Crystal structure of the Bacillus subtilis penicillin-binding protein 4a, and its complex with a peptidoglycan mimetic peptide
Sauvage, Eric ; Duez, Colette ; Herman, Raphaël et al
in Journal of Molecular Biology (2007), 371(2), 528-539
The genome of Bacillus subtilis encodes 16 penicillin-binding proteins (PBPs) involved in the synthesis and/or remodelling of the peptidoglycan during the complex life cycle of this sporulating Gram ... [more ▼]
The genome of Bacillus subtilis encodes 16 penicillin-binding proteins (PBPs) involved in the synthesis and/or remodelling of the peptidoglycan during the complex life cycle of this sporulating Gram-positive rod-shaped bacterium. PBP4a (encoded by the dacC gene) is a low-molecular mass PBP clearly exhibiting in vitro DD-carboxypeptidase activity. We have solved the crystal structure of this protein alone and in complex with a peptide (D-alpha'-aminopymelyl-epsilon-D-alanyl-D-alanine) that mimics the C-terminal end of the Bacillus peptidoglycan stem peptide. PBP4a is composed of three domains: the penicillin-binding domain with a fold similar to the class A 13-lactamase structure and two domains inserted between the conserved motifs 1 and 2 characteristic of the penicillin-recognizing enzymes. The soaking of PBP4a in a solution Of D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine resulted in an adduct between PBP4a and a D-alpha-aminopimelyl-epsilon-D-alanine dipeptide and an unbound D-alanine, i.e. the products of acylation of PBP4a by D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine with the release of a D-alanine. The adduct also reveals a binding pocket specific to the diaminopimelic acid, the third residue of the peptidoglycan stem pentapeptide of B. subtilis. This pocket is specific for this class of PBPs. (C) 2007 Elsevier Ltd. All rights reserved. [less ▲]Detailed reference viewed: 58 (15 ULg)
Structural basis for the actin-binding function of missing-in-metastasis.
; Kerff, Frédéric ; et al
in Structure (2007), 15(2), 145-55
The adaptor protein missing-in-metastasis (MIM) contains independent F- and G-actin binding domains, consisting, respectively, of an N-terminal 250 aa IRSp53/MIM homology domain (IMD) and a C-terminal ... [more ▼]
The adaptor protein missing-in-metastasis (MIM) contains independent F- and G-actin binding domains, consisting, respectively, of an N-terminal 250 aa IRSp53/MIM homology domain (IMD) and a C-terminal WASP-homology domain 2 (WH2). We determined the crystal structures of MIM's IMD and that of its WH2 bound to actin. The IMD forms a dimer, with each subunit folded as an antiparallel three-helix bundle. This fold is related to that of the BAR domain. Like the BAR domain, the IMD has been implicated in membrane binding. Yet, comparison of the structures reveals that the membrane binding surfaces of the two domains have opposite curvatures, which may determine the type of curvature of the interacting membrane. The WH2 of MIM is longer than the prototypical WH2, interacting with all four subdomains of actin. We characterize a similar WH2 at the C terminus of IRSp53 and propose that in these two proteins WH2 performs a scaffolding function. [less ▲]Detailed reference viewed: 9 (0 ULg)
Structure of the class D beta-lactamase OXA-29, a different carbonated dimer
Poster (2006, August)Detailed reference viewed: 3 (0 ULg)
Crystal structure of the actin-binding domain of alpha-actinin 1: evaluating two competing actin-binding models.
; Kerff, Frédéric ; et al
in Journal of Structural Biology (2006), 155(2), 230-8
Alpha-actinin belongs to the spectrin family of actin crosslinking and bundling proteins that function as key regulators of cell motility, morphology and adhesion. The actin-binding domain (ABD) of these ... [more ▼]
Alpha-actinin belongs to the spectrin family of actin crosslinking and bundling proteins that function as key regulators of cell motility, morphology and adhesion. The actin-binding domain (ABD) of these proteins consists of two consecutive calponin homology (CH) domains. Electron microscopy studies on ABDs appear to support two competing actin-binding models, extended and compact, whereas the crystal structures typically display a compact conformation. We have determined the 1.7A resolution structure of the ABD of alpha-actinin 1, a ubiquitously expressed isoform. The structure displays the classical compact conformation. We evaluated the two binding models by surface conservation analysis. The results show a conserved surface that spans both domains and corresponds to two previously identified actin-binding sites (ABS2 and ABS3). A third, and probably less important site, ABS1, is mostly buried in the compact conformation. However, a thorough examination of existing structures suggests a weak and semi-polar binding interface between the two CHs, leaving open the possibility of domain reorientation or opening. Our results are consistent with a two-step binding mechanism in which the ABD interacts first in the compact form observed in the structures, and then transitions toward a higher affinity state, possibly through minor rearrangement of the domains. [less ▲]Detailed reference viewed: 14 (1 ULg)
Actin-bound structures of Wiskott-Aldrich syndrome protein (WASP)-homology domain 2 and the implications for filament assembly.
; Kerff, Frédéric ; et al
in Proceedings of the National Academy of Sciences of the United States of America (2005), 102(46), 16644-9
Wiskott-Aldrich syndrome protein (WASP)-homology domain 2 (WH2) is a small and widespread actin-binding motif. In the WASP family, WH2 plays a role in filament nucleation by Arp2/3 complex. Here we ... [more ▼]
Wiskott-Aldrich syndrome protein (WASP)-homology domain 2 (WH2) is a small and widespread actin-binding motif. In the WASP family, WH2 plays a role in filament nucleation by Arp2/3 complex. Here we describe the crystal structures of complexes of actin with the WH2 domains of WASP, WASP-family verprolin homologous protein, and WASP-interacting protein. Despite low sequence identity, WH2 shares structural similarity with the N-terminal portion of the actin monomer-sequestering thymosin beta domain (Tbeta). We show that both domains inhibit nucleotide exchange by targeting the cleft between actin subdomains 1 and 3, a common binding site for many unrelated actin-binding proteins. Importantly, WH2 is significantly shorter than Tbeta but binds actin with approximately 10-fold higher affinity. WH2 lacks a C-terminal extension that in Tbeta4 becomes involved in monomer sequestration by interfering with intersubunit contacts in F-actin. Owing to their shorter length, WH2 domains connected in tandem by short linkers can coexist with intersubunit contacts in F-actin and are proposed to function in filament nucleation by lining up actin subunits along a filament strand. The WH2-central region of WASP-family proteins is proposed to function in an analogous way by forming a special class of tandem repeats whose function is to line up actin and Arp2 during Arp2/3 nucleation. The structures also suggest a mechanism for how profilin-binding Pro-rich sequences positioned N-terminal to WH2 could feed actin monomers directly to WH2, thereby playing a role in filament elongation. [less ▲]Detailed reference viewed: 27 (0 ULg)
Structural basis of the actin-calponin homology domain interaction.
; Kerff, Frédéric ; Li, Yi et al
in Biophysical Journal (2005), 88(1), 497-497Detailed reference viewed: 4 (0 ULg)
Structural basis of the actin-WH2 domain interaction.
; Kerff, Frédéric ; et al
in Biophysical Journal (2005), 88(1), 10-11Detailed reference viewed: 5 (0 ULg)
Structural basis of protein phosphatase 1 regulation
Terrak, Mohammed ; Kerff, Frédéric ; et al
in Nature (2004), 429(6993), 780-4
The coordinated and reciprocal action of serine/threonine (Ser/Thr) protein kinases and phosphatases produces transient phosphorylation, a fundamental regulatory mechanism for many biological processes ... [more ▼]
The coordinated and reciprocal action of serine/threonine (Ser/Thr) protein kinases and phosphatases produces transient phosphorylation, a fundamental regulatory mechanism for many biological processes. The human genome encodes a far greater number of Ser/Thr protein kinases than of phosphatases. Protein phosphatase 1 (PP1), in particular, is ubiquitously distributed and regulates a broad range of cellular functions, including glycogen metabolism, cell-cycle progression and muscle relaxation. PP1 has evolved effective catalytic machinery but lacks substrate specificity. Substrate specificity is conferred upon PP1 through interactions with a large number of regulatory subunits. The regulatory subunits are generally unrelated, but most possess the RVxF motif, a canonical PP1-binding sequence. Here we reveal the crystal structure at 2.7 A resolution of the complex between PP1 and a 34-kDa N-terminal domain of the myosin phosphatase targeting subunit MYPT1. MYPT1 is the protein that regulates PP1 function in smooth muscle relaxation. Structural elements amino- and carboxy-terminal to the RVxF motif of MYPT1 are positioned in a way that leads to a pronounced reshaping of the catalytic cleft of PP1, contributing to the increased myosin specificity of this complex. The structure has general implications for the control of PP1 activity by other regulatory subunits. [less ▲]Detailed reference viewed: 110 (49 ULg)
PP1 targeting and regulation as revealed by the structure of a PP1 delta-MYPT1 complex.
Terrak, Mohammed ; Kerff, Frédéric ; et al
in Biophysical Journal (2004), 86(1), 172-172Detailed reference viewed: 16 (8 ULg)
Crystal structure of the sensor domain of the BlaR penicillin receptor from Bacillus licheniformis
Kerff, Frédéric ; Charlier, Paulette ; et al
in Biochemistry (2003), 42(44), 12835-12843
As in several staphylococci, the synthesis of the Bacillus licheniformis 749/I beta-lactamase is an inducible phenomenon regulated by a signal-transducing membrane protein BlaR. The C-terminal domain of ... [more ▼]
As in several staphylococci, the synthesis of the Bacillus licheniformis 749/I beta-lactamase is an inducible phenomenon regulated by a signal-transducing membrane protein BlaR. The C-terminal domain of this multimodular protein is an extracellular domain which specifically recognizes beta-lactam antibiotics. When it binds a beta-lactam, a signal is transmitted by the transmembrane region to the intracellular loops. In response, the hydrolytic activity of the BlaR large cytoplasmic L3 loop is induced, and a cascade of reactions is generated, leading to the transcription of the beta-lactamase gene. Here, we describe the crystal structure of the extracellular penicillin-receptor domain of BlaR (residues 346-601) at 2.5 Angstrom resolution in order to understand why this domain, whose folding is very similar to that of class D beta-lactamases, behaves as a highly sensitive penicillin-binding protein rather than a beta-lactamase. Two residues of the BlaR C-terminal domain, Thr452 and Thr542, modify the hydrophobic characteristic of the class D beta-lactamase active site. Both residues seem to be in part responsible for the lack of beta-lactamase activity of the BlaR protein due to the stability of the acyl-enzyme. Although further experimental data are needed to fully understand the transmembrane induction process, the comparison of the BlaR sensor domain structure with those of class D beta-lactamase complexes and penicillin-binding proteins provides interesting elements to hypothesize on possible signal transmission mechanisms. [less ▲]Detailed reference viewed: 31 (5 ULg)
The X-ray structure of the class D beta-lactamase OXA-2 from Salmonella typhymurium
Conference (2002, March 30)Detailed reference viewed: 5 (0 ULg)
The 2.4-A crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin.
Sauvage, Eric ; Kerff, Frédéric ; et al
in Cellular and Molecular Life Sciences : CMLS (2002), 59(7), 1223-32
Penicillin-binding proteins (PBPs) are membrane proteins involved in the final stages of peptidoglycan synthesis and represent the targets of beta-lactam antibiotics. Enterococci are naturally resistant ... [more ▼]
Penicillin-binding proteins (PBPs) are membrane proteins involved in the final stages of peptidoglycan synthesis and represent the targets of beta-lactam antibiotics. Enterococci are naturally resistant to these antibiotics because they produce a PBP, named PBP5fm in Enterococcus faecium, with low-level affinity for beta-lactams. We report here the crystal structure of the acyl-enzyme complex of PBP5fm with benzylpenicillin at a resolution of 2.4 A. A characteristic of the active site, which distinguishes PBP5fm from other PBPs of known structure, is the topology of the loop 451-465 defining the left edge of the cavity. The residue Arg464, involved in a salt bridge with the residue Asp481, confers a greater rigidity to the PBP5fm active site. In addition, the presence of the Val465 residue, which points into the active site, reducing its accessibility, could account for the low affinity of PBP5fm for beta-lactam. This loop is common to PBPs of low affinity, such as PBP2a from Staphylococcus aureus and PBP3 from Bacillus subtilis. Moreover, the insertion of a serine after residue 466 in the most resistant strains underlines even more the determining role of this loop in the recognition of the substrates. [less ▲]Detailed reference viewed: 23 (6 ULg)
Use of a variable incidence angle PIXE arrangement for studying pigment multilayers
Weber, Georges ; ; Strivay, David et al
in Nuclear Instruments & Methods in Physics Research. Section B, Beam Interactions with Materials and Atoms (1998), 139(1-4), 196-201
Particle induced X-ray emission (PIXE) method is used in the field of archeometry and specially to investigate pigment colored multilayers. The tilting of the sample with respect to the incident proton ... [more ▼]
Particle induced X-ray emission (PIXE) method is used in the field of archeometry and specially to investigate pigment colored multilayers. The tilting of the sample with respect to the incident proton beam direction allows to modify the relative contribution of each layer to the fluorescence signal. The experimental results coupled to computer simulations lead to semi-quantitative information about the thickness, the position and the composition of the successive layers. (C) 1998 Elsevier Science B.V. [less ▲]