Ligand-Induced Conformational and Dynamical Changes in a GT-B Glycosyltransferase: Molecular Dynamics Simulations of Heptosyltransferase I Complexes

Abstract

Understanding the dynamical motions and ligand recognition motifs of heptosyltransferase I (HepI) can be critical to discerning the behavior of other glycosyltransferase (GT) enzymes. Prior studies in our lab have demonstrated that GTs in the GT-B structural class, which are characterized by their connection of two Rossman-like domains by a linker region, have conserved structural fold and dynamical motions, despite low sequence homology, therefore making discoveries found in HepI transferable to other GT-B enzymes. Through molecular dynamics simulations and ligand binding free energy analysis of HepI in the apo and bound complexes (for all kinetically relevant combinations of the native substrates/products), we have determined the energetically favored enzymatic pathway for ligand binding and release. Our principal component, dynamic cross correlation, and network analyses of the simulations have revealed correlated motions involving residues within the N-terminal domain communicating with C-terminal domain residues via both proximal amino acid residues and also functional groups of the bound substrates. Analyses of the structural changes, energetics of substrate/product binding, and changes in pK a have elucidated a variety of inter and intradomain interactions that are critical for enzyme catalysis. These data corroborate our experimental observations of protein conformational changes observed in both presteady state kinetic and circular dichroism analyses of HepI. These simulations provided invaluable structural insights into the regions involved in HepI conformational rearrangement upon ligand binding. Understanding the specific interactions governing conformational changes is likely to enhance our efforts to develop novel dynamics disrupting inhibitors against GT-B structural enzymes in the future.

Kinetic Characterization and Computational Modeling of the Escherichia coli Heptosyltransferase II: Exploring the Role of Protein Dynamics in Catalysis for a GT-B Glycosyltransferase

“…Residues 58-64 are analogous to residues 58-70 in HepI which have been previously observed to adopt a more alpha-helical orientation upon ligand binding and has been observed via CD and through crystal structures (Apo: PDB 2GT1, Psuedoternary: PDB 6DFE). 21, 23, 25, 42 In our HepII simulations, residues 58-64 become less dynamic upon presence of the acceptor in both poses (Figure 5B, S11B) which could promote formation of an ordered secondary structure. Additionally, the disordered 60s loop adds a turn to the downstream helix and the helix gains a turn on either side (Figure S15).…”

“…25,65 To elucidate the residues potentially involved in HepII substrate interactions, we performed a sequence alignment of HepI with HepII (HepI PDB: 6DFE and HepII PDB:IPSW; Figure S1), in addition to generating representative multiple sequence alignments (MSAs) of HepI (Figure S2) and HepII (Figure 4) homologues. Previously identified residues in HepI that are involved in substrate binding are indicated 21,25,42 and were used as a guide to identification of residues in a HepII that might be important for catalysis, in addition to examining residues that are highly conserved in a multiple sequence alignment (MSA) of HepII. Based upon these analyses of HepII, we hypothesized FDHLA binding residues to be Pro8, Trp10, His61, Ser90, Lys92, Gly192, Lys195, and Asp269 (Table 1A, S1).…”

“…This information has also allowed us to create a working model for the ternary complex HepII, as previously done for HepI. 42 This working model inspired the use of tryptophan fluorescence to determine the binding affinities of native and non-native donor and acceptor ligands, which also helps substantiate the validity of the ternary structural model of HepII. HepII has a higher catalytic efficiency and a submicromolar affinity for its substrates, which is distinct from prior observations for HepI.…”

“…Missing sidechains were modeled with Prime from the Schrodinger suite. 40–41 The previously developed ternary model of HepI 42 served as template for the placement for ADP-Hep, due to the high degree of sequence similarity occurring in the C-terminal, ADP-Hep binding domain. The C-terminal domains of HepI (residues 179-322) and HepII (residues 182-348) were aligned to aid placement of the ADP-Hep.…”

“…47–49 Ligands were prepared as previously described. 42 Briefly, Atom types were assigned from the second generation Generalized Amber Forcefield (GAFF2) and charges were assigned with the AM1-BCC model. 50–51 PROPKA was utilized in assigning ionization states of titratable sidechains of HepII.…”