title: Structural and functional studies of Hsp70-escort protein--Hep1--of Leishmania braziliensis.
abstract: Hep1 is a mitochondrial Hsp70 (mtHsp70) co-chaperone that presents a zinc finger domain essential for its function. This co-chaperone acts to maintain mtHsp70 in its soluble and functional state. In this work, we have demonstrated that Leishmania braziliensis mtHsp70 (LbmtHsp70) is also dependent on the assistance of Hep1. To understand the L. braziliensis Hep1 (LbHep1) structure-function relationship, we produced LbHep1 and two truncated mutants corresponding to the C-terminal zinc finger domain and the N-terminal region. We observed that LbHep1 is composed of an unfolded N-terminal region and a β-sheet-folded C-terminal domain, which holds the zinc-binding motif. Both LbHep1 and the zinc finger domain construction maintained LbmtHsp70 solubility in co-expression systems after cell lysis. In solution, LbHep1 behaved as a highly elongated monomer, probably due to the unfolded N-terminal region. Furthermore, we also observed that the zinc ion interacted with LbHep1 with high affinity and was critical for LbHep1 structure and stability because its removal from LbHep1 solutions altered the protein structure and stability. In vitro, LbHep1 protected, in sub-stoichiometric fashion, LbmtHsp70 from thermally induced aggregation but did not present intrinsic chaperone activity on model client proteins. Therefore, LbHep1 is a specific chaperone for LbmtHsp70.
authors.affiliation: Institute of Chemistry of São Carlos, University of São Paulo - USP, São Carlos, SP 13560-970, Brazil.
authors.forename: P R
authors.initials: PR
authors.lastname: Dores-Silva
authors.affiliation: Institute of Chemistry of São Carlos, University of São Paulo - USP, São Carlos, SP 13560-970, Brazil.
authors.forename: L L
authors.initials: LL
authors.lastname: Beloti
authors.affiliation: Institute of Chemistry of São Carlos, University of São Paulo - USP, São Carlos, SP 13560-970, Brazil; Post-Graduation Program in Evolutionary Genetics and Molecular Biology, Federal University of São Carlos - UFSCar, São Carlos, SP 13565-905, Brazil.
authors.forename: K
authors.initials: K
authors.lastname: Minari
authors.affiliation: Institute of Chemistry of São Carlos, University of São Paulo - USP, São Carlos, SP 13560-970, Brazil.
authors.forename: S M O
authors.initials: SM
authors.lastname: Silva
authors.affiliation: Institute of Physics, University of São Paulo - USP, São Paulo, SP 05508-090, Brazil.
authors.forename: L R S
authors.initials: LR
authors.lastname: Barbosa
authors.affiliation: Institute of Chemistry of São Carlos, University of São Paulo - USP, São Carlos, SP 13560-970, Brazil. Electronic address: borgesjc@iqsc.usp.br.
authors.forename: J C
authors.initials: JC
authors.lastname: Borges
mesh_terms: D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D007892:Leishmania braziliensis / Q000737:chemistry; D008928:Mitochondria / Q000737:chemistry / Q000235:genetics; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D018840:HSP70 Heat-Shock Proteins; D024101:Mitochondrial Proteins; D018832:Molecular Chaperones
keywords: Biophysical studies; Heat shock proteins; Leishmania braziliensis; mtHsp70