The Conformational Preference of Chemical Cross-linkers Determines the Cross-linking Probability of Reactive Protein Residues

Chemical cross-linking mass spectrometry (XLMS) is an emerging technique in structural biology. Providing the cross-linked peptides are identified by mass spectrometry with high confidence, a distance restraint can be applied between the two reactive protein residues, with the upper bound corresponding to the maximal span of the cross-linker. However, as the upper bound is typically over 20 Å, cross-link distance restraints are unrestrictive and provide a marginal improvement in protein structural refinement. Here we analyze the experimental cross-links for lysine or acidic residues and show that the distribution of Cβ-Cβ′ distances can be described with two overlapping Gaussian species. In addition to the pairwise occurrence probability of the reactive protein residues, we show that the distribution profile of the cross-link distances is determined by the intrinsic conformational propensity of the cross-linker. The cross-linker prefers either a compact or extended conformation and, once attached to a reactive protein residue, predominantly an extended conformation. Consequently, the long-distance Gaussian species occurs at a much higher probability than the short-distance species in the observed cross-links. Together, the probabilistic distribution of the cross-link distance allows the construction of a more restrictive restraint for structural modeling and better use of the XLMS data.