Millisecond Timescale Motions Connect Amino Acid Interaction Networks in Alpha Tryptophan Synthase

Abstract

Tryptophan synthase is a model system for understanding allosteric regulation within enzyme complexes. Amino acid interaction networks were previously delineated in the isolated alpha subunit (αTS) in the absence of the beta subunit (βTS). The amino acid interaction networks were different between the ligand-free enzyme and the enzyme actively catalyzing turnover. Previous X-ray crystallography studies indicated only minor localized changes when ligands bind αTS, and so, structural changes alone could not explain the changes to the amino acid interaction networks. We hypothesized that the network changes could instead be related to changes in conformational dynamics. As such, we conducted nuclear magnetic resonance relaxation studies on different substrate- and products-bound complexes of αTS. Specifically, we collected ¹⁵N R₂ relaxation dispersion data that reports on microsecond-to-millisecond timescale motion of backbone amide groups. These experiments indicated that there are conformational exchange events throughout αTS. Substrate and product binding change specific motional pathways throughout the enzyme, and these pathways connect the previously identified network residues. These pathways reach the αTS/βTS binding interface, suggesting that the identified dynamic networks may also be important for communication with the βTS subunit.

Keywords: allostery; amino acid networks; enzyme dynamics; enzyme regulation; protein NMR; relaxation dispersion; tryptophan synthase.

Figure 1

The structure of αTS. These X-ray crystal structures are from Salmonella typhimurium, which has 85% sequence identity with E.coli αTS used in the NMR experiments. The blue and pink structures represent the ligand-free (PDB 1KFJ) and IGP-bound (PDB 2RHG) states, respectively. Binding of IGP substrate does not substantially change the ground-state αTS structure in the αTS-βTS tryptophan synthase complex.

Figure 2

Conformational exchange events in the E.coli αTS enzyme (A) in the apo resting state, (B) bound to product indole, (C) bound to the product glyceraldehyde-3-phosphate (G3P) and (D) in the working state under catalytic turnover. The working state represents a 4:1 ratio of enzyme bound with substrate indole-3-glycerol phosphate (IGP) to enzyme bound with products indole and G3P (Axe and Boehr, ; Axe et al., 2014). (left) example ¹⁵N R₂ relaxation dispersion curves collected at a ¹H Larmor frequency of 850 MHz for the resonances belonging to Ala9 (black), Ala18 (green), Ile166 (blue), and Ala198 (purple). (middle) locations of conformational exchange according to the R₂ relaxation dispersion experiments plotted as spheres onto the αTS structure. Here, we used the S.typhimurium αTS structure bound to glyceraldehyde-3-phosphate (PDB 2CLK) as it contains resolved β2α2 and β6α6 loops. Purple spheres indicate that associated R₂ relaxation dispersion curves can be fit to two-site exchange, while pink spheres indicate exchange broadening, but the R₂ relaxation dispersion curves cannot be fit reliably to two-site exchange. (right) a comparison of the conformational exchange events in the resting apo state compared to when αTS is bound with ligands. Blue (red) spheres indicate conformational exchange events present in the apo (ligand-bound) state but not in the ligand-bound (apo) state. Most of the amino acid residues associated with a change in conformational dynamics make contact and/or near each other in three-dimensional space. The R₂ relaxation dispersion experiments were conducted at 283 K using a buffer consisting of 50 mM potassium phosphate, pH 7.8, 2 mM DTT, 0.2 mM Na₂EDTA, and 10% ²H₂O, and 0.5–1.0 mM protein with 10 mM indole and/or 20 mM G3P where appropriate.

Figure 3

Comparison of conformational exchange events identified by ¹⁵N R₂ relaxation dispersion experiments and a residue cluster identified by chemical shift covariance analysis (CHESCA) for the (A) apo resting and (B) working states of E. coli αTS. The working state represents active catalytic turnover conditions in which there is a 4:1 ratio of enzyme bound with substrate indole-3-glycerol phosphate (IGP) to enzyme bound with products indole and G3P (Axe and Boehr, ; Axe et al., 2014). Yellow spheres indicate μs-ms timescale conformational exchange events identified by ¹⁵N R₂ relaxation dispersion experiments (i.e., pink and purple spheres in Figure 2), blue spheres indicate residues belonging to the previously identified CHESCA cluster, and green spheres indicate CHESCA cluster residues that show conformational exchange. The same information is presented on (left) the αTS structure and (right) the full TS complex (PDB 2CLK). It should be noted that all NMR data were collected in the absence of βTS. Nonetheless, there are conformational exchange events and CHESCA cluster residues near the βTS-binding interface, which may be important in the context of the full TS complex. There are more CHESCA cluster residues showing conformational exchange in the working state compared to the resting state.

Figure 4

Connections between CHESCA network and exchanging residues in the working state enzyme. Residues that show millisecond conformational exchange according to ¹⁵N R₂ relaxation dispersion studies are boxed in yellow, residues that were previously identified to be CHESCA network residues (Axe et al., 2014) are boxed in blue, and residues that are CHESCA network residues and show conformational exchange are boxed in green. Lines indicate the type of interaction between the residues, including hydrogen bond (pink line), a hydrophobic interaction (cyan line), covalently attached (black line), or within at least 6 Å distance where side chain motion may lead to contact. CHESCA network residues either undergo conformational exchange or interact with residues that undergo conformational exchange.