Identification of the Geometric Requirements for Allosteric Communication between the α- and β-Subunits of Tryptophan Synthase

Abstract

The pyridoxal 5-phosphate-dependent tryptophan synthase ␣ 2 ␤ 2 complex is a paradigmatic protein for substrate channeling and allosteric regulation. The enzymatic activity is modulated by a ligand-mediated equilibrium between open (inactive) and closed (active) conformations of the ␣-and ␤-subunit, predominantly involving the mobile ␣ loop 6 and the ␤-COMM domain that contains ␤ helix 6. The ␣ ligand-triggered intersubunit communication seems to rely on a single hydrogen bond formed between the carbonyl oxygen of ␤Ser-178 of ␤ helix 6 and the NH group of ␣Gly-181 of ␣ loop 6. We investigated whether and to what extent mutations of ␣Gly-181 and ␤Ser-178 affect allosteric regulation by the replacement of ␤Ser-178 with Pro or Ala and of ␣Gly-181 with either Pro to remove the amidic proton that forms the hydrogen bond or Ala, Val, and Phe to analyze the dependence on steric hindrance of the open-closed conformational transition. The ␣ and ␤ activity assays and the equilibrium distribution of ␤-subunit catalytic intermediates indicate that mutations do not significantly influence the intersubunit catalytic activation but completely abolish ligand-induced ␣-to ␤-subunit signaling, demonstrating distinct pathways for ␣-␤-site communication. Limited proteolysis experiments indicate that the removal of the interaction between ␤Ser-178 and ␣Gly-181 strongly favors the more trypsin-accessible open conformation of the ␣-active site. When the hydrogen bond cannot be formed, the ␣-subunit is unable to attain the closed conformation, and consequently, the allosteric signal is aborted at the subunit interface.Regulation of enzyme activity by interaction with metabolites is a key feature for the maintenance of cell life. This goal is achieved through conformational changes that, depending on protein complexity, involve tertiary and/or quaternary states. A common event that accompanies ligand binding to proteins is the transition between open and closed states (Ref. 1 and the references therein). Usually, the closed state is a more active conformation, bringing substrates and catalytic residues in close proximity, conferring special features to the active site environment, and protecting the productive catalysis from side reactions, generally involving water molecules.The ␣ 2 ␤ 2 complex of tryptophan synthase (TS) 1 represents a particularly interesting case (2) because the open to closed transition serves a dual role: the increase in catalytic activity and the allosteric communication between subunits (3-9). The enzyme catalyzes the biosynthesis of L-tryptophan in two steps: the ␣-subunit cleaves indole-3-glycerol phosphate to D-glyceraldehyde-3-phosphate and indole, which, intramolecularly channeled to the ␤-subunit (10, 11), reacts with L-serine in a ␤ replacement reaction. The ␤-subunit contains a pyridoxal 5Ј-phosphate bound to Lys-87 as an internal aldimine (12). During catalysis, a series of chromophoric intermediates are formed, including an external aldimine, an ␣-aminoacrylate and quinonoid species (Scheme 1). The cat...

Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation

“…These positions comprise just 31% of the protein sequence, indicating modest enrichment within residues in the immediate spatial route between the α/β interface and the β-subunit active site. In contrast to the many mutations that have been identified as deleterious to the allosteric communication in TrpS (29,30), the mutations identified here using directed evolution are the first reported to affect allosteric communication and increase the activity of TrpB in isolation.…”

Tryptophan synthase: a mine for enzymologists

“…In the absence of a-subunit allosteric effectors, a-loop6 is disordered and not detectable by X-ray crystallography [12]. Upon binding of a-ligands, such as IPP, GP, IAG, IAD and indoline-G3P adduct, a strong hydrogen bond is formed between the NH of aGly181 and the carbonyl moiety of bSer178 of b-helix6 [13,83,84], resulting in the stabilization of the a-loop6 in a conformation that covers the a-active site (Fig. 1b, c).…”

“…Furthermore, limited proteolysis experiments on the wild-type enzyme and mutants of aGly181 and bSer178 were carried out indicating that, in the absence of the hydrogen bond between aX181 and bX178, a-loop6 remains in the open conformation (Fig. 1c) and regulatory signals to the b-active site are knocked out [84]. To gain insight on the position of a-loop6 in the absence of allosteric ligands, molecular dynamics simulations were carried out on the wild-type enzyme and mutants [85,86].…”

“…This communication is predominantly achieved via the key interaction between aGly181 of the a-loop6 and bSer178 of b-helix6 (Fig. 1b, c) [13,14,[83][84][85]. In the absence of a-subunit allosteric effectors, a-loop6 is disordered and not detectable by X-ray crystallography [12].…”

“…In turn, aThr183 is able to interact with the catalytic residue aAsp60 and moves the ligand towards the other a-subunit catalytic residue aGlu49 [16], favoring catalysis. Several mutants of aGly181, aThr183 and bSer178 were prepared and their functional and regulatory properties characterized [77,[83][84][85][86], indicating their relevance for the allosteric regulation. Furthermore, limited proteolysis experiments on the wild-type enzyme and mutants of aGly181 and bSer178 were carried out indicating that, in the absence of the hydrogen bond between aX181 and bX178, a-loop6 remains in the open conformation (Fig.…”