Identification of novel quaternary domain interactions in the Hsp90 chaperone, GRP94 - PubMed (original) (raw)

Identification of novel quaternary domain interactions in the Hsp90 chaperone, GRP94

Feixia Chu et al. Protein Sci. 2006 Jun.

Abstract

The structural basis for the coupling of ATP binding and hydrolysis to chaperone activity remains a central question in Hsp90 biology. By analogy to MutL, ATP binding to Hsp90 is thought to promote intramolecular N-terminal dimerization, yielding a molecular clamp functioning in substrate protein activation. Though observed in studies with recombinant domains, whether such quaternary states are present in native Hsp90s is unknown. In this study, native subunit interactions in GRP94, the endoplasmic reticulum Hsp90, were analyzed using chemical cross-linking in conjunction with tandem mass spectrometry. We report the identification of two distinct intermolecular interaction sites. Consistent with previous studies, one site comprises the C-terminal dimerization domain. The remaining site represents a novel intermolecular contact between the N-terminal and middle (M) domains of opposing subunits. This N+M domain interaction was present in the nucleotide-empty, ADP-, ATP-, or geldanamycin-bound states and could be selectively disrupted upon addition of synthetic geldanamycin dimers. These results identify a compact, intertwined quaternary conformation of native GRP94 and suggest that intersubunit N+M interactions are integral to the structural biology of Hsp90.

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Figures

Figure 1.

Differential nano-LC-MS analysis strategy to identify intermonomeric cross-linked species from homodimeric protein assemblies. Two polypeptide chains of the dimer complex are colored in green and in blue to distinguish their origin. (Orange sticks) Cross-linking bridges, (sticks with red dots) single-ended cross-links.

Figure 2.

Cross-linking of the GRP94 dimer complex. (A) SDS-PAGE analysis of the cross-linking reaction. (B,C) Total ion chromatograms (TIC) of the tryptic digestion mixture of the cross-linked dimer band (B) and the monomer band (C). (Insets) Mass spectra of the peptides eluted at ∼21 min.

Figure 3.

Structure and low-energy CID spectrum of the cross-linked species with a m/z value of 664.344+ from the GRP94 dimer digest. C-terminal sequence ions that did not contain the cross-linker moiety were labeled in lowercase letters, e.g., yi (blue), and yi′ (red) ions.

Figure 4.

The intermonomeric cross-linked species with a m/z value of 549.094+ were identified only in the covalently linked dimer digest. (A,B) Extract ion chromatograms (XIC) of m/z value 549.09 from the tryptic digestion mixture of the dimer band (A) and the monomer band (B). (Insets) Mass spectra of the peptides eluted at ∼27.8 min.

Figure 5.

Structure and low-energy CID spectrum of the cross-linked species with a m/z value of 549.094+ from the GRP94 dimer digest. C-terminal sequence ions that did not contain the cross-linker moiety were labeled in lowercase letters, e.g., yi (blue), and yi′ (red) ions.

Figure 6.

Comparison of the amount of the intermonomeric cross-linked peptides of GRP94 in complex with geldanamycin, GMD-4c, or GMD-9c. The digestion mixtures of DSS cross-linked dimer were analyzed consecutively on LC-MS. The amount of the cross-linked peptides is represented by the peak intensity of the corresponding peptides.

Figure 7.

A model for GRP94 subunit interaction dynamics. GRP94 N-terminal, middle, and C-terminal domains are annotated as N, M, and C, respectively. (Orange squares) Adenosine nucleotide binding sites, (magenta circles) approximate positions for the clustered residues involved in N+M domain cross-linking reaction. The diameter of the C-terminal domain is ∼30 Å, and the maximal distance the cross-linker (DSS) can span is 12 Å. (A) As illustrated, GRP94 samples both an “open” (Harris et al. 2004) and “closed” conformation, with intersubunit N+M domain cross-linking limited to the “closed” conformation. In this model, the two distinct conformers constitute a pre-existing conformational equilibrium, independent of adenosine nucleotide binding. The addition of bivalent geldanamycin ligand, GMD-4c (orange double arrow), introduces steric hindrance at the intersubunit N+M surface and thereby prevents the intersubunit cross-links between the N-terminal and middle domains. (B) Additional topological representations of the model depicted in A, emphasizing the intersubunit N+M interactions.

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Identification of novel quaternary domain interactions in the Hsp90 chaperone, GRP94 - PubMed (original) (raw)