title: 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.
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich, CH-8093, Zurich, Switzerland.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Roderer
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich, CH-8093, Zurich, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Neuenschwander
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich, CH-8093, Zurich, Switzerland.
authors.forename: Giosiana
authors.initials: G
authors.lastname: Codoni
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich, CH-8093, Zurich, Switzerland.
authors.forename: Severin
authors.initials: S
authors.lastname: Sasso
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich, CH-8093, Zurich, Switzerland.
authors.forename: Marianne
authors.initials: M
authors.lastname: Gamper
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich, CH-8093, Zurich, Switzerland.
authors.forename: Peter
authors.initials: P
authors.lastname: Kast
mesh_terms: D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000378:metabolism; D019943:Amino Acid Substitution; D001483:Base Sequence; D002138:Calibration; D002826:Chorismate Mutase / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods*; D015723:Gene Library; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D011897:Random Allocation
chemical_list: D009097:Multienzyme Complexes; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D002826:Chorismate Mutase