Polymer topology revealed by ion mobility coupled with mass spectrometry.

Hyperbranched and star shaped polymers have raised tremendous interest because of their unusual structural and photochemical properties, which provide them potent applications in various domains, namely in the biomedical field. In this context, the development of adequate tools aiming to probe particular three-dimensional features of such polymers is of crucial importance. In this present work, ion mobility coupled with mass spectrometry was used to experimentally derive structural information related to cationized linear and star shaped poly-ε-caprolactones as a function of their charge state and chain length. Two major conformations were observed and identified using theoretical modeling: (1) near spherical conformations whose sizes are invariant with the polymer topology for long and lightly charged chains and (2) elongated conformations whose sizes vary with the polymer topology for short and highly charged chains. These conformations were further confirmed by collisional activation experiments based on the ejection thresholds of the coordinated cations that vary according to the elongation amplitude of the polymer chains. Finally, a comparison between solution and gas-phase conformations highlights a compaction of the structure with a loss of specific chain arrangements during the ionization and desolvation steps of the electrospray process, fueling the long-time debated question related to the preservation of the analyte structure during the transfer into the mass spectrometer.

Three-dimensional structure of RBcel1, a metagenome-derived psychrotolerant family GH5 endoglucanase.

RBcel1 is an endoglucanase belonging to glycoside hydrolase family 5 subfamily 5 (GH5_5) that was recently identified from a soil metagenome library from the Antarctic. Unlike its closest structural homologue (Cel5A from Thermoascus aurantiacus), this enzyme was reported to be able to catalyze transglycosylation reactions and has putatively been implicated in the bacterial cellulose-synthesis process. Here, the structure of RBcel1 at 1.4 Å resolution, solved by molecular replacement, is reported. The structure and putative substrate-binding site are described and compared with those of other GH5_5 subfamily members.

Critical role of tryptophan 154 for the activity and stability of class D beta-lactamases.

The catalytic efficiency of the class D beta-lactamase OXA-10 depends critically on an unusual carboxylated lysine as the general base residue for both the enzyme acylation and deacylation steps of catalysis. Evidence is presented that the interaction between the indole group of Trp154 and the carboxylated lysine is essential for the stability of the posttranslationally modified Lys70. Substitution of Trp154 by Gly, Ala, or Phe yielded noncarboxylated enzymes which displayed poor catalytic efficiencies and reduced stability when compared to the wild-type OXA-10. The W154H mutant was partially carboxylated. In addition, the maximum values of k(cat) and k(cat)/K(M) were shifted toward pH 7, indicating that the carboxylation state of Lys70 is dependent on the protonation level of the histidine. A comparison of the three-dimensional structures of the different proteins also indicated that the Trp154 mutations did not modify the overall structures of OXA-10 but induced an increased flexibility of the Omega-loop in the active site. Finally, the deacylation-impaired W154A mutant was used to determine the structure of the acyl-enzyme complex with benzylpenicillin. These results indicate a role of the Lys70 carboxylation during the deacylation step and emphasize the importance of Trp154 for the ideal positioning of active site residues leading to an optimum activity.

Conformationally driven gas-phase H/D exchange of dinucleotide negative ions.

Gas-phase hydrogen/deuterium exchange of six deprotonated dinucleotides with CD(3)OD was performed in the second hexapole of a Fourier transform ion-cyclotron resonance (FTICR) mass spectrometer. To complete these experiments, dynamic simulations were carried out to investigate the different conformations adopted by the dinucleotides. In the experimental conditions and in integrating the experimental and theoretical results, H/D exchange was shown to be controlled by hydrogen accessibility and not by the chemical nature of the heteroatom bearing the exchangeable hydrogen. A model including simultaneous H/D exchanges at the experimental time scale was used to reproduce the dinucleotide H/D exchange kinetic plots. The relay mechanism was not relevant for dinucleotides. This allowed the H/D exchange rates to be directly linked to conformations.

NMR structure of Citrobacter freundii AmpD, comparison with bacteriophage T7 lysozyme and homology with PGRP domains.

AmpD is a bacterial amidase involved in the recycling of cell-wall fragments in Gram-negative bacteria. Inactivation of AmpD leads to derepression of beta-lactamase expression, presenting a major pathway for the acquisition of constitutive antibiotic resistance. Here, we report the NMR structure of AmpD from Citrobacter freundii (PDB accession code 1J3G). A deep substrate-binding pocket explains the observed specificity for low molecular mass substrates. The fold is related to that of bacteriophage T7 lysozyme. Both proteins bind zinc at a conserved site and require zinc for amidase activity, although the enzymatic mechanism seems to differ in detail. The structure-based sequence alignment identifies conserved features that are also conserved in the eukaryotic peptidoglycan recognition protein (PGRP) domains, including the zinc-coordination site in several of them. PGRP domains thus belong to the same fold family and, where zinc-binding residues are conserved, may have amidase activity. This hypothesis is supported by the observation that human serum N-acetylmuramyl-L-alanine amidase seems to be identical with a soluble form of human PGRP-L.

Mutational analysis of the catalytic centre of the Citrobacter freundii AmpD N-acetylmuramyl-L-alanine amidase.

Citrobacter freundii AmpD is an intracellular 1,6-anhydro-N-acetylmuramyl-L-alanine amidase involved in both peptidoglycan recycling and beta-lactamase induction. AmpD exhibits a strict specificity for 1,6-anhydromuropeptides and requires zinc for enzymic activity. The AmpD three-dimensional structure exhibits a fold similar to that of another Zn2+ N-acetylmuramyl-L-alanine amidase, the T7 lysozyme, and these two enzymes define a new family of Zn-amidases which can be related to the eukaryotic PGRP (peptidoglycan-recognition protein) domains. In an attempt to assign the different zinc ligands and to probe the catalytic mechanism of AmpD amidase, molecular modelling based on the NMR structure and site-directed mutagenesis were performed. Mutation of the two residues presumed to act as zinc ligands into alanine (H34A and D164A) yielded inactive proteins which had also lost their ability to bind zinc. By contrast, the active H154N mutant retained the capacity to bind the metal ion. Three other residues which could be involved in the AmpD catalytic mechanism have been mutated (Y63F, E116A, K162H and K162Q). The E116A mutant was inactive, but on the basis of the molecular modelling this residue is not directly involved in the catalytic mechanism, but rather in the binding of the zinc by contributing to the correct orientation of His-34. The K162H and K162Q mutants retained very low activity (0.7 and 0.2% of the wild-type activity respectively), whereas the Y63F mutant showed 16% of the wild-type activity. These three latter mutants exhibited a good affinity for Zn ions and the substituted residues are probably involved in the binding of the substrate. We also describe a new method for generating the N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-tripeptide AmpD substrate from purified peptidoglycan by the combined action of two hydrolytic enzymes.

How to remain nonfolded and pliable: the linkers in modular α-amylases as a case study.

The primary structure of linkers in a new class of modular α-amylases constitutes a paradigm of the structural basis that allows a polypeptide to remain nonfolded, extended and pliable. Unfolding is mediated through a depletion of hydrophobic residues and an enrichment of hydrophilic residues, amongst which Ser and Thr are over-represented. An extended and flexible conformation is promoted by the sequential arrangement of Pro and Gly, which are the most abundant residues in these linkers. This is complemented by charge repulsion, charge clustering and disulfide-bridged loops. Molecular dynamics simulations suggest the existence of conformational transitions resulting from a transient and localized hydrophobic collapse, arising from the peculiar composition of the linkers. Accordingly, these linkers should not be regarded as fully disordered, but rather as possessing various discrete structural patterns allowing them to fulfill their biological function as a free energy reservoir for concerted motions between structured domains.

Identification of fragmentation channels of dinucleotides using deuterium labeling.

The fragmentation of the totally deuterated dinucleotide dAT(-) in labile positions (heteroatom-bound hydrogens) was compared for different MS/MS methods: CID, IRMPD, and EID. These experiments allowed us to affirm the coexistence of several fragmentation channels. They can be classified according to the involvement of nonlabile or labile protons in the fragmentation process. Moreover, double resonance experiments were performed in IRMPD and EID. They demonstrated the existence of consecutive fragmentation processes. The probability with which each channel is taken depends on the fragmentation technique used, i.e., the energy and the time scale of the method. The fragmentation channels that involve labile protons requiring peculiar three-dimensional structures are entropically unfavorable and enthalpically favorable. They are more observed in IRMPD and EID. The involvement of labile and, therefore, exchangeable protons in the fragmentation mechanism casts doubt on the use of tandem mass spectrometry to localize incorporated deuteriums in oligonucleotides.

Mercaptophosphonate compounds as broad-spectrum inhibitors of the metallo-beta-lactamases.

Although commercialized inhibitors of active site serine beta-lactamases are currently used in coadministration with antibiotic therapy, no clinically useful inhibitors of metallo-beta-lactamases (MBLs) have yet been discovered. In this paper, we investigated the inhibitory effect of mercaptophosphonate derivatives against the three subclasses of MBLs (B1, B2, and B3). All 14 tested mercaptophosphonates, with the exception of 1a, behaved as competitive inhibitors for the three subclasses. Apart from 13 and 21, all the mercaptophosphonates tested exhibit a good inhibitory effect on the subclass B2 MBL CphA with low inhibition constants (K(i) < 15 muM). Interestingly, compound 18 turned out to be a potent broad spectrum MBL inhibitor. The crystallographic structures of the CphA-10a and CphA-18 complexes indicated that the sulfur atom of 10a and the phosphonato group of 18 interact with the Zn(2+) ion, respectively. Molecular modeling studies of the interactions between compounds 10a and 18 and the VIM-4 (B1), CphA (B2), and FEZ-1 (B3) enzymes brought to light different binding modes depending on the enzyme and the inhibitor, consistent with the crystallographic structures.

Importance of the conserved residues in the peptidoglycan glycosyltransferase module of the class A penicillin-binding protein 1b of Escherichia coli.

The peptidoglycan glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs catalyze glycan chain elongation of the bacterial cell wall. These enzymes belong to the GT51 family, are characterized by five conserved motifs, and have some fold similarity with the phage lambda lysozyme. In this work, we have systematically modified all the conserved amino acid residues of the GT module of Escherichia coli class A PBP1b by site-directed mutagenesis and determined their importance for the in vivo and in vitro activity and the thermostability of the protein. To get an insight into the GT active site of this paradigm enzyme, a model of PBP1b GT domain was constructed based on the available crystal structures (PDB codes 2OLV and 2OLU). The data show that in addition to the essential glutamate residues Glu233 of motif 1 and Glu290 of motif 3, the residues Phe237 and His240 of motif 1 and Gly264, Thr267, Gln271, and Lys274 of motif 2, all located in the catalytic cavity of the GT domain, are essential for the in vitro enzymatic activity of the PBP1b and for its in vivo functioning. Thus, the first three conserved motifs contain most of the residues that are required for the GT activity of the PBP1b. The residues Asp234, Phe237, His240, Thr267, and Gln271 are proposed to maintain the structure of the active site and the positioning of the catalytic Glu233.

Relationship between propeptide pH unfolding and inhibitory ability during ProDer p 1 activation mechanism.

The major allergen Der p 1 of the house dust mite Dermatophagoides pteronyssinus is a papain-like cysteine protease (CA1) produced as an inactive precursor and associated with allergic diseases. The propeptide of Der p 1 exhibits a specific fold that makes it unique in the CA1 propeptide family. In this study, we investigated the activation steps involved in the maturation of the recombinant protease Der p 1 expressed in Pichia pastoris and the interaction of the full-length and truncated soluble propeptides with their parent enzyme in terms of activity inhibition and BIAcore interaction analysis. According to our results, the activation of protease Der p 1 is a multistep mechanism that is characterized by at least two intermediates. The propeptide strongly inhibits unglycosylated and glycosylated recombinant Der p 1 (K(D)=7 nM) at neutral pH. This inhibition is pH dependent. It decreases from pH 7 to pH 4 and can be related to conformational changes of the propeptide characterized by an increase of its flexibility and formation of a molten globule state. Our results indicate that activation of the zymogen at pH 4 is a compromise between activity preservation and propeptide unfolding.

Charge transfer study through the determination of the ionization energies of tetrapeptides X3-Tyr, X = Gly, Ala, or Leu. Influence of the inclusion of one glycine in alanine and leucine containing peptides.

The energies of the fundamental and several excited states of tetrapeptide radical cations were determined at the outer valence Green's function (OVGF) level, at three geometries corresponding to the lowest energy conformations: two for the neutral and one for the cation. The conformations were optimized at the density functional theory level within the B3LYP framework. It was found that, from a purely energetic point of view, a charge initially created on the tyrosine chromophore could migrate without any geometrical change and without further activation once the excited electronic state of the ionized chromophore was formed. This migration could reach the NH(2) terminus for the neutral conformations but should stop at the adjacent peptide link for the cation conformation. These results stress the probable influence of the electronic coupling between the states rather than the existence of a barrier on the charge pathway to explain the difference between the peptides in the charge-transfer process leading to the loss of an iminium [NH(2)=CHR](+) cation. The dissociation energy of the asymptote related to the formation of this NH(2) terminus iminium cation was calculated for few species and it appears that the excess energy available for dissociation is significant when starting from the lowest energy conformations of the neutral or the cation, provided that the charge transfer is effective. It was also found that the amino acids did not conserve their energetic properties and their zero order energy levels turned to a complete new energetic scheme corresponding to the conformation of the peptide.

Ab initio study of the ionization of the DNA bases: ionization potentials and excited states of the cations.

The ionization of the four DNA bases is investigated by means of ab initio calculations. Accurate values of the gas-phase vertical and adiabatic ionization potentials (IP) are obtained at the MP2/6-31G(2d(0.8,alpha(d)),p) level of theory. The need of introducing extra polarization to the standard 6-31G(d,p) basis set is demonstrated by test calculations and an optimal value of alpha(d) = 0.1 is obtained. Ionization to electronically excited radical cations is also considered. The low-lying excited states of the cations are characterized for the first time. The topology of the corresponding potential energy surfaces is qualitatively described in terms of the stationary points (minima and saddle points) located on these surfaces. A conical intersection is characterized for the first time on the ground-state potential energy surface of all cations. It arises from the crossing of the adiabatic surfaces of the ground and first excited state at planar geometries. A nonplanar minimum is observed for the cytosine cation only. The geometry and electronic changes occurring along these surfaces are analyzed, leading to a comparison between the different nucleobase cations. The study of larger ionized systems related to DNA is rendered possible thanks to the optimized medium size basis set proposed in this work, as exemplified by the calculation of the IP of a stacked dimer of guanines.

Catalytic mechanism of the Streptomyces K15 DD-transpeptidase/penicillin-binding protein probed by site-directed mutagenesis and structural analysis.

The Streptomyces K15 penicillin-binding DD-transpeptidase is presumed to be involved in peptide cross-linking during bacterial cell wall peptidoglycan assembly. To gain insight into the catalytic mechanism, the roles of residues Lys38, Ser96, and Cys98, belonging to the structural elements defining the active site cleft, have been investigated by site-directed mutagenesis, biochemical studies, and X-ray diffraction analysis. The Lys38His and Ser96Ala mutations almost completely abolished the penicillin binding and severely impaired the transpeptidase activities while the geometry of the active site was essentially the same as in the wild-type enzyme. It is proposed that Lys38 acts as the catalytic base that abstracts a proton from the active serine Ser35 during nucleophilic attack and that Ser96 is a key intermediate in the proton transfer from the Ogamma of Ser35 to the substrate leaving group nitrogen. The role of these two residues should be conserved among penicillin-binding proteins containing the Ser-Xaa-Asn/Cys sequence in motif 2. Conversion of Cys98 into Asn decreased the transpeptidase activity and increased hydrolysis of the thiolester substrate and the acylation rate with most beta-lactam antibiotics. Cys98 is proposed to play the same role as Asn in motif 2 of other penicilloyl serine transferases in properly positioning the substrate for the catalytic process.

Crp of Streptomyces coelicolor is the third transcription factor of the large CRP-FNR superfamily able to bind cAMP.

The chromosomal inactivation of the unique transcription factor of Streptomyces coelicolor that displays a cyclic-nucleotide-binding domain, Crp(Sco), led to a germination-defective phenotype similar to the mutant of the adenylate cyclase gene (cya) unable to produce cAMP. By means of cAMP affinity chromatography we demonstrate the specific cAMP-binding ability of Crp(Sco), which definitely demonstrate that a Cya/cAMP/Crp system is used to trigger germination in S. coelicolor. However, electromobility shift assays with the purified Crp(Sco)-cAMP complex and the CRP-like cis-acting element of its own promoter failed. Moreover, we were unable to complement an Escherichia coli crp mutant in trans with Crp(Sco). The fact that Vfr from Pseudomonas aeruginosa and GlxR from Corynebacterium glutamicum could complement such an E. coli mutant suggests that the way Crp(Sco) interacts with DNA should mechanistically differ from its most similar members. This hypothesis was further supported by homology modelling of Crp(Sco) that confirmed an unusual organisation of the DNA-binding domain compared to the situation observed in Crp(Eco).