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

B American Society for Mass Spectrometry, 2014 J. Am. Soc. Mass Spectrom. (2015) 26:472Y481 DOI: 10.1007/s13361-014-1048-z RESEARCH ARTICLE Molecular Basis for Structural Heterogeneity of an Intrinsically Disordered Protein Bound to a Partner by Combined ESI-IM-MS and Modeling Annalisa D’Urzo,1 Albert Konijnenberg,2 Giulia Rossetti,3,4 Johnny Habchi,5,6 Jinyu Li,3,7 Paolo Carloni,3 Frank Sobott,2,8 Sonia Longhi,5,6 Rita Grandori1 1 Department of Biotechnology and Biosciences, University of Milano-Bicocca, 20126 Milan, Italy Biomolecular and Analytical Mass Spectrometry group, Department of Chemistry, University of Antwerp, 2020 Antwerpen, Belgium 3 Computational Biophysics, German Research School for Simulation Sciences, and Computational Biomedicine, Institute for Advanced Simulation IAS-5 and Institute of Neuroscience and Medicine INM-9, Forschungszentrum Jülich, 52425 Jülich, Germany 4 Jülich Supercomputing Center, Forschungszentrum Jülich, 52425 Jülich, Germany 5 Architecture et Fonction des Macromolécules Biologiques (AFMB), Aix-Marseille Université, UMR 7257, 13288 Marseille, France 6 CNRS, AFMB UMR 7257, 13288 Marseille, France 7 Institute of Biochemistry and Molecular Biology, RWTH Aachen University, 52057 Aachen, Germany 8 Center for Proteomics (CFP-CeProMa), University of Antwerp, 2020 Antwerpen, Belgium 2 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 NTAIL domain and the phosphoprotein X domain (PXD) 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 NTAIL domain bound to PXD at atomic resolution. Keywords: Conformational ensemble, Native mass spectrometry, Ion mobility, Hydrophobic Interactions, Measles virus, Ntail–Pxd complex Received: 18 July 2014/Revised: 4 November 2014/Accepted: 8 November 2014/Published Online: 16 December 2014 Introduction T he last decade has witnessed an extension to the protein structure-function paradigm, with the progressive understanding of the functional importance of intrinsically disordered Electronic supplementary material The online version of this article (doi:10.1007/s13361-014-1048-z) contains supplementary material, which is available to authorized users. Correspondence to: Sonia Longhi; e-mail: , Rita Grandori; e-mail: proteins (IDPs) or regions (IDRs). These proteins or protein regions lack ordered secondary and tertiary structure under physiological conditions and exist in solution as dynamic and heterogeneous conformational ensembles [1–3]. Approximately 40% of the human proteins are predicted to contain at least one disordered segment of at least 30 amino acids, with as many as 25% of them likely to be disordered from start to end [4]. Predictions on representative genomes from the three kingdoms of life (i.e., bacteria, archaea, and eukaryotes) confirm the ubiquitous character of structural disorder, in spite of significant differences in its relative amount in the three domains [5]. The extent of protein structural disorder tends to increase with A. D’Urzo et al.: Structural Heterogeneity By MS and Modeling biological complexity. This trend could be related to the typical involvement of IDPs in signaling and regulation [6–8]. The structural plasticity of IDPs allows for recognition and binding of multiple partners, resulting in pleiotropic roles of these proteins. Many cases have been described in the literature, in which IDPs acquire ordered conformations upon binding to partners or ligands. Folding coupled to binding can pertain either to specific segments or to the whole protein [9]. Complete folding can lead to well-structured complexes that can be analyzed by conventional techniques such as X-ray crystallography [10–12]. However, increasing evidence shows that many IDPs retain a high degree of structural disorder even in the bound state. These “fuzzy” complexes [13] are stabilized by short, ordered recognition elements, referred to as molecular recognition elements (MoREs), and a large number of highly unstable contacts, leading to a cloud of interconverting conformations around a structured core [9, 10, 13–16]. This staccatotype of interactions is much more difficult to characterize than stable interactions of folded complexes. Yet, it is thought to be relevant for biological function. Fuzzy regions within complexes can harbor regulatory post-translational modification sites, or can mediate interactions with additional partners. They can even, directly or indirectly, interfere with recognition elements, promoting or inhibiting binding. In addition, fuzziness provides a way to reduce the entropic penalty, thereby affording enhanced affinity [15, 17]. Hence, description of the conformational ensembles characterizing fuzzy IDP complexes is essential to the understanding of the molecular processes by which IDPs establish their functional networks. However, the highly heterogeneous nature of IDPs and the fuzziness that is often observed in their bound state makes their structural characterization very challenging. Such investigation demands the combined application of various biophysical methods capable of capturing conformational heterogeneity and identifying metastable states. In this regard, native mass spectrometry based on nano-ESI sources has emerged as a powerful approach, allowing detection of coexisting conformers with distinct global compactness [18, 19]. The average charge state of each component yields an estimate of SASA for the structure in solution, at the moment of transfer to the gas phase [20, 21]. Hyphenation with IM measurements adds a further dimension to species separation and offers estimates of the CCS for each detected structure in the gas phase [22–25]. These techniques conjugate the exceptional analytical power of mass spectrometry with structural description and, therefore, are particularly well suited to the challenges pose (...truncated)