However, many aspects of allostery in TS are still not understood and only a fraction of residues involved in signal propagation from the active site of TrpA to the active site of TrpB (and vice versa) has been identified.Īncestral sequence reconstruction (ASR) is an in silico method that allows one to calculate the most probable sequences of proteins from extinct species with the help of a multiple sequence alignment of extant species and a phylogenetic model ( 12, 13). These properties and a long history of research make the TS an excellent model system for the analysis of allosteric communication within multienzyme complexes ( 9– 11). In order to coordinate these reactions, the TrpA and TrpB subunits mutually activate each other in an allosteric manner ( 8). These subunits catalyze the last 2 steps in the biosynthesis of the essential amino acid tryptophan: TrpA cleaves indole-3-glycerol phosphate (IGP) into glyceraldehyde-3-phosphate (GAP) and the reaction intermediate indole, which is subsequently channeled to the active site of TrpB where it reacts in a pyridoxal phosphate (PLP)-mediated reaction with l-serine to l-tryptophan ( Fig. Tryptophan synthase (TS) is a heterotetrameric αββα complex where a central dimer of 2 β-subunits (TrpB) is surrounded by 2 α-subunits (TrpA) ( 7). It has been shown, for instance, that allosteric signals in protein kinase A ( 4), in imidazole glycerol phosphate synthase ( 5), and in aminodeoxychorismate synthase ( 6) can be disrupted with single point mutations. One possibility to identify such elements is the analysis of specific mutations that disturb communication between the allosteric site and the active site ( 1).

However, allosteric communication between distant sites is likely mediated via multiple paths, which makes it difficult to identify the role of individual structural elements for signal propagation ( 2). Previous studies have indicated that allostery is based on dynamic effects of the protein backbone and of side chains that can be understood by a combination of computational and experimental approaches ( 3, 4). Nevertheless, due to the complexity of the studied systems, there are still plenty of questions open with respect to the structural basis of allostery ( 2). Allosteric communication is central for the regulation of the activity of many enzyme complexes and has been intensely studied with various approaches over the past decades. Our findings demonstrate that ancestral sequence reconstruction can efficiently identify residues contributing to allosteric signal propagation in multienzyme complexes.Īllostery describes the phenomenon that protein function can be altered by the binding of an effector ligand at a site that is topographically distinct from the active site ( 1). The effect of our mutational studies was rationalized by a community analysis based on molecular dynamics simulations. Sequence comparison of these 2 intermediates and iterative rounds of site-directed mutagenesis allowed us to identify 4 of 413 residues from TrpB that are crucial for its allosteric activation by TrpA. Remarkably, the switching from TrpB inhibition to TrpB activation by TrpA occurred between 2 successive TS intermediates. To identify amino acid residues that are responsible for this inversion of the allosteric effect, all 6 evolutionary TrpA and TrpB intermediates that stepwise link LBCA-TS with ncTS were characterized. However, LBCA-TrpA allosterically decreases the activity of LBCA-TrpB, whereas, for example, the modern ncTrpA from Neptuniibacter caesariensis allosterically increases the activity of ncTrpB. LBCA-TS is similar to modern TS by forming a αββα complex with indole channeling taking place.

Previously, the sequences of TrpA and TrpB from the last bacterial common ancestor (LBCA) have been computed by means of ASR and characterized.

We have analyzed this allosteric network by means of ancestral sequence reconstruction (ASR), which is an in silico method to resurrect extinct ancestors of modern proteins. It is characterized by the channeling of the reaction intermediate indole and the mutual activation of the α-subunit TrpA and the β-subunit TrpB via a complex allosteric network. Tryptophan synthase (TS) is a heterotetrameric αββα complex.

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