Nature of allosteric inhibition in glutamate racemase: discovery and characterization of a cryptic inhibitory pocket using atomistic MD simulations and pKa calculations

Abstract

Enzyme inhibition via allostery, in which the ligand binds remotely from the active site, is a poorly understood phenomenon and represents a significant challenge to structure-based drug design. Dipicolinic acid (DPA), a major component of Bacillus spores, is shown to inhibit glutamate racemase from Bacillus anthracis , a monosubstrate/monoproduct enzyme, in a novel allosteric fashion. Glutamate racemase has long been considered an important drug target for its integral role in bacterial cell wall synthesis. The DPA binding mode was predicted via multiple docking studies and validated via site-directed mutagenesis at the binding locus, while the mechanism of inhibition was elucidated with a combination of Blue Native polyacrylamide gel electrophoresis, molecular dynamics simulations, and free energy and pK(a) calculations. Inhibition by DPA not only reveals a novel cryptic binding site but also represents a form of allosteric regulation that exploits the interplay between enzyme conformational changes, fluctuations in the pK(a) values of buried residues and catalysis. The potential for future drug development is discussed.

Figure 1

Experimental results for noncompetitive inhibition of RacE2_WT (a), RacE2_S207A (b) and RacE2_K106A (c) by DPA. Kinetic data was attained via circular dichroism and three independent Michaelis-Menten curves were globally fit to a noncompetitive inhibition model to produce the presented K_i values (further details in Supporting Information).

Figure 2

Superpose of top-docked positions of DPA (space-filling) to the RacE2 dimer (ribbon, 2GZM) as determined by GOLD v4.1 (magenta), Autodock v4 (blue) and FRED v2.2.5 (green) (a). The binding pocket is located at the dimer interface and is composed of residues from both monomers, as detailed by the interaction map (b). After minimization, the backbone contact of Ser207 is swapped for a contact with the beta-hydroxyl group (c). After MD simulation of the top docked complex with Ser207 replaced by Ala, the binding site lacks any contact with the region previously containing Ser207 (d). Letters immediately preceding the residue numbers indicate the monomer, A or B. Ligand interaction maps were constructed using the LigX function of MOE v2009.10.

Figure 3

Superpose of a variety of GR structures in order to highlight the diversity of known allosteric positions. Superpose of DPA bound to B. a. RacE2, UDP-MurNAc-L-Ala bound to E. c. GR (2JFN), and pyrazolopyrimidinedione analogue (aka Compound A) bound to H. p. GR (2JFZ). Only the trace of one monomer is shown for clarity and the perspective is looking at the enzyme face directly opposite of the entrance to the active site. D-glutamate (yellow) is seen in the background bound to the active site, while DPA (green), UDP-MurNAc-L-Ala (magenta), and Compound A (red) are seen in the foreground. All three cryptic binding sites are distinct. Indicated are center of mass distances between molecules. There is no evidence that any one GR structure possesses all of these allosteric pockets. Rather, the figure is meant to illustrate the distinctive positions and identities of these three different effectors relative to the glutamate binding pocket.

Figure 4

BN-PAGE to determine the oligomerization of wild-type and mutant RacE2. Wildtype RacE2 and RacE2_S207A (a) or RacE2_K106A (b) were run side-by-side at concentrations varying from 45 to 180 µg/mL. Albumin and carbonic anhydrase were included as running controls. Arrowheads indicate bands representing the dimer and monomer. Band intensity was quantified via pixel counting and the ratio of monomer to dimer was plotted against protein concentration for RacE2_S207A (solid line = WT, dotted line = mutant; c) and RacE2_K106A (d). Data represents an average of three or more independent trials with standard error shown. Data was additionally fitted to the expression for M/C ratio as a function of total protein concentration and the monomer:dimer equilibrium constant (see Supplementary Methods for derivation of this expression and model fitting parameters).The results indicate that the two mutants do not have any significant effect on the oligomeric equilibrium. Additionally, see Figure S3 for BN-PAGE of RacE2 and running controls with NativeMark ladder.

Figure 5

Ligand interaction maps for glutamate bound to monomer A of the DPA-lacking RacE2 complex (a) and DPA-bound RacE2 complex (b), as well as DPA bound to the cryptic binding site located at the RacE2 dimer interface (c). Maps were generated from the final structures of 20-nanosecond MD simulations using the LigX function of MOE v2009.10. Predicted binding energy of glutamate was averaged over the set of representative structures extracted from MD simulations of the binary (red, n=15) and ternary complex (blue, n=4, d), error bars = SEM. The details of the binding energy calculations are outlined in the Computational Procedures section of Material and Methods.

Figure 6

Distribution of pK_a values calculated for Cys74 (the catalytic base of D → L racemization) for E₂·D-glu₂ and E₂·D-glu₂·DPA. Each structure, selected via QR factorization of a collection of simulation snapshots, was each used in pKa calculation using the MEAD algorithm from the H++ program. Details of parameters employed in these pKa calculations are located in the Computational Procedures section of the Materials and Methods.

Figure 7

Schematic of life cycle of differentiating B. anthracis cell, with Ca²⁺-DPA levels and consequent GR activity indicated.