Crystal Structures of Penicillin-Binding Proteins 4 and 5 from Haemophilus influenzae

We have determined high-resolution apo crystal structures of two low molecular weight penicillin-binding proteins (PBPs), PBP4 and PBP5, from Haemophilus influenzae, one of the most frequently found pathogens in the upper respiratory tract of children. Novel β-lactams with notable antimicrobial activity have been designed, and crystal structures of PBP4 complexed with ampicillin and two of the novel molecules have also been determined. Comparing the apo form with those of the complexes, we find that the drugs disturb the PBP4 structure and weaken X-ray diffraction, to very different extents. PBP4 has recently been shown to act as a sensor of the presence of penicillins in Pseudomonas aeruginosa, and our models offer a clue to the structural basis for this effect. Covalently attached penicillins press against a phenylalanine residue near the active site and disturb the deacylation step. The ready inhibition of PBP4 by β-lactams compared to PBP5 also appears to be related to the weaker interactions holding key residues in a catalytically competent position.     

Unusual Conformation of the SxN Motif in the Crystal Structure of Penicillin-Binding Protein A from Mycobacterium tuberculosis

PBPA from Mycobacterium tuberculosis is a class B-like penicillin-binding protein (PBP) that is not essential for cell growth in M. tuberculosis, but is important for proper cell division in Mycobacterium smegmatis. We have determined the crystal structure of PBPA at 2.05 Å resolution, the first published structure of a PBP from this important pathogen. Compared to other PBPs, PBPA has a relatively small N-terminal domain, and conservation of a cluster of charged residues within this domain suggests that PBPA is more related to class B PBPs than previously inferred from sequence analysis. The C-terminal domain is a typical transpeptidase fold and contains the three conserved active-site motifs characterisitic of penicillin-interacting enzymes. Whilst the arrangement of the SxxK and KTG motifs is similar to that observed in other PBPs, the SxN motif is markedly displaced away from the active site, such that its serine (Ser281) is not involved in hydrogen bonding with residues of the other two motifs. A disulfide bridge between Cys282 (the “x” of the SxN motif) and Cys266, which resides on an adjacent loop, may be responsible for this unusual conformation. Another interesting feature of the structure is a relatively long connection between β5 and α11, which restricts the space available in the active site of PBPA and suggests that conformational changes would be required to accommodate peptide substrate or β-lactam antibiotics during acylation. Finally, the structure shows that one of the two threonines postulated to be targets for phosphorylation is inaccessible (Thr362), whereas the other (Thr437) is well placed on a surface loop near the active site.     

PBP Active Site Flexibility as the Key Mechanism for β-Lactam Resistance in Pneumococci

Penicillin-binding proteins (PBPs), the main targets of β-lactam antibiotics, are membrane-associated enzymes that catalyze the two last steps in the biosynthesis of peptidoglycan. In Streptococcus pneumoniae, a major human pathogen, the surge in resistance to such antibiotics is a direct consequence of the proliferation of mosaic PBP-encoding genes, which give rise to proteins containing tens of mutations. PBP2b is a major drug resistance target, and its modification is essential for the development of high levels of resistance to piperacillin. In this work, we have solved the crystal structures of PBP2b from a wild-type pneumococcal strain, as well as from a highly drug-resistant clinical isolate displaying 58 mutations. Although mutations are present throughout the entire PBP structure, those surrounding the active site influence the total charge and the polar character of the region, while those in close proximity to the catalytic nucleophile impart flexibility onto the β3/β4 loop area, which encapsulates the cleft. The wealth of structural data on pneumococcal PBPs now underlines the importance of high malleability in active site regions of drug-resistant strains, suggesting that active site “breathing” could be a common mechanism employed by this pathogen to prevent targeting by β-lactams.     

Crystal Structures of Penicillin-Binding Protein 3 (PBP3) from Methicillin-Resistant Staphylococcus aureus in the Apo and Cefotaxime‐Bound Forms

Staphylococcus aureus is a widespread Gram‐positive opportunistic pathogen, and a methicillin‐resistant form (MRSA) is particularly difficult to treat clinically. We have solved two crystal structures of penicillin‐binding protein (PBP) 3 (PBP3) from MRSA, the apo form and a complex with the β-lactam antibiotic cefotaxime, and used electrospray mass spectrometry to measure its sensitivity to a variety of penicillin derivatives. PBP3 is a class B PBP, possessing an N-terminal non-penicillin‐binding domain, sometimes called a dimerization domain, and a C-terminal transpeptidase domain. The model shows a different orientation of its two domains compared to earlier models of other class B PBPs and a novel, larger N-domain. Consistent with the nomenclature of “dimerization domain”, the N-terminal region forms an apparently tight interaction with a neighboring molecule related by a 2-fold symmetry axis in the crystal structure. This dimer form is predicted to be highly stable in solution by the PISA server, but mass spectrometry and analytical ultracentrifugation provide unequivocal evidence that the protein is a monomer in solution.     

Fluorescence anisotropy-based measurement of Pseudomonas aeruginosa penicillin-binding protein 2 transpeptidase inhibitor acylation rate constants

High-molecular-weight penicillin-binding proteins (PBPs) are essential integral membrane proteins of the bacterial cytoplasmic membrane responsible for biosynthesis of peptidoglycan. They are the targets of antibacterial β-lactam drugs, including penicillins, cephalosporins, and carbapenems. β-Lactams covalently acylate the active sites of the PBP transpeptidase domains. Because β-lactams are time-dependent inhibitors, quantitative assessment of the inhibitory activity of these compounds ideally involves measurement of their second-order acylation rate constants. We previously described a fluorescence anisotropy-based assay to measure these rate constants for soluble constructs of PBP3 (Anal. Biochem. 439 (2013) 37–43). Here we report the expression and purification of a soluble construct of Pseudomonas aeruginosa PBP2 as a fusion protein with NusA. This soluble PBP2 was used to measure second-order acylation rate constants with the fluorescence anisotropy assay. Measurements were obtained for mecillinam, which reacts specifically with PBP2, and for several carbapenems. The assay also revealed that PBP2 slowly hydrolyzed mecillinam and was used to measure the rate constant for this deacylation reaction.     

Crystal structures of complexes of bacterial DD-peptidases with peptidoglycan-mimetic ligands: the substrate specificity puzzle

The X-ray crystal structures of covalent complexes of the Actinomadura R39 dd-peptidase and Escherichia coli penicillin-binding protein (PBP) 5 with β-lactams bearing peptidoglycan-mimetic side chains have been determined. The structure of the hydrolysis product of an analogous peptide bound noncovalently to the former enzyme has also been obtained. The R39 dd-peptidase structures reveal the presence of a specific binding site for the d-α-aminopimelyl side chain, characteristic of the stem peptide of Actinomadura R39. This binding site features a hydrophobic cleft for the pimelyl methylene groups and strong hydrogen bonding to the polar terminus. Both of these active site elements are provided by amino acid side chains from two separate domains of the protein. In contrast, no clear electron density corresponding to the terminus of the peptidoglycan-mimetic side chains is present when these β-lactams are covalently bound to PBP5. There is, therefore, no indication of a specific side-chain binding site in this enzyme. These results are in agreement with those from kinetics studies published earlier and support the general prediction made at the time of a direct correlation between kinetics and structural evidence. The essential high-molecular-mass PBPs have demonstrated, to date, no specific reactivity with peptidoglycan-mimetic peptide substrates and β-lactam inhibitors and, thus, probably do not possess a specific substrate-binding site of the type demonstrated here with the R39 dd-peptidase. This striking deficiency may represent a sophisticated defense mechanism against low-molecular-mass substrate-analogue inhibitors/antibiotics; its discovery should focus new inhibitor design.     

Trapping of an acyl-enzyme intermediate in a penicillin-binding protein (PBP)-catalyzed reaction

Class A penicillin-binding proteins (PBPs) catalyze the last two steps in the biosynthesis of peptidoglycan, a key component of the bacterial cell wall. Both reactions, glycosyl transfer (polymerization of glycan chains) and transpeptidation (cross-linking of stem peptides), are essential for peptidoglycan stability and for the cell division process, but remain poorly understood. The PBP-catalyzed transpeptidation reaction is the target of β-lactam antibiotics, but their vast employment worldwide has prompted the appearance of highly resistant strains, thus requiring concerted efforts towards an understanding of the transpeptidation reaction with the goal of developing better antibacterials. This goal, however, has been elusive, since PBP substrates are rapidly deacylated. In this work, we provide a structural snapshot of a “trapped” covalent intermediate of the reaction between a class A PBP with a pseudo-substrate, N-benzoyl-d-alanylmercaptoacetic acid thioester, which partly mimics the stem peptides contained within the natural, membrane-associated substrate, lipid II. The structure reveals that the d-alanyl moiety of the covalent intermediate (N-benzoyl-d-alanine) is stabilized in the cleft by a network of hydrogen bonds that place the carbonyl group in close proximity to the oxyanion hole, thus mimicking the spatial arrangement of β-lactam antibiotics within the PBP active site. This arrangement allows the target bond to be in optimal position for attack by the acceptor peptide and is similar to the structural disposition of β-lactam antibiotics with PBP clefts. This information yields a better understanding of PBP catalysis and could provide key insights into the design of novel PBP inhibitors.     

Structural and computational investigations of VIM-7: insights into the substrate specificity of vim metallo-β-lactamases

The presence of metallo-β-lactamases (MBLs) in many clinically important human bacterial pathogens limits treatment options, as these enzymes efficiently hydrolyze nearly all β-lactam antibiotics. VIM enzymes are among the most widely distributed MBLs, but many of the individual VIM subtypes remain poorly characterized. Pseudomonas aeruginosa VIM-7 is the most divergent among VIM-type MBLs in terms of amino acid sequence. Here we present crystal structures of VIM-7 as the native enzyme, with Cys221 oxidized (VIM-7-Ox), and with a sulfur atom bridging the two active-site zinc ions (VIM-7-S). Comparison with VIM-2 and VIM-4 structures suggests an explanation for the reduced catalytic efficiency of VIM-7 against cephalosporins with a positively charged cyclic substituent at the C3 position (e.g., ceftazidime). Kinetic variations are attributed to substitutions in residues 60-66 (that form a loop adjacent to the active site previously implicated in substrate binding) and to the disruption of two hydrogen-bonding clusters through substitutions at positions 218 and 224. Furthermore, the less negatively charged surface of VIM-7 (compared to VIM-2) may also contribute to the reduced hydrolytic efficiency. Docking of the cephalosporins ceftazidime and cefotaxime into the VIM-2 and VIM-7 structures reveals that amino acid substitutions may cause the mode of substrate binding to differ between the two enzymes. Our structures thus provide new insights into the variation in substrate specificity that is evident across this family of clinically important enzymes.     

Fluctuations in nuclear envelope's potential mediate synchronization of early neural activity

Penicillin binding proteins (PBPs) catalyze essential steps in the biosynthesis of peptidoglycan, the main component of the bacterial cell wall. PBPs can harbor two catalytic domains, namely the glycosyltransferase (GT) and transpeptidase (TP) activities, the latter being the target for β-lactam antibiotics. Despite the availability of structural information regarding bi-functional PBPs, little is known regarding the interaction and flexibility between the TP and GT domains. Here, we describe the structural characterization in solution by small angle X-ray scattering (SAXS) of PBP1b, a bi-functional PBP from Streptococcus pneumoniae. The molecule is present in solution as an elongated monomer. Refinement of internal coordinates starting from a homology model yields models in which the two domains are in an extended conformation without any mutual contact compatible with the existence of restricted mobility.     

Selective inhibition of penicillin-binding proteins and its effects on growth and architecture of Staphylococcus aureus

We found that the three high molecular weight penicillin-binding proteins (PBP) 1, 2, and 3 of Staphylococcus aureus could be blocked by the β-lactam antibiotics imipenem, cefotaxime, and mecillinam, respectively. The inhibition of any of these PBPs was not sufficient for an antibacterial effect. Even the simultaneous blocking of PBPs 2 and 3, previously supposed to be the lethal targets of β-lactam antibiotics, did not induce bacteriolysis, nor did the combined saturation of PBPs 2, 3, and 4. Instead, PBP 1 seems to play a key role, because on one hand the combined inhibition of PBP 1 with any of the other high molecular weight PBPs led to bacteriolysis, on the other hand, only inhibition of PBP 1 led to a loss of the ‘splitting system’ of the staphylococcal cross wall, similar to that observed in penicillin G-treated cells earlier.     

Crystal structure of the Bacillus subtilis penicillin-binding protein 4a, and its complex with a peptidoglycan mimetic peptide

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 beta-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.     

Molecular Basis for the Role of Staphylococcus aureus Penicillin Binding Protein 4 in Antimicrobial Resistance

Penicillin binding proteins (PBPs) are membrane-associated proteins that catalyze the final step of murein biosynthesis. These proteins function as either transpeptidases or carboxypeptidases and in a few cases demonstrate transglycosylase activity. Both transpeptidase and carboxypeptidase activities of PBPs occur at the d-Ala-d-Ala terminus of a murein precursor containing a disaccharide pentapeptide comprising N-acetylglucosamine and N-acetyl-muramic acid-l-Ala-d-Glu-l-Lys-d-Ala-d-Ala. β-Lactam antibiotics inhibit these enzymes by competing with the pentapeptide precursor for binding to the active site of the enzyme. Here we describe the crystal structure, biochemical characteristics, and expression profile of PBP4, a low-molecular-mass PBP from Staphylococcus aureus strain COL. The crystal structures of PBP4-antibiotic complexes reported here were determined by molecular replacement, using the atomic coordinates deposited by the New York Structural Genomics Consortium. While the pbp4 gene is not essential for the viability of S. aureus, the knockout phenotype of this gene is characterized by a marked reduction in cross-linked muropeptide and increased vancomycin resistance. Unlike other PBPs, we note that expression of PBP4 was not substantially altered under different experimental conditions, nor did it change across representative hospital- or community-associated strains of S. aureus that were examined. In vitro data on purified recombinant S. aureus PBP4 suggest that it is a β-lactamase and is not trapped as an acyl intermediate with β-lactam antibiotics. Put together, the expression analysis and biochemical features of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.Penicillin binding proteins (PBPs) are critical components of the cell wall synthesis machinery in bacteria. These membrane-associated proteins are broadly classified as low-molecular-mass (LMM) PBPs that are monofunctional d,d-carboxypeptidase enzymes or multimodular high-molecular-mass (HMM) PBPs with multiple functional roles. PBPs, in general, are anchored to the cytoplasmic membrane by a noncleavable pseudo-signal peptide. In the case of the HMM PBPs, the cytoplasmic C-terminal domain binds penicillin and catalyzes peptidoglycan cross-linking, whereas the juxtamembrane N-terminal domain participates in transglycosylation (12). The catalytic penicillin-binding (PB) module also occurs as part of penicillin sensor transducers, such as Staphylococcus aureus MecR and Bacillus licheniformis BlaR (15). The transpeptidase activity in HMM PBPs is based on a conserved lysine residue located in the so-called catalytic S-X-X-K motif, whereas the other conserved S-X-N and K(H)-T(S)-G motifs govern carboxypeptidase activity and bind penicillin (20). The carboxypeptidase domain of PBPs is the target for β-lactam antibiotics in susceptible staphylococci (with penicillin MICs as low as 1 μg/ml).The transpeptidase activity of the PBPs occurs at the d-Ala-d-Ala terminus of a precursor disaccharide pentapeptide comprising N-acetylglucosamine and N-acetyl-muramic acid-l-Ala-d-Ala-l-Lys-d-Ala-d-Ala. This reaction is initiated by acylation involving a nucleophilic attack by the active-site serine on the penultimate d-Ala residue to form an acyl-enzyme complex. The C-terminal d-Ala is subsequently released from the peptide chain, followed by deacylation. In the case of HMM PBPs, deacylation occurs when an amino group on a second peptide substrate acts as an acceptor, resulting in a peptide cross-link between two adjacent peptidoglycan strands. The carboxypeptidase activity of LMM PBPs follows a similar reaction scheme, except that the acceptor in this case is a water molecule. β-Lactam antibiotics mimic the substrates of the PBPs. However, unlike the natural substrate, the β-lactam-PBP acyl adduct is stable and results in irreversible inhibition of PBP function. The β-lactam-PBP acyl adduct has been characterized extensively, with over 50 protein-antibiotic complexes reported to date (37). Thus, in contrast to the nonessential LMM PBPs, HMM PBPs constitute lethal targets for β-lactam antibiotics (6).Staphylococcus aureus is a gram-positive coccus and is one of the leading causes of high morbidity and mortality associated with both community- and hospital-associated infections (42, 46). This coccus shows extensive genomic variation, with over 22% of the genome dedicated to dispensable regions. A genome-scale analysis of a clinical strain of S. aureus is of particular interest in this context, wherein the conversion of a susceptible strain of S. aureus to a multidrug-resistant phenotype was shown to involve just 35 mutations in 13 loci, achieved within 3 months (36). Of the five PBPs in S. aureus, an acquired PBP, PBP2a, is the most extensively examined, as it was noted to be a specific marker for methicillin-resistant S. aureus (MRSA) strains. Among the intrinsic PBPs, PBP1 has been shown to play a key role in cell growth and division (2). PBP2 is a dual-function enzyme with both transglycosylase and transpeptidase activities, and inhibition of this protein leads to restrained peptidoglycan elongation and subsequent leakage of cytoplasmic contents due to cell lysis (34, 40). Inactivation of PBP3 neither changes the muropeptide composition of the cell wall nor significantly decreases the rate of autolysis. However, cells of abnormal size and shape and with disoriented septa are produced when bacteria with inactivated PBP3 are grown with sub-MIC levels of methicillin (29).S. aureus PBP4 is a carboxypeptidase and is needed for the secondary cross-linking of peptidoglycan (19). However, it is not essential for cell growth under laboratory conditions, because mutants of S. aureus defective in PBP4 are viable (48). Overexpression of PBP4 was noted to result in an increase in β-lactam resistance and in greater cross-linking of the peptidoglycan (18). S. aureus PBP4 is similar to other LMM PBPs and is grouped within the superfamily of penicillin-susceptible and penicillin-interacting enzymes. However, homologues of PBP4 have a different phenotype in other species (1, 15). For example, a mutation of PBP4 in Pseudomonas aeruginosa triggers an AmpR-dependent overproduction of the chromosomal β-lactamase AmpC. The P. aeruginosa PBP4 mutant also activates CreBC, a two-component regulator, thereby mediating β-lactam resistance (33). Indeed, S. aureus PBP4 has been suggested to have different functions in strains with different genetic backgrounds (26). However, based on in vitro and genetic data, S. aureus PBP4 is primarily a transpeptidase and has little d,d-carboxypeptidase activity. This is also supported by the observation that increased carboxypeptidase activity decreases cell wall cross-linking due to loss of the free d-Ala-d-Ala termini necessary for transpeptidation (10). In this context, it is pertinent that pbp4 gene knockout strains of S. aureus were more resistant to the glycopeptide antibiotic vancomycin (46).Here we present the biochemical and structural characteristics of PBP4 from S. aureus strain COL. S. aureus PBP4 is a β-lactamase. A comparison of the crystal structure of S. aureus PBP4 in complex with antibiotic with that of its Escherichia coli homologue, PBP5, provides a conformational and biochemical rationale for the β-lactamase activity of PBP4. Monitoring the expression of PBP4 in the MRSA strain COL and representative clinical strains of S. aureus suggested that the expression level of PBP4 does not fluctuate substantially across these strains. Together, these data on the structure, expression, activity, and regulation of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.     

Trapping open and closed forms of FitE—A group III periplasmic binding protein

Periplasmic binding proteins (PBPs) are essential components of bacterial transport systems, necessary for bacterial growth and survival. The two‐domain structures of PBPs are topologically classified into three groups based on the number of crossovers or hinges between the globular domains: group I PBPs have three connections, group II have two, and group III have only one. Although a large number of structures for group I or II PBPs are known, fewer group III PBPs have been structurally characterized. Group I and II PBPs exhibit significant domain motions during transition from the unbound to ligand‐bound form, however, no large conformational changes have been observed to date in group III PBPs. We have solved the crystal structure of a periplasmic binding protein FitE, part of an iron transport system, fit, recently identified in a clinical E. coli isolate. The structure, determined at 1.8 Å resolution, shows that FitE is a group III PBP containing a single α‐helix bridging the two domains. Among the individual FitE molecules present in two crystal forms we observed three different conformations (open, closed, intermediate). Our crystallographic and molecular dynamics results strongly support the notion that group III PBPs also adopt the same Venus flytrap mechanism as do groups I and II PBPs. Unlike other group III PBPs, FitE forms dimers both in solution and in the crystals. The putative siderophore binding pocket is lined with arginine residues, suggesting an anionic nature of the iron‐containing siderophore. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Resistance to β-Lactam Antibiotics Conferred by Point Mutations in Penicillin-Binding Proteins PBP3, PBP4 and PBP6 in Salmonella enterica

Penicillin-binding proteins (PBPs) are enzymes responsible for the polymerization of the glycan strand and the cross-linking between glycan chains as well as the target proteins for β-lactam antibiotics. Mutational alterations in PBPs can confer resistance either by reducing binding of the antibiotic to the active site or by evolving a β-lactamase activity that degrades the antibiotic. As no systematic studies have been performed to examine the potential of all PBPs present in one bacterial species to evolve increased resistance against β-lactam antibiotics, we explored the ability of fifteen different defined or putative PBPs in Salmonella enterica to acquire increased resistance against penicillin G. We could after mutagenesis and selection in presence of penicillin G isolate mutants with amino-acid substitutions in the PBPs, FtsI, DacB and DacC (corresponding to PBP3, PBP4 and PBP6) with increased resistance against β-lactam antibiotics. Our results suggest that: (i) most evolved PBPs became ‘generalists” with increased resistance against several different classes of β-lactam antibiotics, (ii) synergistic interactions between mutations conferring antibiotic resistance are common and (iii) the mechanism of resistance of these mutants could be to make the active site more accessible for water allowing hydrolysis or less binding to β-lactam antibiotics.     

Extracellular-Regulated Kinase 2 Is Activated by the Enhancement of Hinge Flexibility

Protein motions underlie conformational and entropic contributions to enzyme catalysis; however, relatively little is known about the ways in which this occurs. Studies of the mitogen-activated protein kinase ERK2 (extracellular-regulated protein kinase 2) by hydrogen-exchange mass spectrometry suggest that activation enhances backbone flexibility at the linker between N- and C-terminal domains while altering nucleotide binding mode. Here, we address the hypothesis that enhanced backbone flexibility within the hinge region facilitates kinase activation. We show that hinge mutations enhancing flexibility promote changes in the nucleotide binding mode consistent with domain movement, without requiring phosphorylation. They also lead to the activation of monophosphorylated ERK2, a form that is normally inactive. The hinge mutations bypass the need for pTyr but not pThr, suggesting that Tyr phosphorylation controls hinge motions. In agreement, monophosphorylation of pTyr enhances both hinge flexibility and nucleotide binding mode, measured by hydrogen-exchange mass spectrometry. Our findings demonstrate that regulated protein motions underlie kinase activation. Our working model is that constraints to domain movement in ERK2 are overcome by phosphorylation at pTyr, which increases hinge dynamics to promote the active conformation of the catalytic site.     

Structural and functional difference of pheromone binding proteins in discriminating chemicals in the gypsy moth, Lymantria dispar

Pheromone-binding proteins (PBPs) of the gypsy moth, Lymantria dispar L., play an important role in olfaction. Here structures of PBPs were first built by Homology Modeling, and each model of PBPs had seven α-helices and a large hydrophobic cavity including 25 residues for PBP1 and 30 residues for PBP2. Three potential semiochemicals were first screened by CDOCKER program based on the PBP models and chemical database. These chemicals were Palmitic acid n-butyl ester (Pal), Bis(3,4-epoxycyclohexylmethyl) adipate (Bis), L-trans-epoxysuccinyl-isoleucyl-proline methyl ester propylamide (CA-074). The analysis of chemicals docking the proteins showed one hydrogen bond was established between the residues Lys94 and (+)-Disparlure ((+)-D), and л-л interactions were present between Phe36 of PBP1 and (+)-D. The Lys94 of PBP1 formed two and three hydrogen bonds with Bis and CA-074, respectively. There was no residue of PBP2 interacting with these four chemicals except Bis forming one hydrogen bond with Lys121. After simulating the conformational changes of LdisPBPs at pH7.3 and 5.5 by constant pH molecular dynamics simulation in implicit solvent, the N-terminal sequences of PBPs was unfolded, only having five α-helices, and PBP2 had larger binding pocket at 7.3 than PBP1. To investigate the changes of α-helices at different pH, far-UV and near-UV circular dichroism showed PBPs consist of α-helices, and the tertiary structures of PBP1 and PBP2 were influenced at pH7.3 and 5.5. The fluorescence binding assay indicated that PBP1 and PBP2 have similarly binding affinity to (+)-D at pH 5.5 and 7.3, respectively. At pH 5.5, the dissociation constant of the complex between PBP1 and 2-decyl-1-oxaspiro [2.2] pentane (OXP1) was 0.68 ± 0.01 μM, for (+)-D was 5.32 ± 0.11 μM, while PBP2 with OXP1 and (+)-D were 1.88 ± 0.02 μM and 5.54 ± 0.04 μM, respectively. Three chemicals screened had higher affinity to PBP1 than (+)-D except Pal at pH5.5, and had lower affinity than (+)-D at pH7.3. To PBP2, these chemicals had lower affinity than the sex pheromone except Bis at pH 5.5 and pH 7.3. Only PBP1 had higher affinity with Sal than the sex pheromone at pH 5.5. Therefore, the structures of PBP1 and PBP2 had different changes at pH5.5 and 7.3, showing different affinity to chemicals. This study helps understanding the role of PBPs as well as in developing more efficient chemicals for pest control.     

The Solution Structure,Binding Properties,and Dynamics of the Bacterial Siderophore-binding Protein FepB

The periplasmic binding protein (PBP) FepB plays a key role in transporting the catecholate siderophore ferric enterobactin from the outer to the inner membrane in Gram-negative bacteria. The solution structures of the 34-kDa apo- and holo-FepB from Escherichia coli, solved by NMR, represent the first solution structures determined for the type III class of PBPs. Unlike type I and II PBPs, which undergo large “Venus flytrap” conformational changes upon ligand binding, both forms of FepB maintain similar overall folds; however, binding of the ligand is accompanied by significant loop movements. Reverse methyl cross-saturation experiments corroborated chemical shift perturbation results and uniquely defined the binding pocket for gallium enterobactin (GaEnt). NMR relaxation experiments indicated that a flexible loop (residues 225–250) adopted a more rigid and extended conformation upon ligand binding, which positioned residues for optimal interactions with the ligand and the cytoplasmic membrane ABC transporter (FepCD), respectively. In conclusion, this work highlights the pivotal role that structural dynamics plays in ligand binding and transporter interactions in type III PBPs.     

Binding properties of pheromone-binding protein 1 from the common cutworm Spodoptera litura

Pheromone-binding proteins (PBPs) were formerly thought to act as passive pheromone carriers. However, recent studies, particularly in Drosophila melanogaster, suggest that PBPs are involved in the recognition of semiochemicals, thus making ligand-binding studies more meaningful. Previously, we cloned three PBPs from Spodoptera litura (Slit), and showed that SlitPBP1 is much more abundant than the other two, particularly in male antennae. To investigate the ligand specificity of SlitPBP1, we expressed the protein in a bacterial system and performed binding experiments with the three components of the specific sex pheromones (Z9-14:Ac, Z9,E11-14:Ac and Z9,E12-14:Ac), as well as with 26 volatile ligands. The results indicated that SlitPBP1 bound all three sex pheromone components with dissociation constants between 0.6 and 1.1 μM. The same protein also bound with comparable affinities several pheromone analogs, but not plant volatiles. The presence of a double bond was the most important element for a strong binding, while its position and configuration also affected the affinity. Finally, the binding of pheromone components is strongly affected by pH, showing a critical pH value corresponding to isoelectric point of the protein. This suggests that a pH-dependent conformational mechanism might exist in SlitPBP1 for pheromone binding and release.     

Carbenicillin as Inhibitor of β-Lactamase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

CARBENICILLIN was produced as a new, semi-synthetic penicillin with antibacterial activity against Pseudomonas aeruginosa and some other microorganisms¹, but this compound was known to be destroyed by staphylococcal penicillinase². Newsom et al.³ described the substrate profile of a constitutive β-lactamase from one strain of Pseudomonas aeruginosa and reported the hydrolysis of carbenicillin at a rate higher than benzylpenicillin. When compared with the inducible enzyme described by Sabath et al.⁴, it differed both in the substrate profile and the ability to hydrolyse carbenicillin. Lack of activity of the inducible enzyme on carbenicillin was also reported by Garber and Friedman⁵ when studying eight strains of Pseudomonas aeruginosa. Sykes and Richmond⁶ were able to identify three types of β-lactamases among fifty-six strains of Pseudomonas aeruginosa according to induci-bility, substrate profile and activity on carbenicillin. Type I (Sabath et al.⁴) was inducible, highly active on cephaloridine and showed no activity on carbenicillin. Types II (Sykes and Richmond⁷) and III (Newsom et al.³) were constitutive and inactivated carbenicillin at different rates. Only the constitutive enzymes conferred resistance towards carbenicillin. We have investigated the activity on carbenicillin of β-lactamases from strains of Pseudomonas aeruginosa isolated from clinical specimens. Activity on benzylpenicillin and cephaloridine was also studied.