YfiBNR is a tripartite signalling system in Pseudomonas aeruginosa that modulates intracellular c-di-GMP levels in response to signals received in the periplasm. YfiB is an outer membrane lipoprotein and presumed sensor protein that sequesters the repressor protein YfiR. To provide insights into YfiBNR function, we have determined three-dimensional crystal structures of YfiB and YfiR from P. aeruginosa PAO1 alone and as a 1:1 complex. A YfiB(27–168) construct is predominantly dimeric, whereas a YfiB(59–168) is monomeric, indicating that YfiB can dimerize via its N-terminal region. YfiR forms a stable complex with YfiB(59–168), while the YfiR binding interface is obstructed by the N-terminal region in YfiB(27–168). The YfiB-YfiR complex reveals a conserved interaction surface on YfiR that overlaps with residues predicted to interact with the periplasmic PAS domain of YfiN. Comparison of native and YfiR-bound structures of YfiB suggests unwinding of the N-terminal linker region for attachment to the outer membrane. A model is thus proposed for YfiR sequestration at the outer membrane by YfiB. Our work provides the first detailed insights into the interaction between YfiB and YfiR at the molecular level and is a valuable starting point for further functional and mechanistic studies of the YfiBNR signalling system.
Dehydration is one of the key steps in the biosynthesis of mycolic acids and is vital to the growth of Mycobacterium tuberculosis (Mtb). Consequently, stalling dehydration cures tuberculosis (TB). Clinically used anti-TB drugs like thiacetazone (TAC) and isoxyl (ISO) as well as flavonoids inhibit the enzyme activity of the β-hydroxyacyl-ACP dehydratase HadAB complex. How this inhibition is exerted, has remained an enigma for years. Here, we describe the first crystal structures of the MtbHadAB complex bound with flavonoid inhibitor butein, 2’,4,4’-trihydroxychalcone or fisetin. Despite sharing no sequence identity from Blast, HadA and HadB adopt a very similar hotdog fold. HadA forms a tight dimer with HadB in which the proteins are sitting side-by-side, but are oriented anti-parallel. While HadB contributes the catalytically critical His-Asp dyad, HadA binds the fatty acid substrate in a long channel. The atypical double hotdog fold with a single active site formed by MtbHadAB gives rise to a long, narrow cavity that vertically traverses the fatty acid binding channel. At the base of this cavity lies Cys61, which upon mutation to Ser confers drug-resistance in TB patients. We show that inhibitors bind in this cavity and protrude into the substrate binding channel. Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site. The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0181-1) contains supplementary material, which is available to authorized users.
Nisin is a widely used antibacterial lantibiotic polypeptide produced by Lactococcus lactis. NisP belongs to the subtilase family and functions in the last step of nisin maturation as the leader-peptide peptidase. Deletion of the nisP gene in LAC71 results in the production of a non-active precursor peptide with the leader peptide unremoved. Here, the 1.1 Å resolution crystal structure of NisP is reported. The structure shows similarity to other subtilases, which can bind varying numbers of Ca atoms. However, no calcium was found in this NisP structure, and the predicted calcium-chelating residues were placed so as to not allow NisP to bind a calcium ion in this conformation. Interestingly, a short peptide corresponding to its own 635-647 sequence was found to bind to the active site of NisP. Biochemical assays and native mass-spectrometric analysis confirmed that NisP possesses an auto-cleavage site between residues Arg647 and Ser648. Further, it was shown that NisP mutated at the auto-cleavage site (R647P/S648P) had full catalytic activity for nisin leader-peptide cleavage, although the C-terminal region of NisP was no longer cleaved. Expressing this mutant in L. lactis LAC71 did not affect the production of nisin but did decrease the proliferation rate of the bacteria, suggesting the biological significance of the C-terminal auto-cleavage of NisP.
Alginate production in Pseudomonas aeruginosa is regulated by the alternate σ factor AlgU, which in turn is regulated by the MucABCD system. The anti‐σ factor MucA binds AlgU in the cytoplasm and prevents AlgU from binding to the RNA polymerase for transcription. MucB binds MucA in the periplasm and inhibits proteolysis of MucA and subsequent release of AlgU. In this work, we report crystal structures of MucA in complex with AlgU and MucB. A structure of MucB alone reveals the structural changes required for MucA recognition. A unique disulfide bond is identified in MucB, and mutation of this disulfide bond results in a shift from monomer to MucB dimers or tetramers. As MucB tetramers have previously been shown to be unable to bind MucA, this suggests a redox‐sensitive stress response mechanism in MucB. The AlgU–MucA structure reveals a conserved σ factor/anti‐σ factor complex, but AlgU lacks a disulfide bond conserved in many other σ factors. Our structures reveal the molecular basis for MucA recognition by MucB in the periplasm and AlgU in the cytoplasm, thus providing an important step in understanding the mechanisms that regulate a key signal transduction pathway involved in P. aeruginosa pathogenesis.DatabaseThe atomic coordinates and structure factors for MucAcyto–AlgU, MucB, and MucAperi–MucB have been deposited in the Protein Data Bank (PDB) with the accession code 6IN7, 6IN8, and 6IN9, respectively.
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