Bacillus licheniformis BlaR1 L3 loop is a zinc metalloprotease activated by self-proteolysis

In Bacillus licheniformis 749/I, BlaP β-lactamase is induced by the presence of a β-lactam antibiotic outside the cell. The first step in the induction mechanism is the detection of the antibiotic by the membrane-bound penicillin receptor BlaR1 that is composed of two functional domains: a carboxy-terminal domain exposed outside the cell, which acts as a penicillin sensor, and an amino-terminal domain anchored to the cytoplasmic membrane, which works as a transducer-transmitter. The acylation of BlaR1 sensor domain by the antibiotic generates an intramolecular signal that leads to the activation of the L3 cytoplasmic loop of the transmitter by a single-point cleavage. The exact mechanism of L3 activation and the nature of the secondary cytoplasmic signal launched by the activated transmitter remain unknown. However, these two events seem to be linked to the presence of a HEXXH zinc binding motif of neutral zinc metallopeptidases. By different experimental approaches, we demonstrated that the L3 loop binds zinc ion, belongs to Gluzincin metallopeptidase superfamily and is activated by self-proteolysis.     

Crystal structure of a functional dimer of the PhoQ sensor domain

The PhoP-PhoQ two-component system is a well studied bacterial signaling system that regulates virulence and stress response. Catalytic activity of the histidine kinase sensor protein PhoQ is activated by low extracellular concentrations of divalent cations such as Mg2+, and subsequently the response regulator PhoP is activated in turn through a classic phosphotransfer pathway that is typical in such systems. The PhoQ sensor domains of enteric bacteria contain an acidic cluster of residues (EDDDDAE) that has been implicated in direct binding to divalent cations. We have determined crystal structures of the wild-type Escherichia coli PhoQ periplasmic sensor domain and of a mutant variant in which the acidic cluster was neutralized to conservative uncharged residues (QNNNNAQ). The PhoQ domain structure is similar to that of DcuS and CitA sensor domains, and this PhoQ-DcuS-CitA (PDC) sensor fold is seen to be distinct from the superficially similar PAS domain fold. Analysis of the wild-type structure reveals a dimer that allows for the formation of a salt bridge across the dimer interface between Arg-50' and Asp-179 and with nickel ions bound to aspartate residues in the acidic cluster. The physiological importance of the salt bridge to in vivo PhoQ function has been confirmed by mutagenesis. The mutant structure has an alternative, non-physiological dimeric association.     

Crystal structure of the BstDEAD N-terminal domain: a novel DEAD protein from Bacillus stearothermophilus

Most cellular processes requiring RNA structure rearrangement necessitate the action of Asp-Glu-Ala-Asp (DEAD) proteins. Members of the family, named originally for the conserved DEAD amino acid sequence, are thought to disrupt RNA structure and facilitate its rearrangement by unwinding short stretches of duplex RNA. BstDEAD is a novel 436 amino acid representative of the DEAD protein family from Bacillus stearothermophilus that contains all eight conserved motifs found in DEAD proteins and is homologous with other members of the family. Here, we describe the 1.85 A resolution structure of the N-terminal domain (residues 1-211) of BstDEAD (BstDEAD-NT). Similar to the corresponding domains of related helicases, BstDEAD-NT adopts a parallel alpha/beta structure with RecA-like topology. In general, the conserved motifs superimpose on closely related DEAD proteins and on more distantly related helicases such as RecA. This affirms the current belief that the core helicase domains, responsible for mechanistic activity, are structurally similar in DEAD proteins. In contrast, however, the so-called Walker A P-loop, which binds the beta- and gamma-phosphates of ATP, adopts a rarely seen closed conformation that would sterically block ATP binding. The closed conformation may be indicative of a general regulatory feature among DEAD proteins (and RNA helicases) that differs from that used by DNA helicases. BstDEAD also contains a unique extension of approximately 60 residues at the C terminus that is highly basic, suggesting that it might bind nucleic acids and, in so doing, confer specificity to the helicase activity of the core region.     

Crystal structure of the glutamate receptor GluA1 N-terminal domain

The AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) subfamily of iGluRs (ionotropic glutamate receptors) is essential for fast excitatory neurotransmission in the central nervous system. The malfunction of AMPARs (AMPA receptors) has been implicated in many neurological diseases, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. The active channels of AMPARs and other iGluR subfamilies are tetramers formed exclusively by assembly of subunits within the same subfamily. It has been proposed that the assembly process is controlled mainly by the extracellular ATD (N-terminal domain) of iGluR. In addition, ATD has also been implicated in synaptogenesis, iGluR trafficking and trans-synaptic signalling, through unknown mechanisms. We report in the present study a 2.5 ? (1 ?=0.1 nm) resolution crystal structure of the ATD of GluA1. Comparative analyses of the structure of GluA1-ATD and other subunits sheds light on our understanding of how ATD drives subfamily-specific assembly of AMPARs. In addition, analysis of the crystal lattice of GluA1-ATD suggests a novel mechanism by which the ATD might participate in inter-tetramer AMPAR clustering, as well as in trans-synaptic protein-protein interactions.     

Crystal structures of the Apo and penicillin-acylated forms of the BlaR1 beta-lactam sensor of Staphylococcus aureus

Staphylococcus aureus is among the most prevalent and antibiotic-resistant of pathogenic bacteria. The resistance of S. aureus to prototypal beta-lactam antibiotics is conferred by two mechanisms: (i) secretion of hydrolytic beta-lactamase enzymes and (ii) production of beta-lactam-insensitive penicillin-binding proteins (PBP2a). Despite their distinct modes of resistance, expression of these proteins is controlled by similar regulation systems, including a repressor (BlaI/MecI) and a multidomain transmembrane receptor (BlaR1/MecR1). Resistance is triggered in response to a covalent binding event between a beta-lactam antibiotic and the extracellular sensor domain of BlaR1/MecR1 by transduction of the binding signal to an intracellular protease domain capable of repressor inactivation. This study describes the first crystal structures of the sensor domain of BlaR1 (BlaRS) from S. aureus in both the apo and penicillin-acylated forms. The structures show that the sensor domain resembles the beta-lactam-hydrolyzing class D beta-lactamases, but is rendered a penicillin-binding protein due to the formation of a very stable acyl-enzyme. Surprisingly, conformational changes upon penicillin binding were not observed in our structures, supporting the hypothesis that transduction of the antibiotic-binding signal into the cytosol is mediated by additional intramolecular interactions of the sensor domain with an adjacent extracellular loop in BlaR1.     

Crystal structure of the C-type lectin-like domain from the human hematopoietic cell receptor CD69

CD69, one of the earliest specific antigens acquired during lymphoid activation, acts as a signal-transducing receptor involved in cellular activation events, including proliferation and induction of specific genes. CD69 belongs to a family of receptors that modulate the immune response and whose genes are clustered in the natural killer (NK) gene complex. The extracellular portion of these receptors represent a subfamily of C-type lectin-like domains (CTLDs), which are divergent from true C-type lectins and are referred to as NK-cell domains (NKDs). We have determined the three-dimensional structure of human CD69 NKD in two different crystal forms. CD69 NKD adopts the canonical CTLD fold but lacks the features involved in Ca(2+) and carbohydrate binding by C-type lectins. CD69 NKD dimerizes noncovalently, both in solution and in crystalline state. The dimer interface consists of a hydrophobic, loosely packed core, surrounded by polar interactions, including an interdomain beta sheet. The intersubunit core shows certain structural plasticity that may facilitate conformational rearrangements for binding to ligands. The surface equivalent to the binding site of other members of the CTLD superfamily reveals a hydrophobic patch surrounded by conserved charged residues that probably constitutes the CD69 ligand-binding site.     

Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain

The glucagon-like peptide-1 receptor (GLP-1R) belongs to Family B1 of the seven-transmembrane G protein-coupled receptors, and its natural agonist ligand is the peptide hormone glucagon-like peptide-1 (GLP-1). GLP-1 is involved in glucose homeostasis, and activation of GLP-1R in the plasma membrane of pancreatic beta-cells potentiates glucose-dependent insulin secretion. The N-terminal extracellular domain (nGLP-1R) is an important ligand binding domain that binds GLP-1 and the homologous peptide Exendin-4 with differential affinity. Exendin-4 has a C-terminal extension of nine amino acid residues known as the "Trp cage", which is absent in GLP-1. The Trp cage was believed to interact with nGLP-1R and thereby explain the superior affinity of Exendin-4. However, the molecular details that govern ligand binding and specificity of nGLP-1R remain undefined. Here we report the crystal structure of human nGLP-1R in complex with the antagonist Exendin-4(9-39) solved by the multiwavelength anomalous dispersion method to 2.2A resolution. The structure reveals that Exendin-4(9-39) is an amphipathic alpha-helix forming both hydrophobic and hydrophilic interactions with nGLP-1R. The Trp cage of Exendin-4 is not involved in binding to nGLP-1R. The hydrophobic binding site of nGLP-1R is defined by discontinuous segments including primarily a well defined alpha-helix in the N terminus of nGLP-1R and a loop between two antiparallel beta-strands. The structure provides for the first time detailed molecular insight into ligand binding of the human GLP-1 receptor, an established target for treatment of type 2 diabetes.     

Structure, function, and fate of the BlaR signal transducer involved in induction of beta-lactamase in Bacillus licheniformis.

The membrane-spanning protein BlaR is essential for the induction of beta-lactamase in Bacillus licheniformis. Its nature and location were confirmed by the use of an antiserum specific for its carboxy-terminal penicillin sensor, its function was studied by genetic dissection, and the structure of the penicillin sensor was derived from hydrophobic cluster analysis of the amino acid sequence by using, as a reference, the class A beta-lactamases with known three-dimensional structures. During the first 2 h after the addition of the beta-lactam inducer, full-size BlaR, bound to the plasma membrane, is produced, and then beta-lactamase is produced. By 2 h after induction, BlaR is present in various (membrane-bound and cytosolic) forms, and there is a gradual decrease in beta-lactamase production. The penicillin sensors of BlaR and the class D beta-lactamases show strong similarities in primary structures. They appear to have the same basic spatial disposition of secondary structures as that of the class A beta-lactamases, except that they lack several alpha helices and, therefore, have a partially uncovered five-stranded beta sheet and a more readily accessible active site. Alterations of BlaR affecting conserved secondary structures of the penicillin sensor and specific sites of the transducer annihilate beta-lactamase inducibility.     

Crystal structure of uroporphyrinogen decarboxylase from Bacillus subtilis

Uroporphyrinogen decarboxylase (UROD) is a branch point enzyme in the biosynthesis of the tetrapyrroles. It catalyzes the decarboxylation of four acetate groups of uroporphyrinogen III to yield coproporphyrinogen III, leading to heme and chlorophyll biosynthesis. UROD is a special type of nonoxidative decarboxylase, since no cofactor is essential for catalysis. In this work, the first crystal structure of a bacterial UROD, Bacillus subtilis UROD (UROD(Bs)), has been determined at a 2.3 A resolution. The biological unit of UROD(Bs) was determined by dynamic light scattering measurements to be a homodimer in solution. There are four molecules in the crystallographic asymmetric unit, corresponding to two homodimers. Structural comparison of UROD(Bs) with eukaryotic URODs reveals a variation of two loops, which possibly affect the binding of substrates and release of products. Structural comparison with the human UROD-coproporphyrinogen III complex discloses a similar active cleft, with five invariant polar residues (Arg29, Arg33, Asp78, Tyr154, and His322) and three invariant hydrophobic residues (Ile79, Phe144, and Phe207), in UROD(Bs). Among them, Asp78 may interact with the pyrrole NH groups of the substrate, and Arg29 is a candidate for positioning the acetate groups of the substrate. Both residues may also play catalytic roles.     

Crystal structure of the dimeric coiled-coil domain of the cytosolic nucleic acid sensor LRRFIP1

LRRFIP1 binds cytoplasmic double-stranded DNA and RNA and interacts with FLI, the mammalian homolog of Drosophila flightless I, through a highly conserved 87-amino acid domain. Upon binding nucleic acid ligands, LRRFIP1 recruits and activates β-catenin, leading to the IRF3-dependent production of type I interferon. However, the molecular mechanism of LRRFIP1 signaling is not well understood. Here we show that the FLI-interacting domain of LRRFIP1 forms a classic parallel, homodimeric coiled coil with 10 heptad repeats and 22 helical turns. The coiled coil domain is also a dimer in solution. However, a longer LRRFIP1 construct spanning the coiled coil and DNA binding domains assembles into higher order oligomers in solution. The structure of LRRFIP1-CC constitutes a valuable tool for probing the mechanism of LRRFIP1 signaling and for structural studies of larger LRRFIP1 constructs.     

Purification and structure elucidation of the N-acetylbacillosamine-containing polysaccharide from Bacillus licheniformis ATCC 9945.

The exopolysaccharide of Bacillus licheniformis ATCC 9945 (formerly B. subtilis ATCC 9945) contains among other glycoses 4-acetamido-2-amino-2,4,6-trideoxy-D-glucose, termed N-acetylbacillosamine (Bac2N4NAc). A similar diamino glycose, 2-acetamido-4-amino-2,4,6-trideoxy-D-glucose, was found in a surface layer (S-layer) glycoprotein preparation of Clostridium symbiosum HB25. Electron microscopic studies, however, showed that B. licheniformis ATCC 9945 is not covered with an S-layer lattice, indicating that the N-acetylbacillosamine present in that organism might be a constituent of a cell wall-associated polymer. For elucidation of the structure of the N-acetylbacillosamine-containing polysaccharide, it was purified from a trichloroacetic acid extract of B. licheniformis ATCC 9945 cells. Using different hydrolysis protocols and a hydrolysate of the S-layer glycoprotein preparation from C. symbiosum HB25 as reference, the purified polysaccharide was found to contain 2,4-diamino-2,4,6-trideoxy-glucose, 2-acetamido-2-deoxy-glucose, 2-acetamido-2-deoxy-galactose and galactose in a molar ratio of 1 : 1 : 1 : 2. One- and two-dimensional NMR spectroscopy, including 800 MHz proton magnetic resonance measurements, in combination with chemical modification and degradation experiments, revealed that the polysaccharide consists of identical pyruvylated pentasaccharide repeating units with the structure: [-->3)-[(S)Py-(3,4)-beta-D-Galp-(1-->6)]-alpha-D-GlcpNAc-(1-->3)-beta-D-Bacp2N4NAc-(1-->3)-[(S)Py-(3,4)-beta-D-Galp-(1-->6)]-beta-D-GalpNAc-(1-->](n)     

Crystal structure of a prolactin receptor antagonist bound to the extracellular domain of the prolactin receptor

The crystal structure of the complex between an N-terminally truncated G129R human prolactin (PRL) variant and the extracellular domain of the human prolactin receptor (PRLR) was determined at 2.5A resolution by x-ray crystallography. This structure represents the first experimental structure reported for a PRL variant bound to its cognate receptor. The binding of PRL variants to the PRLR extracellular domain was furthermore characterized by the solution state techniques, hydrogen exchange mass spectrometry, and NMR spectroscopy. Compared with the binding interface derived from mutagenesis studies, the structural data imply that the definition of PRL binding site 1 should be extended to include residues situated in the N-terminal part of loop 1 and in the C terminus. Comparison of the structure of the receptor-bound PRL variant with the structure reported for the unbound form of a similar analogue ( Jomain, J. B., Tallet, E., Broutin, I., Hoos, S., van Agthoven, J., Ducruix, A., Kelly, P. A., Kragelund, B. B., England, P., and Goffin, V. (2007) J. Biol. Chem. 282, 33118-33131 ) demonstrates that receptor-induced changes in the backbone of the four-helix bundle are subtle, whereas large scale rearrangements and structuring occur in the flexible N-terminal part of loop 1. Hydrogen exchange mass spectrometry data imply that the dynamics of the four-helix bundle in solution generally become stabilized upon receptor interaction at binding site 1.     

Lysine Nzeta-decarboxylation switch and activation of the beta-lactam sensor domain of BlaR1 protein of methicillin-resistant Staphylococcus aureus

The integral membrane protein BlaR1 of methicillin-resistant Staphylococcus aureus senses the presence of β-lactam antibiotics in the milieu and transduces the information to the cytoplasm, where the biochemical events that unleash induction of antibiotic resistance mechanisms take place. We report herein by two-dimensional and three-dimensional NMR experiments of the sensor domain of BlaR1 in solution and by determination of an x-ray structure for the apo protein that Lys-392 of the antibiotic-binding site is posttranslationally modified by N(ζ)-carboxylation. Additional crystallographic and NMR data reveal that on acylation of Ser-389 by antibiotics, Lys-392 experiences N(ζ)-decarboxylation. This unique process, termed the lysine N(ζ)-decarboxylation switch, arrests the sensor domain in the activated ("on") state, necessary for signal transduction and all the subsequent biochemical processes. We present structural information on how this receptor activation process takes place, imparting longevity to the antibiotic-receptor complex that is needed for the induction of the antibiotic-resistant phenotype in methicillin-resistant S. aureus.     

Extracellular serine proteinase from Bacillus licheniformis

The serine proteinase from B. licheniformis was purified by affinity chromatography on the sorbent obtained by attachment of p-(omega-aminomethyl)-phenylboronic acid via an amino group to CH-Sepharose. The use of this sorbent specific to the serine proteinases active sites resulted in a 35-fold purification of the enzyme with an apparent activity yield of 288%. Such a high activity yield is due to a removal of the enzyme inhibitors. The N-terminal sequence of B. licheniformis extracellular serine proteinase traced for 35 amino acid residues coincides with that of subtilisin Carlberg, a serine proteinase presumed to be secreted by a B. subtilis strain. Since the amino acid composition as well as the functional properties of these two enzymes did not reveal any noticeable differences, it was assumed that both proteinases are very similar, if not identical. This conclusion leads to reconsideration of the existing concept on an extremely fast rate of subtilisin evolution. Three multiple forms of B. licheniformis extracellular serine proteinase were found to differ only in their net charges, presumably as a result of partial deamidation of Asn or Gln residues within their structure.     

Evidence of an intramolecular interaction between the two domains of the BlaR1 penicillin receptor during the signal transduction

The BlaR1 protein is a penicillin-sensory transducer involved in the induction of the Bacillus licheniformis beta-lactamase. The amino-terminal domain of the protein exhibits four transmembrane segments (TM1-TM4) that form a four-alpha-helix bundle embedded in the plasma bilayer. The carboxyl-terminal domain of 250 amino acids (BlaR-CTD) fused at the carboxyl end of TM4 possesses the amino acid sequence signature of penicillin-binding proteins. This membrane topology suggests that BlaR-CTD and the BlaR-amino-terminal domain are responsible for signal reception and signal transduction, respectively. With the use of phage display experiments, we highlight herein an interaction between BlaR-CTD and the extracellular, 63-amino acid L2 loop connecting TM2 and TM3. This interaction does not occur in the presence of penicillin. This result suggests that binding of the antibiotic to BlaR1 might entail the release of the interaction between L2 and BlaR-CTD, causing a motion of the alpha-helix bundle and transfer of the information to the cytoplasm of the cell. In addition, fluorescence spectroscopy, CD, and Fourier transform IR spectroscopy experiments indicate that in contrast to the behavior of the corresponding Staphylococcus aureus protein, the beta-lactam antibiotic does not induce a drastic conformational change in B. licheniformis BlaR-CTD.     

The penicillin sensory transducer, BlaR, involved in the inducibility of β-lactamase synthesis in Bacillus licheniformis is embedded in the plasma membrane via a four-α-helix bundle

Prediction studies, conformational analyses and membrane-topology mapping lead to the conclusion that the penicillin sensory transducer, BlaR, involved in the inducibility of β-lactamase synthesis in Bacillus licheniformis , is embedded in the plasma membrane bilayer via four transmembrane segments TM1–TM4 that form a four-α-helix bundle. The extracellular 262-amino-acid-residue polypeptide, S340–R601, that is fused at the carboxy end of TM4, possesses the amino acid sequence signature of a penicilloyl serine transferase. It probably functions as penicillin sensor. As an independent entity, this polypeptide behaves as a high-affinity penicillin-binding protein. As a component of the full-size BlaR, it adopts a different conformation presumably because of interactions with the extracellular 63-amino-acid-residue P53–S115 loop that connects TM2 and TM3. Reception of the penicillin-induced signal requires a precise conformation of the sensor but it does not involve penicilloylation of the serine residue S402 of motif STYK. Signal transmission through the plasma membrane by the four-α-helix bundle may proceed in a way comparable to that of the aspartate receptor, Tar. Signal emission in the cytosol by the intracellular 189-amino-acid-residue Y134–K322 loop that connects TM3 and TM4, may proceed via the activation of a putative metallopeptidase.