B-I00 to R.G.]. Funding for open access charge: Foundation for Polish Science [TEAM,POIR.04.04.00-00-5C75/17-00].Peer reviewe

Two-step mechanism of J-domain action in driving Hsp70 function.

J-domain proteins (JDPs), obligatory Hsp70 cochaperones, play critical roles in protein homeostasis. They promote key allosteric transitions that stabilize Hsp70 interaction with substrate polypeptides upon hydrolysis of its bound ATP. Although a recent crystal structure revealed the physical mode of interaction between a J-domain and an Hsp70, the structural and dynamic consequences of J-domain action once bound and how Hsp70s discriminate among its multiple JDP partners remain enigmatic. We combined free energy simulations, biochemical assays and evolutionary analyses to address these issues. Our results indicate that the invariant aspartate of the J-domain perturbs a conserved intramolecular Hsp70 network of contacts that crosses domains. This perturbation leads to destabilization of the domain-domain interface-thereby promoting the allosteric transition that triggers ATP hydrolysis. While this mechanistic step is driven by conserved residues, evolutionarily variable residues are key to initial JDP/Hsp70 recognition-via electrostatic interactions between oppositely charged surfaces. We speculate that these variable residues allow an Hsp70 to discriminate amongst JDP partners, as many of them have coevolved. Together, our data points to a two-step mode of J-domain action, a recognition stage followed by a mechanistic stage

Analysis of Reconstituted Tripartite Complex Supports Avidity-based Recruitment of Hsp70 by Substrate Bound J-domain Protein

Dataset for manuscript "Analysis of Reconstituted Tripartite Complex Supports Avidity-based Recruitment of Hsp70 by Substrate Bound J-domain Protein":- molecular dynamics trajectories and structural model- GraphPad Prism files for all figures in main text and supplementary data</p