Conserved central domains control the quaternary structure of type I and type II Hsp40 molecular chaperones.

Heat shock protein (Hsp)40s play an essential role in protein metabolism by regulating the polypeptide binding and release cycle of Hsp70. The Hsp40 family is large, and specialized family members direct Hsp70 to perform highly specific tasks. Type I and Type II Hsp40s, such as yeast Ydj1 and Sis1, are homodimers that dictate functions of cytosolic Hsp70, but how they do so is unclear. Type I Hsp40s contain a conserved, centrally located cysteine-rich domain that is replaced by a glycine- and methionine-rich region in Type II Hsp40s, but the mechanism by which these unique domains influence Hsp40 structure and function is unknown. This is the case because high-resolution structures of full-length forms of these Hsp40s have not been solved. To fill this void, we built low-resolution models of the quaternary structure of Ydj1 and Sis1 with information obtained from biophysical measurements of protein shape, small-angle X-ray scattering, and ab initio protein modeling. Low-resolution models were also calculated for the chimeric Hsp40s YSY and SYS, in which the central domains of Ydj1 and Sis1 were exchanged. Similar to their human homologs, Ydj1 and Sis1 each has a unique shape with major structural differences apparently being the orientation of the J domains relative to the long axis of the dimers. Central domain swapping in YSY and SYS correlates with the switched ability of YSY and SYS to perform unique functions of Sis1 and Ydj1, respectively. Models for the mechanism by which the conserved cysteine-rich domain and glycine- and methionine-rich region confer structural and functional specificity to Type I and Type II Hsp40s are discussed.

[1]  C. Ramos.  A spectroscopic‐based laboratory experiment for protein conformational studies * , 2004, Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology.

[2]  E. Craig,et al.  Role of Pam16's degenerate J domain in protein import across the mitochondrial inner membrane. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[3]  R. Wickner,et al.  [URE3] Prion Propagation in Saccharomyces cerevisiae: Requirement for Chaperone Hsp104 and Curing by Overexpressed Chaperone Ydj1p , 2000, Molecular and Cellular Biology.

[4]  D. Cyr.  Cooperation of the molecular chaperone Ydj1 with specific Hsp70 homologs to suppress protein aggregation. , 1995, FEBS letters.

[5]  S. Wickner,et al.  In Vivo Modulation of a DnaJ Homolog, CbpA, by CbpM , 2007, Journal of bacteriology.

[6]  Dmitri I Svergun,et al.  Global rigid body modeling of macromolecular complexes against small-angle scattering data. , 2005, Biophysical journal.

[7]  Dmitri I. Svergun,et al.  Determination of the regularization parameter in indirect-transform methods using perceptual criteria , 1992 .

[8]  E. Garí,et al.  Cyclin Cln3 is retained at the ER and released by the J chaperone Ydj1 in late G1 to trigger cell cycle entry. , 2007, Molecular cell.

[9]  C. Georgopoulos,et al.  Genetic analysis of two genes, dnaJ and dnaK, necessary for Escherichia coli and bacteriophage lambda DNA replication , 1978, Molecular and General Genetics MGG.

[10]  D I Svergun,et al.  Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. , 1999, Biophysical journal.

[11]  F. Hartl,et al.  Regulation of the Heat-shock Protein 70 Reaction Cycle by the Mammalian DnaJ Homolog, Hsp40* , 1996, The Journal of Biological Chemistry.

[12]  Peter Schuck,et al.  Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems. , 2002, Biophysical journal.

[13]  O. Glatter,et al.  19 – Small-Angle X-ray Scattering , 1973 .

[14]  H. Edelhoch,et al.  Spectroscopic determination of tryptophan and tyrosine in proteins. , 1967, Biochemistry.

[15]  William J. Welch,et al.  ATP-induced protein Hsp70 complex dissociation requires K+ but not ATP hydrolysis , 1993, Nature.

[16]  E. Craig,et al.  In Vivo Bipartite Interaction Between the Hsp40 Sis1 and Hsp70 in Saccharomyces cerevisiae , 2005, Genetics.

[17]  F. Hartl,et al.  Molecular Chaperones in the Cytosol: from Nascent Chain to Folded Protein , 2002, Science.

[18]  S. Lindquist,et al.  Rnq1: an epigenetic modifier of protein function in yeast. , 2000, Molecular cell.

[19]  G. Weber,et al.  Pressure dissociation and conformational drift of the beta dimer of tryptophan synthase. , 1986, Biochemistry.

[20]  P E Wright,et al.  Solution structure of the cysteine-rich domain of the Escherichia coli chaperone protein DnaJ. , 2000, Journal of molecular biology.

[21]  K. Arndt,et al.  The yeast SIS1 protein, a DnaJ homolog, is required for the initiation of translation , 1993, Cell.

[22]  K. Arndt,et al.  Characterization of SIS1, a Saccharomyces cerevisiae homologue of bacterial dnaJ proteins , 1991, The Journal of cell biology.

[23]  E. Craig,et al.  Specificity of class II Hsp40 Sis1 in maintenance of yeast prion [RNQ+]. , 2003, Molecular biology of the cell.

[24]  P. Schuck,et al.  Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. , 2000, Biophysical journal.

[25]  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.

[26]  T. Lithgow,et al.  The J‐protein family: modulating protein assembly, disassembly and translocation , 2004, EMBO reports.

[27]  C. L. Oliveira,et al.  Low resolution structure and stability studies of human GrpE#2, a mitochondrial nucleotide exchange factor. , 2006, Archives of biochemistry and biophysics.

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

[29]  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.

[30]  H. Orland,et al.  Partially folded states of proteins: characterization by X-ray scattering. , 1995, Journal of molecular biology.

[31]  D. Cyr,et al.  Protein Folding Activity of Hsp70 Is Modified Differentially by the Hsp40 Co-chaperones Sis1 and Ydj1* , 1998, The Journal of Biological Chemistry.

[32]  F. Hartl,et al.  The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system DnaK, DnaJ, and GrpE. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Dmitri I. Svergun,et al.  Uniqueness of ab initio shape determination in small-angle scattering , 2003 .

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

[35]  C. Georgopoulos,et al.  Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[36]  D. Cyr,et al.  The Hdj‐2/Hsc70 chaperone pair facilitates early steps in CFTR biogenesis , 1999, The EMBO journal.

[37]  P. Casey,et al.  Farnesylation of YDJ1p is required for function at elevated growth temperatures in Saccharomyces cerevisiae. , 1992, The Journal of biological chemistry.

[38]  C. Ramos,et al.  Protein folding assisted by chaperones. , 2005, Protein and peptide letters.

[39]  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.

[40]  Peter V. Konarev,et al.  MASSHA – a graphics system for rigid-body modelling of macromolecular complexes against solution scattering data , 2001 .

[41]  B. Sha,et al.  Direct interactions between molecular chaperones heat-shock protein (Hsp) 70 and Hsp40: yeast Hsp70 Ssa1 binds the extreme C-terminal region of yeast Hsp40 Sis1. , 2002, The Biochemical journal.

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

[43]  B. Bukau,et al.  The Human DnaJ Homologue dj2 Facilitates Mitochondrial Protein Import and Luciferase Refolding , 1997, The Journal of cell biology.

[44]  D. Cyr,et al.  Eukaryotic homologues of Escherichia coli dnaJ: a diverse protein family that functions with hsp70 stress proteins. , 1993, Molecular biology of the cell.

[45]  Walter Neupert,et al.  Why Do We Still Have a Maternally Inherited Mitochondrial DNA ? Insights from Evolutionary Medicine , 2007 .

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

[47]  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.

[48]  F. Hartl,et al.  Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding , 1992, Nature.

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

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

[51]  R. Wickner,et al.  [URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae. , 1994, Science.

[52]  T. Laue.  Biophysical studies by ultracentrifugation. , 2001, Current opinion in structural biology.

[53]  Dmitri I. Svergun,et al.  Automated matching of high- and low-resolution structural models , 2001 .

[54]  D. Cyr,et al.  The crystal structure of the peptide-binding fragment from the yeast Hsp40 protein Sis1. , 2000, Structure.

[55]  D. Cyr,et al.  Regulation of Hsp70 function by a eukaryotic DnaJ homolog. , 1992, Journal of Biological Chemistry.

[56]  Douglas M. Cry,et al.  Cooperation of the molecular chaperone Ydj1 with specific Hsp70 homologs to suppress protein aggregation , 1995 .

[57]  W. Miller,et al.  A time-efficient, linear-space local similarity algorithm , 1991 .