In Vivo Conformational Dynamics of Hsp90 and Its Interactors

Abstract

Summary Hsp90 belongs to a family of some of the most highly expressed heat shock proteins that function as molecular chaperones to protect the proteome not only from the heat shock, but from other misfolding events. As many client proteins of Hsp90 are involved in oncogenesis, this chaperone has been in focus of intense research efforts. Yet, we lack structural information for how Hsp90 interacts with co-chaperones and client proteins. Here, we develop a mass spectrometry based approach that allows quantitative measurements of in vitro and in vivo effects of small molecule inhibitors on Hsp90 conformation, and interaction with co-chaperones and client proteins. From this analysis we were able to derive structural models for how Hsp90 engages its interaction partners in vivo and how different drugs affect these structures. Additionally, the methodology described here offers a new approach to probe the effects of virtually any inhibitor treatment on the proteome level.

Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry

“…Chemical cross-linking provides evidence of proximal solventaccessible sites on proteins in vivo and is a means to predict and assemble models of protein structures (34,35,39). It is important to note that the chemical cross-linking workflow in this study was performed on whole, functional mitochondria.…”

“…Functional, respiring mitochondria were cross-linked in situ using a PIR cross-linker, biotin-aspartate proline-PIR n-hydroxyphthalimide (BDP-NHP) (33)(34)(35)39), and were assembled into an interactome consisting of 2,427 nonredundant cross-linked peptide pairs across 11 individual murine samples (Dataset S1). We identified 327 unique mitochondrial proteins, 31.3% of the murine mitochondrial proteome (327 of 1,042 proteins; MitoCarta 2.0) (Fig.…”

“…We did not observe links between complex III and complex IV, as is consistent with studies that showed these two complexes do not coimmunoprecipitate from Black-6 murine mitochondria, such as those used here, because of a mutation in the scaffolding protein SCAF1 (60). The in situ cross-linked peptides from whole, respiring mitochondria presented here establish a final conformation of complex I-complex III that is highly similar to the cryo-EM model and a set of powerful molecular probes that can be used in the future to quantify conditional response on supercomplex assembly in functional mitochondria (35).…”

“…S5). Identification of potentially dynamic or transient interactions are particularly interesting, because it was established previously that crosslinked peptide abundances change in a conformation-dependent manner (35); thus future studies could harness the ATIF1-ATPA/B links to elucidate condition-and conformation-specific flexing of the ATIF1 helix.…”

“…These capabilities can uncover novel functions derived from protein-protein interactions, and crosslinks have provided data complementary to cryo-EM and crystallography to enhance the understanding of the structure and function of complex systems (27). Second, large-scale chemical cross-linking analyses applied to living systems can enable the determination of protein interactions and conformational changes in complex, dynamic native environments, including the quantification of changes in response to pharmacological intervention (35). In the present study protein interaction reporter (PIR)-based XL-MS was performed using a peptide-based chemical crosslinking molecule for unambiguous identification of cross-linked peptides (33,34,(36)(37)(38).…”