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Title

Structural analysis of the interactions between hsp70 chaperones and the yeast DNA replication protein Orc4p.

AuthorsMoreno-del Álamo, María ; Sánchez-Gorostiaga, Alicia; Serrano-López, Ana; Prieto, Alicia ; Cuéllar, Jorge; Martín-Benito, Jaime; Valpuesta, José M.; Giraldo, R.
KeywordsYeast
Hsp70 chaperones
DNA replication
Macromolecular assemblies
Saccharomyces cerevisiae
Origin recognition complex
Escherichia coli
HSP70 Heat-Shock Proteins
Protein binding
Issue Date15-Oct-2010
PublisherElsevier
CitationJournal of Molecular Biology, 403(1): 24-39 (2010)
AbstractHsp70 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 (IL4) in the initiator specific motif (ISM) in Orc4p is the main target for DnaK/Hsp70. The three-dimensional electron microscopy reconstruction of a stable Orc4p2–DnaK complex suggests that the C-terminal substrate-binding domain in the chaperone clamps the AAA+ IL4 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 IL4 selectively disrupts Orc4p interaction with Orc2p. Allelic substitution of ORC4 by mutants in each residue of IL4 results in lethal (I184A) or thermosensitive (L185A and L186A) initiation-defective phenotypes in vivo. The interplay between Hsp70 chaperones and the Orc4p-IL4 motif might have an adaptor role in the sequential, stoichiometric assembly of ScORC subunits.
DescriptionYou need the Java runtime environment to see the additional information in this article as Protein Structures.
Publisher version (URL)http://www.sciencedirect.com/science/article/pii/S0022283610008843
URIhttp://hdl.handle.net/10261/41510
DOI10.1016/j.jmb.2010.08.022
ISSN1089-8638
Appears in Collections:(CIB) Artículos
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