Ismail Moarefi

Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine

The adaptor protein Hop mediates the association of the molecular chaperones Hsp70 and Hsp90. The TPR1 domain of Hop specifically recognizes the C-terminal heptapeptide of Hsp70 while the TPR2A domain binds the C-terminal pentapeptide of Hsp90. Both sequences end with the motif EEVD. The crystal structures of the TPR-peptide complexes show the peptides in an extended conformation, spanning a groove in the TPR domains. Peptide binding is mediated by electrostatic interactions with the EEVD motif, with the C-terminal aspartate acting as a two-carboxylate anchor, and by hydrophobic interactions with residues upstream of EEVD. The hydrophobic contacts with the peptide are critical for specificity. These results explain how TPR domains participate in the ordered assembly of Hsp70-Hsp90 multichaperone complexes.

Structure of the molecular chaperone prefoldin: unique interaction of multiple coiled coil tentacles with unfolded proteins.

Prefoldin (GimC) is a hexameric molecular chaperone complex built from two related classes of subunits and present in all eukaryotes and archaea. Prefoldin interacts with nascent polypeptide chains and, in vitro, can functionally substitute for the Hsp70 chaperone system in stabilizing non-native proteins for subsequent folding in the central cavity of a chaperonin. Here, we present the crystal structure and characterization of the prefoldin hexamer from the archaeum Methanobacterium thermoautotrophicum. Prefoldin has the appearance of a jellyfish: its body consists of a double beta barrel assembly with six long tentacle-like coiled coils protruding from it. The distal regions of the coiled coils expose hydrophobic patches and are required for multivalent binding of nonnative proteins.

The protein import motor of mitochondria: a targeted molecular ratchet driving unfolding and translocation

Unfolding and import of preproteins into mitochondria are facilitated by a molecular motor in which heat shock protein 70 (Hsp70) in the matrix plays an essential role. Here we present two different experimental approaches to analyze mechanisms underlying this function of Hsp70. First, preproteins containing stretches of glutamic acid (polyE) or glycine (polyG) repeats in front of folded domains were imported into mitochondria. This occurred although Hsp70 cannot pull on these stretches to unfold the folded domains, since it does not bind to polyE and polyG. Secondly, preproteins containing titin immunoglobulin (Ig)‐like domains were imported into mitochondria, despite the fact that forces of >200 pN are required to mechanically unfold these domains. Since molecular motors generate forces of ∼5 pN, Hsp70 could not promote unfolding of the Ig‐like domains by mechanical pulling. Our observations suggest that Hsp70 acts as an element of a Brownian ratchet, which mediates unfolding and translocation of preproteins across the mitochondrial membranes.

Structural basis for SH3 domain-mediated high-affinity binding between Mona/Gads and SLP-76

SH3 domains are protein recognition modules within many adaptors and enzymes. With more than 500 SH3 domains in the human genome, binding selectivity is a key issue in understanding the molecular basis of SH3 domain interactions. The Grb2‐like adaptor protein Mona/Gads associates stably with the T‐cell receptor signal transducer SLP‐76. The crystal structure of a complex between the C‐terminal SH3 domain (SH3C) of Mona/Gads and a SLP‐76 peptide has now been solved to 1.7 Å. The peptide lacks the canonical SH3 domain binding motif P–x–x–P and does not form a frequently observed poly‐proline type II helix. Instead, it adopts a clamp‐like shape around the circumfence of the SH3C β‐barrel. The central R–x–x–K motif of the peptide forms a 310 helix and inserts into a negatively charged double pocket on the SH3C while several other residues complement binding through hydrophobic interactions, creating a short linear SH3C binding epitope of uniquely high affinity. Interestingly, the SH3C displays ion‐dependent dimerization in the crystal and in solution, suggesting a novel mechanism for the regulation of SH3 domain functions.

Prediction of Novel Bag-1 Homologs Based on Structure/Function Analysis Identifies Snl1p as an Hsp70 Co-chaperone in Saccharomyces cerevisiae

Polypeptide binding by the chaperone Hsp70 is regulated by its ATPase activity, which is itself regulated by co-chaperones including the Bag domain nucleotide exchange factors. Here, we tested the functional contribution of residues in the Bag domain of Bag-1M that contact Hsp70. Two point mutations, E212A and E219A, partially reduced co-chaperone activity, whereas the point mutation R237A completely abolished activity in vitro. Based on the strict positional conservation of the Arg-237 residue, several Bag domain proteins were predicted from various eukaryotic genomes. One candidate, Snl1p from Saccharomyces cerevisiae, was confirmed as a Bag domain co-chaperone. Snl1p bound specifically to the Ssa and Ssb forms of yeast cytosolic Hsp70, as revealed by two-hybrid screening and co-precipitations from yeast lysate. In vitro, Snl1p also recognized mammalian Hsp70 and regulated the Hsp70 ATPase activity identically to Bag-1M. Point mutations in Snl1p that disrupted the conserved residues Glu-112 and Arg-141, equivalent to Glu-212 and Arg-237 in Bag-1M, abolished the interaction with Hsp70 proteins. In live yeast, mutated Snl1p could not substitute for wild-type Snl1p in suppressing the lethal defect caused by truncation of the Nup116p nuclear pore component. Thus, Snl1p is the first Bag domain protein identified in S. cerevisiae, and its interaction with Hsp70 is essential for biological activity.

From autoinhibition to inhibition in trans: the Raf-1 regulatory domain inhibits Rok-α kinase activity

The mechanism by which Raf-1 antagonizes Rok-α to promote migration and tumorigenesis is revealed.

Rational Development of Cell‐Penetrating High Affinity SH3 Domain Binding Peptides That Selectively Disrupt the Signal Transduction of Crk Family Adapters

CHRISTIAN KARDINAL,a GUIDO POSERN,a JIE ZHENG,b BEATRICE S. KNUDSEN,c AMGEN PEPTIDE TECHNOLOGY GROUP,d ISMAIL MOAREFI,e AND STEPHAN M. FELLERa,f aLaboratory of Molecular Oncolology, MSZ, University of Würzburg, Würzburg, Germany bDepartment of Structural Biology, St. Jude Childrens Hospital, Memphis, Tennessee cDepartment of Pathology, New York Hospital, New York, New York dBoulder, Colorado eMax Planck Institute for Biochemistry, Martinsried, Germany