A comprehensive investigation of the impact of cross-linker backbone structure on protein dynamics analysis: A case study with Pin1

Understanding protein structure is essential for elucidating its function. Cross-linking mass spectrometry (XL-MS) has been widely recognized as a powerful tool for analyzing protein complex structures. However, the effect of cross-linker backbone structure on protein dynamic conformation analysis remains less understood. In this study, we investigated the impact of cross-linker backbone structure on resolving the dynamic conformations of Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1), which features a blend of relatively steady intradomain structures and dynamic interdomain regions. Three cross-linkers with varying arm lengths and different oxygen-containing backbones, Disuccinimidyl tartrate (DST), Bis(succinimidyl) di(ethylene glycol) (BS(PEG)₂), and Disuccinimidyl dihydroxydodecanedioate (DSDHD), were selected based on the theoretical inter-lysine distances within Pin1. By employing all-atom molecular dynamics (MD) simulations and solution nuclear magnetic resonance (NMR), we characterized the kinetic properties of cross-linkers and their perturbations to the protein structure. Additionally, we systematically evaluated the capability of cross-linkers with different backbones to analyze the structure and interdomain dynamics of Pin1. The results suggest that BS(PEG)₂, with its optimal arm length and ability to rapidly transition between compact and extended states, provides more interdomain dynamic conformational information of Pin1, while achieving a comparable level of intradomain structural detail to that obtained with the shorter cross-linker DST. Overall, this study highlights the critical role of cross-linker backbone structure in structural analysis of protein dynamics using mass spectrometry.