A potent anti-dengue human antibody preferentially recognizes the conformation of E protein monomers assembled on the virus surface

Dengue virus (DENV), which consists of four serotypes (DENV1-4), infects over 400million people annually. Previous studies have indicated most human monoclonal antibodies (HMAbs) from dengue patients are cross-reactive and poorly neutralizing. Rare neutralizing HMAbs are usually serotype-specific and bind to quaternary structure-dependent epitopes. We determined the structure of DENV1 complexed with Fab fragments of a highly potent HMAb 1F4 to 6Å resolution by cryo-EM. Although HMAb 1F4 appeared to bind to virus and not E proteins in ELISAs in the previous study, our structure showed that the epitope is located within an envelope (E) protein monomer, and not across neighboring E proteins. The Fab molecules bind to domain I (DI), and DI-DII hinge of the E protein. We also showed that HMAb 1F4 can neutralize DENV at different stages of viral entry in a cell type and receptor dependent manner. The structure reveals the mechanism by which this potent and specific antibody blocks viral infection.Subject Categories Microbiology, Virology & Host Pathogen Interaction; Immunolog

Structure and mechanism of action of two bacterial enzymes : MltE from Escherichia coli and AspB from Bacillus sp. YM55-1

Het proefschrift beschrijft de kristalstructuren en reactiemechanismen van twee verschillende bacteriële enzymen, de lytische transglycosylase MltE uit Escherichia coli en de aspartase AspB uit Bacillus sp. YM55-1. MltE verbreekt de beta-1,4-glycosidische binding tussen N-acetylmuraminezuur and N-acetylglucosamine residuën in het bacteriële celwand materiaal peptidoglycan. MltE wordt gedacht een rol te spelen in het bacteriële celwand metabolisme, waardoor het een potentieel doelwit is voor antibacteriële middelen. MltE is één van de weinige endolytische transglycosylasen van E. coli. Kristalstructuren van MltE zonder substraat, met gebonden chitopentaose, en van een ternair complex met de inhibitor bulgecin en een murodipeptide maakten het mogelijk om in detail de interacties van het enzym met peptidoglycan fragmenten te bestuderen. In combinatie met plaatsgerichte mutagenese experimenten verklaren de structuren waarom MltE endolytische activiteit heeft en hoe het de reactie katalyseert. Het tweede enzyme, de aspartase AspB uit Bacillus sp. YM55-1, katalyseert de omzetting van L-aspartaat in fumaraat en ammonia. Aspartases worden gebruikt als biokatalysatoren voor de industriële productie van enantiozuiver L-aspartaat, een belangrijke bouwstof voor de synthese van voedseladditieven en zoetstoffen. Echter, het precieze katalytische mechanism van het enzym is lange tijd onduidelijk gebleven wegens gebrek aan informatie over hoe substraat en product aan het enzym binden. Door kristalstructuren van AspB op te helderen in aan- en afwezigheid van het substraat L-aspartaat hebben we nu de aminozuren kunnen definiëren die verantwoordelijk zijn voor de katalyse. In combinatie met plaatsgerichte mutagenese en enzym kinetiek experimenten kan het werkingsmechanisme van het enzym nu volledig verklaard worden. In this thesis, crystal structures and reaction mechanisms of two different bacterial enzymes are described. The first enzyme is the lytic transglycosylase MltE from Escherichia coli, which cleaves the beta-1,4-glycosidic bonds between N-acetylmuramic acid and Nacetylglucosamine residues in the bacterial cell wall material peptidoglycan. The enzyme is thought to function in bacterial cell wall turn-over, remodeling and maintenance, which makes it a potential target for antibacterials. MltE is distinct because it is one of the few endoacting lytic transglycosylases of E. coli. The crystal structures of MltE in a substrate-free state, in a binary complex with chitopentaose, and in a ternary complex with the glycopeptide inhibitor bulgecin A and a murodipeptide allowed a detailed analysis of the saccharidebinding interactions. In combination with site-directed mutagenesis studies the structures explain why MltE is an endo-acting enzyme and how it catalyzes the reaction. The second enzyme is the aspartase AspB from Bacillus sp. YM55-1, which catalyzes the reversible deamination of L-aspartate into fumarate and ammonia. Aspartases are used as biocatalysts for the industrial production of enantiopure L-aspartate, an important starting compound for the synthesis of food additives and artificial sweeteners. However, their precise catalytic mechanism has remained elusive because of lack of information on the binding mode of substrate, product or substrate analogs. Crystal structures of AspB in an unliganded state and with bound L-aspartate have now revealed the residues responsible for catalysis. Accompanying site directed mutagenesis and enzyme kinetics experiments allowed to fully explain the mechanism of action of this enzyme.

The development of therapeutic antibodies against dengue virus

10.1016/j.antiviral.2016.01.002Antiviral Research1287-1

On the Mechanism of Peptidoglycan Binding and Cleavage by the endo-Specific Lytic Transglycosylase MltE from Escherichia coli

The lytic transglycosylase MltE from Escherichia coli is a periplasmic, outer membrane-attached enzyme that cleaves the β-1,4-glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine residues in the cell wall peptidoglycan, producing 1,6-anhydromuropeptides. Here we report three crystal structures of MltE: in a substrate-free state, in a binary complex with chitopentaose, and in a ternary complex with the glycopeptide inhibitor bulgecin A and the murodipeptide N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-Glu. The substrate-bound structures allowed a detailed analysis of the saccharide-binding interactions in six subsites of the peptidoglycan-binding groove (subsites −4 to +2) and, combined with site-directed mutagenesis analysis, confirmed the role of Glu64 as catalytic acid/base. The structures permitted the precise modeling of a short glycan strand of eight saccharide residues, providing evidence for two additional subsites (+3 and +4) and revealing the productive conformational state of the substrate at subsites −1 and +1, where the glycosidic bond is cleaved. Full accessibility of the peptidoglycan-binding groove and preferential binding of an N-acetylmuramic acid residue in a 4C1 chair conformation at subsite +2 explain why MltE shows only endo- and no exo-specific activity toward glycan strands. The results further indicate that catalysis of glycosidic bond cleavage by MltE proceeds via distortion toward a sofa-like conformation of the N-acetylmuramic acid sugar ring at subsite −1 and by anchimeric assistance of the sugar’s N-acetyl group, as shown previously for the lytic transglycosylases Slt70 and MltB.

Structural Basis for the Catalytic Mechanism of Aspartate Ammonia Lyase

Aspartate ammonia lyases (or aspartases) catalyze the reversible deamination of L-aspartate into fumarate and ammonia. The lack of crystal structures of complexes with substrate, product, or substrate analogues so far precluded determination of their precise mechanism of catalysis. Here, we report crystal structures of AspB, the aspartase from Bacillus sp. YM55-1, in an unliganded state and in complex with L-aspartate at 2.4 and 2.6 Å resolution, respectively. AspB forces the bound substrate to adopt a high-energy, enediolate-like conformation that is stabilized, in part, by an extensive network of hydrogen bonds between residues Thr101, Ser140, Thr141, and Ser319 and the substrate’s β-carboxylate group. Furthermore, substrate binding induces a large conformational change in the SS loop (residues G317SSIMPGKVN326) from an open conformation to one that closes over the active site. In the closed conformation, the strictly conserved SS loop residue Ser318 is at a suitable position to act as a catalytic base, abstracting the Cβ proton of the substrate in the first step of the reaction mechanism. The catalytic importance of Ser318 was confirmed by site-directed mutagenesis. Site-directed mutagenesis of SS loop residues, combined with structural and kinetic analysis of a stable proteolytic AspB fragment, further suggests an important role for the small C-terminal domain of AspB in controlling the conformation of the SS loop and, hence, in regulating catalytic activity. Our results provide evidence supporting the notion that members of the aspartase/fumarase superfamily use a common catalytic mechanism involving general base-catalyzed formation of a stabilized enediolate intermediate.

Aspartase/Fumarase Superfamily: A Common Catalytic Strategy Involving General Base-Catalyzed Formation of a Highly Stabilized aci-Carboxylate Intermediate

Members of the aspartase/fumarase superfamily share a common tertiary and quaternary fold, as well as a similar active site architecture; the superfamily includes aspartase, fumarase, argininosuccinate lyase, adenylosuccinate lyase, δ-crystallin, and 3-carboxy-cis,cis-muconate lactonizing enzyme (CMLE). These enzymes all process succinyl-containing substrates, leading to the formation of fumarate as the common product (except for the CMLE-catalyzed reaction, which results in the formation of a lactone). In the past few years, X-ray crystallographic analysis of several superfamily members in complex with substrate, product, or substrate analogues has provided detailed insights into their substrate binding modes and catalytic mechanisms. This structural work, combined with earlier mechanistic studies, revealed that members of the aspartase/fumarase superfamily use a common catalytic strategy, which involves general base-catalyzed formation of a stabilized aci-carboxylate (or enediolate) intermediate and the participation of a highly flexible loop, containing the signature sequence GSSxxPxKxN (named the SS loop), in substrate binding and catalysis.

On the Mechanism of Peptidoglycan Binding and Cleavage by the <i>endo</i>-Specific Lytic Transglycosylase MltE from <i>Escherichia coli</i>

The lytic transglycosylase MltE from <i>Escherichia coli</i> is a periplasmic, outer membrane-attached enzyme that cleaves the β-1,4-glycosidic bonds between <i>N</i>-acetylmuramic acid and <i>N</i>-acetylglucosamine residues in the cell wall peptidoglycan, producing 1,6-anhydromuropeptides. Here we report three crystal structures of MltE: in a substrate-free state, in a binary complex with chitopentaose, and in a ternary complex with the glycopeptide inhibitor bulgecin A and the murodipeptide <i>N</i>-acetylglucosaminyl-<i>N</i>-acetylmuramyl-l-Ala-d-Glu. The substrate-bound structures allowed a detailed analysis of the saccharide-binding interactions in six subsites of the peptidoglycan-binding groove (subsites −4 to +2) and, combined with site-directed mutagenesis analysis, confirmed the role of Glu64 as catalytic acid/base. The structures permitted the precise modeling of a short glycan strand of eight saccharide residues, providing evidence for two additional subsites (+3 and +4) and revealing the productive conformational state of the substrate at subsites −1 and +1, where the glycosidic bond is cleaved. Full accessibility of the peptidoglycan-binding groove and preferential binding of an <i>N</i>-acetylmuramic acid residue in a ⁴C₁ chair conformation at subsite +2 explain why MltE shows only <i>endo</i>- and no <i>exo</i>-specific activity toward glycan strands. The results further indicate that catalysis of glycosidic bond cleavage by MltE proceeds via distortion toward a sofa-like conformation of the <i>N</i>-acetylmuramic acid sugar ring at subsite −1 and by anchimeric assistance of the sugar’s <i>N</i>-acetyl group, as shown previously for the lytic transglycosylases Slt70 and MltB