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. 2007 Nov 9;28(3):422-33.
doi: 10.1016/j.molcel.2007.08.022.

Structural basis of J cochaperone binding and regulation of Hsp70

Jianwen Jiang  1 , E Guy Maes, Alexander B Taylor, Liping Wang, Andrew P Hinck, Eileen M Lafer, Rui Sousa
Affiliations

Structural basis of J cochaperone binding and regulation of Hsp70

Jianwen Jiang et al. Mol Cell. .

Abstract

The many protein processing reactions of the ATP-hydrolyzing Hsp70s are regulated by J cochaperones, which contain J domains that stimulate Hsp70 ATPase activity and accessory domains that present protein substrates to Hsp70s. We report the structure of a J domain complexed with a J responsive portion of a mammalian Hsp70. The J domain activates ATPase activity by directing the linker that connects the Hsp70 nucleotide binding domain (NBD) and substrate binding domain (SBD) toward a hydrophobic patch on the NBD surface. Binding of the J domain to Hsp70 displaces the SBD from the NBD, which may allow the SBD flexibility to capture diverse substrates. Unlike prokaryotic Hsp70, the SBD and NBD of the mammalian chaperone interact in the ADP state. Thus, although both nucleotides and J cochaperones modulate Hsp70 NBD:linker and NBD:SBD interactions, the intrinsic persistence of those interactions differs in different Hsp70s and this may optimize their activities for different cellular roles.

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Figures

Figure 1
Figure 1. Stimulation of Hsc70 ATPase by free and disulfide-linked J domains
A: Single-turnover ATPase rates for 1 μM Hsc70, Hsc70ΔC (aa 1-554), NBD (aa 1-386) or NBD_Linker (aa 1-394) alone or with 10 μM added J domain (“+ J”). Error bars are ±s.e. for n=3–6. B: As in A, but with R171C substituted NBD or NBD_Linker with D876C J domain either added in solution (at 10 or 500 μM, as indicated) or cross-linked to the Hsc70 (“X J”).
Figure 2
Figure 2. The auxilin J domain:NBD_Linker complex
A: Auxilin J Domain:NBD_Linker complex with J domain (cyan) in ribbon representation and NBD_Linker rendered as a transparent surface (green; with aa 383–390 in magenta) with the path of the polypeptide chain shown as a coil and the bound nucleotide in stick representation. B: Model from A rotated as indicated. In yellow on the J domain are regions corresponding to those mapped by NMR (in the polyoma virus T-antigen) to be involved in interaction with Hsc70 (Garimella et al., 2006). C: The region indicated by the box in B expanded to identify residues important for the J domain:Hsc70 interaction. These are labeled with white lettering on the surface of the Hsc70, which is colored green, red, and blue for carbon, oxygen, and nitrogen atoms, respectively, and with black lettering on the J domain with stick representations of the side-chains of relevant J domain residues colored cyan, red, and blue for carbon, oxygen, and nitrogen atoms, respectively.
Figure 3
Figure 3. Effects of J domain on the ATPase rates of WT and mutant Hsc70ΔC enzymes
Experimental conditions as in fig. 1A, but with J domain (+J) added at 20 μM. Error bars are ±s.e. for n=3.
Figure 4
Figure 4. J domain induced changes in linker conformation may activate ATPase through interactions with Y371 and I181
A: Structures of the J domain (cyan) and Hsc70 residues 371–389, 181, and 187 with the linker in the ‘Out’ conformation. Hsc70 linker residues 383–389 and 371–382+181+187 are in magenta and green, respectively. The ED around the illustrated Hsc70 residues is contoured at 0.5 σ. B: As in A, but with the linker in the ‘In’ conformation and extending to residue 390; average B-factors for linker residues 383–389 (“Out”) or 383–390 (“In”) are 55 and 56, respectively, while the average B-factor for residues 3–382 of the NBD is 28. C: Effects of J domain on the ATPase rates of WT and mutant Hsc70ΔC enzymes. Experimental conditions as in fig. 1, but with Hsc70ΔC and J domain (+J) at 10 and 25 μM, respectively. Error bars are ±s.e. for n=3.
Figure 5
Figure 5. Elements of transmission of the J signal to the NBD
A: Stick representations of the AMPPNP, Hsc70 residues 172–181, 383–390 (“Linker”) from the J Domain:NBD_Linker structure are shown superimposed on the AMPPNP and residues 172–181 from an isolated NBD complexed with AMPPNP (pdb 1NGJ (Flaherty et al., 1994)). Alignment of the ribose, α- and β-phosphates of the nucleotides from each structure was optimized. Nitrogens, oxygens, and phosphates are colored blue, red, and orange, respectively. The AMPPNP γ-phosphate in J-domain complexed structure has swung towards E175. Carbons from the J-domain complexed or uncomplexed NBD are colored, respectively, green and magenta. Ribbon representaions of J-domain residues 874–895 and NBD residues 3–386 are shown in cyan and light grey, respectively. B. ATPase rates of WT or E715S NBD_Linker in the absence or presence (‘+J’) of 10 μM J domain.
Figure 6
Figure 6. J domain stimulation of Hsc70 ATPase activity requires displacement of the SBD from the NBD
A. Left: Structure of the 2-domain Hsc70 (Hsc70ΔC: pdb 1YUW(Jiang et al., 2005)) with the SBD (aa 397-554) in orange, the NBD (aa 1-382) in green, the interdomain linker (aa 383-396) in magenta, and residues K524 and D152 in red. Right: Structure of the NBD_Linker:J domain complex with the J domain in cyan and NBD and linker (extending to aa 390) colored as on the left. The SBD and linker (aa 392-554) from the left-hand structure are placed to suggest how the SBD would have to move away from the NBD to allow the J domain to bind and to allow the linker to interact with the NBD. B: Rates of ATP hydrolysis for WT or D152C/K524C 2-domain (aa 1-554) Hsc70 under oxidizing (“Ox”) or reducing (“Red”=2 mM DTT) conditions in the absence or presence of J domain. The inset gel of D152C/K524C resolved on SDS PAGE under reducing or oxidizing conditions shows that ~80% of the enzyme forms a faster migrating, disulfide bonded species in the oxidizing environment.
Figure 7
Figure 7
NMR spectra reveal interactions between the Hsc70 NBD and SBD in the ADP state. HSQC spectrum of 2-domain (aa 1-554) Hsc70 (black with folded peaks in gray) superimposed on that of the NBD (aa 1-386; red outlines with folded peaks in purple) in 1 mM ADP.
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