Distance restraints from crosslinking mass spectrometry: Mining a molecular dynamics simulation database to evaluate lysine–lysine distances

Abstract

Integrative structural biology attempts to model the structures of protein complexes that are challenging or intractable by classical structural methods (due to size, dynamics, or heterogeneity) by combining computational structural modeling with data from experimental methods. One such experimental method is chemical crosslinking mass spectrometry (XL-MS), in which protein complexes are crosslinked and characterized using liquid chromatography-mass spectrometry to pinpoint specific amino acid residues in close structural proximity. The commonly used lysinereactive N-hydroxysuccinimide ester reagents disuccinimidylsuberate (DSS) and bis(sulfosuccinimidyl)suberate (BS 3 ) have a linker arm that is 11.4 Å long when fully extended, allowing C a (alpha carbon of protein backbone) atoms of crosslinked lysine residues to be up to~24 Å apart. However, XL-MS studies on proteins of known structure frequently report crosslinks that exceed this distance. Typically, a tolerance of~3 Å is added to the theoretical maximum to account for this observation, with limited justification for the chosen value. We used the Dynameomics database, a repository of high-quality molecular dynamics simulations of 807 proteins representative of diverse protein folds, to investigate the relationship between lysine-lysine distances in experimental starting structures and in simulation ensembles. We conclude that for DSS/BS 3 , a distance constraint of 26-30 Å between C a atoms is appropriate. This analysis provides a theoretical basis for the widespread practice of adding a tolerance to the crosslinker length when comparing XL-MS results to structures or in modeling. We also discuss the comparison of XL-MS results to MD simulations and known structures as a means to test and validate experimental XL-MS methods.

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

“…Eleven of the 13 distances are within 30 Å, a typical benchmark (34)(35)(36)(37)(38)(39), and the full distance distribution (SI Appendix, Fig. S2) matches the typical distributions found for this cross-linker (25,37,(39)(40)(41).…”

Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

“…33 We first incubated the purified complex with increasing concentrations of either the DSS or BS 3 chemical crosslinker, which covalently conjugates 2 free amine groups over a Ca-Ca distance of approximately 24 to 30 A . 34 Addition of crosslinker led to the appearance of a high molecular mass band above the 250-kDa molecular mass marker, a size that corresponded well to the calculated total mass of the mini pentameric assembly (»350 kDa) (Fig. S2).…”

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

“…XL-MS provides definitive binary interaction data (e.g., subunit A is close in space to subunit B) and spatial restraints between proteins with a resolution of several amino acids at the primary sequence level (limited by the location of crosslinkable residues). These restraints are in the range 7-30 Å, with a median distance of approximately 15 Å for the most commonly used lysine-reactive reagents [14,61,62] and slightly shorter for carboxyl-reactive hydrazides and zero-length crosslinks [18]. This allows the determination of the proximity and, if multiple crosslinks in complementary regions are found, the relative orientation of the subunits.…”