Functional Mapping of Protein-Protein Interactions in an Enzyme Complex by Directed Evolution

Abstract

The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM) of Mycobacterium tuberculosis is poorly active on its own, but becomes >100-fold more efficient upon formation of a complex with the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS). The crystal structure of the enzyme complex revealed involvement of C-terminal MtCM residues with the MtDS interface. Here we employed evolutionary strategies to probe the tolerance to substitution of the C-terminal MtCM residues from positions 84–90. Variants with randomized positions were subjected to stringent selection in vivo requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQδ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQδ sequences, was intolerant to mutation, whereas Leu88 and Gly89 exhibited a preference for small and hydrophobic residues in functional MtCM-MtDS complexes. In the absence of MtDS, selection under relaxed conditions identifies positions 84–86 as MtCM integrity determinants, suggesting that the more C-terminal residues function in the activation by MtDS. Several MtCM variants, purified using a novel plasmid-based T7 RNA polymerase gene expression system, showed that a diminished ability to physically interact with MtDS correlates with reduced activatability and feedback regulatory control by Tyr and Phe. Mapping critical protein-protein interaction sites by evolutionary strategies may pinpoint promising targets for drugs that interfere with the activity of protein complexes.

Complex Formation between Two Biosynthetic Enzymes Modifies the Allosteric Regulatory Properties of Both

“…Although these observations of the complex have been made from in vitro experiments, the in vivo importance of the interaction and activity boost has been recently exposed in an elegant directed evolution study where the activity of MtuCM and the activity boost afforded by MtuDAH7PS are reported by an E. coli selection system (16). Similarly, interactions between DAH7PS and CM are reported for pairings from a number of organisms including Corynebacterium glutamicum, Amycolaptoposis methanolica, and Brevibacterium flavum (27)(28)(29)(30).…”

“…Whereas MtuDAH7PS does not directly contribute residues to the MtuCM catalytic site, several MtuCM residues, most notably from the loop between helix 1 and 2, are repositioned on complex formation. In addition, residues of the C-terminal tail have been observed to be important for activating complex formation (15), and, moreover, activation by Mtu-DAH7PS of MtuCM variants appears to be correlated with Phe and Tyr inhibition (16). These rearrangements enhance the maximal rate and significantly lower the Michaelis constant of MtuCM.…”

“…2). More recently the C-terminal region of the MtuCM has been demonstrated to be important for the interaction with and activation by MtuDAH7PS (16).…”

Inter-Enzyme Allosteric Regulation of Chorismate Mutase in Corynebacterium glutamicum: Structural Basis of Feedback Activation by Trp

“…The DS activity of MtDS is synergistically inhibited by the end products of the pathway (Phe, Tyr, and Trp), whereas the CM activity of the MtCM-MtDS complex, but not of MtCM alone, is synergistically inhibited by Phe and Tyr (32)(33)(34)(35)(36). In an evolutionary study on the MtCM-MtDS complex, we 6 could show that the activation factor by MtDS of MtCM variants is directly correlated with their response to the feedback inhibitors Tyr and Phe (37). interface (36).…”

Remote Control by Inter-Enzyme Allostery: A Novel Paradigm for Regulation of the Shikimate Pathway

“…Remarkably, the DS activity in this bacterium is not regulated by aromatic amino acids [27]. Recent reports on shikimate pathway enzymes in Mycobacterium tuberculosis have shown that nature also uses an ingenious alternative strategy for allosteric control of the flux through the shikimate pathway [28][29][30].…”