title: Structural analysis of the interactions between hsp70 chaperones and the yeast DNA replication protein Orc4p.
abstract: Hsp70 chaperones, besides their role in assisting protein folding, are key modulators of protein disaggregation, being consistently found as components of most macromolecular assemblies isolated in proteome-wide affinity purifications. A wealth of structural information has been recently acquired on Hsp70s complexed with Hsp40 and NEF co-factors and with small hydrophobic target peptides. However, knowledge of how Hsp70s recognize large protein substrates is still limited. Earlier, we reported that homologue Hsp70 chaperones (DnaK in Escherichia coli and Ssa1-4p/Ssb1-2p in Saccharomyces cerevisiae) bind strongly, both in vitro and in vivo, to the AAA+ domain in the Orc4p subunit of yeast origin recognition complex (ORC). ScORC is the paradigm for eukaryotic DNA replication initiators and consists of six distinct protein subunits (ScOrc1p-ScOrc 6p). Here, we report that a hydrophobic sequence (IL(4)) in the initiator specific motif (ISM) in Orc4p is the main target for DnaK/Hsp70. The three-dimensional electron microscopy reconstruction of a stable Orc4p(2)-DnaK complex suggests that the C-terminal substrate-binding domain in the chaperone clamps the AAA+ IL(4) motif in one Orc4p molecule, with the substrate-binding domain lid subdomain wedging apart the other Orc4p subunit. Pairwise co-expression in E. coli shows that Orc4p interacts with Orc1/2/5p. Mutation of IL(4) selectively disrupts Orc4p interaction with Orc2p. Allelic substitution of ORC4 by mutants in each residue of IL(4) results in lethal (I184A) or thermosensitive (L185A and L186A) initiation-defective phenotypes in vivo. The interplay between Hsp70 chaperones and the Orc4p-IL(4) motif might have an adaptor role in the sequential, stoichiometric assembly of ScORC subunits.
authors: Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas - CSIC, C/ Ramiro de Maeztu, 9, E-28040 Madrid, Spain.
authors: María Moreno-Del
authors: MM
authors: Álamo
authors: Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas - CSIC, C/ Ramiro de Maeztu, 9, E-28040 Madrid, Spain.
authors: Alicia
authors: A
authors: Sánchez-Gorostiaga
authors: Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas - CSIC, C/ Ramiro de Maeztu, 9, E-28040 Madrid, Spain.
authors: Ana M
authors: AM
authors: Serrano
authors: Department of Environmental Biology, Centro de Investigaciones Biológicas - CSIC, C/ Ramiro de Maeztu, 9, E-28040 Madrid, Spain.
authors: Alicia
authors: A
authors: Prieto
authors: Department of Macromolecular Structures, Centro Nacional de Biotecnología - CSIC, C/ Darwin, 3, E-28049 Madrid, Spain.
authors: Jorge
authors: J
authors: Cuéllar
authors: Department of Macromolecular Structures, Centro Nacional de Biotecnología - CSIC, C/ Darwin, 3, E-28049 Madrid, Spain.
authors: Jaime
authors: J
authors: Martín-Benito
authors: Department of Macromolecular Structures, Centro Nacional de Biotecnología - CSIC, C/ Darwin, 3, E-28049 Madrid, Spain.
authors: José M
authors: JM
authors: Valpuesta
authors: Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas - CSIC, C/ Ramiro de Maeztu, 9, E-28040 Madrid, Spain. Electronic address: rgiraldo@cib.csic.es.
authors: Rafael
authors: R
authors: Giraldo
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D021621:Imaging, Three-Dimensional; D050296:Microbial Viability; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D051738:Origin Recognition Complex / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D050505:Mutant Proteins; C497219:ORC4 protein, S cerevisiae; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; C063842:dnaK protein, E coli