Protonation state of the S-O-NH crosslink between lysine and cysteine

A new putative oxygen-bridged crosslink between lysine and cysteine was experimentally identified in a transaldolase from the Gram-negative bacterium Neisseria gonorrhoeae (NgTAL). This crosslink was proposed to function as a reversible redox switch. Although it has generally been represented in its neutral S-O-NH form, its protonation state remains uncertain. Importantly, protonation may influence both the physicochemical properties of the crosslink and the structure of the surrounding protein environment. In the present study, we used a multiscale computational approach combining QM-cluster, QM/MM, and molecular dynamics simulations to investigate the preferred protonation state of the Cys38-S-O-NH-Lys8 crosslink and the structural consequences of nitrogen protonation. QM-cluster calculations identified the nitrogen centre as having the highest proton affinity (PA) and gas-phase basicity (GPB) across all dielectric environments considered. When the crystallographic water molecules were included explicitly, proton transfer through the water network yielded the N-protonated S-O-NH₂ ⁺ species, while no stable S- or O-protonated crosslink was obtained. The QM/MM calculations also favoured nitrogen protonation, giving PA and GPB values of 1700.2 and 1685.5 kJ mol^-1, respectively. Furthermore, the optimized QM/MM structure revealed an extended hydrogen-bonding network connecting the protonated crosslink to Glu93 through the water molecules. Three independent 200 ns MD simulations further showed that the protonated crosslink maintains a persistent association with Glu93, through a direct hydrogen bond (∼69% occupancy) or a one-water bridge (∼18%), whereas the neutral crosslink most often lacked an interaction with Glu93 (∼66%). Nitrogen protonation also shifted the preferred Glu93 side-chain conformation towards a rotameric state resembling the crystallographic structure and modestly altered the global conformational dynamics of the protein. Collectively, these results consistently support assignment of the NgTAL crosslink as predominantly N-protonated, S-O-NH₂ ⁺. Its stabilization arises from the combined influence of the local water network, Glu93, and the surrounding protein environment.