Analysis of the impact of molecular motions on the efficiency of XL-MS and the distance restraints in hybrid structural biology

Covalent cross-link mapping by mass spectrometry (XL-MS) is rapidly becoming the most widely used method of hybrid structural biology. We investigated the impact of incremental variations of cross-linker length have on the depth of XL-MS interrogation of protein-protein complexes, and assessed the role molecular motions in solution play in generation of cross-link-derived distance restraints. Supplementation of a popular NHS-ester cross-linker, DSS, with 2 reagents shorter or longer by CH2-CH2, increased the number of non-reductant cross-links by ~50%. Molecular dynamics simulations of these cross-linkers revealed 3 individual, partially overlapping ranges of motion, consistent with partially overlapping sets of cross-links formed by each reagent. Similar simulations elucidated protein fold-specific ranges of motions for the reactive and backbone atoms from rigid and flexible target domains. Together these findings create a quantitative framework for generation of cross-linker- and protein fold-specific distance restraints for XL-MS-guided protein-protein docking.