Nucleotide sequence of the PSE-4 carbenicillinase gene and correlations with the Staphylococcus aureus PC1 beta-lactamase crystal structure

The nucleotide sequence of the PSE-4 beta-lactamase gene from Pseudomonas aeruginosa strain Dalgleish has been determined. The structural gene encodes a polypeptide product of 252 amino acids with an estimated molecular mass of 29,246 Da for the mature form of the protein. The PSE-4 gene has limited homology with other beta-lactamases at the DNA level. An alignment of all known class A beta-lactamases permitted as to identify specific residues important for enzyme structure and function. To confirm observations based on the linear sequences, we designed a new molecular model for PSE-4 beta-lactamase based on x-ray data from the Staphylococcus aureus PC1 beta-lactamase at 2.0-A resolution. The structural similarities between PSE-4 and class A beta-lactamases are more extensive than indicated by earlier biochemical studies. The combined structural and sequence information now available for a series of beta-lactamases identifies conserved residues in these molecules, giving insight of their divergence and ancestry. Analysis of the PSE-4 flanking DNA sequences revealed an integration site common to antibiotic resistance genes inserted into transposons of the Tn21 family with the target integration sequence AAGTT.     

Insights into the molecular basis of tick-borne encephalitis from multiplatform metabolomics

BackgroundTick-borne encephalitis virus (TBEV) is the most prevalent arbovirus, with a tentative estimate of 10,000 to 10,500 infections occurring in Europe and Asia every year. Endemic in Northeast China, tick-borne encephalitis (TBE) is emerging as a major threat to public health, local economies and tourism. The complicated array of host physiological changes has hampered elucidation of the molecular mechanisms underlying the pathogenesis of this disease.Methodology/Principle findingsSystem-level characterization of the serum metabolome and lipidome of adult TBEV patients and a healthy control group was performed using liquid chromatography tandem mass spectrometry. By tracking metabolic and lipid changes during disease progression, crucial physiological changes that coincided with disease stages could be identified. Twenty-eight metabolites were significantly altered in the sera of TBE patients in our metabolomic analysis, and 14 lipids were significantly altered in our lipidomics study. Among these metabolites, alpha-linolenic acid, azelaic acid, D-glutamine, glucose-1-phosphate, L-glutamic acid, and mannose-6-phosphate were altered compared to the control group, and PC(38:7), PC(28:3;1), TAG(52:6), etc. were altered based on lipidomics. Major perturbed metabolic pathways included amino acid metabolism, lipid and oxidative stress metabolism (lipoprotein biosynthesis, arachidonic acid biosynthesis, leukotriene biosynthesis and sphingolipid metabolism), phospholipid metabolism and triglyceride metabolism. These metabolites were significantly perturbed during disease progression, implying their latent utility as prognostic markers.Conclusions/SignificanceTBEV infection causes distinct temporal changes in the serum metabolome and lipidome, and many metabolites are potentially involved in the acute inflammatory response and immune regulation. Our global analysis revealed anti- and pro-inflammatory processes in the host and changes to the entire metabolic profile. Relationships between metabolites and pathologies were established. This study provides important insight into the pathology of TBE, including its pathology, and lays the foundation for further research into putative markers of TBE disease.     

Insights into the molecular basis of polyglutamine neurodegeneration from studies of a spinocerebellar ataxia type 7 mouse model

Spinocerebellar ataxia type 7 (SCA7) is one member of a growing list of neurodegenerative disorders that are all caused by CAG repeat expansions that produce disease by encoding elongated polyglutamine tracts in a variety of apparently unrelated proteins. In this review, we provide an overview of our efforts to determine the molecular basis of polyglutamine neurotoxicity in SCA7 by modeling this polyglutamine repeat disorder in mice. We discuss how our SCA7 mouse model develops a phenotype that is reminiscent of the retinal and cerebellar disease pathology seen in human patients. All of these findings are considered in the context of numerous other models of polyglutamine disease pathology in mice and other organisms, together with various other in vitro and biochemical studies. We present the competing hypotheses of polyglutamine disease pathogenesis, and explain how our studies of SCA7 brainstem and retinal degeneration using this mouse model have yielded insights into possible mechanisms and pathways of polyglutamine disease pathology. In addition to illustrating how our SCA7 mouse model has allowed us to develop and advance notions of disease pathogenesis, we propose a model of polyglutamine molecular pathology that attempts to integrate the key observations in the field. We close by describing why our SCA7 mouse model should be useful for the next phase of polyglutamine disease research--the development of therapies, and predict that this stage of experimentation will continue to rely heavily on the mouse.     

Insights into the mechanisms underlying CFTR channel activity, the molecular basis for cystic fibrosis and strategies for therapy

Mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) cause CF (cystic fibrosis), a fatal genetic disease commonly leading to airway obstruction with recurrent airway inflammation and infection. Pulmonary obstruction in CF has been linked to the loss of CFTR function as a regulated Cl- channel on the lumen-facing membrane of the epithelium lining the airways. We have learned much about the molecular basis for nucleotide- and phosphorylation-dependent regulation of channel activity of the normal (wild-type) version of the CFTR protein through electrophysiological studies. The major CF-causing mutation, F508del-CFTR, causes the protein to misfold and be retained in the ER (endoplasmic reticulum). Importantly, recent studies in cell culture have shown that retention in the ER can be 'corrected' through the application of certain small-molecule modulators and, once at the surface, the altered channel function of the major mutant can be 'potentiated', pharmacologically. Importantly, two such small molecules, a 'corrector' (VX-809) and a 'potentiator' (VX-770) compound are undergoing clinical trial for the treatment of CF. In this chapter, we describe recent discoveries regarding the wild-type CFTR and F508del-CFTR protein, in the context of molecular models based on X-ray structures of prokaryotic ABC (ATP-binding cassette) proteins. Finally, we discuss the promise of small-molecule modulators to probe the relationship between structure and function in the wild-type protein, the molecular defects caused by the most common mutation and the structural changes required to correct these defects.     

Insights into the molecular basis of the NOD2 signalling pathway

The cytosolic pattern recognition receptor NOD2 is activated by the peptidoglycan fragment muramyl dipeptide to generate a proinflammatory immune response. Downstream effects include the secretion of cytokines such as interleukin 8, the upregulation of pro-interleukin 1β, the induction of autophagy, the production of antimicrobial peptides and defensins, and contributions to the maintenance of the composition of the intestinal microbiota. Polymorphisms in NOD2 are the cause of the inflammatory disorder Blau syndrome and act as susceptibility factors for the inflammatory bowel condition Crohn''s disease. The complexity of NOD2 signalling is highlighted by the observation that over 30 cellular proteins interact with NOD2 directly and influence or regulate its functional activity. Previously, the majority of reviews on NOD2 function have focused upon the role of NOD2 in inflammatory disease or in its interaction with and response to microbes. However, the functionality of NOD2 is underpinned by its biochemical interactions. Consequently, in this review, we have taken the opportunity to address the more ‘basic’ elements of NOD2 signalling. In particular, we have focused upon the core interactions of NOD2 with protein factors that influence and modulate the signal transduction pathways involved in NOD2 signalling. Further, where information exists, such as in relation to the role of RIP2, we have drawn comparison with the closely related, but functionally discrete, pattern recognition receptor NOD1. Overall, we provide a comprehensive resource targeted at understanding the complexities of NOD2 signalling.     

Insights into the molecular basis of a bispecific antibody's target selectivity

Bispecific antibodies constitute a valuable class of therapeutics owing to their ability to bind 2 distinct targets. Dual targeting is thought to enhance biological efficacy, limit escape mechanisms, and increase target selectivity via a strong avidity effect mediated by concurrent binding to both antigens on the surface of the same cell. However, factors that regulate the extent of target selectivity are not well understood. We show that dual targeting alone is not sufficient to promote efficient target selectivity, and report the substantial roles played by the affinity of the individual arms, overall avidity and valence. More particularly, various monovalent bispecific IgGs composed of an anti-CD70 moiety paired with variants of the anti-CD4 mAb ibalizumab were tested for preferential binding and selective depletion of CD4⁺/CD70⁺ T cells over cells expressing only one of the target antigens that resulted from antibody dependent cell-mediated cytotoxicity. Variants exhibiting reduced CD4 affinity showed a greater degree of target selectivity, while the overall efficacy of the bispecific molecule was not affected.     

Insights into the molecular basis of a bispecific antibody's target selectivity

Bispecific antibodies constitute a valuable class of therapeutics owing to their ability to bind 2 distinct targets. Dual targeting is thought to enhance biological efficacy, limit escape mechanisms, and increase target selectivity via a strong avidity effect mediated by concurrent binding to both antigens on the surface of the same cell. However, factors that regulate the extent of target selectivity are not well understood. We show that dual targeting alone is not sufficient to promote efficient target selectivity, and report the substantial roles played by the affinity of the individual arms, overall avidity and valence. More particularly, various monovalent bispecific IgGs composed of an anti-CD70 moiety paired with variants of the anti-CD4 mAb ibalizumab were tested for preferential binding and selective depletion of CD4⁺/CD70⁺ T cells over cells expressing only one of the target antigens that resulted from antibody dependent cell-mediated cytotoxicity. Variants exhibiting reduced CD4 affinity showed a greater degree of target selectivity, while the overall efficacy of the bispecific molecule was not affected.     

Mechanistic features of Salmonella typhimurium propionate kinase (TdcD): Insights from kinetic and crystallographic studies

Short-chain fatty acids (SCFAs) play a major role in carbon cycle and can be utilized as a source of carbon and energy by bacteria. Salmonella typhimurium propionate kinase (StTdcD) catalyzes reversible transfer of the γ-phosphate of ATP to propionate during l-threonine degradation to propionate. Kinetic analysis revealed that StTdcD possesses broad ligand specificity and could be activated by various SCFAs (propionate > acetate ≈ butyrate), nucleotides (ATP ≈ GTP > CTP ≈ TTP; dATP > dGTP > dCTP) and metal ions (Mg^2 + ≈ Mn^2 + > Co^2 +). Inhibition of StTdcD by tricarboxylic acid (TCA) cycle intermediates such as citrate, succinate, α-ketoglutarate and malate suggests that the enzyme could be under plausible feedback regulation. Crystal structures of StTdcD bound to PO₄ (phosphate), AMP, ATP, Ap4 (adenosine tetraphosphate), GMP, GDP, GTP, CMP and CTP revealed that binding of nucleotide mainly involves hydrophobic interactions with the base moiety and could account for the broad biochemical specificity observed between the enzyme and nucleotides. Modeling and site-directed mutagenesis studies suggest Ala88 to be an important residue involved in determining the rate of catalysis with SCFA substrates. Molecular dynamics simulations on monomeric and dimeric forms of StTdcD revealed plausible open and closed states, and also suggested role for dimerization in stabilizing segment 235–290 involved in interfacial interactions and ligand binding. Observation of an ethylene glycol molecule bound sufficiently close to the γ-phosphate in StTdcD complexes with triphosphate nucleotides supports direct in-line phosphoryl transfer.     

Evaluation of inhibition of the carbenicillin-hydrolyzing beta-lactamase PSE-4 by the clinically used mechanism-based inhibitors

Characterization of the biochemical steps in the inactivation chemistry of clavulanic acid, sulbactam and tazobactam with the carbenicillin-hydrolyzing beta-lactamase PSE-4 from Pseudomonas aeruginosa is described. Although tazobactam showed the highest affinity to the enzyme, all three inactivators were excellent inhibitors for this enzyme. Transient inhibition was observed for the three inactivators before the onset of irreversible inactivation of the enzyme. Partition ratios (k(cat)/k(inact)) of 11, 41 and 131 were obtained with clavulanic acid, tazobactam and sulbactam, respectively. Furthermore, these values were found to be 14-fold, 3-fold and 80-fold lower, respectively, than the values obtained for the clinically important TEM-1 beta-lactamase. The kinetic findings were put in perspective by determining the computational models for the pre-acylation complexes and the immediate acyl-enzyme intermediates for all three inactivators. A discussion of the pertinent structural factors is presented, with PSE-4 showing subtle differences in interactions with the three inhibitors compared to the TEM-1 enzyme.     

Insights into the structural basis of the pH-dependent dimer-tetramer equilibrium through crystallographic analysis of recombinant Diocleinae lectins

The structural ground underlying the pH-dependency of the dimer-tetramer transition of Diocleinae lectins was investigated by equilibrium sedimentation and X-ray crystal structure determination of wild-type and site-directed mutants of recombinant lectins. Synthetic genes coding for the full-length alpha-chains of the seed lectins of Dioclea guianensis (termed r-alphaDguia) and Dioclea grandiflora (termed r-alphaDGL) were designed and expressed in Escherichia coli. This pioneering approach, which will be described in detail in the present paper, yielded recombinant lectins displaying carbohydrate-binding activity, dimer-tetramer equilibria and crystal structures indistinguishable from their natural homologues. Conversion of the pH-stable tetrameric r-alphaDGL into a structure exhibiting pH-dependent dimer-tetramer transition was accomplished through mutations that abolished the interdimeric interactions at the central cavity of the tetrameric lectins. Both the central and the peripheral interacting regions bear structural information for formation of the canonical legume lectin tetramer. We hypothesize that the strength of the ionic contacts at these sites may be modulated by the pH, leading to dissociation of those lectin structures that are not locked into a pH-stable tetramer through interdimeric contacts networking the central cavity loops.     

Insights into the molecular basis of thermal stability from the structure determination of Pyrococcus furiosus gluatamate dehydrogenase

Abstract: The structure determination of the glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus has been completed at 2.2 Å resolution. The structure has been compared with the glutamate dehydrogenases from the mesophiles Clostridium symbiosum, Escherichia coli and Neurospora crassa . This comparison has revealed that the hyperthermophilic enzyme contains a striking series of networks of ion-pairs which are formed by regions of the protein which contain a high density of charged residues. Such regions are not found in the mesophilic enzymes and the number and extent of ion-pair formation is much more limited. The ion-pair networks are clustered at both inter domain and inter subunit interfaces and may well represent a major stabilising feature associated with the adaptation of enzymes to extreme temperatures.     

Structural and kinetic studies on beta-lactamase K1 from Klebsiella aerogenes.

beta-Lactamase K1 from Klebsiella aerogenes 1082E hydrolyses both penicillins and cephalosporins comparably and is inhibited by mercurials but not by cloxacillin. These properties distinguish it from those other beta-lactamases that have been allotted to classes on the basis of their amino sequences. beta-Lactamase K1 has been isolated by affinity chromatography; its composition shows resemblances to class A beta-lactamases. Moreover, the N-terminal sequence is similar to those of class A beta-lactamases: there is about 30% identity over the first 32 residues. Furthermore, a putative active-site octapeptide has been isolated and its sequence is similar to the region around the active-site serine residue in class A beta-lactamases. There is one thiol group in beta-lactamase K1; it is not essential for activity. The pH-dependence of kcat. and kcat./Km for the hydrolysis of benzylpenicillin by beta-lactamase K1 were closely similar, suggesting that the rate-determining step is cleavage of the beta-lactam ring.     

Insights into the structure and dynamics of the dinuclear zinc beta-lactamase site from Bacteroides fragilis

Herein, we report quantum chemical calculations and molecular dynamics (MD) simulations of the dinuclear form of the Bacteroides fragilis zinc beta-lactamase. We studied four different configurations which differ in the protonation state of the Asp103 residue and in the presence or absence of a Zn1-OH-Zn2 bridge. The flexibility of the Zn1-OH-Zn2 bridge was studied by means of quantum mechanical (QM) calculations on cluster models while the relative stabilities of the different configurations were estimated from QM linear scaling calculations on the enzyme. Contacts between important residues (Cys104, Asp69, Lys185, etc.), the solvation of the zinc ions, and the conformation of the active site beta-hairpin loop were characterized by the MD analyses. The influence of the buried sodium ion close to the Zn2 position was investigated by carrying out a secondary simulation where the sodium ion was replaced with an internal water molecule. The comparative structural analyses among the different MD trajectories augmented with energetic calculations have demonstrated that the B. fragilis protein efficiently binds the internal Na(+) ion observed crystallographically. Moreover, we found that when Asp103 is unprotonated, a rigid Zn1-OH-Zn2 bridge results, while for neutral Asp103, a fluctuating Zn1-Zn2 distance was possible via the breaking and formation of the Zn1-OH-Zn2 bridge. The mechanistic implications of these observations are discussed in detail.     

X-ray crystallographic and kinetic studies of human sorbitol dehydrogenase

Sorbitol dehydrogenase (hSDH) and aldose reductase form the polyol pathway that interconverts glucose and fructose. Redox changes from overproduction of the coenzyme NADH by SDH may play a role in diabetes-induced dysfunction in sensitive tissues, making SDH a therapeutic target for diabetic complications. We have purified and determined the crystal structures of human SDH alone, SDH with NAD(+), and SDH with NADH and an inhibitor that is competitive with fructose. hSDH is a tetramer of identical, catalytically active subunits. In the apo and NAD(+) complex, the catalytic zinc is coordinated by His69, Cys44, Glu70, and a water molecule. The inhibitor coordinates the zinc through an oxygen and a nitrogen atom with the concomitant dissociation of Glu70. The inhibitor forms hydrophobic interactions to NADH and likely sterically occludes substrate binding. The structure of the inhibitor complex provides a framework for developing more potent inhibitors of hSDH.     

A kinetic model for the molecular basis of the contractile activity of Acanthamoeba myosins IA and IB

Acanthamoeba myosins IA and IB are single-headed, monomeric molecules consisting of one heavy chain and one light chain. Both have high actin-activated Mg2+-ATPase activity, when the heavy chain is phosphorylated, but neither seems to be able to form the bipolar filaments that are generally thought to be required for actomyosin-dependent contractility. In this paper, we show that, at fixed F-actin concentration, the actin-activated Mg2+-ATPase activities of myosins IA and IB increase about 5-fold in specific activity in a cooperative manner as the myosin concentration is increased. The myosin concentration range over which this cooperative change occurs depends on the actin concentration. More myosin I is required for the cooperative increase in activity at high concentrations of F-actin. The cooperative increase in specific activity at limiting actin concentrations is caused by a decrease in the KATPase for F-actin. The high and low KATPase states of the myosin have about the same Vmax at infinite actin concentration. Both myosins are completely bound to the F-actin long before the Vmax values are reached. Therefore, much of the actin activation must be the result of interactions between F-actin and actomyosin. These kinetic data can be explained by a model in which the cooperative shift of myosin I from the high KATPase to the low KATPase state results from the cross-linking of actin filaments by myosin I. Cross-linking might occur either through two actin-binding sites on a single molecule or by dimers or oligomers of myosin I induced to form by the interaction of myosin I monomers with the actin filaments. The ability of Acanthamoeba myosins IA and IB to cross-link actin filaments is demonstrated in the accompanying paper (Fujisaki, H., Albanesi, J.P., and Korn, E.D. (1985) J. Biol. Chem. 260, 11183-11189).     

Crystal structure of Thermotoga maritima alpha-L-fucosidase. Insights into the catalytic mechanism and the molecular basis for fucosidosis

Fucosylated glycoconjugates are involved in numerous biological events, and alpha-l-fucosidases, the enzymes responsible for their processing, are therefore of crucial importance. Deficiency in alpha-l-fucosidase activity is associated with fucosidosis, a lysosomal storage disorder characterized by rapid neurodegeneration, resulting in severe mental and motor deterioration. To gain insight into alpha-l-fucosidase function at the molecular level, we have determined the crystal structure of Thermotoga maritima alpha-l-fucosidase. This enzyme assembles as a hexamer and displays a two-domain fold, composed of a catalytic (beta/alpha)(8)-like domain and a C-terminal beta-sandwich domain. The structures of an enzyme-product complex and of a covalent glycosyl-enzyme intermediate, coupled with kinetic and mutagenesis studies, allowed us to identify the catalytic nucleophile, Asp(244), and the Br?nsted acid/base, Glu(266). Because T. maritima alpha-l-fucosidase occupies a unique evolutionary position, being far more closely related to the mammalian enzymes than to any other prokaryotic homolog, a structural model of the human enzyme was built to document the structural consequences of the genetic mutations associated with fucosidosis.     

Insights into the ClC-4 transport mechanism from studies of Zn2+ inhibition

The CLC family of chloride channels and transporters is a functionally diverse group of proteins important in a wide range of physiological processes. ClC-4 and ClC-5 are localized to endosomes and seem to play roles in the acidification of these compartments. These proteins were recently shown to function as Cl⁻/H⁺ antiporters. However, relatively little is known about the detailed mechanism of CLC-mediated Cl⁻/H⁺ antiport, especially for mammalian isoforms. We attempted to identify molecular tools that might be useful in probing structure-function relationships in these proteins. Here, we record currents from human ClC-4 (hClC-4) expressed in Xenopus oocytes, and find that Zn²⁺ inhibits these currents, with an apparent affinity of ∼50 μM. Although Cd²⁺ has a similar effect, Co²⁺ and Mn²⁺ do not inhibit hClC-4 currents. In contrast, the effect of Zn²⁺ on the ClC-0 channel, Zn²⁺-mediated inhibition of hClC-4 is minimally voltage-dependent, suggesting an extracellular binding site for the ion. Nine candidate external residues were tested; only mutations of three consecutive histidine residues, located in a single extracellular loop, significantly reduced the effect of Zn²⁺, with one of these making a larger contribution than the other two. An analogous tri-His sequence is absent from ClC-0, suggesting a fundamentally different inhibitory mechanism for the ion on hClC-4. Manipulations that alter transport properties of hClC-4, varying permeant ions as well as mutating the “gating glutamate”, dramatically affect Zn²⁺ inhibition, suggesting the involvement of a heretofore unexplored part of the protein in the transport process.