Two distinct classes of cochaperones compete for the EEVD motif in heat shock protein 70 to tune its chaperone activities

Abstract

Chaperones of the heat shock protein 70 (Hsp70) family engage in protein–protein interactions with many cochaperones. One “hotspot” for cochaperone binding is the EEVD motif, found at the extreme C terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat domain cochaperones, such as the E3 ubiquitin ligase CHIP. In addition, the EEVD motif also interacts with a structurally distinct domain that is present in class B J-domain proteins, such as DnaJB4. These observations suggest that CHIP and DnaJB4 might compete for binding to Hsp70’s EEVD motif; however, the molecular determinants of such competition are not clear. Using a collection of EEVD-derived peptides, including mutations and truncations, we explored which residues are critical for binding to both CHIP and DnaJB4. These results revealed that some features, such as the C-terminal carboxylate, are important for both interactions. However, CHIP and DnaJB4 also had unique preferences, especially at the isoleucine position immediately adjacent to the EEVD. Finally, we show that competition between these cochaperones is important in vitro, as DnaJB4 limits the ubiquitination activity of the Hsp70–CHIP complex, whereas CHIP suppresses the client refolding activity of the Hsp70–DnaJB4 complex. Together, these data suggest that the EEVD motif has evolved to support diverse protein–protein interactions, such that competition between cochaperones may help guide whether Hsp70-bound proteins are folded or degraded. Chaperones of the heat shock protein 70 (Hsp70) family engage in protein–protein interactions with many cochaperones. One “hotspot” for cochaperone binding is the EEVD motif, found at the extreme C terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat domain cochaperones, such as the E3 ubiquitin ligase CHIP. In addition, the EEVD motif also interacts with a structurally distinct domain that is present in class B J-domain proteins, such as DnaJB4. These observations suggest that CHIP and DnaJB4 might compete for binding to Hsp70’s EEVD motif; however, the molecular determinants of such competition are not clear. Using a collection of EEVD-derived peptides, including mutations and truncations, we explored which residues are critical for binding to both CHIP and DnaJB4. These results revealed that some features, such as the C-terminal carboxylate, are important for both interactions. However, CHIP and DnaJB4 also had unique preferences, especially at the isoleucine position immediately adjacent to the EEVD. Finally, we show that competition between these cochaperones is important in vitro, as DnaJB4 limits the ubiquitination activity of the Hsp70–CHIP complex, whereas CHIP suppresses the client refolding activity of the Hsp70–DnaJB4 complex. Together, these data suggest that the EEVD motif has evolved to support diverse protein–protein interactions, such that competition between cochaperones may help guide whether Hsp70-bound proteins are folded or degraded. Members of the heat shock protein 70 (Hsp70) family of molecular chaperones play a critical role in maintaining protein homeostasis (aka proteostasis). These chaperones bind to misfolded or unfolded proteins and direct them to diverse processes such as protein folding, translocation, complex formation, and degradation (1Mayer M.P. Bukau B. Hsp70 chaperones: Cellular functions and molecular mechanism.Cell. Mol. Life Sci. 2005; 62: 670-684Google Scholar, 2Kim Y.E. Hipp M.S. Bracher A. Hayer-Hartl M. Hartl F.U. Molecular chaperone functions in protein folding and proteostasis.Annu. Rev. Biochem. 2013; 82: 323-355Google Scholar). Remarkably, this diversity of functions is enabled by a relatively simple structure: Hsp70s are composed of a nucleotide-binding domain (NBD), a substrate-binding domain (SBD), and an ⍺-helical lid domain (Fig. 1A) (3Mayer M.P. Gierasch L.M. Recent advances in the structural and mechanistic aspects of Hsp70 molecular chaperones.J. Biol. Chem. 2019; 294: 2085-2097Google Scholar, 4Zuiderweg E.R. Hightower L.E. Gestwicki J.E. The remarkable multivalency of the Hsp70 chaperones.Cell Stress Chaperones. 2017; 22: 173-189Google Scholar). In eukaryotes, many cytosolic Hsp70s also bear a C-terminal unstructured region terminating in a conserved EEVD motif. These various domains are in allosteric communication; for example, ATPase activity in the NBD causes a conformational change that regulates binding of “client” proteins in the SBD (5Slepenkov S.V. Witt S.N. Kinetics of the reactions of the Escherichia coli molecular chaperone DnaK with ATP: Evidence that a three-step reaction precedes ATP hydrolysis.Biochemistry. 1998; 37: 1015-1024Google Scholar, 6Swain J.F. Dinler G. Sivendran R. Montgomery D.L. Stotz M. Gierasch L.M. Hsp70 chaperone ligands control domain association via an allosteric mechanism mediated by the interdomain linker.Mol. Cell. 2007; 26: 27-39Google Scholar). Hsp70s rarely work alone. Rather, the diversity of Hsp70’s functions is imparted by cochaperones, such as J-domain proteins (JDPs) (7Rosenzweig R. Nillegoda N.B. Mayer M.P. Bukau B. The Hsp70 chaperone network.Nat. Rev. Mol. Biol. 2019; Scholar, The chaperone proteins as of Rev. Mol. Biol. A. The of Hsp70 molecular Mol. and tetratricopeptide repeat domain proteins A. G. Hartl F.U. of in the of the Scholar). of these cochaperones, such as and bind Hsp70s and of to client binding R. Mayer M.P. Molecular mechanism of ATP by Hsp70 Cell. Scholar, of the J-domain in the of with Sci. A. 1998; Scholar, a of Hsp70 chaperone 1998; Scholar, Gestwicki J.E. of to heat shock protein 70 (Hsp70) distinct in Biol. Chem. Scholar). In addition, cochaperones also as Hsp70 and to example, some and proteins Hsp70’s to protein degradation the chaperone activity of Scholar, M. R. to Chem. Scholar, of a tetratricopeptide protein that interacts with heat shock proteins and regulates chaperone Cell. Biol. Scholar). between Hsp70 and cochaperones, mediated by a of direct protein–protein interactions is critical for the diversity of the chaperone R. Gestwicki J.E. interactions in the molecular chaperone Chem. Scholar, Hsp70 to many Biochem. Sci. 2017; Scholar). of this is that are Hsp70 for cochaperones to such that the cochaperones compete for to distinct is important to Hsp70 cochaperones. of cochaperone binding is the EEVD motif that is present at the extreme C terminus of cytoplasmic Hsp70s M.P. R. of a motif in Hsp70 that ATPase binding and with Scholar, domains of protein and Stress Chaperones. Scholar). cytoplasmic also a conserved C-terminal EEVD motif B. A. in Sci. Scholar). However, the Hsp70 and at to the EEVD (Fig. we the in which the C-terminal is whereas the is the In this the cytoplasmic Hsp70s an isoleucine at whereas the cytoplasmic a The binding of and to cochaperones and interactions of the and domains of the C terminus of Scholar, M. M. M. of the CHIP E3 ubiquitin ligase and a Cell. 2005; Scholar, A. M. Molecular of the of with Sci. Scholar, binding by Sci. Scholar). structural and a in the domain interactions to both the of the and the terminus A. R. B. In and of the protein Scholar, In of tetratricopeptide repeat proteins in and as of Scholar). of these has that some cochaperones for Hsp70’s or Gestwicki J.E. binding of tetratricopeptide repeat proteins to heat shock protein 70 (Hsp70) and heat shock protein is by and Scholar, E.R. Gestwicki J.E. The molecular chaperone Hsp70 protein by binding the tetratricopeptide repeat Biol. Chem. Scholar, R. repeat Molecular of the critical for binding and Biol. Chem. Scholar, between folding and degradation for client role for Scholar, A. B. G. Hartl F.U. by and of in Hsp70 Biol. Chem. Scholar, D.L. domains of the are important for Hsp70 Biol. Chem. Scholar). mutations in Hsp70’s motif to the for binding to proteins, such as protein and C terminus of protein A. B. G. Hartl F.U. by and of in Hsp70 Biol. Chem. Scholar, M. M. Gestwicki J.E. for C the E3 ubiquitin ligase CHIP to Chem. Biol. 2019; Scholar). has that some class B also bind to Hsp70’s EEVD motif M. as revealed by the of the domain in complex with the Scholar, for client and activity of 2019; Scholar, M. M. Nillegoda N.B. Bukau B. R. proteins to Scholar). are in and and are for conserved J-domain of the of of the domain the various of Stress Chaperones. Scholar, and of and of chaperone Stress Chaperones. 1998; Scholar, M. Escherichia coli and heat shock proteins ATPase activity of Sci. A. Scholar, M. The of the Escherichia coli protein the ATPase activity of DnaK and are for Biol. Chem. which Hsp70s the interdomain between the NBD and SBD E.R. A. Mayer M.P. Gestwicki J.E. A. in the Hsp70 chaperone Chem. 2013; Scholar). This an the and is for the of Hsp70’s ATPase activity (7Rosenzweig R. Nillegoda N.B. Mayer M.P. Bukau B. The Hsp70 chaperone network.Nat. Rev. Mol. Biol. 2019; Scholar, The chaperone proteins as of Rev. Mol. Biol. Scholar, R. Mayer M.P. Molecular mechanism of ATP by Hsp70 Cell. Scholar, of the molecular with cochaperone Sci. A. 1998; Scholar). In to the class B are by a domains C-terminal domains and and a domain (Fig. 1A) The chaperone proteins as of Rev. Mol. Biol. Scholar). and with Hsp70 for client and activity of 2019; Scholar, B. The of the with Scholar, of residues in the that in Biol. Chem. Scholar, B. of the binding of protein 2005; to and them to the is also the of between class B and the EEVD motif B. The of the with Scholar, of a motif in by a Sci. A. Scholar). This in the as as the (Fig. M. as revealed by the of the domain in complex with the Scholar, for client and activity of 2019; Scholar, M. M. Nillegoda N.B. Bukau B. R. proteins to Scholar, B. of and Hsp70 C-terminal Scholar, The domain of binding for a distinct of chaperone Cell. Biol. 1998; Scholar). binding of to the EEVD motif is for client and activity of 2019; Scholar, B. interactions between molecular chaperones protein 70 and Hsp70 the extreme C-terminal region of is the EEVD is by mutations in or of the of the Hsp70 to is M.P. R. of a motif in Hsp70 that ATPase binding and with Scholar, for client and activity of 2019; Scholar, M. M. Nillegoda N.B. Bukau B. R. proteins to Scholar, M. M. A. of and interactions in of the Mol. Biol. Scholar, of class and class B with Scholar). the EEVD has also to of the by the such that binding to the EEVD ATPase client and of by Hsp70 M. M. Nillegoda N.B. Bukau B. R. proteins to Scholar, Nillegoda N.B. G. A. Bukau B. Molecular of by Scholar, R. A. Bukau B. of by the Hsp70 Biol. Chem. Scholar). with the binding of domains to the EEVD motif, is known the molecular determinants of the residues that to of Hsp70s by class B M. as revealed by the of the domain in complex with the Scholar, for client and activity of 2019; Scholar, M. M. Nillegoda N.B. Bukau B. R. proteins to Scholar, B. of and Hsp70 C-terminal Scholar, M. M. A. of and interactions in of the Mol. Biol. Scholar, of class and class B with a for the has not we the determinants of the the class B DnaJB4. Using and we found that DnaJB4 to the Hsp70 motif not Using and we also found that DnaJB4 the terminus and has for the this we an in Hsp70’s EEVD and to the of the that this with DnaJB4 is critical for both ATPase and client refolding also that DnaJB4 with the of the Hsp70–CHIP complex in ubiquitination and CHIP the chaperone functions of the Hsp70–DnaJB4 complex. Together, these suggest that competition between distinct of cochaperones the of Hsp70 the molecular determinants of the complex, we the of an EEVD to M. as revealed by the of the domain in complex with the with M. A. A. A. A. A. R. A. protein for the to a of the complex. the between the domains (Fig. we that this to which residues in the EEVD motif might important for we that a of residues the DnaJB4 and the EEVD terminus DnaJB4 also the of the isoleucine or DnaJB4 In in the such as and and not as to direct interactions. the we to residues in the EEVD motif that important with of DnaJB4. to this we a the residues of and interactions with by In this DnaJB4 with a of (Fig. that this is that for the by M. M. Nillegoda N.B. Bukau B. R. proteins to Scholar). However, is that the and in the are for the of the J-domain to this we the B. L.M. of structural and that the of protein with in the Biol. Chem. to DnaJB4 and binding to the This had as both and the DnaJB4 with (Fig. the of the we binding to a of the class family of and found that had (Fig. the also to bind which is an class B cochaperone that (Fig. cytosolic Hsp70s and in EEVD whether DnaJB4 also bind motif, we to the C of the cytoplasmic chaperones the Hsp70 and and (Fig. binding to DnaJB4 In the of DnaJB4 an of (Fig. with or a of to in the that bind to DnaJB4. to DnaJB4 this we the as in the the as the In this and to DnaJB4 whereas and not (Fig. not of the by the at of that the to the whether the to the between binding and peptides, we the in the for and binding to DnaJB4 by this to binding to DnaJB4 (Fig. these data show a direct between DnaJB4 and chaperone C that is for the Hsp70 that DnaJB4 interacts with the Hsp70 we to the molecular determinants of this The C-terminal is for EEVD binding to domain cochaperones CHIP M. M. Gestwicki J.E. for C the E3 ubiquitin ligase CHIP to Chem. Biol. 2019; we whether the might also for binding to DnaJB4. In competition of the binding (Fig. that is a critical we to of the the or binding to DnaJB4 (Fig. of the however, and of the These are with structural the of Hsp70’s C terminus are to to the and the C-terminal an important that the residues are for the with we an of the to the of The found at an binding (Fig. this that the to of DnaJB4 (Fig. This in the the isoleucine is by and in DnaJB4 The that to the at and with a the Together, these suggest that the and the are important for binding to DnaJB4 and that especially and to binding is known that domain cochaperones the and the terminus in the EEVD motif E.R. Hightower L.E. Gestwicki J.E. The remarkable multivalency of the Hsp70 chaperones.Cell Stress Chaperones. 2017; 22: 173-189Google Scholar, M. M. M. of the CHIP E3 ubiquitin ligase and a Cell. 2005; Scholar, R. Molecular mechanism of the of protein by terminus of protein Biol. Chem. Scholar). we found that the are also critical for binding DnaJB4 we that cochaperones, such as compete for binding in However, not whether DnaJB4 and CHIP the at an in this we to repeat the binding with CHIP. This an important the M. M. Gestwicki J.E. for C the E3 ubiquitin ligase CHIP to Chem. Biol. 2019; and we to direct with in the DnaJB4 Using the with an C terminus we that the important for binding to CHIP in (Fig. we the to show that the and are important for binding CHIP (Fig. these results with the In these we that the and which are conserved in the EEVD are for binding to CHIP. In these are in binding to DnaJB4 and the is to with in of these residues might by the to the at for binding to both CHIP and we at this to CHIP the isoleucine in residues and to for this whereas and residues (Fig. to and to for and residues at the residues and for the isoleucine is to a by residues and of DnaJB4 we that or may and by interactions with in this however, is to this these suggest that DnaJB4 and CHIP molecular to bind the EEVD motif. work by the that the of Hsp70’s EEVD with class B is important for chaperone M. M. A. of and interactions in of the Mol. Biol. Scholar, of class and class B with and structural revealed that this is mediated by an allosteric of that M. M. Nillegoda N.B. Bukau B. R. proteins to Scholar, Nillegoda N.B. G. A. Bukau B. Molecular of by Scholar). we to of to these in this we a of in which the EEVD motif In addition, we a of the critical isoleucine which not binding to DnaJB4 These in ATPase and refolding we the ATPase of the to a M.P. R. of a motif in Hsp70 that ATPase binding and with Scholar, M. M. A. of and interactions in of the Mol. Biol. had ATPase activity with the However, had activity this for the of DnaJB4 binding the we the of DnaJB4 to ATPase activity by the DnaJB4 the ATPase activity of by at a of DnaJB4 to the (Fig. that class B M. M. A. of and interactions in of the Mol. Biol. Scholar). an of with a of and that the of the is important for ATP the of the in refolding (Fig. in which both and in the to with DnaJB4. the EEVD is to client refolding by such that the that this activity example, DnaJB4 may the motif with the motif, to of with with that the not the to with in client we refolding by in the of which not engage with the EEVD motif M. M. Nillegoda N.B. Bukau B. R. proteins to with with (Fig. that the EEVD is for with such as DnaJB4. The complex of Hsp70s with CHIP is known to the ubiquitination and degradation of client proteins M. M. M. of the CHIP E3 ubiquitin ligase and a Cell. 2005; Scholar, The CHIP regulates protein mediated by Biol. Scholar). the complex of Hsp70s and DnaJB4 is with functions The chaperone proteins as of Rev. Mol. Biol. Scholar). we that competition between them might these distinct this we ubiquitination CHIP to Hsp70 or in with R. M. between Hsp70 and CHIP regulates ubiquitination of client Scholar, R. proteins as of the ubiquitin ligase CHIP terminus of Biol. Chem. 2013; both Hsp70 and by the of (Fig. DnaJB4 to these in of Hsp70 DnaJB4 had an important whether this competition might also the ubiquitination of Hsp70-bound we ubiquitination reactions the Hsp70 client A. M. G. A. R. and Hsp70 degradation and Mol. (Fig. we ubiquitination of and by CHIP that by the of that competition for the EEVD motif by DnaJB4 the activity of CHIP. we explored whether CHIP might to ATPase we found that of CHIP to DnaJB4 of ATPase activity (Fig. This with in which CHIP to the activity of in M. R. Mayer M.P. CHIP in protein by Hsp70-bound Scholar). of CHIP may to DnaJB4 between Hsp70 and mediated by both the and the of CHIP to client refolding by the complex. of CHIP folding reactions that is a (Fig. The of CHIP to client with ATPase is by including as a that bind to unfolded as as for in the as an of the ubiquitin ligase Biol. Cell. 2005; Scholar, functions of the E3 ubiquitin ligase Biol. Chem. 2007; Scholar). binding to chaperone C is a of domain cochaperones A. B. G. Hartl F.U. by and of in Hsp70 Biol. Chem. we that proteins may compete with and the of DnaJB4 to we found that cochaperone interactions in protein Biol. Chem. 1998; refolding in the of DnaJB4 (Fig. This with that refolding by M. M. The the folding Scholar). a cochaperone that also a refolding by by to ATP A. Hartl F.U. domains and a domain to the chaperone 22: Scholar). Together, these that proteins and DnaJB4 compete for the EEVD motif to of Hsp70 and chaperone that the of the is Molecular chaperones are to including and of Hsp70 family the chaperone Biol. Chem. Scholar, M. of and the chaperone Biol. Chem. Scholar). some to in the binding to cochaperones. of the the Hsp70 EEVD motif is known to binding of CHIP Gestwicki J.E. binding of tetratricopeptide repeat proteins to heat shock protein 70 (Hsp70) and heat shock protein is by and Scholar, R. B. C-terminal of Hsp70 and regulates binding to CHIP and to protein 2013; Scholar). this in as the of to the in a of the of an for CHIP (Fig. In the had binding to DnaJB4 (Fig. that might a CHIP not DnaJB4. This is also by of the binding for the EEVD motif to CHIP or DnaJB4 (Fig. M. M. Gestwicki J.E. for C the E3 ubiquitin ligase CHIP to Chem. Biol. 2019; and M. as revealed by the of the domain in complex with the to the EEVD motif is an binding by Sci. Scholar). In this binding the is in a with the and of this is to M. M. Gestwicki J.E. for C the E3 ubiquitin ligase CHIP to Chem. Biol. 2019; Scholar). to the EEVD motif a with the relatively to M. as revealed by the of the domain in complex with the Scholar). of this is to binding to with the Together, these results suggest that especially of the of to binding at this the of the EEVD motif to these domains the that molecular by CHIP and DnaJB4 molecular of Hsp70 with cochaperones a diverse of functions to this molecular a in the is to and a complex between Hsp70 and cochaperones This is a are A. The of Hsp70 molecular Mol. The chaperone proteins as of Rev. Mol. Biol. and cochaperone A. R. B. In and of the protein and these are with the cytosolic Hsp70s A. The diverse of the show distinct an of unique are the of is this of in and and the that many Hsp70 M. A. B. of a Scholar, A. M.P. J.F. M. A. M. B. is an that Scholar, M. G. B. B. chaperone the of protein homeostasis Scholar). is important to which cochaperones might compete and which molecular determinants are to of these we the domain proteins and class B the Hsp70 EEVD motif and This of especially important to these cochaperones functions of with the client to a and CHIP client M. M. A. of and interactions in of the Mol. Biol. Scholar, M. R. Mayer M.P. CHIP in protein by Hsp70-bound Scholar). competition for binding the EEVD to the by the Hsp70 we of Hsp70 functions and that distinct of cochaperones the of via competition for the EEVD motif. the of Hsp70 to bind CHIP is to that for the EEVD motif and of a to which bind at this the we found that CHIP has a for the EEVD motif DnaJB4 and CHIP is is and has of a tetratricopeptide protein that interacts with heat shock proteins and regulates chaperone Cell. Biol. Scholar, B. the activity of of Scholar). These observations suggest that CHIP is for binding to the EEVD motif, client client however, is of of and family heat and in Stress Chaperones. 26: Scholar, protein a mechanism 2019; Scholar). In addition, we a of a is to the EEVD motif, whereas DnaJB4 via to DnaJB4 binding CHIP. This that CHIP not to client as might B. of the cochaperone CHIP folding activity in Cell. Biol. Scholar). Finally, we also binding of DnaJB4 to the the (Fig. B and and a of ubiquitination by DnaJB4 (Fig. the of and might also which are and the chaperones are Finally, the of the EEVD interactions with proteins and are not clear. Hsp70 functions of binding A. Gestwicki J.E. The interactions of molecular chaperones with client are Biol. Chem. the and cochaperone are to important both which are and allosteric are This may ATPase of the is in binding to the may to a of allosteric to the Together, these suggest might and to cochaperone and which are in Hsp70 functions are In addition, this also that are and cochaperones in CHIP and which of competition for the EEVD client proteins are also to these example, to bind and of for client and activity of 2019; Scholar, B. The of the with Scholar, of a motif in by a Sci. A. such that to compete with Hsp70’s EEVD motif. the of unfolded by may EEVD binding to class B the of Hsp70–CHIP the interacts with a of that are by M. M. Gestwicki J.E. for C the E3 ubiquitin ligase CHIP to Chem. Biol. 2019; Scholar). activity that in an and some of these the EEVD motif. EEVD binding to CHIP a C-terminal (Fig. however, we found that EEVD binding to DnaJB4 not this (Fig. that CHIP is for by may not These for in the of in that are for the role of these in functions J.E. and for molecular chaperone Biol. Chem. 2019; 294: Scholar). of this the to between we in the that the EEVD motif to CHIP and DnaJB4 with distinct structural the of DnaJB4 for the and the and suggest that might EEVD binding to this cochaperone the the for a and in binding to not for a for These is that the of cochaperones in the EEVD motif. DnaJB4 and both in Escherichia coli a with and of at the to with and at in binding with by and by and the to a The with binding by and with The protein with a to with and to the and to at and to The protein binding and to to The protein by an in and and in coli a with and of at an of to and with at in binding with by and by and the to The with binding and protein with with and to the and reaction at The protein to a with and the with and B with with CHIP in coli a with and and in to at to with and by in binding with and The by and the to The with binding and and the in the by with at The to to and The protein by in CHIP in and at R. A. M. interactions with the chaperone that Mol. Biol. R. A. M. interactions with the chaperone that Mol. Biol. Gestwicki J.E. binding of tetratricopeptide repeat proteins to heat shock protein 70 (Hsp70) and heat shock protein is by and Gestwicki J.E. binding of tetratricopeptide repeat proteins to heat shock protein 70 (Hsp70) and heat shock protein is by and and Gestwicki J.E. binding of tetratricopeptide repeat proteins to heat shock protein 70 (Hsp70) and heat shock protein is by and as by with a to the terminus via a to and an at the C in to and at in at a of in at an of and an of with a in data binding data and the competition data to and to control to of or with various of DnaJB4 or or CHIP in in binding binding CHIP binding The and to at for to for the to compete with the with DnaJB4 and in binding The and to at for to of CHIP and with in CHIP The and to at for to with a in a (3Mayer M.P. Gierasch L.M. Recent advances in the structural and mechanistic aspects of Hsp70 molecular chaperones.J. Biol. Chem. 2019; 294: 2085-2097Google 70 in reactions to and to The and of in binding data between and 70 to and to a in DnaJB4 the ATPase the as Gestwicki J.E. of to heat shock protein 70 (Hsp70) distinct in Biol. Chem. Scholar, M. A. E.R. Gestwicki J.E. the complex in of Chem. Biol. Scholar). In and various of DnaJB4 to and the reactions by of to for at which and with at and a of to the to and as to a the ATP by DnaJB4 binding and the of DnaJB4 binding to and the ATPase activity of as Gestwicki J.E. of to heat shock protein 70 (Hsp70) distinct in Biol. Chem. Scholar). in for at and a in an ATP and of DnaJB4 and the reaction with the of competition with of and DnaJB4 with a of CHIP or of In the to for at in the and a of to and CHIP DnaJB4 of and ATP ATP and in ubiquitination CHIP DnaJB4 to at for to the reactions by of in to for a of CHIP and at for chaperone for in and to for by to and by a the or by with of ubiquitination by in which to a for in ubiquitination with to of ubiquitination as M. M. Gestwicki J.E. for C the E3 ubiquitin ligase CHIP to Chem. Biol. 2019; Scholar). proteins and by of of for at The reaction by of and by and a by to and to a for at in in in at for in in in for at for in and an at and and as data are in the data are the or The that of with the of this as a the of A. and G. M. and G. M. and and A. M. and M. and G. M. and G. M. and A. G. G. The support the G. and The is the of the and not the of the of

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