Molecular Basis for Structural Heterogeneity of an Intrinsically Disordered Protein Bound to a Partner by Combined ESI-IM-MS and Modeling

Abstract

Abstract. Intrinsically disordered proteins (IDPs) form biologically active complexes that can retain a high degree of conformational disorder, escaping structural characterization by conventional approaches. An example is offered by the complex between the intrinsically disordered N TAIL domain and the phosphoprotein X domain (P XD ) from measles virus (MeV). Here, distinct conformers of the complex are detected by electrospray ionization-mass spectrometry (ESI-MS) and ion mobility (IM) techniques yielding estimates for the solvent-accessible surface area (SASA) in solution and the average collision cross-section (CCS) in the gas phase. Computational modeling of the complex in solution, based on experimental constraints, provides atomic-resolution structural models featuring different levels of compactness. The resulting models indicate high structural heterogeneity. The intermolecular interactions are predominantly hydrophobic, not only in the ordered core of the complex, but also in the dynamic, disordered regions. Electrostatic interactions become involved in the more compact states. This system represents an illustrative example of a hydrophobic complex that could be directly detected in the gas phase by native mass spectrometry. This work represents the first attempt to modeling the entire N TAIL domain bound to P XD at atomic resolution.

Ensemble Methods Enable a New Definition for the Solution to Gas-Phase Transfer of Intrinsically Disordered Proteins

“…19 Overall, gas-phase methods appear uniquely suited to meet the challenge of describing the conformations and dynamics of flexible proteins as evidenced by the increasing number of publications in this area in recent years. [20][21][22][23][24] Nevertheless, recent studies describing the collapse of disordered structures in the absence of solvent have raised questions over the fidelity to which gas-phase methods can capture the solution conformations of these proteins. 25,26 Recent shifts in our understanding of how flexible species escape electrospray droplets during ESI also add to the complexity of describing the solution to gas-phase transfer of IDPs.…”

“…This ability of SAXS allows us to investigate the accuracy to which ESI charge state distributions represent the conformational space that is occupied by IDPs in solution. 24,[29][30][31][32] We find dramatically different structural depictions of the IDPs depending on the technique employed to characterize these proteins. In solution the IDP ensembles largely describe unimodal distributions of conformers whereas gas-phase methods describe a conformational space that is characterized by 3 distinct subpopulations broadly defined as compact (c), intermediate (i) and extended (e) with increasing Ω and charge.…”

Mass spectrometry-enabled structural biology of membrane proteins

“…It is important to note, however, that the range of complexes which can be studied by native MS methods is restricted to those that survive intact in the gas phase which, in turn, is determined by the nature of the non-covalent interactions involved in complex formation. For example, since hydrophobic interactions are weakened in the gas phase, macromolecular complexes that rely on hydrophobic contacts for assembly may not survive intact during the transfer to the gas phase [82,83]. In addition, the loss of a solvation shell may result in structural reorganization, as evidenced for water soluble proteins, including intrinsically disordered proteins or complexes with 'open' topologies as found in extended polyproteins, such as IgGs or ring-like subunit assemblies [76,81,163].…”

“…[90] with permission. may be too labile, and will not retain their native fold during nESI, although this is not always the case [82,83]. In the analysis of water soluble proteins using native MS, gentle ionization and instrument conditions are typically used to preserve non-covalent interactions [60].…”

Identification and Structural Characterization of an Intermediate in the Folding of the Measles Virus X Domain

“…In measles virus (a member of the Morbillivirus genus), the C-terminal region of phosphoprotein P is a globular domain of 49 amino acids, called X domain (XD), 2 composed of three ␣-helices organized as an anti-parallel bundle (12). Previous investigations on measles virus XD have suggested this domain to be structurally heterogeneous, populating at least two alternative conformations under native conditions (13). This feature is consistent with the findings of Kingston et al (14), who suggested that the native state of XD from mumps virus (a Rubulavirus member) represents an example of folding induced by crystal packing effects.…”