Solution X-ray scattering study of a full-length class A penicillin-binding protein.

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 ß-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.

Expression of highly toxic genes in E. coli: special strategies and genetic tools.

Escherichia coli (E. coli) remains the most efficient widely-used host for recombinant protein production. Well-known genetics, high transformation efficiency, cultivation simplicity, rapidity and inexpensiveness are the main factors that contribute to the selection of this host. With the advent of the post-genomic era has come the need to express in this bacterium a growing number of genes originating from different organisms. Unfortunately, many of these genes severely interfere with the survival of E. coli cells. They lead to bacteria death or cause significant defects in bacteria growth that dramatically decrease expression capabilities. In this paper, we review special strategies and genetics tools successfully used to express, in E. coli, highly toxic genes. Suppression of basal expression from leaky inducible promoters, suppression of read-through transcription from cryptic promoters, tight control of plasmids copy numbers and proteins production as inactive (but reversible) forms are among the solutions presented and discussed. Special expression vectors and modified E. coli strains are listed and their effectiveness illustrated with key examples, some of which are related to our study of the highly toxic phage T4 restriction endoribonuclease RegB. We mainly selected those strategies and tools that permit E. coli normal growth until the very moment of highly toxic gene induction. Expression then occurs efficiently before cells die. Because they do not target a particular toxic effect, these strategies and tools can be used to express a wide variety of highly toxic genes.

Evidence for an alpha-helix --> pi-bulge helicity modulation for the neu/erbB-2 membrane-spanning segment. A 1H NMR and circular dichroism study.

The 35-residue peptide corresponding to the very hydrophobic transmembrane region of the tyrosine kinase receptor neu, Neu(TM35), has been synthesized. The peptide can be solubilized in millimolar concentrations in TFE or incorporated into an SDS-water micellar solution or into well-hydrated DMPC/DCPC bicelles. In all these media, circular dichroism demonstrated that the peptide adopts a helical structure for about 80% of its amino acids. The peptide is monomeric below 2 mM in TFE, as also determined by variable concentration experiments. The three-dimensional solution structure in TFE has been obtained by homonuclear proton NMR and shows a well-defined alpha-helix from residues 4 to 21, then a pi-bulge from Ile(22) to Gly(28), and a final short alpha-helix from positions 29 to 32. This experimental finding is in agreement with structures predicted recently by molecular dynamics calculations in a vacuum [Sajot, N., and Genest, M. (2000) Eur. Biophys. J. 28, 648-662]. The biological implications of a possible retention of this structure in a membrane environment are finally discussed.

Role of the substrate conformation and of the S1 protein in the cleavage efficiency of the T4 endoribonuclease RegB.

The T4 endoribonuclease RegB is involved in the inactivation of the phage early messengers. It cuts specifically in the middle of GGAG sequences found in early messenger intergenic regions but not GGAG sequences located in coding sequences or in late messengers. In vitro RegB activity is very low but is enhanced by a factor up to 100 by the ribosomal protein S1. In the absence of clear sequence motif distinguishing substrate and non-substrate GGAG-containing RNAs, we postulated the existence of a structural determinant. To test this hypothesis, we correlated the structure, probed by NMR spectroscopy, with the cleavage propensity of short RNA molecules derived from an artificial substrate. A kinetic analysis of the cleavage was performed in the presence and absence of S1. In the absence of S1, RegB efficiently hydrolyses substrates in which the last G of the GGAG motif is located in a short stem between two loops. Both strengthening and weakening of this structure strongly decrease the cleavage rate, indicating that this structure constitutes a positive cleavage determinant. Based on our results and those of others, we speculate that S1 favors the formation of the structure recognized by RegB and can thus be considered a "presentation protein."

NMR analysis of CYP1(HAP1) DNA binding domain-CYC1 upstream activation sequence interactions: recognition of a CGG trinucleotide and of an additional thymine 5 bp downstream by the zinc cluster and the N-terminal extremity of the protein.

The DNA binding domain of the yeast transcriptional activator CYP1(HAP1) contains a zinc-cluster structure. The structures of the DNA binding domain-DNA complexes of two other zinc-cluster proteins (GAL4 and PPR1) have been studied by X-ray crystallography. Their binding domains present, besides the zinc cluster, a short linker peptide and a dimerization element. They recognize, as homodimers, two rotationally symmetric CGG trinucleotides, the linker peptide and the dimerization element playing a crucial role in binding specificity. Surprisingly, CYP1 recognizes degenerate forms of a direct repeat, CGGnnnTAnCGGnnnTA, and the role of its linker is under discussion. To better understand the binding specificity of CYP1, we have studied, by NMR, the interaction between the CYP1(55-126) peptide and two DNA fragments derived from the CYC1 upstream activation sequence 1B. Our data indicate that CYP1(55-126) interacts with a CGG and with a thymine 5 bp downstream. The CGG trinucleotide is recognized by the zinc cluster in the major groove, as for GAL4 and PPR1, and the thymine is bound in the minor groove by the N-terminal region, which possesses a basic stretch of arginyl and lysyl residues. This suggests that the CYP1(55-126) N-terminal region could play a role in the affinity and/or specificity of the interaction with its DNA targets, in contrast to GAL4 and PPR1.

A novel model for the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most frequent mutation is the deletion of F508 in the first nucleotide binding fold (NBF1). It induces a perturbation in the folding of NBF1, which impedes posttranslational maturation of CFTR. Determination of the three-dimensional structure of NBF1 would help to understand this defect. We present a novel model for NBF1 built from the crystal structure of bovine mitochondrial F1-ATPase protein. This model gives a reasonable interpretation of the effect of mutations on the maturation of the protein and, in agreement with the CD data, leads to reconsideration of the limits of NBF1 within CFTR.

Insight into cystic fibrosis by structural modelling of CFTR first nucleotide binding fold (NBF1).

Cystic fibrosis is a human monogenic genetic disease caused by mutations in the cystic fibrosis (CF) gene, which encodes a membrane protein which functions as a channel: the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The most frequent mutation, a deletion of phenylalanine F508 (delta F508), is located in the first nucleotide binding domain of CFTR: NBF1. This mutation leads to a folding defect in NBF1, responsible for an incomplete maturation of CFTR. The absence of CFTR at the surface of epithelial cells causes the disease. Determination of the three-dimensional (3D) structure of NBF1 is a key step to understanding the alterations induced by the mutation. In the absence of any experimental data, we have chosen to build a 3D model for NBF1. This model was built by homology modelling starting from F1-ATPase, the only protein of known 3D structure in the ATP binding cassette (ABC) family. This new model defines the central and critical position of F508, predicted in the hydrophobic core of NBF1. F508 indeed could be involved in hydrophobic interactions to ensure a correct folding pathway. Moreover, this model enables the localization of the LSGGQ sequence (a highly conserved sequence in the ABC family) in a loop, at the surface of the protein. This reinforces the hypothesis of its role for mediation of domain-domain interactions of functional significance for the channel regulation. Finally, the model also allows redefinition of the ends of NBF1 within the CFTR sequence. These extremities are defined by the secondary structure elements that are involved in the NBF1 fold. They lead to reconsideration of the C-terminal limit which was initially defined by the end of exon 12.

What function for human lithostathine?: structural investigations by three-dimensional structure modeling and high-resolution NMR spectroscopy.

Human lithostathine is a 144-residue protein, expressed in various organs and pathologies. Several biological functions have been proposed for this protein. Among others, inhibition of nucleation and growth of CaCO3 crystals in the pancreas and bacterial aggregation has retained attention, because lithostathine presents high sequence similarities with calcium-dependent (or C-type) lectins. To study its structure-function relationship and compare it with that of C-type lectins, we have built a model for lithostathine. This model is derived from the only two C-type lectins of known structures: rat mannose binding protein and human E-selectin. An original strategy, inspired by that proposed by Havel and Snow, was designed for model building. We have undertaken NMR studies on the natural protein. Although complete structure determination has not yet been achieved, the NMR studies did confirm the main characteristics of the model. From analysis of the proposed model, we concluded that lithostathine is not expected to present sugar- or calcium-binding properties. Therefore, the mechanisms of bacterial aggregation and inhibition of CaCO3 nucleation and growth have not yet been elucidated.

Solution structure of bovine angiogenin by 1H nuclear magnetic resonance spectroscopy.

The three-dimensional structure of bovine angiogenin has been determined using two- and three-dimensional proton NMR spectroscopy. The solution structure is very close to that recently determined by X-ray diffraction analysis. This structure appears well defined, even if five loops and one helix exhibit greater flexibility. Analysis of the active site geometry confirms the position of the Glu-118 residue which obstructs the pyrimidine binding site. There is no experimental evidence of an unobstructed conformation of angiogenin in solution. In addition, it appears that the Glu-118 and Ser-119 residues and the cell receptor binding loop may play an important role in the differences of C-terminal fragment organization and ribonucleolytic activity observed between angiogenins and ribonuclease A.

1H, 15N resonance assignment and three-dimensional structure of CYP1 (HAP1) DNA-binding domain.

CYP1(HAP1) is a transcriptional activator involved in the aerobic metabolism of the yeast Saccharomyces cerevisiae. The amino acid sequence of its DNA-binding domain suggests that it belongs to the "zinc cluster" class. This region is indeed characterized by a pattern known to form a bimetal thiolate cluster where two zinc ions are coordinated by six cysteine residues. Structures of two such domains, those from GAL4 and PPR1, have been solved as complexes with DNA. These domains consist of the zinc cluster connected to a dimerization helix by a linker peptide. They recognize, as a dimer, an inverted repeat of a CGG motif that is separated by a specific number of bases. Interestingly, the specificity of that interaction seems not to be due to the interaction between the cluster region and the DNA but rather to a fine tune between the structure of the linker peptide and the number of base-pairs separating the two CGGs. However, the CYP1 target sites fail to display such a consensus sequence. One of the two CGG sites is poorly conserved and some experiments suggest a direct rather than an inverted repeat. Using 1H, 15N and 113Cd NMR spectroscopy, we have undertaken the analysis of the structural properties of the CYP1(56-126) fragment that consists of the zinc-cluster region, the linker peptide and a part of the dimerization helix. We have demonstrated that the six cysteine residues of the peptide chelate two cadmium ions as in GAL4 and PPR1. Fifteen structures of the zinc-cluster region (residues 60 to 100) were calculated, the linker peptide and the dimerization helix being unstructured under the conditions of our study. This region possesses the same overall fold as in GAL4 and PPR1, and most of the side-chains involved in the interaction with DNA are structurally conserved. This suggests that the CYP1 zinc-cluster region recognizes a CGG triplet in the same way as GAL4 and PPR1. In this case, the particular properties of CYP1 seem to be due to the structure of the linker peptide and/or of the dimerization helix.

Proton resonance assignments and secondary structure of bovine angiogenin.

Angiogenins are 14-kDa proteins able to induce blood vessel growth in various preparations and are thus thought to be involved in the development of solid tumors. Angiogenins have significant similarities with extracellular ribonuclease and possess a characteristic nuclease activity against large RNA molecules. These proteins are also able to induce second-messenger pathways. We have undertaken the determination of the three-dimensional structure of bovine angiogenin by using nuclear magnetic resonance (NMR) spectroscopy. Since this protein was directly purified from cow milk, it was not possible to enrich angiogenin with 13C or 15N isotopes. However, extensive use of two-dimensional and three-dimensional proton NMR experiments enabled us to identify all but four spin systems and to assign all corresponding proton resonances. Identification of most backbone-backbone nuclear Overhauser enhancements led to the characterization of the secondary structure elements of the protein. Comparison with the structure of ribonuclease A and analysis of the location of conserved residues confirmed that the two molecules have very similar structures.

Synthesis of charybdotoxin and of two N-terminal truncated analogues. Structural and functional characterisation.

Charybdotoxin and two N-terminal truncated peptides, corresponding to the 2-37 and 7-37 sequences, were obtained by stepwise solid-phase synthesis using N alpha-t-butyloxycarbonyl and benzyltype side-chain protection. While this strategy was generally useful, the S-acetamidomethyl protecting group used for the six cysteines was not completely stable under HF treatment and its subsequent removal by mercury(II) treatment was neither complete nor devoid of side reactions. The completely deprotected native and truncated sequences were folded efficiently in the presence of glutathione and were finally purified by high-pressure liquid chromatography with overall yields of 4.0-5.0%. Each protein was characterised chemically, structurally and functionally. 1H-NMR spectroscopy was used and a complete assignment of all the protons of the three synthetic proteins was achieved. NMR data show that synthetic charybdotoxin is indistinguishable from the natural protein. The two truncated proteins contain the same elements of secondary structure and a similar overall three-dimensional structure, in agreement with circular dichroic measurements. The shortest analogue, however, may have local structural perturbations and/or higher flexibility. Biological activity on dog epithelial Ca(2+)-activated K+ channels and on rat brain synaptosomal voltage-dependent K+ channels show that synthetic charybdotoxin was as potent as the natural toxin on both channels. For both channels, deletion of the first amino acid, 5-oxoproline (pyroglutamic acid) decreased only slightly the potency of the inhibitor, while deletion of the entire 1-6 segment reduced potency much more. We conclude that the N-terminal region of charybdotoxin plays a functional role in tuning the toxin's biological activity but is not essential for the folding and stability of the structure. The structure of the shortest analogue represents an interesting example of how a well organised and stable alpha/beta fold can be engineered with only 31 amino acid residues.

Purification and characterization of a scorpion defensin, a 4kDa antibacterial peptide presenting structural similarities with insect defensins and scorpion toxins.

Insect defensins are a group of inducible small-sized antibacterial peptides with three intramolecular disulfide bridges. NMR studies have recently shown that they share striking structural similarities with scorpion toxins. We have investigated in a scorpion species, Leiurus quinquestriatus, the potential presence of antibacterial molecules and report the isolation and structural characterization of a novel insect defensin homologue, which we refer to as scorpion defensin. This peptide shows a remarkably high degree of sequence homology with a defensin recently characterized in a species belonging to the ancient insect order of the Odonata with which it defines a novel ancient subclass of defensins. The scorpion defensin has in common with the scorpion toxins a consensus sequence Cys-[...]-Cys-Xaa-Xaa-Xaa-Cys-[...]-Gly-Xaa-Cys-[...]-Cys-Xaa-Cys present in all scorpion toxins characterized so far.

Three-dimensional solution structure of a curaremimetic toxin from Naja nigricollis venom: a proton NMR and molecular modeling study.

The solution conformation of toxin alpha from Naja nigricollis (61 amino acids and four disulfides), a snake toxin which specifically blocks the activity of the nicotinic acetylcholine receptor (AcChoR), has been determined using nuclear magnetic resonance spectroscopy and molecular modeling. The solution structures were calculated using 409 distance and 73 dihedral angle restraints. The average atomic rms deviation between the eight refined structures and the mean structure is approximately 0.5 A for the backbone atoms. The overall folding of toxin alpha consists of three major loops which are stabilized by three disulfide bridges and one short C terminal loop stabilized by a fourth disulfide bridge. All the disulfides are grouped in the same region of the molecule, forming a highly constrained structure from which the loops protrude. As predicted, this structure appears to be very similar to the 1.4-A resolution crystal structure of another snake neurotoxin, namely, erabutoxin b from Laticauda semifasciata. The atomic rms deviation for the backbone atoms between the solution and crystal structures is approximately 1.7 A. The minor differences which are observed between the two structures are partly related to the deletion of one residue from the chain of toxin alpha. It is notable that, although the two toxins differ from each other by 16 amino acid substitutions, their side chains have an essentially similar spatial organization. However, most of the side chains which constitute the presumed AcChoR binding site for the curaremimetic toxins are poorly resolved in toxin alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of side-chain organization on a refined model of charybdotoxin: structural and functional implications.

The spatial organization of side chains on a refined model of charybdotoxin is presented. First, the structural role of two groups of well-defined, low-accessible side chains (Thr3, Val5, Val16, Leu20, Cys33 and Leu20, His21, Thr23, Cys17, Cys35) is discussed. These side chains are conserved in three out of the five known scorpion toxins acting on K+ channels. Interestingly, they are not conserved in scyllatoxin which presents a slightly different secondary structure organization. Second, the spatial organization of all positively charged residues is analyzed. Comparison with the results presented by Park and Miller [(1992) Biochemistry (preceding paper in this issue)] shows that all functionally important positive residues are located on the beta-sheet side of the toxin. These results are different from those obtained by Auguste et al. [(1992) Biochemistry 31, 648-654] on scyllatoxin, which blocks a different type of K+ channel. This study shows, in fact, that functionally important positive residues are located on the helix side of the toxin. Thus, charybdotoxin and scyllatoxin, which present the same global fold, interact with two different classes of K+ channels by two different parts of the motif.

Refined structure of charybdotoxin: common motifs in scorpion toxins and insect defensins.

Conflicting three-dimensional structures of charybdotoxin (Chtx), a blocker of K+ channels, have been previously reported. A high-resolution model depicting the tertiary structure of Chtx has been obtained by DIANA and X-PLOR calculations from new proton nuclear magnetic resonance (NMR) data. The protein possesses a small triple-stranded antiparallel beta sheet linked to a short helix by two disulfides and to an extended fragment by one disulfide, respectively. This motif also exists in all known structures of scorpion toxins, irrespective of their size, sequence, and function. Strikingly, antibacterial insect defensins also adopt this folding pattern.

Three-dimensional structure of natural charybdotoxin in aqueous solution by 1H-NMR. Charybdotoxin possesses a structural motif found in other scorpion toxins.

A 600-MHz proton NMR study of natural charybdotoxin, a toxin acting on K+ channels, is reported. The unambiguous sequential assignment of all the protons of the toxin was achieved. The analysis of NOEs and of backbone coupling constants showed the existence of an alpha-helix (residues 10-19) and of an antiparallel beta-sheet in the 26-35 part. Three-dimensional structures were generated by distance geometry, using a set of 114 interresidual calibrated constraints (63 sequential, 47 medium and long range, 4 hydrogen bonds) and 29 phi angles. These structures show that charybdotoxin is composed of a beta-sheet linked to an alpha-helix by two disulphide bridges and to an extended fragment by the third disulphide bridge. Comparison with the other known structures of long and short scorpion toxins shows that this structural motif is common to all these proteins.

Topography of toxin-acetylcholine receptor complexes by using photoactivatable toxin derivatives.

We have defined the molecular environment of a snake neurotoxin interacting with the high- and low-affinity binding sites of the nicotinic acetylcholine receptor (AcChoR). This was done by photocoupling reactions using three toxin derivatives with photoactivatable moieties on Lys-15, Lys-47, and Lys-51. Competition data showed that Lys-47 belongs to the toxin-AcChoR interacting domain whereas the other two residues are excluded from it. We first tentatively determined the threshold of covalent coupling, indicative of the proximity between the photoactivatable probes and subunits, by quantifying the coupling occurring between the same derivatives and a model compound (i.e., a toxin-specific monoclonal antibody). We then (i) quantified the coupling yields occurring when both binding sites of AcChoR were occupied by the toxin derivatives, (ii) discriminately quantified the coupling yields at the high-affinity binding site, and (iii) deduced the coupling yields at the low-affinity binding site. In the high-affinity site, the probes on Lys-15 and Lys-47 predominantly reacted with the high-affinity site of the AcChoR alpha subunit whereas the probe on Lys-51 reacted with the delta subunit. In the low-affinity site, the probe on Lys-47 predominantly reacted with the low-affinity site of the alpha chain and the beta chain whereas those on Lys-15 and Lys-51 reacted with the gamma and delta chains, respectively. A three-dimensional model showing a unique organization of AcChoR bound to two toxin molecules is presented.