Identification of a consensus motif in substrates bound by a Type I Hsp40

Protein aggregation is a hallmark of a large and diverse number of conformational diseases. Molecular chaperones of the Hsp40 family (Escherichia coli DnaJ homologs) recognize misfolded disease proteins and suppress the accumulation of toxic protein species. Type I Hsp40s are very potent at suppressing protein aggregation and facilitating the refolding of damaged proteins. Yet, the molecular mechanism for the recognition of nonnative polypeptides by Type I Hsp40s such as yeast Ydj1 is not clear. Here we computationally identify a unique motif that is selectively recognized by Ydj1p. The motif is characterized by the consensus sequence GX[LMQ]{P}X{P}{CIMPVW}, where [XY] denotes either X or Y and {XY} denotes neither X nor Y. We further verify the validity of the motif by site-directed mutagenesis and show that substrate binding by Ydj1 requires recognition of this motif. A yeast proteome screen revealed that many proteins contain more than one stretch of residues that contain the motif and are separated by varying numbers of amino acids. In light of our results, we propose a 2-site peptide-binding model and a plausible mechanism of peptide presentation by Ydj1p to the chaperones of the Hsp70 family. Based on our results, and given that Ydj1p and its human ortholog Hdj2 are functionally interchangeable, we hypothesize that our results can be extended to understanding human diseases.

[1]  E. Craig,et al.  Functional Specificity Among Hsp70 Molecular Chaperones , 1997, Science.

[2]  E. Wanker,et al.  Hsp70 and hsp40 chaperones can inhibit self-assembly of polyglutamine proteins into amyloid-like fibrils. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[3]  E. Fortunato,et al.  Farnesylation of Ydj1 is required for in vivo interaction with Hsp90 client proteins. , 2008, Molecular biology of the cell.

[4]  Jingzhi Li,et al.  Structure-based mutagenesis studies of the peptide substrate binding fragment of type I heat-shock protein 40. , 2005, The Biochemical journal.

[5]  F. Hartl,et al.  Molecular chaperones as modulators of polyglutamine protein aggregation and toxicity , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Bernd Bukau,et al.  The Hsp70 and Hsp60 Chaperone Machines , 1998, Cell.

[7]  A. Hinck,et al.  Structural basis of J cochaperone binding and regulation of Hsp70. , 2007, Molecular cell.

[8]  H. Fischer,et al.  Low Resolution Structural Study of Two Human HSP40 Chaperones in Solution , 2005, Journal of Biological Chemistry.

[9]  C. Fan,et al.  The Type I Hsp40 Zinc Finger-like Region Is Required for Hsp70 to Capture Non-native Polypeptides from Ydj1* , 2005, Journal of Biological Chemistry.

[10]  A. Caplan,et al.  Characterization of YDJ1: a yeast homologue of the bacterial dnaJ protein , 1991, The Journal of cell biology.

[11]  D. Masison,et al.  A role for cytosolic hsp70 in yeast [PSI(+)] prion propagation and [PSI(+)] as a cellular stress. , 2000, Genetics.

[12]  C. Link,et al.  Interaction of intracellular beta amyloid peptide with chaperone proteins. , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[13]  R. Melki,et al.  Molecular Chaperones and the Assembly of the Prion Ure2p in Vitro* , 2008, Journal of Biological Chemistry.

[14]  L. Itzhaki,et al.  Hsp40 Interacts Directly with the Native State of the Yeast Prion Protein Ure2 and Inhibits Formation of Amyloid-like Fibrils* , 2007, Journal of Biological Chemistry.

[15]  Y. Chernoff,et al.  Modulation of Prion-dependent Polyglutamine Aggregation and Toxicity by Chaperone Proteins in the Yeast Model* , 2005, Journal of Biological Chemistry.

[16]  Shuangye Yin,et al.  Eris: an automated estimator of protein stability , 2007, Nature Methods.

[17]  Kwang-Hwi Cho,et al.  BIOPHYSICS AND COMPUTATIONAL BIOLOGY , 2009 .

[18]  R. Sousa,et al.  Structural basis of interdomain communication in the Hsc70 chaperone. , 2005, Molecular cell.

[19]  L Rensing,et al.  Chaperones in cell cycle regulation and mitogenic signal transduction: a review , 2000, Cell proliferation.

[20]  Feng Ding,et al.  Modeling backbone flexibility improves protein stability estimation. , 2007, Structure.

[21]  D. Cyr,et al.  Regulation of Hsp70 function by a eukaryotic DnaJ homolog. , 1992, The Journal of biological chemistry.

[22]  Soojin Lee,et al.  Identification of Essential Residues in the Type II Hsp40 Sis1 That Function in Polypeptide Binding* , 2002, The Journal of Biological Chemistry.

[23]  Bernd Bukau,et al.  Multistep mechanism of substrate binding determines chaperone activity of Hsp70 , 2000, Nature Structural Biology.

[24]  C. Georgopoulos,et al.  Role of the major heat shock proteins as molecular chaperones. , 1993, Annual review of cell biology.

[25]  M. Cheetham,et al.  Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function. , 1998, Cell stress & chaperones.

[26]  Jingzhi Li,et al.  The crystal structure of the C-terminal fragment of yeast Hsp40 Ydj1 reveals novel dimerization motif for Hsp40. , 2005, Journal of molecular biology.

[27]  S. Lindquist,et al.  The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. , 1993, Annual review of genetics.

[28]  E. Craig,et al.  Heat shock proteins: molecular chaperones of protein biogenesis , 1993, Microbiological reviews.

[29]  R. Melki,et al.  Molecular chaperones and the assembly of the prion Sup35p, an in vitro study , 2006, The EMBO journal.

[30]  C. Link,et al.  Interaction of intracellular β amyloid peptide with chaperone proteins , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[31]  Soojin Lee,et al.  Exchangeable chaperone modules contribute to specification of type I and type II Hsp40 cellular function. , 2003, Molecular biology of the cell.

[32]  S. R. Terlecky,et al.  A role for a 70-kilodalton heat shock protein in lysosomal degradation of intracellular proteins. , 1989, Science.

[33]  F. Hartl,et al.  Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria , 1989, Nature.

[34]  W. Kabsch,et al.  Dictionary of protein secondary structure: Pattern recognition of hydrogen‐bonded and geometrical features , 1983, Biopolymers.

[35]  J. Reinstein,et al.  Mechanism of regulation of hsp70 chaperones by DnaJ cochaperones. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[36]  T. Langer,et al.  DnaJ-like proteins: molecular chaperones and specific regulators of Hsp70. , 1994, Trends in biochemical sciences.

[37]  Soojin Lee,et al.  Mechanisms for regulation of Hsp70 function by Hsp40 , 2003, Cell stress & chaperones.

[38]  Jingzhi Li,et al.  The crystal structure of the yeast Hsp40 Ydj1 complexed with its peptide substrate. , 2003, Structure.

[39]  Jason C. Young,et al.  More than folding: localized functions of cytosolic chaperones. , 2003, Trends in biochemical sciences.

[40]  Feng Ding,et al.  Emergence of Protein Fold Families through Rational Design , 2006, PLoS Comput. Biol..

[41]  Jingzhi Li,et al.  Peptide substrate identification for yeast Hsp40 Ydj1 by screening the phage display library , 2004, Biological Procedures Online.

[42]  J. Richmond,et al.  Variant-specific [PSI+] infection is transmitted by Sup35 polymers within [PSI+] aggregates with heterogeneous protein composition. , 2008, Molecular biology of the cell.

[43]  C. L. Oliveira,et al.  Conserved central domains control the quaternary structure of type I and type II Hsp40 molecular chaperones. , 2008, Journal of molecular biology.

[44]  F. Hartl,et al.  Molecular chaperone functions of heat-shock proteins. , 1993, Annual review of biochemistry.

[45]  G. Blobel,et al.  70K heat shock related proteins stimulate protein translocation into microsomes , 1988, Nature.

[46]  Peter M. Douglas,et al.  The Type I Hsp40 Ydj1 Utilizes a Farnesyl Moiety and Zinc Finger-like Region to Suppress Prion Toxicity* , 2009, Journal of Biological Chemistry.

[47]  F. Hartl,et al.  A zinc finger‐like domain of the molecular chaperone DnaJ is involved in binding to denatured protein substrates. , 1996, The EMBO journal.

[48]  H. Pelham.  Speculations on the functions of the major heat shock and glucose-regulated proteins , 1986, Cell.

[49]  Y. Chernoff,et al.  Huntingtin toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1 , 2002, The Journal of cell biology.

[50]  H. Kampinga,et al.  Hsp70 and Hsp40 Chaperone Activities in the Cytoplasm and the Nucleus of Mammalian Cells* , 1997, The Journal of Biological Chemistry.