Atomic-resolution chemical characterization of (2x)72-kDa tryptophan synthase via four- and five-dimensional 1H-detected solid-state NMR.

NMR chemical shifts provide detailed information on the chemical properties of molecules, thereby complementing structural data from techniques like X-ray crystallography and electron microscopy. Detailed analysis of protein NMR data, however, often hinges on comprehensive, site-specific assignment of backbone resonances, which becomes a bottleneck for molecular weights beyond 40 to 45 kDa. Here, we show that assignments for the (2x)72-kDa protein tryptophan synthase (665 amino acids per asymmetric unit) can be achieved via higher-dimensional, proton-detected, solid-state NMR using a single, 1-mg, uniformly labeled, microcrystalline sample. This framework grants access to atom-specific characterization of chemical properties and relaxation for the backbone and side chains, including those residues important for the catalytic turnover. Combined with first-principles calculations, the chemical shifts in the ß-subunit active site suggest a connection between active-site chemistry, the electrostatic environment, and catalytically important dynamics of the portal to the ß-subunit from solution.

Mutation of ßGln114 to Ala Alters the Stabilities of Allosteric States in Tryptophan Synthase Catalysis.

The tryptophan synthase (TS) bienzyme complexes found in bacteria, yeasts, and molds are pyridoxal 5'-phosphate (PLP)-requiring enzymes that synthesize l-Trp. In the TS catalytic cycle, switching between the open and closed states of the α- and ß-subunits via allosteric interactions is key to the efficient conversion of 3-indole-d-glycerol-3'-phosphate and l-Ser to l-Trp. In this process, the roles played by ß-site residues proximal to the PLP cofactor have not yet been fully established. ßGln114 is one such residue. To explore the roles played by ßQ114, we conducted a detailed investigation of the ßQ114A mutation on the structure and function of tryptophan synthase. Initial steady-state kinetic and static ultraviolet-visible spectroscopic analyses showed the Q to A mutation impairs catalytic activity and alters the stabilities of intermediates in the ß-reaction. Therefore, we conducted X-ray structural and solid-state nuclear magnetic resonance spectroscopic studies to compare the wild-type and ßQ114A mutant enzymes. These comparisons establish that the protein structural changes are limited to the Gln to Ala replacement, the loss of hydrogen bonds among the side chains of ßGln114, ßAsn145, and ßArg148, and the inclusion of waters in the cavity created by substitution of the smaller Ala side chain. Because the conformations of the open and closed allosteric states are not changed by the mutation, we hypothesize that the altered properties arise from the lost hydrogen bonds that alter the relative stabilities of the open (ßT state) and closed (ßR state) conformations of the ß-subunit and consequently alter the distribution of intermediates along the ß-subunit catalytic path.

Toho-1 ß-lactamase: backbone chemical shift assignments and changes in dynamics upon binding with avibactam.

Backbone chemical shift assignments for the Toho-1 ß-lactamase (263 amino acids, 28.9 kDa) are reported based on triple resonance solution-state NMR experiments performed on a uniformly 2H,13C,15N-labeled sample. These assignments allow for subsequent site-specific characterization at the chemical, structural, and dynamical levels. At the chemical level, titration with the non-ß-lactam ß-lactamase inhibitor avibactam is found to give chemical shift perturbations indicative of tight covalent binding that allow for mapping of the inhibitor binding site. At the structural level, protein secondary structure is predicted based on the backbone chemical shifts and protein residue sequence using TALOS-N and found to agree well with structural characterization from X-ray crystallography. At the dynamical level, model-free analysis of 15N relaxation data at a single field of 16.4 T reveals well-ordered structures for the ligand-free and avibactam-bound enzymes with generalized order parameters of ~ 0.85. Complementary relaxation dispersion experiments indicate that there is an escalation in motions on the millisecond timescale in the vicinity of the active site upon substrate binding. The combination of high rigidity on short timescales and active site flexibility on longer timescales is consistent with hypotheses for achieving both high catalytic efficiency and broad substrate specificity: the induced active site dynamics allows variously sized substrates to be accommodated and increases the probability that the optimal conformation for catalysis will be sampled.

Backbone assignments and conformational dynamics in the S. typhimurium tryptophan synthase α-subunit from solution-state NMR.

Backbone assignments for the isolated α-subunit of Salmonella typhimurium tryptophan synthase (TS) are reported based on triple resonance solution-state NMR experiments on a uniformly 2H,13C,15N-labeled sample. From the backbone chemical shifts, secondary structure and random coil index order parameters (RCI-S2) are predicted. Titration with the 3-indole-D-glycerol 3'-phosphate analog, N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), leads to chemical shift perturbations indicative of conformational changes from which an estimate of the dissociation constant is obtained. Comparisons of the backbone chemical-shifts, RCI-S2 values, and site-specific relaxation times with and without F9 reveal allosteric changes including modulation in secondary structures and loop rigidity induced upon ligand binding. A comparison is made to the X-ray crystal structure of the α-subunit in the full TS αßßα bi-enzyme complex and to two new X-ray crystal structures of the isolated TS α-subunit reported in this work.