Identification of key hinge residues important for nucleotide-dependent allostery in E. coli Hsp70/DnaK.
Identification of key hinge residues important for nucleotide-dependent allostery in E. coli Hsp70/DnaK.
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DOI：
10.1371/journal.pcbi.1003279
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发表时间：
2013
影响因子：
4.3
通讯作者：
Carlson HA
中科院分区：
文献类型：
--
作者：
Ung PM;Thompson AD;Chang L;Gestwicki JE;Carlson HA
DnaK is a molecular chaperone that has important roles in protein folding. The hydrolysis of ATP is essential to this activity, and the effects of nucleotides on the structure and function of DnaK have been extensively studied. However, the key residues that govern the conformational motions that define the apo, ATP-bound, and ADP-bound states are not entirely clear. Here, we used molecular dynamics simulations, mutagenesis, and enzymatic assays to explore the molecular basis of this process. Simulations of DnaK's nucleotide-binding domain (NBD) in the apo, ATP-bound, and ADP/Pi-bound states suggested that each state has a distinct conformation, consistent with available biochemical and structural information. The simulations further suggested that large shearing motions between subdomains I-A and II-A dominated the conversion between these conformations. We found that several evolutionally conserved residues, especially G228 and G229, appeared to function as a hinge for these motions, because they predominantly populated two distinct states depending on whether ATP or ADP/Pi was bound. Consistent with the importance of these “hinge” residues, alanine point mutations caused DnaK to have reduced chaperone activities in vitro and in vivo. Together, these results clarify how sub-domain motions communicate allostery in DnaK. DnaK belongs to the highly conserved heat shock protein 70 (Hsp70) family, a group of ATP-dependent molecular chaperones that regulates proteostasis. Studies have suggested that global movements of the subdomains in the nucleotide-binding domain (NBD) of DnaK regulate its catalytic activity. However, there is less known about the key residues involved in these subdomain motions and whether these residues might also regulate inter-domain allostery with the substrate-binding domain (SBD). To examine the motions in the NBD, dynamics simulations of DnaK's NBD in the apo, ATP-bound, and ADP/Pi-bound states were performed. Through essential dynamics and torsion angle analyses, we identified motions and highly conserved hinge residues between subdomains IIA and IIB that are likely to be important for nucleotide cycling and for communicating the nucleotide state to the SBD. Supporting this model, mutating these conserved hinge residues affected ATPase activity and chaperone functions in vitro and in bacteria, suggesting their importance in the nucleotide-dependent motions in DnaK.
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影响因子：
4
作者：
Chafekar, Sidhartha M.;Wisen, Susanne;Thompson, Andrea D.;Echeverria, AnaLisa;Walter, Gladis M.;Evans, Christopher G.;Makley, Leah N.;Gestwicki, Jason E.;Duennwald, Martin L.
通讯作者：
Duennwald, Martin L.
影响因子：
5.6
作者：
Bhattacharya, Akash;Kurochkin, Alexander V.;Yip, Grover N. B.;Zhang, Yongbo;Bertelsen, Eric B.;Zuiderweg, Erik R. P.
通讯作者：
Zuiderweg, Erik R. P.
影响因子：
64.5
作者：
Albanèse, V;Yam, AYW;Frydman, J
通讯作者：
Frydman, J
影响因子：
4.3
作者：
Chiappori, Federica;Merelli, Ivan;Morra, Giulia
通讯作者：
Morra, Giulia
DOI：
10.1073/pnas.0903503106
发表时间：
2009-05-26
影响因子：
11.1
作者：
Bertelsen, Eric B.;Chang, Lyra;Zuiderweg, Erik R. P.
通讯作者：
Zuiderweg, Erik R. P.