Ligand-Induced Formation of a Transient Tryptophan Synthase Complex with αββ Subunit Stoichiometry

The prototypical tryptophan synthases form a stable heterotetrameric alpha beta beta alpha complex in which the constituting TrpA and TrpB1 subunits activate each other in a bidirectional manner. The hyperthermophilic archaeon Sulfolobus solfataricus does not contain a TrpB1 protein but instead two members of the phylogenetically distinct family of TrpB2 proteins, which are encoded within (sTrpB2i) and outside (sTrpB2a) the tryptophan operon. It has previously been shown that sTrpB2a does not functionally or structurally interact with sTrpA, whereas sTrpB2i substantially activates sTrpA in a unidirectional manner. However, in the absence of catalysis, no physical complex between sTrpB2i and sTrpA could be detected. In order to elucidate the structural requirements for complex formation, we have analyzed the interaction between sTrpA (alpha-monomer) and sTrpB2i (beta beta-dimer) by means of spectroscopy, analytical gel filtration, and analytical ultracentrifugation, as well as isothermal titration calorimetry. In the presence of the TrpA ligand glycerol 3-phosphate (GP) and the TrpB substrate L-serine, sTrpA and sTrpB2i formed a physical complex with a thermodynamic dissociation constant of about 1 mu M, indicating that the affinity between the alpha- and beta beta-subunits is weaker by at least 1 order of magnitude than the affinity between the corresponding subunits of prototypical tryptophan synthases. The observed stoichiometry of the complex was 1 subunit of sTrpA per 2 subunits of sTrpB2i, which corresponds to a alpha beta beta quaternary structure and testifies to a strong negative cooperativity for the binding of the alpha-monomers to the beta beta-dimer. The analysis of the interaction between sTrpB2i and sTrpA in the presence of several substrate, transition state, and product analogues suggests that the alpha beta beta complex remains stable during the whole catalytic cycle and disintegrates into alpha- and beta beta-subunits upon the release of the reaction product tryptophan. The formation of a transient tryptophan synthase complex, together with the observed low affinity of sTrpB2i for L-serine, couples the rate of tryptophan biosynthesis in S. solfataricus to the cytosolic availability of L-serine.