In Silico Identification and Experimental Validation of Distal Activity-Enhancing Mutations in Tryptophan Synthase

Abstract

Allostery is a central mechanism for the regulation of multi-enzyme complexes. The mechanistic basis that drives allosteric regulation is poorly understood but harbors key information for enzyme engineering. In the present study, we focus on the tryptophan synthase complex that is composed of TrpA and TrpB subunits, which allosterically activate each other. Specifically, we develop a rational approach for identifying key amino acid residues of TrpB distal from the active site. Those residues are predicted to be crucial for shifting the inefficient conformational ensemble of the isolated TrpB to a productive ensemble through intra-subunit allosteric effects. The experimental validation of the conformationally driven TrpB design demonstrates its superior stand-alone activity in the absence of TrpA, comparable to those enhancements obtained after multiple rounds of experimental laboratory evolution. Our work evidences that the current challenge of distal active site prediction for enhanced function in computational enzyme design has become within reach.

Dynamic allosteric networks drive adenosine A1receptor activation and G-protein coupling

“…[29] First, we use the get Residue Interaction eNergies and Networks (gRINN) [29] tool to calculate the pairwise residue interaction energies along the A1R-ADO conformational ensemble and obtain the mean interaction energy matrix (IEM). Second, we processed the mean IEM into the shortest-path map (SPN) [26,30,31] tool to construct and visualize the PEN graph. The analysis of the PEN associated with the A1R-ADO ensemble shows that the extracellular region can communicate with the intracellular region through multiple energy pathways (see Figure 2 A).…”

“…A complete understanding of allosteric modulation involves the decoding of the communication pathways that dynamically couples distinct protein sites. Despite the difficulties, network theory has been successfully applied to uncover the allosteric communication pathways in protein complexes [23, 26], including GPCRs [27, 28]. In order to trace down the allosteric pathways in A 1 R-ADO, we relied on the protein energy networks (PEN) approach.…”

“…[30] First, we use the get Residue Interaction eNergies and Networks (gRINN)[30] tool to calculate the pairwise residue interaction energies along the A 1 R-ADO conformational ensemble and obtain the mean interaction energy matrix (IEM). Second, we processed the mean IEM into the shortest-path map (SPN) [27, 31, 32] tool to construct and visualize the PEN graph.…”

“…To study the allosteric communication by means of protein energy networks (PEN) we processed the normalized mean IEM into to Shortest Path Map (SPM) tool. [27, 31, 32] The SPM algorithm first constructs a network graph in which only the pair of residues with a normalized mean interaction energy (IE) higher than 0.1 are considered as nodes. The length of the edge connecting pair of residues (nodes) is drawn according to their normalized mean IE value (d ij =-log |IE ij |).…”

Allosteric rescue of catalytically impaired ATP phosphoribosyltransferase variants links protein dynamics to active-site electrostatic preorganisation

“…29 Therefore, any structural or conformational differences that may lead to allosteric rescue of R56A-PaHisGS catalysis are not captured in the static view of the crystal structure. The curious conformational behavior of the activated dimer was further explored using the shortest path map (SPM) approach, 39 which enables the identification of pairs of residues in both the active site and distal positions 40 which could be expected to in turn facilitate key interactions between R56 and PRPP across the dimer. This is in overall agreement with the allosteric activation mechanism gleaned from the crystal structures of PaHisGS and PaATPPRT.…”