Interdomain interaction through helices A and B of DnaK peptide binding domain

Self Cite

We examined the effect of deletion of different segments in the helical subdomain (the so-called "lid") of the DnaK peptide-binding domain on peptide binding and protein stability. At 25°C, wt DnaK and the deletion mutant proteins are able to stably bind peptides with similar affinity. However, at physiological (37°C) and stress (42°C) temperatures, removal of the N-terminal half of ␣B and the rest of the lid drastically decreases the ability of the protein to bind substrates. Differential scanning calorimetry and infrared spectroscopy show that this behavior is accompanied by destabilization of the peptide-binding domain. Our data suggest that the reversible interaction between the lid and ␤-sandwich subdomains of DnaK peptide-binding domain is required for the stabilization of the loops that form the peptide-binding site, which in turn modulates the protein affinity for peptide substrates. This interaction might have functional implications because it could prevent rebinding of the peptide substrate, which would be forced to fold.

Section: Resultssupporting

confidence: 82%

mentioning

confidence: 98%

The Lid Subdomain of DnaK Is Required for the Stabilization of the Substrate-binding Site

Moro¹,

Fernández‐Sáiz²,

Muga

2004

Self Cite

“…C, upon rapid mixing with ATP, fp5 release follows the same kinetic profile in DnaK(1-631) (red), D628A (green), and wild type DnaK (blue). D, DnaK(1-631) (red), D628A (green), and wild type (blue) display the same characteristic blue shift and intensity quench of Trp fluorescence upon ATP binding, indicating that they undergo productive interdomain docking (7,41). The lower and more lightly shaded traces are from measurements after ATP addition.…”

Section: Dnak C-terminal Region Is Not Required For Binding Of a Peptmentioning

confidence: 95%

“…Nucleotide-binding domain residue Trp-102 undergoes fluorescence emission changes upon binding of ATP and in the presence of the SBD lid and is therefore a probe for functional ATP-induced interdomain docking (41). The physical proximity of the disordered C-terminal tail to the structured SBD lid suggests that C-terminal mutation might alter lid interaction with the nucleotide-binding domain.…”

Section: Dnak C-terminal Region Is Not Required For Binding Of a Peptmentioning

confidence: 99%

Conserved, Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

Smock

Blackburn

Gierasch

2011

The 70-kDa heat shock proteins (Hsp70s) function as molecular chaperones through the allosteric coupling of their nucleotide-and substrate-binding domains, the structures of which are highly conserved. In contrast, the roles of the poorly structured, variable length C-terminal regions present on Hsp70s remain unclear. In many eukaryotic Hsp70s, the extreme C-terminal EEVD tetrapeptide sequence associates with co-chaperones via binding to tetratricopeptide repeat domains. It is not known whether this is the only function for this region in eukaryotic Hsp70s and what roles this region performs in Hsp70s that do not form complexes with tetratricopeptide repeat domains. We compared C-terminal sequences of 730 Hsp70 family members and identified a novel conservation pattern in a diverse subset of 165 bacterial and organellar Hsp70s. Mutation of conserved C-terminal sequence in DnaK, the predominant Hsp70 in Escherichia coli, results in significant impairment of its protein refolding activity in vitro without affecting interdomain allostery, interaction with co-chaperones DnaJ and GrpE, or the binding of a peptide substrate, defying classical explanations for the chaperoning mechanism of Hsp70. Moreover, mutation of specific conserved sites within the DnaK C terminus reduces the capacity of the cell to withstand stresses on protein folding caused by elevated temperature or the absence of other chaperones. These features of the C-terminal region support a model in which it acts as a disordered tether linked to a conserved, weak substrate-binding motif and that this enhances chaperone function by transiently interacting with folding clients.The ubiquitously distributed Hsp70 family of molecular chaperones shepherd newly synthesized polypeptide chains, protect cells from stress-induced protein aggregation, assist in protein translocation across membranes, and regulate assembly and disassembly of macromolecular complexes. These physiological functions are accomplished by a two-domain allosteric mechanism in which cycles of ATP binding and hydrolysis in the N-terminal nucleotide-binding domain (NBD) 2 control the binding and release of hydrophobic polypeptide segments in the substrate-binding domain (SBD) (1-3). Although we have gained an increasingly detailed picture of this allosteric transition in Hsp70 chaperones and modes of substrate interaction (4 -7), it is striking that there is much less known about the function of the extreme C-terminal unstructured region (Fig. 1). Binding of the C-terminal region of mammalian Hsc70 to substrate was suggested in earlier work (8, 9), and more recently, the C-terminal segment of eukaryotic cytoplasmic Hsp70s was found to contain a conserved tetratricopeptide repeat (TPR) domain interaction motif that mediates binding with Chip, Hip, or Hop co-chaperones that facilitate the assembly of a variety of Hsp70 complexes (3). Even though bacteria lack homologs of these Hsp70-interacting TPR domain proteins, many, including the extensively characterized E. coli Hsp70 DnaK, have a disord...

“…As can be deduced from the crystal structure of bovine Hsc70, mutations N175A/D176A and E240A/V241A affect close contact ionic and hydrophobic interactions that directly connect the NBD with helix A of the SBD (N175A/D176A and E240A/ V241A) and with an adjacent part of the SBD ␤-sheet subdomain (E240A/V241A). Both structural elements have been implicated in the allosteric mechanism because helix A forms the basis of the ␣-helical lid that covers the peptide-binding pocket formed by the ␤-sheet subdomain (54,55). Additionally, apart from directly disrupting molecular contacts between NBD and SBD, the mutations could affect interdomain communication by interfering with the insertion of the hydrophobic domain linker segment into a hydrophobic cleft between subdomains IA and IIA of the NBD.…”

Section: Discussionmentioning

confidence: 99%

Impaired Interdomain Communication in Mitochondrial Hsp70 Results in the Loss of Inward-directed Translocation Force

Becker

Krayl

Strub

et al. 2009

The essential mitochondrial Hsp70 (mtHsp70) is required for the import of mitochondrial preproteins into the matrix compartment. The translocation-specific activity of mtHsp70 is coordinated by its interaction with specific partner proteins, forming the import motor complex that provides the energy for unfolding and complete translocation of precursor polypeptide chains. A major biochemical characteristic of Hsp70-type chaperones is their nucleotide-regulated affinity to polypeptide substrates. To study the role of this allosteric regulation in the course of preprotein translocation, we have generated specific mtHsp70 mutations located within or close to the interface between the nucleotide-binding and the substrate-binding domains. Mitochondria isolated from the mtHsp70 mutants displayed severely reduced import efficiencies in vitro. Two of the mutants exhibited strong growth defects in vivo and were significantly impaired in the generation of an inward-directed, ATPdependent import force on precursor proteins in transit. The biochemical properties of these two mutant proteins were consistent with defects in the transfer of conformational signals to the substrate-binding domain, resulting in a prolonged and enhanced interaction with imported substrate proteins. Furthermore, interference with the allosteric mechanism resulted in defects of translocation-specific partner protein interaction. We conclude that even a partial disruption of the interdomain communication in the mtHsp70 chaperone results in an almost complete breakdown of its translocation-driving properties.Chaperones of the 70-kDa family (Hsp70) are ubiquitous proteins that occur in all species (except archaea) and fulfill crucial cellular functions under normal and stress conditions (1). Eukaryotic cells contain several Hsp70 paralogs that are generally involved in all stages of protein biogenesis, ranging from biosynthesis at the ribosomes, intracellular transport, and eventually proteolysis (2-4). Mitochondria of the model organism Saccharomyces cerevisiae contain three types of Hsp70 proteins, encoded by the genes SSC1, SSQ1, and SSC3 (ECM10) (5). Ssc3 is expressed at very low levels under normal growth conditions and its cellular function is so far unknown. Ssq1 has been shown to assist the assembly of iron/sulfur cluster cofactors in mitochondria. The most abundant and functionally important mitochondrial Hsp70 is Ssc1, generally referred to as mtHsp70. It performs most of the typical chaperone functions in terms of protein folding and quality control for polypeptides localized in the mitochondrial matrix compartment (5-7). In addition, Ssc1 is required for the import of all mitochondrial precursor proteins destined for the matrix (8). In yeast cells, deletion of SSC1 results in a lethal phenotype, a property that has been directly attributed to its crucial role in the import process. Mitochondrial preprotein translocation comprises a series of consecutive steps (9): after synthesis at cytosolic ribosomes, the N-terminal targeting signal of a p...