Molecular Mechanisms Underlying the Loop-Closing Dynamics of β-1,4 Galactosyltransferase 1

Abstract

The β-1,4 galactosylation catalyzed by β-1,4 galactosyltransferases (β4Gal-Ts) is not only closely associated with diverse physiological and pathological processes in humans but also widely applied in the N-glycan modification of protein glycoengineering. The loop-closing process of β4Gal-Ts is an essential intermediate step intervening in the binding events of donor substrate (UDP-Gal/Mn²⁺) and acceptor substrate during its catalytic cycle, with a significant impact on the galactosylation activities. However, the molecular mechanisms in regulating loop-closing dynamics are not entirely clear. Here, we construct Markov state models (MSMs) based on approximately 20 μs of all-atom molecular dynamics simulations to explore the loop-closing dynamics for β-1,4 galactosyltransferase 1 (β4Gal-T1). Our MSM reveals five key metastable states of β4Gal-T1 upon substrate binding, indicating that the entire conformational transition occurs on a time scale of ∼10 μs. Moreover, a regulatory mechanism involving six conserved residues (R187, H190, F222, W310, I341, and D346) among β4Gal-Ts is validated to account for the loop-closing dynamics of the C-loop and W-loop by site-directed mutagenesis and enzymatic activity assays, exhibiting high consistency with our computational predictions. Overall, our research proposes detailed atomic-level insight into the loop-closing dynamics of the C-loop and W-loop on β4Gal-T1, contributing to a deeper understanding of catalytic mechanisms of β-1,4 galactosylation.

Figure 1

Catalytic cycle of β4Gal-T1 and initial conformational states defining its loop-closing process. (a) Schematic diagram of the biochemical reaction catalyzed by β4Gal-T1, transferring Gal from UDP-Gal to GlcNAc. The entire catalytic cycle of β4Gal-T1 is split into seven steps, and the open state and closed state involved in the loop-closing process are labeled out in red. (b) Constructed models of β4Gal-T1 bound with UDP-Gal/Mn²⁺ (UDP-Gal-T1) with C-loop and W-loop in the open state and the closed state. For both states, C-loop and W-loop are depicted in green and magenta cartoon with residues H343 and W310 shown in sticks. Substrates of Mn²⁺ and UDP-Gal are shown in purple spheres and bright cyan sticks, respectively. Key hydrogen bonds are shown in black dash.

Figure 2

MSM identifies five metastable states during the loop-closing process. (a) Free energy landscape of MD data projected onto the top two tICs, with five metastable states indicated at their respective positions. (b) Representative conformations of the five metastable states, depicted by 20 randomly selected structures from each macrostate. The corresponding populations among the MD data are labeled below each metastable state. For each state, C-loop, W-loop, and other domains are depicted in green, magenta, and gray cartoon, respectively, while substrates of UDP-Gal and Mn²⁺ are hidden for a clear view.

Figure 3

Kinetic network and key substrate interactions of five metastable states. (a) Diagram illustrating the kinetic network of the five metastable states derived from our MSM analysis. Each node represents a metastable state, with the MFPT for transitions indicated above the arrows. Arrow thickness is proportional to the transition flux. (b) Representative structures for each macrostate, highlighting key interactions among C-loop, W-loop, and substrates. For each state, C-loop and W-loop are depicted in green and magenta cartoon with key residues shown in sticks. Substrates of Mn²⁺ and UDP-Gal are shown in purple sphere and bright cyan sticks, respectively. Key hydrogen bonds are shown in a black dash.

Figure 4

Key structural features for five metastable states during the loop-closing process. (a) Key structural regions for statistical analysis, including Cα atoms of the α-helix residues Q354-R358 (green spheres); the C-loop (green cartoon); the W-loop (magenta cartoon); UDP-Gal (cyan sticks) and Mn²⁺ (purple spheres). (b) RMSD values of the Cα atoms on the C-loop and W-loop for each macrostate by referring to coordinates of the initial closed structure. (c) Contact numbers between β4GalT1 and UDP-Gal for each macrostate. (d) α-Helix propensities of residues Q354 to R358 for each macrostate. (e) Solvent-accessible surface area (SASA) of the C-loop and W-loop for each macrostate. (f) Number of water molecules around UDP-Gal, with a cutoff distance of 3.5 Å. The mean value and the standard error are calculated based on all microstates within each macrostate.

Figure 5

Hydrogen bonding occupancy and structural dynamics involved in substrate binding for five metastable states. (a) Five key hydrogen bonds (HBs) at the closed state: H1, hydrogen bonding between R187 and the P₂O₅^2– moiety of UDP-Gal; H2, hydrogen bonding between W310 and the P₂O₅^2– moiety of UDP-Gal; H3, hydrogen bonding between R187 and D346; H4, hydrogen bonding between N349 and F222; H5, hydrogen bonding between D346 and N349. (b) Hydrogen bond occupancies of H1, H2, H3, H4, and H5 for each metastable state. (c) MD data projection onto two coordinates: the first tIC and the radius of gyration (Rg) for the W-loop and C-loop residues. The colored density plot shows the distribution of conformations in metastable states S1–S5. (d) RMSF values for each residue in the C-loop and W-loop. (e) Rotation of the residue W310, measured by the dihedral angle defined by the central O atom of the P₂O₅^2– moiety (A1), the carbonyl C atom of W310 (A2), the Cα atom of W310 (A3), and the Cγ atom of W310 (A4). (f) Distributions of the defined dihedral angle in (e) for each metastable state.

Figure 6

Schematic diagram illustrating the molecular mechanism underlying the loop-closing dynamics of β4GalT1. The entire dynamics are depicted in six sequential panels, representing the open state, substrate binding, C-loop fluctuation, C-loop translocation, C-loop/W-loop changes, and the closed state. Conformational changes of β4GalT1 are depicted by loops around the substrate-binding site, key residues, and substrates. C-loop, W-loop and other surrounding loops are shown in green, magenta, and gray curves, respectively. Residues R358, I341, D346, R187, H190, F222, W310 and Mn²⁺ (colored spheres) form interactions with UDP-Gal moieties including uridine (cyan hexagon), P₂O₅^2– (orange rectangle), and galactosyl (cyan triangle) during the loop-closing process. Some residues on the C-loop show highly flexible coils (green wavy lines) or have changeable secondary structure from coil to helix (green rectangles) at different steps during the loop-closing process. The π–π stacking and hydrogen bonding interactions are shown in yellow and red dash, respectively.