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.

Structural Proteomics Methods to Interrogate the Conformations and Dynamics of Intrinsically Disordered Proteins

“…It was found that the KID domain of p27 (p27 KID ), which folds upon binding to Cdk2/cyclin A, causes the rigid dimer to become more dynamic. As expected, full-length p27 (p27 FL ) is tamed upon binding, with the most extended conformations no longer being accessible upon complex formation ( Beveridge et al, 2019b ). In this work, the strength of IMMS is its ability to visualize both the bound and unbound form of an IDP, without the preference for the bound form that is displayed by many techniques.…”

A stochastic dynamic mass spectrometric diffusion method and its application to 3D structural analysis of the analytes

“…Because of this, as has been shown from our research, so far, the D SD parameter accounts for the multidimensional molecular conformation and electronic structure of the analyte ion. We may add that even with the employment of some of the most prominent MS methods, for instance, the ion mobility mass spectrometry, where the method operates within the spans of the measurement times between minutes and μs to ms, the MS structural information is mainly limited to 2D analysis as has been shown from the accumulated research more recently (Fuller et al, 2018;Beveridge et al, 2019;Chen et al, 2019;Woodall et al, 2019). The methodological efforts consisting of the computation of collision cross section (CCS) yields, in fact, to energetics of analyte ions, presumably corresponding to a 3D molecular conformation, which is con-nected with the drift time and the velocity of the MS ions via the Mason-Schamp equation.…”

Untangling Hydrogen Bond Networks with Ion Mobility Spectrometry and Quantum Chemical Calculations: A Case Study on H+XPGG

“…A number of groups are now using ion mobility spectrometry-mass spectrometry (IMS-MS) to study biomolecular structure and dynamics. − In IMS, the unique ion mobilities are determined by measuring the time required to traverse a drift tube filled with an inert buffer gas. The mobility measurements can be related to the ions’ orientationally averaged collision cross section, which effectively reports on an ion’s shape .…”