Staphylococcus aureus Penicillin-Binding Protein 2 Can Use Depsi-Lipid II Derived from Vancomycin-Resistant Strains for Cell Wall Synthesis.

Vancomycin-resistant Staphylococcus aureus (S. aureus) (VRSA) uses depsipeptide-containing modified cell-wall precursors for the biosynthesis of peptidoglycan. Transglycosylase is responsible for the polymerization of the peptidoglycan, and the penicillin-binding protein 2 (PBP2) plays a major role in the polymerization among several transglycosylases of wild-type S. aureus. However, it is unclear whether VRSA processes the depsipeptide-containing peptidoglycan precursor by using PBP2. Here, we describe the total synthesis of depsi-lipid I, a cell-wall precursor of VRSA. By using this chemistry, we prepared a depsi-lipid II analogue as substrate for a cell-free transglycosylation system. The reconstituted system revealed that the PBP2 of S. aureus is able to process a depsi-lipid II intermediate as efficiently as its normal substrate. Moreover, the system was successfully used to demonstrate the difference in the mode of action of the two antibiotics moenomycin and vancomycin.

Murein (peptidoglycan) binding property of the essential cell division protein FtsN from Escherichia coli.

The binding of the essential cell division protein FtsN of Escherichia coli to the murein (peptidoglycan) sacculus was studied. Soluble truncated variants of FtsN, including the complete periplasmic part of the protein as well as a variant containing only the C-terminal 77 amino acids, did bind to purified murein sacculi isolated from wild-type cells. FtsN variants lacking this C-terminal region showed reduced or no binding to murein. Binding of FtsN was severely reduced when tested against sacculi isolated either from filamentous cells with blocked cell division or from chain-forming cells of a triple amidase mutant. Binding experiments with radioactively labeled murein digestion products revealed that the longer murein glycan strands (>25 disaccharide units) showed a specific affinity to FtsN, but neither muropeptides, peptides, nor short glycan fragments bound to FtsN. In vivo FtsN could be cross-linked to murein with the soluble disulfide bridge containing cross-linker DTSSP. Less FtsN, but similar amounts of OmpA, was cross-linked to murein of filamentous or of chain-forming cells compared to levels in wild-type cells. Expression of truncated FtsN variants in cells depleted in full-length FtsN revealed that the presence of the C-terminal murein-binding domain was not required for cell division under laboratory conditions. FtsN was present in 3,000 to 6,000 copies per cell in exponentially growing wild-type E. coli MC1061. We discuss the possibilities that the binding of FtsN to murein during cell division might either stabilize the septal region or might have a function unrelated to cell division.

Expression and characterization of the isolated glycosyltransferase module of Escherichia coli PBP1b.

The enzymes involved in the biosynthesis of peptidoglycan are targets for the development of new antibiotics. The bifunctional high molecular weight (HMW) penicillin-binding proteins (PBPs), which contain both glycosyltransferase (GTase) and transpeptidase (TPase) activities, are particularly attractive targets because of their extracellular location. However, there is limited mechanistic or structural information about the GTase modules of these enzymes. In this paper, we describe the overexpression and characterization of the GTase module of Escherichia coli PBP1b, a paradigm of the HMW PBPs. We define the C-terminal boundary of the GTase module and show that the isolated module can be overexpressed at significantly higher levels than the full-length protein. The catalytic efficiency and other characteristics of the isolated module are comparable in most respects to the full-length enzyme. This work lays the groundwork for mechanistic and structural analysis of GTase modules.

Structural determinants required to target penicillin-binding protein 3 to the septum of Escherichia coli.

In Escherichia coli, cell division is mediated by the concerted action of about 12 proteins that assemble at the division site to presumably form a complex called the divisome. Among these essential division proteins, the multimodular class B penicillin-binding protein 3 (PBP3), which is specifically involved in septal peptidoglycan synthesis, consists of a short intracellular M1-R23 peptide fused to a F24-L39 membrane anchor that is linked via a G40-S70 peptide to an R71-I236 noncatalytic module itself linked to a D237-V577 catalytic penicillin-binding module. On the basis of localization analyses of PBP3 mutants fused to green fluorescent protein by fluorescence microscopy, it appears that the first 56 amino acid residues of PBP3 containing the membrane anchor and the G40-E56 peptide contain the structural determinants required to target the protein to the cell division site and that none of the putative protein interaction sites present in the noncatalytic module are essential for the positioning of the protein to the division site. Based on the effects of increasing production of FtsQ or FtsW on the division of cells expressing PBP3 mutants, it is suggested that these proteins could interact. We postulate that FtsQ could play a role in regulating the assembly of these division proteins at the division site and the activity of the peptidoglycan assembly machineries within the divisome.

The basis for resistance to beta-lactam antibiotics by penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus.

Penicillin-binding protein 2a (PBP2a) of Staphylococcus aureus is refractory to inhibition by available beta-lactam antibiotics, resulting in resistance to these antibiotics. The strains of S. aureus that have acquired the mecA gene for PBP2a are designated as methicillin-resistant S. aureus (MRSA). The mecA gene was cloned and expressed in Escherichia coli, and PBP2a was purified to homogeneity. The kinetic parameters for interactions of several beta-lactam antibiotics (penicillins, cephalosporins, and a carbapenem) and PBP2a were evaluated. The enzyme manifests resistance to covalent modification by beta-lactam antibiotics at the active site serine residue in two ways. First, the microscopic rate constant for acylation (k2) is attenuated by 3 to 4 orders of magnitude over the corresponding determinations for penicillin-sensitive penicillin-binding proteins. Second, the enzyme shows elevated dissociation constants (Kd) for the non-covalent pre-acylation complexes with the antibiotics, the formation of which ultimately would lead to enzyme acylation. The two factors working in concert effectively prevent enzyme acylation by the antibiotics in vivo, giving rise to drug resistance. Given the opportunity to form the acyl enzyme species in in vitro experiments, circular dichroism measurements revealed that the enzyme undergoes substantial conformational changes in the course of the process that would lead to enzyme acylation. The observed conformational changes are likely to be a hallmark for how this enzyme carries out its catalytic function in cross-linking the bacterial cell wall.

Mixed quantum mechanical/molecular mechanical (QM/MM) study of the deacylation reaction in a penicillin binding protein (PBP) versus in a class C beta-lactamase.

The origin of the substantial difference in deacylation rates for acyl-enzyme intermediates in penicillin-binding proteins (PBPs) and beta-lactamases has remained an unsolved puzzle whose solution is of great importance to understanding bacterial antibiotic resistance. In this work, accurate, large-scale mixed ab initio quantum mechanical/molecular mechanical (QM/MM) calculations have been used to study the hydrolysis of acyl-enzyme intermediates formed between cephalothin and the dd-peptidase of Streptomyces sp. R61, a PBP, and the Enterobacter cloacae P99 cephalosporinase, a class C beta-lactamase. Qualitative and, in the case of P99, quantitative agreement was achieved with experimental kinetics. The faster rate of deacylation in the beta-lactamase is attributed to a more favorable electrostatic environment around Tyr150 in P99 (as compared to that for Tyr159 in R61) which facilitates this residue's function as the general base. This is found to be in large part accomplished by the ability of P99 to covalently bind the ligand without concurrent elimination of hydrogen bonds to Tyr150, which proves not to be the case with Tyr159 in R61. This work provides an essential foundation for further work in this area, such as selecting mutations capable of converting the PBP into a beta-lactamase.

The importance of a critical protonation state and the fate of the catalytic steps in class A beta-lactamases and penicillin-binding proteins.

Beta-lactamases and penicillin-binding proteins are bacterial enzymes involved in antibiotic resistance to beta-lactam antibiotics and biosynthetic assembly of cell wall, respectively. Members of these large families of enzymes all experience acylation by their respective substrates at an active site serine as the first step in their catalytic activities. A Ser-X-X-Lys sequence motif is seen in all these proteins, and crystal structures demonstrate that the side-chain functions of the serine and lysine are in contact with one another. Three independent methods were used in this report to address the question of the protonation state of this important lysine (Lys-73) in the TEM-1 beta-lactamase from Escherichia coli. These techniques included perturbation of the pK(a) of Lys-73 by the study of the gamma-thialysine-73 variant and the attendant kinetic analyses, investigation of the protonation state by titration of specifically labeled proteins by nuclear magnetic resonance, and by computational treatment using the thermodynamic integration method. All three methods indicated that the pK(a) of Lys-73 of this enzyme is attenuated to 8.0-8.5. It is argued herein that the unique ground-state ion pair of Glu-166 and Lys-73 of class A beta-lactamases has actually raised the pK(a) of the active site lysine to 8.0-8.5 from that of the parental penicillin-binding protein. Whereas we cannot rule out that Glu-166 might activate the active site water, which in turn promotes Ser-70 for the acylation event, such as proposed earlier, we would like to propose as a plausible alternative for the acylation step the possibility that the ion pair would reconfigure to the protonated Glu-166 and unprotonated Lys-73. As such, unprotonated Lys-73 could promote serine for acylation, a process that should be shared among all active-site serine beta-lactamases and penicillin-binding proteins.

The D,D-carboxypeptidase PBP3 organizes the division process of Streptococcus pneumoniae.

Bacterial division requires the co-ordination of membrane invagination, driven by the constriction of the FtsZ-ring, and concomitant cell wall synthesis, performed by the high-molecular-weight penicillin-binding proteins (HMW PBPs). Using immunofluorescence techniques, we show in Streptococcus pneumoniae that this co-ordination requires PBP3, a D,D-carboxypeptidase that degrades the substrate of the HMW PBPs. In a mutant deprived of PBP3, the apparent rings of HMW PBPs and that of FtsZ are no longer co-localized. In wild-type cells, PBP3 is absent at the future division site and present over the rest of the cell surface, implying that the localization of the HMW PBPs at mid-cell depends on the availability of their substrate. FtsW, a putative translocase of the substrate of the PBPs, forms an apparent ring that is co-localized with the septal HMW PBPs throughout the cell cycle of wild-type cells. In particular, the constriction of the FtsW-ring occurs after that of the FtsZ-ring, with the same delay as the constriction of the septal PBP-rings. However, in the absence of PBP3, FtsW remains co-localized with FtsZ in contrast to the HMW PBPs. Our work reveals an unexpected complexity in the relationships between the division proteins. The consequences of the absence of PBP3 indicate that the peptidoglycan composition is central to the co-ordination of the division process.

On the transcriptional regulation of methicillin resistance: MecI repressor in complex with its operator.

Bacterial resistance to antibiotics poses a serious worldwide public health problem due to the high morbidity and mortality caused by infectious diseases. Most hospital-onset infections are associated with methicillin-resistant Staphylococcus aureus (MRSA) strains that have acquired multiple drug resistance to beta-lactam antibiotics. In a response to antimicrobial stress, nearly all clinical MRSA isolates produce beta-lactamase (BlaZ) and a penicillin-binding protein with low affinity for beta-lactam antibiotics (PBP2a, also known as PBP2' or MecA). Both effectors are regulated by homologous signal transduction systems consisting of a sensor/transducer and a transcriptional repressor. MecI (methicillin repressor) blocks mecA but also blaZ transcription and that of itself and the co-transcribed sensor/transducer. The structure of MecI in complex with a cognate operator double-stranded DNA reveals a homodimeric arrangement with a novel C-terminal spiral staircase dimerization domain responsible for dimer integrity. Each protomer interacts with the DNA major groove through a winged helix DNA-binding domain and specifically recognizes the nucleotide sequence 5'-Gua-Thy-Ade-X-Thy-3'. This results in an unusual convex bending of the DNA helix. The structure of this first molecular determinant of methicillin resistance in complex with its target DNA provides insights into its regulatory mechanism and paves the way for new antimicrobial strategies against MRSA.

Comparison of high-performance liquid chromatography and fluorophore-assisted carbohydrate electrophoresis methods for analyzing peptidoglycan composition of Escherichia coli.

Currently, reversed-phase high-performance liquid chromatography (HPLC) is the method of choice for determining the types and amounts of muropeptide subunits comprising bacterial peptidoglycan. Although effective and sensitive, the technique does not lend itself to high throughput screening, and its complexity and equipment requirements may dissuade some investigators from pursuing certain types of cell wall experiments. Previously, we showed that muropeptides can be labeled with a fluorescent dye and separated by fluorophore-assisted carbohydrate electrophoresis (FACE), a simple and rapid gel procedure that might serve as a prelude to more intense analysis by HPLC. To validate the utility of FACE, we used both techniques to perform a side-by-side analysis of the peptidoglycan of eight mutants and their Escherichia coli parent strain. FACE and HPLC both detected the seven major muropeptides, which represent more than 95% of the total muropeptides present in this organism. In addition, FACE returned the same relative and quantitative results in 92% of 72 measurements, indicating that the procedure gives an accurate overview of peptidoglycan composition. The results also suggest a possible biochemical activity for the AmpC and AmpH proteins of E. coli, and the use of FACE as an in vitro enzyme assay detected possible substrate preferences for the endopeptidase penicillin binding protein 4.

Relationship between penicillin-binding protein patterns and beta-lactamases in clinical isolates of Bacteroides fragilis with different susceptibility to beta-lactam antibiotics.

This study examines the role of the penicillin-binding proteins (PBPs) of Bacteroides fragilis in the mechanism of resistance to different beta-lactam antibiotics. Six of the eight strains used were beta-lactamase-positive by the nitrocefin assay. These strains displayed reduced susceptibility to imipenem (MIC, 2-16 mg l(-1)) and some of them were resistant to the actions of ampicillin, cefuroxime, cephalexin, cefoxitin and piperacillin. When studying specific enzymic activity, the capacity to degrade cefuroxime was only detected in strains AK-4, R212 and 0423 and the capacity to degrade cephalexin was only detected in strains R212 and 2013E; no specific activity was detected on imipenem. Metallo-beta-lactamase activity was only detected in strains AK-2 and 119, despite the fact that the cfiA gene was identified in four strains (AK-2, 2013E, 119 and 7160). The cepA gene was detected in six of the eight strains studied. Three high-molecular-mass PBPs were detected in all strains; however, in some cases, PBP2Bfr and/or PBP3Bfr appeared as a faint band. PBP4Bfr and PBP5Bfr were detected in six strains. PBP6Bfr only was detected in B. fragilis strains AK-2, 0423, 119 and 7160. By analysis of the sequence of B. fragilis chromosomal DNA and comparison with genes that are known to encode PBPs in Escherichia coli, six genes that encode PBP-like proteins were detected in the former organism. The gene that encodes the PBP2 orthologue of E. coli (pbpABfr, PBP3Bfr) was sequenced in six of the eight strains and its implications for resistance were examined. Differences in the PBP3Bfr amino acid sequences of strains AK-2 and 119 and their production of beta-lactamases indicate that these differences are not involved in the mechanism of resistance to imipenem and/or cephalexin.

Interactions between penicillin-binding proteins (PBPs) and two novel classes of PBP inhibitors, arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-ones.

Several non-beta-lactam compounds were active against various gram-positive and gram-negative bacterial strains. The MICs of arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-ones were lower than those of ampicillin and cefotaxime for methicillin-resistant Staphylococcus aureus MI339 and vancomycin-resistant Enterococcus faecium EF12. Several compounds were found to inhibit the cell wall synthesis of S. aureus and the last two steps of peptidoglycan biosynthesis catalyzed by ether-treated cells of Escherichia coli or cell wall membrane preparations of Bacillus megaterium. The effects of the arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-one derivatives on E. coli PBP 3 and PBP 5, Streptococcus pneumoniae PBP 2xS (PBP 2x from a penicillin-sensitive strain) and PBP 2xR (PBP 2x from a penicillin-resistant strain), low-affinity PBP 2a of S. aureus, and the Actinomadura sp. strain R39 and Streptomyces sp. strain R61 DD-peptidases were studied. Some of the compounds exhibited inhibitory activities in the 10 to 100 microM concentration range. The inhibition of PBP 2xS by several of them appeared to be noncompetitive. The dissociation constant for the best inhibitor (Ki = 10 microM) was not influenced by the presence of the substrate.

A novel beta-lactamase activity from a penicillin-binding protein of Treponema pallidum and why syphilis is still treatable with penicillin.

Treponema pallidum, the causative agent of syphilis, is sensitive to penicillins. Yet, an abundant membrane-bound protein of this organism, Tp47, turns over penicillins. It is shown herein that the turnover process is a hydrolytic reaction that results in the corresponding penicilloates, products that have their beta-lactam bonds hydrolyzed. This is the reaction of beta-lactamases, bona fide resistance enzymes to beta-lactam antibiotics. Remarkably, the x-ray structure of Tp47 bears no resemblance to any other beta-lactamases or the related penicillin-binding proteins. Furthermore, evidence is presented that the reaction of Tp47 takes place in the absence of the zinc ion and does not involve intermediary acyl enzyme species. Hence, the beta-lactamase activity of Tp47 is the fifth known mechanism for turnover of beta-lactam antibiotics. Tp47 also exhibits a penicillin binding reaction, in the process of which the enzyme is covalently modified in the active site. The two reactions take place in two different active sites, and the events of the beta-lactamase activity are over 2,000-fold more rapid than the penicillin binding reaction. The level of beta-lactamase activity is high and is held back only by a strong product-inhibition component to the catalytic process. If natural selection would result in a mutant variant of Tp47 that overcomes product inhibition for the beta-lactamase activity, a novel bona fide resistance to penicillins will emerge in Treponema, which will be a disconcerting clinical development. The physiological functions of Tp47 are not known, but it is likely that this is at least a bifunctional enzyme involved in the processing of the Treponema peptidoglycan as a substrate.

Overexpression and enzymatic characterization of Neisseria gonorrhoeae penicillin-binding protein 4.

The penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes involved in cell wall biosynthesis, and are the targets of the beta-lactam antibiotics. The low molecular mass Neisseria gonorrhoeae PBP 4 (NG PBP 4) is the fourth PBP revealed in the gonococcal genome. NG PBP 4 was cloned, overexpressed, purified, and characterized for beta-lactam binding, DD-carboxypeptidase activity, acyl-donor substrate specificity, transpeptidase activity, inhibition by a number of active site directed reagents, and pH profile. NG PBP 4 was efficiently acylated by penicillin (30,000 m-1.s-1). Against a set of five alpha- and epsilon-substituted l-Lys-D-Ala-D-Ala substrates, NG PBP 4 exhibited wide variation in specificity with a preference for N epsilon-acylated substrates, suggesting a possible preference for crosslinked pentapeptide substrates in the cell wall. Substrates with an N epsilon-Cbz group demonstrated pronounced substrate inhibition. NG PBP 4 showed 30-fold higher activity against the depsipeptide Lac-ester substrate than against the analogous peptide substrate, an indication that k2 (acylation) is rate determining for carboxypeptidase activity. No transpeptidase activity was apparent in a model transpeptidase reaction. Among a number of active site-directed agents, N-chlorosuccinimide, elastinal, iodoacetamide, iodoacetic acid, and phenylglyoxal gave substantial inhibition, and methyl boronic acid gave modest inhibition. The pH profile for activity against Ac2-l-Lys-D-Ala-d-Ala (kcat/Km) was bell-shaped, with pKa values at 6.9 and 10.1. Comparison of the enzymatic properties of NG PBP 4 with other DD-carboxypeptidases highlights both similarities and differences within these enzymes, and suggests the possibility of common mechanistic roles for the two highly conserved active site lysines in Class A and C low molecular mass PBPs.

Neisseria gonorrhoeae penicillin-binding protein 3 exhibits exceptionally high carboxypeptidase and beta-lactam binding activities.

A soluble form of penicillin-binding protein 3 (PBP 3) from Neisseria gonorrhoeae was expressed and purified from Escherichia coli and characterized for its interaction with beta-lactam antibiotics, its catalytic properties with peptide and peptidoglycan substrates, and its role in cell viability and morphology. PBP 3 had an unusually high k(2)/K' value relative to other PBPs for acylation with penicillin (7.7 x 10(5) M(-1) s(-1)) at pH 8.5 at 25 degrees C and hydrolyzed bound antibiotic very slowly (k(3) < 4.6 x 10(-5) s(-1), t(1/2) > 230 min). PBP 3 also demonstrated exceptionally high carboxypeptidase activity with a k(cat) of 580 s(-1) and a k(cat)/K(m) of 1.8 x 10(5) M(-1) s(-1) with the substrate N(alpha)-Boc-N(epsilon)-Cbz-L-Lys-D-Ala-D-Ala. This is the highest k(cat) value yet reported for a PBP or other serine peptidases. Activity against a approximately D-Ala-D-Lac peptide substrate was approximately 2-fold lower than against the analogous approximately D-Ala-D-Ala peptide substrate, indicating that deacylation is rate determining for both amide and ester hydrolysis. The pH dependence profiles of both carboxypeptidase activity and beta-lactam acylation were bell-shaped with maximal activity at pH 8.0-8.5. PBP 3 displayed weak transpeptidase activity in a model transpeptidase reaction but was active as an endopeptidase, cleaving dimeric peptide cross-links. Deletion of PBP 3 alone had little effect on viability, growth rate, and morphology of N. gonorrhoeae, although deletion of both PBP 3 and PBP 4, the other low-molecular-mass PBP in N. gonorrhoeae, resulted in a decreased growth rate and marked morphological abnormalities.

Growth and division of Streptococcus pneumoniae: localization of the high molecular weight penicillin-binding proteins during the cell cycle.

The bacterial peptidoglycan, the main component of the cell wall, is synthesized by the penicillin-binding proteins (PBPs). We used immunofluorescence microscopy to determine the cellular localization of all the high molecular weight PBPs of the human pathogen Streptococcus pneumoniae, for a wild type and for several PBP-deficient strains. Progression through the cell cycle was investigated by the simultaneous labelling of DNA and the FtsZ protein. Our main findings are: (i) the temporal dissociation of cell wall synthesis, inferred by the localization of PBP2x and PBP1a, from the constriction of the FtsZ-ring; (ii) the localization of PBP2b and PBP2a at duplicated equatorial sites indicating the existence of peripheral peptidoglycan synthesis, which implies a similarity between the mechanism of cell division in bacilli and streptococci; (iii) the abnormal localization of some class A PBPs in PBP-defective mutants which may explain the apparent redundancy of these proteins in S. pneumoniae.

3-alkoxy-5-isoxazolidinones mimic beta-lactams.

3-Alkoxy-5-isoxazolidinones mimic the action of beta-lactams. They bind to the penicillin-binding proteins. They inhibit Class A, Class B, and Class D beta-lactams. They inhibit the growth of Bacillus subtilis. We give a novel synthesis for these compounds.

Crystal structure of wild-type penicillin-binding protein 5 from Escherichia coli: implications for deacylation of the acyl-enzyme complex.

Penicillin-binding protein 5 (PBP 5) of Escherichia coli functions as a d-alanine carboxypeptidase (CPase), cleaving d-alanine from the C terminus of cell wall peptides. Like all PBPs, PBP 5 forms a covalent acyl-enzyme complex with beta-lactam antibiotics; however, PBP 5 is distinguished by its high rate of deacylation of the acylenzyme complex (t(1/2) approximately 10 min). A Gly105 --> Asp mutation in PBP 5 markedly impairs deacylation with only minor effects on acylation, and abolishes CPase activity. We have determined the three-dimensional structure of a soluble form of wild-type PBP 5 at 1.85-A resolution and have also refined the structure of the G105D mutant form of PBP 5 to 1.9-A resolution. Comparison of the two structures reveals that the major effect of the mutation is to disorder a loop comprising residues 74-90 that sits atop the SXN motif of the active site. Deletion of the 74-90 loop in wild-type PBP 5 markedly diminished the deacylation rate of penicillin G with a minimal impact on acylation, and abolished CPase activity. These effects were very similar to those observed in the G105D mutant, reinforcing the idea that this mutation causes disordering of the 74-90 loop. Mutation of two consecutive serines within this loop, which hydrogen bond to Ser110 and Asn112 in the SXN motif, had marked effects on CPase activity, but not beta-lactam antibiotic binding or hydrolysis. These data suggest a direct role for the SXN motif in deacylation of the acyl-enzyme complex and imply that the functioning of this motif is modulated by the 74-90 loop.

Solution structural study of BlaI: implications for the repression of genes involved in beta-lactam antibiotic resistance.

beta-Lactamase and penicillin-binding protein PBP2' mediate staphylococcal resistance to beta-lactam antibiotics, which are otherwise highly clinically effective. Two repressors (BlaI and MecI) regulate expression of these inducible proteins. Here, we present the first solution structure of the 82 amino acid residue DNA-binding domain of Bacillus licheniformis BlaI which is very similar in primary sequence to the medically significant Staphyloccocal BlaI and MecI proteins. This structure is composed of a compact core of three alpha-helices and a three-stranded beta-sheet typical of the winged helix protein (WHP) family. The protein/DNA complex was studied by NMR chemical shift comparison between the free and complexed forms of BlaI. Residues involved in DNA interaction were identified and a WHP canonical model of interaction with the operators is proposed. In this model, specific contacts occur between the base-pairs of the TACA motif and conserved amino acid residues of the repressor helix H3. These results help toward understanding the repression and induction mechanism of the genes coding for beta-lactamase and PBP2'.

Is use of the hydrophobic moment a sound basis for predicting the structure-function relationships of membrane interactive alpha-helices?

Amphiphilic alpha-helices play a fundamental role in protein membrane association and show a segregation of polar and apolar amino acid residues. Based on correlations between amphiphilic properties and biological function, a number of theoretical approaches have been developed, which quantify alpha-helix amphiphilicity and then attempt to assign function. The most commonly used measure of amphiphilicity is the hydrophobic moment, < microH >, which, when used in conjunction with an alpha-helix's mean hydrophobicity, < H >, has been used to classify membrane interactive amphiphilic alpha-helices as either surface active or transmembrane. Here, the predictive efficacy of plot methodology is reviewed by examining published data, which compare the function of known membrane interactive amphiphilic alpha-helices to that assigned by this methodology. The results of this review are discussed in relation to the reliability of < microH > as a quantifier of alpha-helical amphiphilicity, and the ability of < microH > and < H > to describe alpha-helical structure / function relationships. It is concluded that hydrophobic moment plot methodology is not a generally reliable predictor of alpha-helical structure / function relationships. It appears that the inefficacy of plot methodology is primarily due to the inability of the plot diagram to accommodate the heterogeneity of the alpha-helical classes it attempts to define. However, the predictive efficacy of the methodology appears to be improved if other alpha-helical parameters are also considered when assigning alpha-helical function. It is suggested that the conventional methodology should be seen only as an indicator for the assignation of structure / function relationships, providing a guide to future experimental investigations.