Structural dynamics of the human COP9 signalosome revealed by cross-linking mass spectrometry and integrative modeling

Significance Structural plasticity is a critical property of many protein complexes that has been challenging to study using conventional structural biology tools. Cross-linking mass spectrometry (XL-MS) has become an emergent technology for elucidating architectures of large protein complexes. While effective, current XL-MS methods mostly rely on lysine reactive cross-linking chemistry and have limited capacity in fully defining dynamic structures of protein complexes. Here, we have developed an integrated structural approach based on three MS-cleavable cross-linkers with distinct chemistries. This approach enabled us to obtain highly reliable and comprehensive cross-link data that significantly facilitate integrative structural modeling of dynamic protein complexes. In addition, it has been successfully applied to the COP9 signalosome to determine its structural dynamics associated with its function. The COP9 signalosome (CSN) is an evolutionarily conserved eight-subunit (CSN1–8) protein complex that controls protein ubiquitination by deneddylating Cullin-RING E3 ligases (CRLs). The activation and function of CSN hinges on its structural dynamics, which has been challenging to decipher by conventional tools. Here, we have developed a multichemistry cross-linking mass spectrometry approach enabled by three mass spectometry-cleavable cross-linkers to generate highly reliable cross-link data. We applied this approach with integrative structure modeling to determine the interaction and structural dynamics of CSN with the recently discovered ninth subunit, CSN9, in solution. Our results determined the localization of CSN9 binding sites and revealed CSN9-dependent structural changes of CSN. Together with biochemical analysis, we propose a structural model in which CSN9 binding triggers CSN to adopt a configuration that facilitates CSN–CRL interactions, thereby augmenting CSN deneddylase activity. Our integrative structure analysis workflow can be generalized to define in-solution architectures of dynamic protein complexes that remain inaccessible to other approaches.

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