pubs.acs.org/Biochemistry Published on Web 11/23/2010 r 2010 American Chemical Society 10842 Biochemistry 2010, 49, 10842–10853 DOI: 10.1021/bi1016815 Ligand-Induced Formation of a Transient Tryptophan Synthase Complex with Rββ Subunit Stoichiometry † Alexander Ehrmann, ‡ Klaus Richter, § Florian Busch, ‡ Julia Reimann, ) Sonja-Verena Albers, ) and Reinhard Sterner* ,‡ ‡ Institute of Biophysics and Physical Biochemistry, University of Regensburg, Universit € atsstrasse 31, D-93053 Regensburg, Germany, § Center for Integrated Protein Science Munich at the Department Chemie, Technische Universit € at M€ unchen, Lichtenbergstrasse 4, D-85747 Garching, Germany, and ) Molecular Biology of Archaea, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse 10, D-35043 Marburg, Germany Received October 19, 2010; Revised Manuscript Received November 17, 2010 ABSTRACT: The prototypical tryptophan synthases form a stable heterotetrameric RββR 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 (R-monomer) and sTrpB2i (ββ-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 μM, indicating that the affinity between the R- and ββ-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 Rββ quaternary structure and testifies to a strong negative cooperativity for the binding of the R-monomers to the ββ-dimer. The analysis of the interaction between sTrpB2i and sTrpA in the presence of several substrate, transition state, and product analogues suggests that the Rββ complex remains stable during the whole catalytic cycle and disintegrates into R- and ββ-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. Tryptophan synthase (TS) 1 is one of the best studied multi- enzyme complexes, which has been used as a model to investigate the structural basis and the functional consequences of protein- protein interactions (1-3). The prototypical TS, as for example found in the mesophilic bacteria Salmonella typhimurium and Escherichia coli, is a stable heterotetrameric RββR complex. The TrpA subunit cleaves IGP into glyceraldehyde 3-phosphate (GA3P) and indole, which migrates through a hydrophobic channel to the active site of the associated TrpB1 subunit where it condenses with L-serine to L-tryptophan in a pyridoxal phosphate (PLP) dependent reaction (Figure 1). Isolated TrpA and TrpB1 proteins form stable but poorly active R-monomers and ββ-homodimers, which in the context of the RββR complex mutually stimulate each other. The allosteric coupling of the TrpA and TrpB1 re- actions is transmitted via conformational changes, which mainly involve the flexible βR-loops 2 and 6 of TrpA and the “COMM” domain of TrpB1 (4, 5). Both subunits can switch between open (catalytically inactive) and closed (catalytically active) conforma- tions (6, 7) whose equilibrium is influenced by TrpA and TrpB ligands (8, 9), pH (9, 10), temperature (8, 9), organic solvents (11), and hydrostatic pressure (12). Furthermore, the TrpB1 subunit of TS from S. typhimurium can bind a monovalent cation at a distance of 8 A ˚  from the PLP cofactor, which stabilizes the open conformation (13, 14). The trpA and trpB1 genes are located next to each other within the tryptophan operon. Recently, a new family of trpB2 genes was identified in the genomes of several microorganisms and plants (15, 16). This family can be further divided into the trpB2i (located within the tryptophan operon) and the trpB2o/trpB2a (located outside the operon) subfamilies. The deduced amino acid sequence identities within and between the TrpB1 and TrpB2 families are about 60% and 30%, respectively (15, 16). Accord- ingly, within the COG (clusters of orthologous groups of proteins) database (17), the TrpB1 proteins belong to a different group (COG 0133) than the TrpB2 proteins, which have been classified as “alternative” TS β subunits (COG 1350). In order to † This work was sponsored by the German Research Foundation DFG (BA3943/1-1). J.R. and S.-V.A. were supported by the Dutch Science Organization NWO (VIDI Grant 864.05.005) and intramural funds of the Max Planck Society. *To whom correspondence should be addressed: telephone, þ49-941- 943-3015; fax, þ49-941-943-2813; e-mail, Reinhard.Sterner@biologie. uni-regensburg.de. 1 Abbreviations: TS, tryptophan synthase; TrpA and TrpB1, R- and β 2 -subunits of canonical TS complexes; tmTrpA and tmTrpB1, R- and β 2 -subunits of TS from Thermotoga maritima; sTrpA and sTrpB2i, R- and β 2 -subunits of TS from Sulfolobus solfataricus.