The Stories Tryptophans Tell: Exploring Protein Dynamics of Heptosyltransferase I from Escherichia coli

Abstract

Heptosyltransferase I (HepI) catalyzes the addition of L-glycero-β-D-manno-heptose onto Kdo2-Lipid A, as part of the biosynthesis of the core region of lipopolysaccharide (LPS). Gram-negative bacteria with gene knockouts of HepI have reduced virulence and enhanced susceptibility to hydrophobic antibiotics, making design of inhibitors against HepI of interest. Since HepI protein dynamics are partially rate-limiting, disruption of protein dynamics might provide a new strategy for inhibiting HepI. Discerning the global mechanism of HepI is anticipated to aid development of inhibitors of LPS biosynthesis. Herein, dynamic protein rearrangements involved in the HepI catalytic cycle were probed by combining mutagenesis with intrinsic tryptophan fluorescence and circular dichroism analyses. Using wild-type and mutant forms of HepI, multiple dynamic regions were identified via monitoring fluorescence wavelength changes. Interestingly, Trp residues (Trp199 and Trp217) in the C-terminal domain (which binds ADP-Heptose) are in a more hydrophobic environment upon ODLA binding to the N-terminal domain. These residues are adjacent to the ADP-Heptose binding site (with Trp217 in van der Waals contact with the Adenine ring of ADP-Heptose), suggesting that the two binding sites interact to report on the occupancy state of the enzyme. ODLA binding was also accompanied by a significant stabilization of HepI (heating to 95 °C fails to denature the protein when in the presence of ODLA). These results suggest that conformational rearrangements, from an induced fit model of substrate binding to HepI, are important for catalysis, and the disruption of these dynamics may serve as novel mechanism for inhibiting this and other glycosyltransferase enzymes.

Insights into Heptosyltransferase I Catalysis and Inhibition through the Structure of Its Ternary Complex

“…While the structure of WaaC in complex with a donor analog, ADP-2-fluoro-D-gluco-heptose (4, Figure 1C) is available (PDB: 2H1H) (Grizot et al, 2006), all attempts to date to obtain structural insights into the acceptor binding site have been unsuccessful, most likely due to the complex nature and chemical/physical properties of ReLPS (5,Figure 1C). Evidence for substantial conformational changes and protein stabilization upon acceptor binding has been obtained through fluorescence studies, which also yielded insights into the dynamics of this process (Cote et al, 2017;Czyzyk et al, 2013). These findings reinforce the need for a structure of a ternary complex to be used in inhibitor design, especially since donor analog-based inhibitors studied so far have proved to be modest binders (Grizot et al, 2006).…”

“…Such an arrangement facilitates the changes in enzyme conformation that are needed to effect catalysis. Indeed, conformational changes induced by acceptor, but not donor, binding have been proposed on the basis of increases in the thermal stability of the enzyme (Cote et al, 2017) as well as on changes in intrinsic fluorescence that are thought to arise from movement of tryptophan residues (Czyzyk et al, 2013). These conformational changes seem to have their origin more in the binding of acceptor than of donor, as seen by comparison of the structure of the complex of WaaC plus ADP2F-gluco-heptose (4) with that 7with the surrounding basic amino acids.…”

Conserved conformational hierarchy across functionally divergent glycosyltransferases of the GT-B structural superfamily as determined from microsecond molecular dynamics

“…HepI N-terminal domain residues 1-152 with C-terminal domain residues 180-322) demonstrate predominantly negatively correlated motions. Specific examination of correlations to residues in HepI that are known to participate in conformational changes associated with binding of the sugar acceptor ligand (including Lys 64, Arg 63 and Arg 120), 15,[26][27] identified a series of glycine and proline residues distant from the binding sites of either substrate (>25 Å away from the binding site of the sugar acceptor but within 15 Å of the sugar donor binding site) with strong negative correlated motions (Pro 216, Pro 240, Gly 280 and Gly 288). Each of these residues was individually mutated, with prolines being introduced where the protein originally had a glycine, and vice versa to yield mutant proteins with the greatest possible perturbation of their conformational flexibility at each position.…”

“…5 Further evidence of conformational changes in GT-Bs upon substrate binding comes from intrinsic tryptophan fluorescence (ITF) experiments of the Gram-negative bacterial enzyme Heptosyltransferase I (HepI, inverting, EC: 2.4.99.B6). 15 HepI catalyzes the addition of the first monosaccharide to the nascent polysaccharide core of the outer membrane lipopolysaccharide (LPS). [16][17][18][19] Sole addition of a Heptose sugar to the acceptor substrate (O-deacylated E. coli Kdo2-lipid A, ODLA) analog resulted in a spectral blue-shift of the enzyme's fluorescence profile.…”

The Glycosyltransferases of LPS Core: A Review of Four Heptosyltransferase Enzymes in Context

“…This suggests that the ODLA induced change in HepI must occur prior to chemistry [ 61 ]. Subsequently, work investigated which Trp residue(s) play(s) a role in the observed blue shift so as to better understand conformational change(s) that occur [ 71 ]. In this work, most of the eight Trp residues were mutated to phenylalanine (Phe).…”