Abstract
Networks of noncovalent amino acid interactions propagate allosteric signals throughout proteins. Tryptophan synthase (TS) is an allosterically controlled bienzyme in which the indole product of the alpha subunit (αTS) is transferred through a 25 Å hydrophobic tunnel to the active site of the beta subunit (βTS). Previous nuclear magnetic resonance and molecular dynamics simulations identified allosteric networks in αTS important for its function. We show here that substitution of a distant, surface-exposed network residue in αTS enhances tryptophan production, not by activating αTS function, but through dynamically controlling the opening of the indole channel and stimulating βTS activity. While stimulation is modest, the substitution also enhances cell growth in a tryptophan-auxotrophic strain of Escherichia coli compared to complementation with wild-type αTS, emphasizing the biological importance of the network. Surface-exposed networks provide new opportunities in allosteric drug design and protein engineering, and hint at potential information conduits through which the functions of a metabolon or even larger proteome might be coordinated and regulated.
Highlights
Allosteric regulation of protein function is critical for several biological processes, including metabolism, oxygen transport and signal transduction (Wodak et al, 2019)
The cells supplemented with plasmid but not tryptophan experienced a delay in the start of the growth phase
We evaluated frustration for a number of complexes, including Tryptophan synthase (TS) bound with the αTS substrate mimic N-[1H-indol-3-yl-acetyl] aspartic acid (PDB 1K3U), TS bound with the αTS transition state analog 4-(2hydroxy-4-fluorophenylthio)-butylphosphonic acid (PDB 1C9D), TS bound with αTS inhibitor F9 and L-tryptophan in the beta site (PDB 5CGQ) and TS bound with the βTS quinoid intermediate (PDB 3CEP)
Summary
Allosteric regulation of protein function is critical for several biological processes, including metabolism, oxygen transport and signal transduction (Wodak et al, 2019). Amino acid interaction networks have been proposed to connect different functional sites on a protein to propagate these allosteric signals (Dokholyan, 2016). Such networks might bridge across proteins in multi-enzyme complexes, allowing efficient coordination of various enzyme functions (Cong et al, 2019; East et al, 2020). A better understanding of these protein-spanning networks would provide insight into how protein function is allosterically regulated in multi-enzyme complexes, and may offer novel avenues for protein engineering (Lee et al, 2008; Reynolds, et al, 2011; Gorman et al, 2019) and drug design (Nussinov and Chung, 2015; Greener and Sternberg, 2018).
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