The Catalytic Aspartate Is Protonated in the Michaelis Complex Formed between Trypsin and an in Vitro Evolved Substrate-like Inhibitor: A REFINED MECHANISM OF SERINE PROTEASE ACTION.

The mechanism of serine proteases prominently illustrates how charged amino acid residues and proton transfer events facilitate enzyme catalysis. Here we present an ultrahigh resolution (0.93 A) x-ray structure of a complex formed between trypsin and a canonical inhibitor acting through a substrate-like mechanism. The electron density indicates the protonation state of all catalytic residues where the catalytic histidine is, as expected, in its neutral state prior to the acylation step by the catalytic serine. The carboxyl group of the catalytic aspartate displays an asymmetric electron density so that the O(delta2)-C(gamma) bond appears to be a double bond, with O(delta2) involved in a hydrogen bond to His-57 and Ser-214. Only when Asp-102 is protonated on O(delta1) atom could a density functional theory simulation reproduce the observed electron density. The presence of a putative hydrogen atom is also confirmed by a residual mF(obs) - DF(calc) density above 2.5 sigma next to O(delta1). As a possible functional role for the neutral aspartate in the active site, we propose that in the substrate-bound form, the neutral aspartate residue helps to keep the pK(a) of the histidine sufficiently low, in the active neutral form. When the histidine receives a proton during the catalytic cycle, the aspartate becomes simultaneously negatively charged, providing additional stabilization for the protonated histidine and indirectly to the tetrahedral intermediate. This novel proposal unifies the seemingly conflicting experimental observations, which were previously seen as either supporting the charge relay mechanism or the neutral pK(a) histidine theory

Substrate-bound outward-open structure of a Na+-coupled sialic acid symporter reveals a new Na+ site

Many pathogenic bacteria utilise sialic acids as an energy source or use them as an external coating to evade immune detection. As such, bacteria that colonise sialylated environments deploy specific transporters to mediate import of scavenged sialic acids. Here, we report a substrate-bound 1.95 Å resolution structure and subsequent characterisation of SiaT, a sialic acid transporter from Proteus mirabilis. SiaT is a secondary active transporter of the sodium solute symporter (SSS) family, which use Na+ gradients to drive the uptake of extracellular substrates. SiaT adopts the LeuT-fold and is in an outward-open conformation in complex with the sialic acid N-acetylneuraminic acid and two Na+ ions. One Na+ binds to the conserved Na2 site, while the second Na+ binds to a new position, termed Na3, which is conserved in many SSS family members. Functional and molecular dynamics studies validate the substrate-binding site and demonstrate that both Na+ sites regulate N-acetylneuraminic acid transport

The Sodium Sialic Acid Symporter From Staphylococcus aureus Has Altered Substrate Specificity

Mammalian cell surfaces are decorated with complex glycoconjugates that terminate with negatively charged sialic acids. Commensal and pathogenic bacteria can use host-derived sialic acids for a competitive advantage, but require a functional sialic acid transporter to import the sugar into the cell. This work investigates the sodium sialic acid symporter (SiaT) from Staphylococcus aureus (SaSiaT). We demonstrate that SaSiaT rescues an Escherichia coli strain lacking its endogenous sialic acid transporter when grown on the sialic acids N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc). We then develop an expression, purification and detergent solubilization system for SaSiaT and demonstrate that the protein is largely monodisperse in solution with a stable monomeric oligomeric state. Binding studies reveal that SaSiaT has a higher affinity for Neu5Gc over Neu5Ac, which was unexpected and is not seen in another SiaT homolog. We develop a homology model and use comparative sequence analyses to identify substitutions in the substrate-binding site of SaSiaT that may explain the altered specificity. SaSiaT is shown to be electrogenic, and transport is dependent upon more than one Na+ ion for every sialic acid molecule. A functional sialic acid transporter is essential for the uptake and utilization of sialic acid in a range of pathogenic bacteria, and developing new inhibitors that target these transporters is a valid mechanism for inhibiting bacterial growth. By demonstrating a route to functional recombinant SaSiaT, and developing the in vivo and in vitro assay systems, our work underpins the design of inhibitors to this transporter

The Sodium Sialic acid symporter from Staphylococcus aureus has altered substrate specificity

© 2018 North, Wahlgren, Remus, Scalise, Kessans, Dunevall, Claesson, Soares da Costa, Perugini, Ramaswamy, Allison, Indiveri, Friemann and Dobson. Mammalian cell surfaces are decorated with complex glycoconjugates that terminate with negatively charged sialic acids. Commensal and pathogenic bacteria can use host-derived sialic acids for a competitive advantage, but require a functional sialic acid transporter to import the sugar into the cell. This work investigates the sodium sialic acid symporter (SiaT) from Staphylococcus aureus (SaSiaT). We demonstrate that SaSiaT rescues an Escherichia coli strain lacking its endogenous sialic acid transporter when grown on the sialic acids N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc). We then develop an expression, purification and detergent solubilization system for SaSiaT and demonstrate that the protein is largely monodisperse in solution with a stable monomeric oligomeric state. Binding studies reveal that SaSiaT has a higher affinity for Neu5Gc over Neu5Ac, which was unexpected and is not seen in another SiaT homolog. We develop a homology model and use comparative sequence analyses to identify substitutions in the substrate-binding site of SaSiaT that may explain the altered specificity. SaSiaT is shown to be electrogenic, and transport is dependent upon more than one Na+ion for every sialic acid molecule. A functional sialic acid transporter is essential for the uptake and utilization of sialic acid in a range of pathogenic bacteria, and developing new inhibitors that target these transporters is a valid mechanism for inhibiting bacterial growth. By demonstrating a route to functional recombinant SaSiaT, and developing the in vivo and in vitro assay systems, our work underpins the design of inhibitors to this transporter

Substrate-bound outward-open structure of a Na+-coupled sialic acid symporter reveals a new Na+ site

© 2018 The Author(s). Many pathogenic bacteria utilise sialic acids as an energy source or use them as an external coating to evade immune detection. As such, bacteria that colonise sialylated environments deploy specific transporters to mediate import of scavenged sialic acids. Here, we report a substrate-bound 1.95 Å resolution structure and subsequent characterisation of SiaT, a sialic acid transporter from Proteus mirabilis. SiaT is a secondary active transporter of the sodium solute symporter (SSS) family, which use Na+gradients to drive the uptake of extracellular substrates. SiaT adopts the LeuT-fold and is in an outward-open conformation in complex with the sialic acid N-acetylneuraminic acid and two Na+ions. One Na+binds to the conserved Na2 site, while the second Na+binds to a new position, termed Na3, which is conserved in many SSS family members. Functional and molecular dynamics studies validate the substrate-binding site and demonstrate that both Na+sites regulate N-acetylneuraminic acid transport

Structural basis of bacterial defense against g-type lysozyme-based innate immunity

Gram-negative bacteria can produce specific proteinaceous inhibitors to defend themselves against the lytic action of host lysozymes. So far, four different lysozyme inhibitor families have been identified. Here, we report the crystal structure of the Escherichia coli periplasmic lysozyme inhibitor of g-type lysozyme (PliG-Ec) in complex with Atlantic salmon g-type lysozyme (SalG) at a resolution of 0.95 Å, which is exceptionally high for a complex of two proteins. The structure reveals for the first time the mechanism of g-type lysozyme inhibition by the PliG family. The latter contains two specific conserved regions that are essential for its inhibitory activity. The inhibitory complex formation is based on a double 'key-lock' mechanism. The first key-lock element is formed by the insertion of two conserved PliG regions into the active site of the lysozyme. The second element is defined by a distinct pocket of PliG accommodating a lysozyme loop. Computational analysis indicates that this pocket represents a suitable site for small molecule binding, which opens an avenue for the development of novel antibacterial agents that suppress the inhibitory activity of PliG

Elucidation of a sialic acid metabolism pathway in mucus-foraging Ruminococcus gnavus unravels mechanisms of bacterial adaptation to the gut

N-acetylneuraminic acid (Neu5Ac), the most abundant sialic acid form in humans, is commonly found in a terminal location on colonic mucins glycans where it is a much-coveted source of nutrients for gut bacteria. The mucin-foraging strategy of the human gut symbiont Ruminococcus gnavus is associated with the expression of an intramolecular trans-sialidase (IT-sialidase) that targets and cleaves off terminal α2–3 -linked Neu5Ac from glycoproteins, releasing 2,7-anhydro-Neu5Ac instead of Neu5Ac. Using a combination of in silico, molecular, biochemical and structural approaches, we have unravelled a unique metabolic pathway leading to the transport and metabolism of 2,7-anhydro-Neu5Ac which is underpinned by the exquisite specificity of the sialic acid transporter. The substrate binding protein, which forms part of a sialic acid transporter (SAT2) in R. gnavus ATCC29149, is specific to 2,7-anhydro-Neu5Ac, as shown by fluorescence spectroscopy, isothermal titration calorimetry (ITC), and saturation transfer difference nuclear magnetic resonance spectroscopy (STD NMR). Once inside the cell, 2,7-anhydro-Neu5Ac is converted into Neu5Ac via a novel enzymatic reaction catalysed by an oxidoreductase, RgNanOx. Following this conversion, Neu5Ac is then catabolised into N-acetylmannosamine (ManNAc) and pyruvate via the action of a Neu5Ac specific aldolase that is structurally and biochemically typical of NanA-like enzymes, as shownby X-ray crystallography of RgNanA wild-type and site-directed active site mutant K167A in complex with Neu5Ac. We confirmed the importance of this metabolic pathway in vivo by generating a R. gnavus nan cluster deletion mutant that lost the ability to grow on sialylated substrates. We showed that in gnotobiotic mice colonised with R. gnavus wild-type and mutant strains, the fitness of the nan mutant was significantly impaired as compared to the wild-type strain with a reduced ability to colonise the mucus layer. Overall, our study revealed a novel sialic acid pathway in bacteria, which has significant implications for the spatial adaptation of mucin-foraging gut symbionts in health and disease