Ion Mobility Mass Spectrometry Measures the Conformational Landscape of p27 and its Domains and how this is Modulated upon Interaction with Cdk2/cyclin A

Abstract

Intrinsically disordered proteins have been reported to undergo 'disorder to order' transitions upon binding to their partners in the cell. The extent of the ordering on binding and the lack of order prior to binding is difficult to visualize with classical structure determination methods. Binding of p27 to the Cdk2/cyclin A complex is accompanied by partial folding of p27 in the KID domain, with the retention of dynamic behaviour for function, particularly in the C-terminal half of the protein, positioning it as an exemplary system to probe conformational diversity. Here we employ native ion mobility with mass spectrometry (IM-MS) to measure the intrinsic dynamic properties of p27, both in isolation and within the trimeric complex with Cdk2/cyclin A. This stepwise approach reveals the conformational distributions of the constituent proteins and how they are restructured on complex formation; the trimeric Cdk2/cyclin A/p27-KID complex possesses significant structural heterogeneity cf. Cdk2/cyclin A. These findings support the formation of a fuzzy complex in which both the N and C termini of p27 interact with Cdk2/cyclin A in multiple closely associated states.

Tandem-trapped ion mobility spectrometry/mass spectrometry (tTIMS/MS): a promising analytical method for investigating heterogenous samples

“…As we pointed out in the Introduction to this review, IMS/MS should be ideally suited to study structures of biological systems. Indeed, many applications of IMS/MS, ranging from studies of peptide and protein assemblies 17,18,[34][35][36][37][38]49,154 to proteins 19,[39][40][41][42][43][44] and protein complexes, [45][46][47][48][49][50] showcase the tremendous potential of IMS/MS for the field of structural biology. Nevertheless, the application of IMS/MS to study structures of protein systems remains challenging.…”

“…29 Moreover, through measurements of momentum transfer cross sections, IMS/MS provides information related to the conformation of detected ions. 16,[30][31][32][33] These abilities prompted an array of structural studies of biological problems with IMS/MS, from peptide assemblies 17,18,[34][35][36][37][38] to proteins 19,[39][40][41][42][43][44] and protein complexes. [45][46][47][48][49][50] Additionally, Clemmer demonstrated how specific isomers of the small protein ubiquitin can be isolated from a mixture of isomers and selectively interrogated by coupling collisional-activation with consecutive IMS-separation and mobility-selection steps (tandem-IMS).…”