Mechanisms of protein sequence divergence and incompatibility.

Alignments of orthologous protein sequences convey a complex picture. Some positions are utterly conserved whilst others have diverged to variable degrees. Amongst the latter, many are non-exchangeable between extant sequences. How do functionally critical and highly conserved residues diverge? Why and how did these exchanges become incompatible within contemporary sequences? Our model is phosphoglycerate kinase (PGK), where lysine 219 is an essential active-site residue completely conserved throughout Eukaryota and Bacteria, and serine is found only in archaeal PGKs. Contemporary sequences tested exhibited complete loss of function upon exchanges at 219. However, a directed evolution experiment revealed that two mutations were sufficient for human PGK to become functional with serine at position 219. These two mutations made position 219 permissive not only for serine and lysine, but also to a range of other amino acids seen in archaeal PGKs. The identified trajectories that enabled exchanges at 219 show marked sign epistasis - a relatively small loss of function with respect to one amino acid (lysine) versus a large gain with another (serine, and other amino acids). Our findings support the view that, as theoretically described, the trajectories underlining the divergence of critical positions are dominated by sign epistatic interactions. Such trajectories are an outcome of rare mutational combinations. Nonetheless, as suggested by the laboratory enabled K219S exchange, given enough time and variability in selection levels, even utterly conserved and functionally essential residues may change

A schematic PGK phylogenetic tree.

<p>Shown are 175 PGK protein sequences rooted with Glycerate kinase from <i>Neisseria meningitides</i> (Detailed analysis, a complete tree (620 sequences) and alignment are given as Supplementary information). All bacterial sequences (in green) and eukaryotic sequences (blue) have lysine at position 219. None of the archeal sequences (in red) harbors a lysine at 219, with the most abundant amino acids being serine (45/73) and threonine (12/73). Redundancy threshold was increased to 93% to create a minimal set of sequences.</p

Evolved variant G4-v4 is highly permissive to exchanges at position 219.

<p>(a) Libraries encoding all 20 possible amino acids at position 219 were generated for wild-type hPGK, and it's evolved variant G4-v4. Aprox. 500 transformants from each library were plated on glucose selection plates. Only 4% of the wild-type library clones were viable, all carrying lysine at 219 (amino acids are denoted with single letter codes). In contrast, 26% of the G4-v4 library transformants were viable. These were variable in colony size and growth rates and carried a range of amino acids. With the exception of glycine (smallest colonies and lowest growth rate), the amino acids tolerated by variant G4-v4 are all observed within archaeal PGKs. Conversely, leucine is the only amino acid observed in archaeal PGKs (rarely though, 5/72) but not in G4-v4's viable clones. (b) The growth rates of the G4-v4 variants carrying different amino acids at position 219. The values comprise are average of 12 parallel measurements. Also shown is the growth rate for wild type hPGK for comparison.</p

Representative variants underlining the directed evolution of hPGK- serine 219.

<p>Noted are sequence changes, growth rates and <i>k_cat/K_M</i> values of representative variants along the laboratory trajectory. Variants annotation: G stands for the round number from which a variant was isolated, and v denotes the variant's sequential number within this round. The left column denotes the sequence composition and rate parameters of wild-type hPGK. In bold are mutations that appeared at ≥90% of the variants by the last round (near complete fixation). Mutations that were not fixed appear in standard letters and their percentage of occurrence is denoted in brackets (no percentage is indicated for mutations that appeared only once in >100 variants).</p>a<p>Note that <i>k_cat/K_M</i> for wild type hPGK we measured was ∼11-fold lower than previously reported <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003665#pgen.1003665-Szabo1" target="_blank">[18]</a>. The origins of this difference are unknown, yet the rates for various mutants were measured by us under the very same conditions as wild-type, thus providing consistency within the reported set. The ± mark represents the S.D within 3 independent measurements.</p

Growth rates and PGK cell lysate activities.

<p>Plotted are growth rates (open symbols, right Y axis) and levels of PGK enzymatic activity in crude cell lysates (closed symbols, left Y axis) as a function of the expression inducer (AHT) concentration. Shown are the parental <i>E. coli</i> strain (squares) and its <i>pgk</i> knockout (Δ<i>pgk</i>). The latter was supplemented with the pZA plasmid encoding wild-type hPGK (lysine 219; triangles), or its K219S mutant (diamonds). Cells were grown in glycerol-succinate media supplemented with 5 mM glucose and increasing AHT concentrations. Cell suspensions were normalized to the same cell density, lysed, and PGK activity levels in the lysates were determined. The lysate activities were normalized to the activity of the parental <i>E. coli</i> strain MG1655 (3±0.34×10⁵ µmole product/min/mg dry cells). Growth rates are presented as the inverse of the shortest doubling time among time intervals <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003665#pgen.1003665-Sasson1" target="_blank">[46]</a> (the shortest doubling time was observed with <i>E. coli</i> MG1655, and was 63±2.8 mins). Error bars represent the standard deviation of ≥3 independent measurements (growth, lysis, and assays).</p

Epistatic effects of mutations underline divergence and incompatibility at position 219.

<p>(<b>a</b>) The engineered double mutant, I239M plus D304E, exhibits similar growth rate to the evolved variant G4-v4 with both lysine (in blue) and serine at 219 (in red). Thus, the 3 additional mutations in G4-v4 play a minor role (also indicated by I239M and D304E being the only mutations that were fixed; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003665#pgen-1003665-t001" target="_blank">Table 1</a>). Starting from the permissive state represented by the double mutant, both reversions, I239M and D304E, exhibit sign epistasis with respect to position 219: they improve growth at the background of lysine 219 but decrease growth with serine at 219. With respect to each other, M239I and E304D are nearly-additive at the background of lysine 219, but interact with strong negative epistasis at the background of serine 219. (<b>b</b>) All possible trajectories underlying the lysine-serine exchange. The first letter in each triplet represents position 219, the second represents 239, and the third, position 403. The wild-type and the K219S mutant are labeled. (<b>c</b>) Sign epistasis restricts the PGK accessible divergence routes. Under selection to maintain wild-type-like activity levels, no routes are accessible. Further, the few accessible routes under weak selection for PGK's activity (all arrows) boil down to a single route (solid arrows) under more stringent selection (growth rate ≤0.0125 min⁻¹ vs. wild-type hPGK (0.0156 min⁻¹)). Error bars represent S.D of 12 independent measurements. Note that a direct serine-lysine transition, i.e., by a single nucleotide exchange, is impossible. The most likely bridging intermediate is arginine, which seems to confer the highest growth rate at the background of the transition variant G4-v4 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003665#pgen-1003665-g004" target="_blank">Fig. 4b</a>).</p

Growth rates and catalytic efficiencies of the evolved variants.

<p>(a) Growth rates of the evolved serine 219 hPGK variants and of their lysine 219 counterparts. (b) <i>k_cat/K_M</i> values of the same variants (log scale). Error bars represent S.D of 8 measurements. Each double headed dashed arrow connects a 219 serine variant with its 219 lysine counterpart.</p

AcpM, the Meromycolate Extension Acyl Carrier Protein of <i>Mycobacterium tuberculosis</i>, Is Activated by the 4′-Phosphopantetheinyl Transferase PptT, a Potential Target of the Multistep Mycolic Acid Biosynthesis

Modification of acyl carrier proteins (ACP) or domains by the covalent binding of a 4′-phosphopantetheine (4′-PP) moiety is a fundamental condition for activation of fatty acid synthases (FASes) and polyketide synthases (PKSes). Binding of 4′-PP is mediated by 4′ phosphopantetheinyl transfersases (PPTases). <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) possesses two essential PPTases: acyl carrier protein synthase (<i>Mtb</i> AcpS), which activates the multidomain fatty acid synthase I (FAS I), and <i>Mtb</i> PptT, an Sfp-type broad spectrum PPTase that activates PKSes. To date, it has not been determined which of the two <i>Mtb</i> PPTases, AcpS or PptT, activates the meromycolate extension ACP, <i>Mtb</i> AcpM, en route to the production of mycolic acids, the main components of the mycobacterial cell wall. In this study, we tested the enzymatic activation of a highly purified <i>Mtb</i> apo-AcpM to <i>Mtb</i> holo-AcpM by either <i>Mtb</i> PptT or <i>Mtb</i> AcpS. By using SDS-PAGE band shift assay and mass spectrometry analysis, we found that <i>Mtb</i> PptT is the PPTase that activates <i>Mtb</i> AcpM. We measured the catalytic activity of <i>Mtb</i> PptT toward CoA, using an activation assay of a blue pigment synthase, BpsA (a nonribosomal peptide synthase, NRPS). BpsA activation by <i>Mtb</i> PptT was inhibited by <i>Mtb</i> apo<i>-</i>AcpM through competition for CoA, in accord with <i>Mtb</i> AcpM activation. A structural model of the putative interaction between <i>Mtb</i> PptT and <i>Mtb</i> AcpM suggests that both hydrophobic and electrostatic interactions stabilize this complex. To conclude, activation of <i>Mtb</i> AcpM by <i>Mtb</i> PptT reveals a potential target of the multistep mycolic acid biosynthesis

Overall Survival and Clinical Characteristics of BRCA‐Associated Cholangiocarcinoma: A Multicenter Retrospective Study

BackgroundBiliary tract malignancies, in particular cholangiocarcinomas (CCA), are rare tumors that carry a poor prognosis. BRCA2 mutation carriers have an increased risk of developing CCA with a reported relative risk of ∼5 according to the Breast Cancer Linkage Consortium. In addition to this risk, there are potential therapeutic implications in those harboring somatic and/or germline (GL) BRCA mutations. Therefore, it is important to define the clinical characteristics of GL/somatic BRCA1/2 variants in CCA patients.Materials and methodsWe performed a multicenter retrospective analysis of CCA patients diagnosed between January 2000 and December 2013 with GL or somatic variants in BRCA1/2 genes detected by GL mutations testing and/or by tumor next generation sequencing. Cases were identified from clinical databases at participating institutions. Data including demographics, clinical history, surgical procedures, and systemic chemotherapy or radiation were extracted from patients' records.ResultsOverall, 18 cases were identified: 5 carriers of GL BRCA1/2 mutations (4 BRCA2; 1 BRCA1) and 13 harboring somatic variations (7 BRCA1; 6 BRCA2). Mean age at diagnosis was 60, SD ± 10 years (range 36-75 years), with male and female prevalence rates of 61.2% and 38.8%, respectively. Stage at diagnosis was I (n = 4), II (n = 3), III (n = 3), and IV (n = 8). Six patients had extrahepatic CCA and the rest intrahepatic CCA. Thirteen patients received platinum-based therapy and four were treated with poly ADP ribose polymerase inhibitors, of whom one experienced sustained disease response with a progression-free survival of 42.6 months. Median overall survival from diagnosis for patients with stage I/II in this study was 40.3 months (95% confidence interval [CI], 6.73-108.15) and with stages III/IV was 25 months (95% CI, 15.23-40.57).ConclusionBRCA-associated CCA is uncommon. This multicenter retrospective study provides a thorough clinical analysis of a BRCA-associated CCA cohort, which can serve as a benchmark for future development and design of expanded analyses and clinical trials.Implications for practiceBRCA-associated CCA is uncommon but a very important subtype of hepatic malignancies, due to its rising prevalence. Better clinical characterization of this subtype might allow application of targeted therapy for CCA patients with germline or somatic mutations in BRCA1/2 genes, especially due to previously reported success of such therapies in other BRCA-associated malignancies. Thus this study, first of its kind, provides a basis for future multi-centered analyses in larger cohorts, as well as clinical trials. Additionally, this study emphasizes the importance of both germline and somatic genotyping for all CCA patients