Abstract
Tryptophan synthase (TS) is a heterotetrameric alpha beta beta alpha complex. It is characterized by the channeling of the reaction intermediate indole and the mutual activation of the alpha-subunit TrpA and the beta-subunit TrpB via a complex allosteric network. We have analyzed this allosteric network by means of ancestral sequence reconstruction (ASR), which is an in silico method to resurrect ...
Abstract
Tryptophan synthase (TS) is a heterotetrameric alpha beta beta alpha complex. It is characterized by the channeling of the reaction intermediate indole and the mutual activation of the alpha-subunit TrpA and the beta-subunit TrpB via a complex allosteric network. 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. Previously, the sequences of TrpA and TrpB from the last bacterial common ancestor (LBCA) have been computed by means of ASR and characterized. LBCA-TS is similar to modern TS by forming a appa complex with indole channeling taking place. 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. 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. Remarkably, the switching from TrpB inhibition to TrpB activation by TrpA occurred between 2 successive TS intermediates. 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. The effect of our mutational studies was rationalized by a community analysis based on molecular dynamics simulations. Our findings demonstrate that ancestral sequence reconstruction can efficiently identify residues contributing to allosteric signal propagation in multienzyme complexes.
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