Functional mapping of protein-protein interactions in an enzyme complex by directed evolution

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.ISSN:1932-620

DAHP synthase and chorismate mutase reactions and structure of the MtCM-MtDS complex.

<p>(A) The shikimate pathway starts out with the condensation of d-erythrose-4-phosphate <b>1</b> and phospho<i>enol</i>pyruvate <b>2</b> to form d-<i>arabino</i>-heptulosonate-7-phosphate (DAHP) <b>3</b> catalyzed by DAHP synthase. DAHP is processed in six further enzymatic steps to chorismate <b>4</b>. Chorismate mutase (CM) catalyzes the pericyclic Claisen rearrangement from <b>4</b><i>via</i> the presumed bicyclic transition state <b>5</b> to prephenate <b>6</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone.0116234-Sogo1" target="_blank">[46]</a>. A mimic of this transition state, Bartlett’s <i>endo</i>-oxabicyclic dicarboxylic acid transition state analog <b>7</b> is the best known inhibitor of most CMs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone.0116234-Bartlett1" target="_blank">[47]</a>. (B) Hetero-octameric complex between MtCM and MtDS. MtDS forms a tetrameric core (shown in surface representation) which is flanked by two MtCM dimers (cartoon mode with α-helices represented as cylinders featuring <b>7</b> as a stick model with grey carbons in the active sites) that clamp the MtDS tetramerization interface (PDB: 2W1A) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone.0116234-Sasso1" target="_blank">[10]</a>.</p

Comparison of free and MtDS-complexed MtCM.

<p>Superimposition in cartoon mode of free MtCM (white, PDB: 2VKL, active site liganded with malate) and MtCM from the complex with MtDS (MtCM subunits in blue/orange, PDB: 2W1A, active site liganded with <b>7</b> shown as sticks with black carbons). MtDS is omitted for clarity. (A) Overview with labeled N and C termini. Only the active site ligand of MtCM in PDB: 2W1A, the transition state analog <b>7</b>, is shown. (B) Wall-eyed stereogram of the superimposed active sites, with both liganded malate (sticks with green carbons) and <b>7</b>. Side chains of some active site residues, which change location upon complex formation, are shown as sticks <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone.0116234-Sasso1" target="_blank">[10]</a>.</p

Overview of the plasmid-based T7 RNA polymerase gene expression system.

<p>(A) Map and relevant promoter sequence of library plasmid pKT-CM. This plasmid was used for both <i>in</i><i>vivo</i> selection (relying on P<i>_tet</i>, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone-0116234-g004" target="_blank">Fig. 4B</a>) and <i>in</i><i>vitro</i> overproduction of the MtCM variants (using P_T7). The sequence of the P<i>_tet</i>P_T7 tandem promoter is given with the binding sites for the Tet-responsive TetR repressor highlighted in bold italics and the start codons of the reading frames in bold roman type <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone.0116234-Bertrand1" target="_blank">[49]</a>; underlined are relevant restriction sites, the ribosomal binding site (RBS<i>_tet</i>), −35 and −10 regions of P<i>_tet</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone.0116234-Bertrand1" target="_blank">[49]</a> and the RBS_T7 and promoter P_T7 from phage T7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone.0116234-Rosenberg1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone.0116234-Imburgio1" target="_blank">[50]</a>; start and direction of transcription is marked by an arrowhead <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone.0116234-Neuenschwander2" target="_blank">[22]</a>. (B) Map and relevant promoter sequence of the T7 RNA polymerase plasmid pT7POLTS. The plasmid carries the p15A origin of replication derived from pACYC184 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone.0116234-Chang1" target="_blank">[51]</a>, <i>cat</i> encoding chloramphenicol acetyltransferase, and a P<i>_tet</i> controlled T7 RNA polymerase gene (<i>T7pol</i>) translationally fused at its 3′ end to the sequence for the SsrA degradation tag. In the absence of Tet, TetR binding to its operator sites (highlighted as in panel A) blocks gene expression from P<i>_tet</i> and any T7 RNA polymerase produced due to low-level leaky transcription is effectively eliminated by SsrA-mediated Clp proteolysis, thereby suppressing basal polymerase activity. Provision of Tet releases TetR from the operator, resulting in intracellular polymerase levels higher than can be degraded efficiently by the Clp proteases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone.0116234-Andersen1" target="_blank">[41]</a>. For efficient translation, the alternative RBS_alt can be used. The accumulating polymerase then directs massive transcription from P_T7 controlled genes, such as <i>aroQ_δ</i> on pKT-CM. The entire nucleotide sequence of pT7POLTS is provided as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone.0116234.s002" target="_blank">S2 Fig</a>.</p

Amino acid distribution patterns in MtCM variants before and after selection experiments.

<p>Column colors correspond to the randomized positions 84 (blue), 85 (red), 86 (green), 87 (purple), 88 (cyan), 89 (orange), and 90 (light blue). Side chains are ordered according to increasing volume <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone.0116234-Chothia1" target="_blank">[48]</a>; an asterisk denotes a stop codon. The absolute number of codons compiled at each position is indicated in parentheses next to the wild-type residue. The absolute numbers of individual residues found at every position are, in addition to the graphical representation of the relative frequencies shown here, listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone.0116234.s003" target="_blank">S1 Table</a>. (A) Amino acid residues found under non-selective conditions (M9c +FY). The percentages (and standard deviations σ_n-1) of the 4 individual nucleotides averaged over every randomized position were 25.9 (±7.3)%, 26.5 (±7.5)%, 21.6 (±6.3)%, and 26.0 (±6.0)% for A, C, G, and T, respectively, in the analyzed sample set. (B) Preferred residues selected under a stringent regime (M9c, no inducer added) in the presence of the complex partner MtDS (in KA12/pKIMP-ACG). (C) Residue patterns emerging in selected MtCM variants grown in the absence of MtDS under conditions where free wild-type MtCM can complement the CM deficiency (in KA12/pKIMP-UAUC, plated on M9c +F +Tet^{500 ng/mL}). (D) Correlated amino acid distribution patterns. Positive values indicate a residue preference in the complexed MtCM, whereas negative values (columns in white) show residues frequently observed in functional free MtCM. To simplify the arbitrary graphical summary, and to avoid division by 0, the value at each position represents the ratio of [number of a particular amino acid found in complexed MtCMs +1]/[number of the same amino acid found in the free MtCMs +1] −1.</p

Comparison of phylogenetic and experimental amino acid conservation patterns at the interface between MtCM and MtDS.

<p>Conservation patterns in MtCM are illustrated with the crystal structure of the MtCM-MtDS complex (PDB: 2W1A). The color code for individual residues indicates the level of sequence conservation (black, 100%; red, ≥75%; orange, ≥50%; yellow, ≥33%; white, <33% identity). (A) Overview with MtCM positions colored as in the phylogenetic alignment in Fig. 3B. MtDS is shown in wheat surface representation, MtCM in cartoon mode. Fully conserved residues (black) are generally clustered around the active site with bound <b>7</b> (shown as space-filling model with green carbons), whereas the solvent-exposed residues typically show low conservation (white and yellow). (B, C) Wall-eyed stereogram of a close-up of the contact area between the C-terminal region of MtCM and MtDS, with ligand <b>7</b> depicted in the top left corner as in (A). The C-terminal loop hooks onto the MtDS surface. In (B), the phylogenetic color coding as in (A) is used for the segment from positions 84–90, which is highlighted with dotted spheres for the individual side chains. In (C), the color coding for the same segment represents the conservation pattern found by experiment in selected MtCM variants in the presence of MtDS.</p

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

<div><p>The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM) of <i>Mycobacterium tuberculosis</i> 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-<i>arabino</i>-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 <i>in vivo</i> requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQ_δ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQ_δ 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.</p></div

Correlation between MtDS interactions and sensitivity to feedback inhibition of MtCM variants.

<p>The plot shows a graphical correlation of the salient features of the selected MtCM library variants (and the wild type) from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone-0116234-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone-0116234-g008" target="_blank">Fig. 8</a>. All variants that are activated by MtDS by more than 20 fold also show high sensitivity of the complex to feedback inhibition by Phe and Tyr (<i>i.e.</i> show low residual activity +Phe/Tyr). Conversely, lower sensitivity to feedback inhibition correlates with poor activation by MtDS and with a lower potential to shift the MtDS-band on native PAGE. Circle size and color represent a full (large, red), partial (intermediate, orange), or no shift of the MtDS band (small, yellow).</p

Chorismate mutase activity of wild-type MtCM (wt) and selected MtCM library variants.^a.

a<p>Details to the assay conditions and calculation of individual parameters and their standard deviations are provided in Materials and Methods.</p>b<p>An asterisk indicates premature termination (the last residue is Leu88 for variants 1–6 and 2–8).</p>c<p>Catalytic efficiency of MtCM variant alone (in the absence of MtDS).</p>d<p>The apparent activation factor was estimated as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone.0116234-Sasso1" target="_blank">[10]</a>, as the ratio of CM initial velocities of the MtCM-MtDS complex (<i>v</i>_{0 (MtDS+MtCM)}), normalized by MtCM-variant and chorismate concentrations, over <i>k</i>_cat/<i>K</i>_m for free MtCM.</p>e<p>Ratio of initial velocity <i>v</i>_{0 (MtDS+MtCM)} as in footnote ^c, but measured in the presence of 100 µM Phe and 100 µM Tyr, divided by <i>v</i>_{0 (MtDS+MtCM)} obtained in the absence of these feedback inhibitors.</p><p>Chorismate mutase activity of wild-type MtCM (wt) and selected MtCM library variants.^a.</p

Selection system for MtCM variants that can be activated by MtDS.

<p>The selection system used is based on <i>E. coli</i> strain KA12 lacking the endogenous <i>pheA</i> and <i>tyrA</i> genes, which encode CM-prephenate dehydratase (PDT) and CM-prephenate dehydrogenase (PDH), respectively. Plasmid pKIMP-UAUC has the p15A-derived origin of replication (<i>ori</i>_p15A) and carries *<i>tyrA</i> for a monofunctional PDH and <i>pheC</i> for a monofunctional PDT, in addition to <i>cat</i> providing chloramphenicol resistance <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116234#pone.0116234-Kast1" target="_blank">[21]</a>. Plasmid pKIMP-ACG additionally contains the <i>aroG</i> gene encoding MtDS. (A) Performance of wild-type MtCM (encoded by <i>aroQ_δ</i> on plasmid pKTNTET) without MtDS (pKIMP-UAUC) or with MtDS (pKIMP-ACG) in comparison to a negative control (pKTCTET-0, lacking <i>aroQ_δ</i>). Growth was assessed on M9c-based minimal plates in the presence or absence of Phe (F) and Tyr (Y). Colony size was scored either as++(good growth),+(moderate growth), – (poor growth), or 0 (no trace of growth) as a function of the added Tet concentration, the inducer of the P<i>_tet</i> promoter upstream of <i>aroQ_δ</i>. (B) Schematic representation of the redesigned selection system. The <i>aroQ_δ</i> gene encoding an MtCM library variant is provided on plasmid pKT-CM that has otherwise the same structure as pKTNTET (<i>bla</i>, β-lactamase for ampicillin resistance; its <i>ori</i>_pUC is compatible with <i>ori</i>_p15A). Under stringent selection conditions (M9c), host cells transformed with both pKIMP-ACG and a pKT-CM library plasmid can only produce enough prephenate and consequently Phe and Tyr needed for growth if the encoded MtCM variant can engage in a productive complex with MtDS (wide green arrow). Transformants having insufficient CM activity (thin, light green arrow) require exogenously added F and Y for growing on M9c minimal plates.</p