Molecular Mechanisms\nUnderlying the Loop-Closing Dynamics\nof β‑1,4 Galactosyltransferase 1

The β-1,4 galactosylation catalyzed by β-1,4\ngalactosyltransferases\n(β4Gal-Ts) is not only closely associated with diverse physiological\nand pathological processes in humans but also widely applied in the <i>N</i>-glycan modification of protein glycoengineering. The loop-closing\nprocess of β4Gal-Ts is an essential intermediate step intervening\nin the binding events of donor substrate (UDP-Gal/Mn<sup>2+</sup>)\nand acceptor substrate during its catalytic cycle, with a significant\nimpact on the galactosylation activities. However, the molecular mechanisms\nin regulating loop-closing dynamics are not entirely clear. Here,\nwe construct Markov state models (MSMs) based on approximately 20\nμs of all-atom molecular dynamics simulations to explore the\nloop-closing dynamics for β-1,4 galactosyltransferase 1 (β4Gal-T1).\nOur MSM reveals five key metastable states of β4Gal-T1 upon\nsubstrate binding, indicating that the entire conformational transition\noccurs on a time scale of ∼10 μs. Moreover, a regulatory\nmechanism involving six conserved residues (R187, H190, F222, W310,\nI341, and D346) among β4Gal-Ts is validated to account for the\nloop-closing dynamics of the C-loop and W-loop by site-directed mutagenesis\nand enzymatic activity assays, exhibiting high consistency with our\ncomputational predictions. Overall, our research proposes detailed\natomic-level insight into the loop-closing dynamics of the C-loop\nand W-loop on β4Gal-T1, contributing to a deeper understanding\nof catalytic mechanisms of β-1,4 galactosylation.