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.