Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

Abstract

In recent years, chemical crosslinking of protein complexes and the identification of crosslinked residues by mass spectrometry (XL-MS; sometimes abbreviated as CX-MS) has become an important technique bridging mass spectrometry (MS) and structural biology. By now, XL-MS is well established and supported by publicly available resources as a convenient and versatile part of the structural biologist's toolbox. The combination of XL-MS with cryoelectron microscopy (cryo-EM) and/or integrative modeling is particularly promising to study the topology and structure of large protein assemblies. Among the targets studied so far are proteasomes, ribosomes, polymerases, chromatin remodelers, and photosystem complexes. Here we provide an overview of recent advances in XL-MS, the current state of the field, and a cursory outlook on future challenges. Chemical Crosslinking as a Tool for Structural BiologyStructural biology makes use of many different techniques to elucidate the 3D structures of proteins and protein complexes. While high-resolution structures have traditionally been obtained by X-ray crystallography, cryo-EM is increasingly able to also generate (near) atomic-resolution models. In recent years, techniques and applications of MS have also rapidly progressed. Earlier studies were largely focused on the large-scale identification and quantification of proteins, whereas recent methods also support queries into the composition, stoichiometry, and spatial arrangement of subunits in a complex. These developments have now further progressed toward generating information that contributes, as part of hybrid structural strategies, to the structure elucidation of large molecular assemblies including protein complexes that perform essential processes in the cell. XL-MS is a particularly powerful mass spectrometric technique in this respect, because it provides several layers of information. Identifying protein-protein contacts through XL-MS confirms physical proximity between subunits because the proteins must be close enough in space to be crosslinked. Localizing the side chains that are connected restricts this proximity to certain regions (e.g., domains or even single helices or loops). Finally, the structure of the connected side chains and the crosslinker moiety impart a distance restraint that can be used for molecular modeling purposes because an upper Trends Chemical crosslinking followed by the mass spectrometric analysis of cross linked peptides (XL MS) identifies con tact sites between residues within a single or between multiple proteins.The application of XL MS to many bio logically relevant molecular machines has been shown, with a rapidly growing number of successful studies reported in the past 2 3 years.Crosslinking data are useful in integra tive modeling workflows by providing distance restraints on the surface of folded proteins and complexes. XL MS has been shown to be particularly powerful in combination with 3D cryo electron microscopy. Sciences ; 41 (2016), 1. -S. 20-32 https://dx.doi.org/10.1016...

Mass spectrometry-based cross-linking study shows that the Psb28 protein binds to cytochrome b 559 in Photosystem II

“…To identify the Psb28 binding site, we used an isotope-encoded chemical cross-linker and MS to capture covalently the proteinprotein interactions in PSII (23)(24)(25). We used on-the-fly precursor-ion selection that takes advantage of the isotopic "fingerprint" of cross-linked peptides to facilitate identification (26).…”

Structural prediction of protein models using distance restraints derived from cross-linking mass spectrometry data

“…Chemical cross-linking mass spectrometry (XL-MS) is an increasingly popular method with high analytical sensitivity that complements high-resolution structural approaches such as X-ray crystallography (XRC) and electron microscopy2. XL-MS provides information that two specific amino acids, present in either the same protein or in different proteins, can come within a certain distance of each other in 3D space in solution.…”

Elucidation of host–pathogen protein–protein interactions to uncover mechanisms of host cell rewiring

“…XL-MS thus yields fixed distance restraints between bound residues, suggesting direct physical intra-protein or inter-protein interactions between crosslinked peptides belonging to the same or distinct proteins respectively [56] (see Figure 1). Chemical crosslinking reactions can be performed on purified protein samples [57] using GFP epitope tags [58], on cell lysates [59] or on living cells such as on the pathogen Pseudomonas aeruginosa [60].…”