(Journal of molecular biology[TA]) articles in PubMed
- An Adaptive mutation in Enterococcus faecium LiaR Associated with Antimicrobial Peptide Resistance Mimics Phosphorylation and Stabilizes LiaR in an Activated State. [Journal Article]
- J Mol Biol 2016 Sep 23JM
- The cyclic antimicrobial lipopeptide daptomycin triggers the LiaFSR membrane stress response pathway in enterococci and many other Gram-positive organisms. LiaR is the response regulator that, upon p...
The cyclic antimicrobial lipopeptide daptomycin triggers the LiaFSR membrane stress response pathway in enterococci and many other Gram-positive organisms. LiaR is the response regulator that, upon phosphorylation, binds in a sequence specific manner to DNA to regulate transcription in response to membrane stress. In clinical settings, nonsusceptibility to daptomycin by Enterococcus faecium is correlated frequently with a mutation in LiaR of Trp73 to Cys (LiaR(W73C)). We have determined the structure of the activated Enterococcus faecium LiaR protein at 3.2Å resolution and, in combination with solution studies, show that activation of LiaR induces formation of a LiaR dimer that increases LiaR affinity at least 40-fold for the extended regulatory regions upstream of the liaFSR and liaXYZ operons. In vitro, LiaR(W73C) induces phosphorylation-independent dimerization of LiaR and provides a biochemical basis for non-susceptibility to daptomycin by upregulation of the LiaFSR regulon. A comparison of the Enterococcus faecalis LiaR, Enterococcus faecium LiaR and the LiaR homolog from Staphylococcus aureus (VraR) and the mutations associated with daptomycin resistance suggest that physicochemical properties such as oligomerization state and DNA specificity, though tuned to the biology of each organism, share some features that could be targeted for new antimicrobials.
- New Insights into the Phage Genetic Switch: Effects of Bacteriophage Lambda Operator Mutations on DNA Looping and Regulation of PR, PL, and PRM. [Journal Article]
- J Mol Biol 2016 Sep 23JM
- One of the best understood systems in genetic regulatory biology is the so-called "genetic switch" that determines the choice g the phage encoded CI repressor binds co-operatively to tripartite opera...
One of the best understood systems in genetic regulatory biology is the so-called "genetic switch" that determines the choice g the phage encoded CI repressor binds co-operatively to tripartite operators, OL and OR , in a defined pattern, thus blocking transcription at two lytic promoters, PL and PR, and auto-regulating the promoter, PRM, which directs CI synthesis by the prophage. Fine-tuning of the maintenance of lysogeny is facilitated by interactions between CI dimers bound to OR and OL through formation of a loop by the intervening DNA segment. By using a purified in vitro transcription system, we have genetically dissected the roles of individual operator sites in the formation of the DNA loop and thus have gained several new and unexpected insights into the system. First, although both OR and OL are tripartite, the presence of only a single active CI binding site in one of the two operators is sufficient for DNA loop formation. Second, in PL, unlike in PR, the promoter distal operator site, OL3, is sufficient to directly repress PL. Third, DNA looping mediated by the formation of CI-octamers arising through the interaction of pairs of dimers bound to adjacent operator sites in OR and OL does not require OR and OL to be aligned "in register"; that is CI bound to "out-of-register" sub-operators, e.g. OL1~Ol2 and OR2~OR3 can also mediate loop formation. Finally, based on an examination of the mechanism of activation of PRM when only OR1 or OR2 are wild-type, we hypothesize that RNAP bound at PR interferes with DNA loop formation. Thus, the formation of DNA loops involves potential interactions between proteins bound at numerous cis-acting sites, which therefore very subtly contribute to the regulation of the "switch".
- Making sense of the yeast sphingolipid pathway. [Review]
- J Mol Biol 2016 Sep 21JM
- Sphingolipids (SL) and their metabolites play key roles both as structural components of membranes and as signalling molecules. Many of the key enzymes and regulators of SL metabolism were discovered...
Sphingolipids (SL) and their metabolites play key roles both as structural components of membranes and as signalling molecules. Many of the key enzymes and regulators of SL metabolism were discovered using the yeast Saccharomyces cerevisiae, and based on the high degree of conservation, a number of mammalian homologs were identified. Although yeast continues to be an important tool for SL research, the complexity of SL structure and nomenclature often hampers the ability of new researchers to grasp the subtleties of yeast SL biology and to discover modulators of this intricate pathway. Moreover, the emergence of lipidomics by mass spectrometry has enabled rapid identification of SL species in yeast and rendered analysis of SL composition under various physiological and pathophysiological conditions readily amenable. However, the complex nomenclature of the identified species renders much of the data inaccessible to non-specialists. In this review, we focus on parsing both the classical SL nomenclature and the nomenclature normally used during mass spectrometry analysis, which should facilitate understanding yeast SL data and might shed light on biological processes in which SLs are involved. Finally, we discuss a number of putative roles of various yeast SL species.
- Computationally Designed Armadillo Repeat Proteins for Modular Peptide Recognition. [Journal Article]
- J Mol Biol 2016 Sep 21JM
- Armadillo repeat proteins (ArmRP) recognize their target peptide in extended conformation and bind, in a first approximation, two residues per repeat. They may thus form the basis for building a modu...
Armadillo repeat proteins (ArmRP) recognize their target peptide in extended conformation and bind, in a first approximation, two residues per repeat. They may thus form the basis for building a modular system, in which each repeat is complementary to a piece of the target peptide. Accordingly, preselected repeats could be assembled into specific binding proteins on demand and thereby avoid the traditional generation of every new binding molecule by an independent selection from a library. Stacked armadillo repeats, each consisting of 42 amino acids arranged in three α-helices, build an elongated superhelical structure. Here, we analyzed curvature variations in natural ArmRPs, and identified a repeat pair from yeast importin-α as having the optimal curvature geometry to be complementary to a peptide over its whole length. We employed a symmetric in silico design to obtain a uniform sequence for a stackable repeat while maintaining the desired curvature geometry. Computationally designed armadillo repeat proteins (dArmRPs) had to be stabilized by mutations to remove regions of higher flexibility, which were identified by molecular dynamics (MD) simulations in explicit solvent. Using an N-capping repeat from the consensus-design approach, two different crystal structures of dArmRP were determined. Although the experimental structures of dArmRP deviated from the designed curvature, the insertion of the most conserved binding pockets of natural ArmRPs onto the surface of dArmRPs resulted in binders against the expected peptide with low nanomolar affinities, similar to the binders from the consensus-design series.
- Structural Insights into the Inhibitory Mechanism of an Antibody against B7-H6, a Stress-Induced Cellular Ligand for the Natural Killer Cell Receptor NKp30. [Journal Article]
- J Mol Biol 2016 Sep 20JM
- Antibodies have been shown to block signaling through cell surface receptors using several mechanisms. The two most common are binding to the ligand-binding site of the receptor and, conversely, bind...
Antibodies have been shown to block signaling through cell surface receptors using several mechanisms. The two most common are binding to the ligand-binding site of the receptor and, conversely, binding to the receptor-binding site of the ligand. Here we investigated the inhibitory mechanism of an antibody (17B1.3) against human B7-H6, a stress-induced cellular ligand for the natural killer (NK) cell receptor NKp30. Binding of this antibody to B7-H6, a transmembrane protein expressed on tumor and other stressed cells, but not on normal cells, prevents NK cell activation via NKp30. We determined the crystal structure of antibody 17B1.3 in complex with the ectodomain of B7-H6 to 2.5Å resolution. Surprisingly, 17B1.3 binds to a site on B7-H6 that is completely distinct from the binding site for NKp30, such that 17B1.3 does not block the NKp30-B7-H6 interaction. We then asked whether 17B1.3 prevents signaling by binding to a putative site for B7-H6 dimerization. However, structure-based mutations designed to disrupt potential B7-H6 dimerization through this site did not diminish NKp30-mediated cell activation. We conclude that the bulky 17B1.3 antibody most likely acts by sterically interfering with close cell-cell contacts at the NK cell-target cell interface that are required for NK cell activation. A similar inhibitory mechanism may apply to other antibodies, including therapeutic antibodies, that block signaling through cell surface receptors whose ligands are also cell surface proteins.
- Allosteric regulation points control the conformational dynamics of the molecular chaperone Hsp90. [Journal Article]
- J Mol Biol 2016 Sep 20JM
- Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone responsible for the activation, maturation and trafficking of several hundred client proteins in the cell. It is well known that ...
Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone responsible for the activation, maturation and trafficking of several hundred client proteins in the cell. It is well known that (but not understood how) residues far away from Hsp90's nucleotide binding pocket can regulate its ATPase activity, a phenomenon called allosteric regulation. Here, the computational design of allosteric mutations based was combined with in vitro and in vivo experiments to unravel nucleotide-responsive hot spots in the regulation of Hsp90. With this approach, we identified both activating and inhibiting regulation points and show that changes in those amino acids affect the conformational dynamics and ATPase activity of Hsp90 in vitro. Our observations that activating mutations loosen and inhibiting mutations ridgify the protein explain for the first time, how Hsp90 changes in response to allosteric mutations. Additionally, mutations of these allosteric regulation points can be controlled by the interplay with Hsp90 co-chaperones, thus providing cells with an efficient mechanism of modifying Hsp90's intrinsic properties via different layers of regulation. Altogether, our results show that a framework for transmitting conformational information exists in the Hsp90 structure.
- Computational redesign of thioredoxin is hypersensitive towards minor conformational changes in the backbone template. [Journal Article]
- J Mol Biol 2016 Sep 19JM
- Despite the development of powerful computational tools, the full-sequence design of proteins still remains a challenging task. To investigate the limits and capabilities of computational tools, we c...
Despite the development of powerful computational tools, the full-sequence design of proteins still remains a challenging task. To investigate the limits and capabilities of computational tools, we conducted a study of the ability of the program Rosetta to predict sequences that recreate the authentic fold of thioredoxin. Focusing on the influence of conformational details in the template structures, we based our study on 8 experimentally determined template structures and generated 120 designs from each. For experimental evaluation, we chose 6 sequences from each of the 8 templates by objective criteria. The 48 selected sequences were evaluated based on their progressive ability to: (1) produce soluble protein in Escherichia coli, (2) yield stable monomeric protein, and (3) the ability of the stable, soluble proteins to adopt the target fold. Of the 48 designs, we were able to synthesize 32, 20 of which resulted in soluble protein. Of these, only two were sufficiently stable to be purified. An X-ray crystal structure was solved for one of the designs, revealing a close resemblance to the target structure. We found a significant difference between the eight template structures to realize the above three criteria despite their high structural similarity. Thus, in order to improve the success rate of computational full-sequence design methods, we recommend that multiple template structures are used. Furthermore, this study shows that special care should be taken when geometry optimizing a structure prior to computational design when using a method that is based on rigid conformations.
- Mapping of the communication-mediating interface in nonribosomal peptide synthetases using a genetically encoded photocrosslinker supports an upside-down helix-hand motif. [Journal Article]
- J Mol Biol 2016 Sep 16JM
- Nonribosomal peptide synthetases (NRPSs) are large modular protein templates that assemble bioactive peptides, many of which possess therapeutic importance. Protein-protein interactions between subun...
Nonribosomal peptide synthetases (NRPSs) are large modular protein templates that assemble bioactive peptides, many of which possess therapeutic importance. Protein-protein interactions between subunits of bacterial NRPSs are essential for proper template formation. The structural basis of the typical subunit interface between epimerization (E) and condensation (C) domains is only poorly understood. Conflicting helix-helix and helix-hand models were previously proposed. Here, the genetically encoded photocrosslinker p-benzoylphenylalanine (BpF) was incorporated into the C-terminal communication-mediating domain (COM) of GrsA. Using the partner elongation module TycB1 to form a dipeptide product, we could correlate the ability to form covalent crosslinks with the functional module interaction. Pertubation of the module interaction with the large side chain of BpF in a scan at 19 positions demonstrated the importance of three hydrophobic residues in an α-helical arrangement. Mapping of covalent crosslinks using tandem mass spectrometry revealed residues from the interior of the C domain as part of the protein interface; a finding not predicted by the helix-helix model. The E domain of GrsA was found to be important for the interaction. Together with multiple sequence analyses and structural modeling our results suggest an upside-down helix-hand model in which the C-terminal COM-helix is embedded in a hand motif with a hydrophobic core in a reversed orientation compared to a previous proposal. Our results provide a more detailed and the first direct structural understanding of the COM domain interaction and will contribute to successful biocombinatorial engineering attempts in the design of artificial NRPS templates.
- Structure and function of AmtR in Mycobacterium smegmatis: Implications for post-transcriptional regulation of urea metabolism through a small antisense RNA. [Journal Article]
- J Mol Biol 2016 Sep 15JM
- Soil-dwelling bacteria of the phylum actinomycetes generally harbor either GlnR or AmtR as global regulators of nitrogen metabolism. Mycobacterium smegmatis harbors both these canonical regulators; G...
Soil-dwelling bacteria of the phylum actinomycetes generally harbor either GlnR or AmtR as global regulators of nitrogen metabolism. Mycobacterium smegmatis harbors both these canonical regulators; GlnR regulates the expression of key genes involved in nitrogen metabolism, while the function and signal transduction pathway of AmtR in M. smegmatis remains largely unknown. Here we report the structure and function of the M. smegmatis AmtR and describe the role of AmtR in the regulation of nitrogen metabolism in response to nitrogen availability. To determine the function of AmtR in M. smegmatis, we performed genome-wide expression profiling comparing the wild-type versus an ∆amtR mutant and identified significant changes in the expression of 11 genes, including an operon involved in urea degradation. An AmtR-consensus binding motif (CTGTC-N4-GACAG) was identified in the promoter region of this operon and ligand-independent high-affinity AmtR binding was validated by both electrophoretic mobility shift assays and SPR measurements. We confirmed the transcription of a cis-encoded small RNA complementary to the gene encoding AmtR under nitrogen excess and propose a post-transcriptional regulatory mechanism for AmtR. The three-dimensional X-ray structure of AmtR at 2.0Å revealed an overall TetR-like dimeric structure and alignment of the M. smegmatis AmtR and Corynebacterium glutamicum AmtR regulatory domains showed poor structural conservation providing a potential explanation for the lack of M. smegmatis AmtR interaction with the adenylylated PII protein. Taken together our data suggest an AmtR (repressor)/GlnR (activator) competitive binding mechanism for transcriptional regulation of urea metabolism that is controlled by a cis-encoded small antisense RNA.
New Search Next
- The power of force: Insights into the protein folding process using single-molecule force spectroscopy. [Review]
- J Mol Biol 2016 Sep 14JM
- One of the major challenges in modern biophysics is observing and understanding conformational changes during complex molecular processes, from the fundamental protein folding to the function of mole...
One of the major challenges in modern biophysics is observing and understanding conformational changes during complex molecular processes, from the fundamental protein folding to the function of molecular machines. Single molecule techniques have been one of the major driving forces of the huge progress attained in the last few years. Recent advances in resolution of the experimental setups, aided by theoretical developments and molecular dynamics simulations, have revealed a much higher degree of complexity inside these molecular processes than previously reported using traditional ensemble measurements. This review sums up the evolution of these developments and gives an outlook to prospective discoveries.