Modeling Flexible Protein Structure With AlphaFold2 and Crosslinking Mass Spectrometry.

We propose a pipeline that combines AlphaFold2 (AF2) and crosslinking mass spectrometry (XL-MS) to model the structure of proteins with multiple conformations. The pipeline consists of two main steps: ensemble generation using AF2 and conformer selection using XL-MS data. For conformer selection, we developed two scores-the monolink probability score (MP) and the crosslink probability score (XLP)-both of which are based on residue depth from the protein surface. We benchmarked MP and XLP on a large dataset of decoy protein structures and showed that our scores outperform previously developed scores. We then tested our methodology on three proteins having an open and closed conformation in the Protein Data Bank: Complement component 3 (C3), luciferase, and glutamine-binding periplasmic protein, first generating ensembles using AF2, which were then screened for the open and closed conformations using experimental XL-MS data. In five out of six cases, the most accurate model within the AF2 ensembles-or a conformation within 1 Å of this model-was identified using crosslinks, as assessed through the XLP score. In the remaining case, only the monolinks (assessed through the MP score) successfully identified the open conformation of glutamine-binding periplasmic protein, and these results were further improved by including the "occupancy" of the monolinks. This serves as a compelling proof-of-concept for the effectiveness of monolinks. In contrast, the AF2 assessment score was only able to identify the most accurate conformation in two out of six cases. Our results highlight the complementarity of AF2 with experimental methods like XL-MS, with the MP and XLP scores providing reliable metrics to assess the quality of the predicted models. The MP and XLP scoring functions mentioned above are available at https://gitlab.com/topf-lab/xlms-tools.

Native mass spectrometry for the investigation of protein structural (dis)order.

A central challenge in structural biology is represented by dynamic and heterogeneous systems, as typically represented by proteins in solution, with the extreme case of intrinsically disordered proteins (IDPs) [1-3]. These proteins lack a specific three-dimensional structure and have poorly organized secondary structure. For these reasons, they escape structural characterization by conventional biophysical methods. The investigation of these systems requires description of conformational ensembles, rather than of unique, defined structures or bundles of largely superimposable structures. Mass spectrometry (MS) has become a central tool in this field, offering a variety of complementary approaches to generate structural information on either folded or disordered proteins [4-6]. Two main categories of methods can be recognized. On one side, conformation-dependent reactions (such as cross-linking, covalent labeling, H/D exchange) are exploited to label molecules in solution, followed by the characterization of the labeling products by denaturing MS [7-11]. On the other side, non-denaturing ("native") MS can be used to directly explore the different conformational components in terms of geometry and structural compactness [12-16]. All these approaches have in common the capability to conjugate protein structure investigation with the peculiar analytical power of MS measurements, offering the possibility of assessing species distributions for folding and binding equilibria and the combination of both. These methods can be combined with characterization of noncovalent complexes [17, 18] and post-translational modifications [19-23]. This review focuses on the application of native MS to protein structure and dynamics investigation, with a general methodological section, followed by examples on specific proteins from our laboratory.