ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step of bacterial glycogen and plant starch biosynthesis, the most common carbon storage polysaccharides in nature. A major challenge is to understand how AGPase activity is regulated by metabolites in the energetic flux within the cell. Here we report crystal structures of the homotetrameric AGPase from Escherichia coli in complex with its physiological positive and negative allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP, and sucrose in the active site. FBP and AMP bind to partially overlapping sites located in a deep cleft between glycosyltransferase A-like and left-handed β helix domains of neighboring protomers, accounting for the fact that sensitivity to inhibition by AMP is modulated by the concentration of the activator FBP. We propose a model in which the energy reporters regulate EcAGPase catalytic activity by intra-protomer interactions and inter-protomer crosstalk, with a sensory motif and two regulatory loops playing a prominent role.

Bacterial capsules have evolved to be at the forefront of the cell envelope, making them an essential element of bacterial biology. Efforts to understand the Mycobacterium tuberculosis (Mtb) capsule began more than 60 years ago, but the relatively recent development of mycobacterial genetics combined with improved chemical and immunological tools have revealed a more refined view of capsule molecular composition. A glycogen-like α-glucan is the major constituent of the capsule, with lower amounts of arabinomannan and mannan, proteins and lipids. The major Mtb capsular components mediate interactions with phagocytes that favor bacterial survival. Vaccination approaches targeting the mycobacterial capsule have proven successful in controlling bacterial replication. Although the Mtb capsule is composed of polysaccharides of relatively low complexity, the concept of antigenic variability associated with this structure has been suggested by some studies. Understanding how Mtb shapes its envelope during its life cycle is key to developing anti-infective strategies targeting this structure at the host–pathogen interface.

The evolution of metabolic pathways is a major force behind natural selection. In the spotlight of such process lies the structural evolution of the enzymatic machinery responsible for the central energy metabolism. Specifically, glycogen metabolism has emerged to allow organisms to save available environmental surplus of carbon and energy, using dedicated glucose polymers as a storage compartment that can be mobilized at future demand. The origins of such adaptive advantage rely on the acquisition of an enzymatic system for the biosynthesis and degradation of glycogen, along with mechanisms to balance the assembly and disassembly rate of this polysaccharide, in order to store and recover glucose according to cell energy needs. The first step in the classical bacterial glycogen biosynthetic pathway is carried out by the adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase. This allosteric enzyme synthesizes ADP-glucose and acts as a point of regulation. The second step is carried out by the glycogen synthase, an enzyme that generates linear α-(1→4)-linked glucose chains, whereas the third step catalyzed by the branching enzyme produces α-(1→6)-linked glucan branches in the polymer. Two enzymes facilitate glycogen degradation: glycogen phosphorylase, which functions as an α-(1→4)-depolymerizing enzyme, and the debranching enzyme that catalyzes the removal of α-(1→6)-linked ramifications. In this work, we rationalize the structural basis of glycogen metabolism in bacteria to the light of the current knowledge. We describe and discuss the remarkable progress made in the understanding of the molecular mechanisms of substrate recognition and product release, allosteric regulation and catalysis of all those enzymes.

e Enterovirus 71 (EV71) is a picornavirus that causes outbreaks of hand, foot, and mouth disease (HFMD), primarily in the Asia-Pacific area. Unlike coxsackievirus A16, which also causes HFMD, EV71 induces severe neuropathology leading to high fatalities, especially among children under the age of 6 years. Currently, no established vaccines or treatments are available against EV71 infection. The monoclonal antibody MA28-7 neutralizes only specific strains of EV71 that have a conserved glycine at amino acid VP1-145, a surface-exposed residue that maps to the 5-fold vertex and that has been implicated in receptor binding. The cryo-electron microscopy structure of a complex between EV71 and the Fab fragment of MA28-7 shows that only one Fab fragment occupies each 5-fold vertex. A positively charged patch, which has also been implicated in receptor binding, lies within the Fab footprint. We identify the strain-specific epitope of EV71 and discuss the possible neutralization mechanisms of the antibody. E nterovirus 71 (EV71) infection causes outbreaks of hand, foot, and mouth disease (HFMD), predominantly in the Asia-Pacific region (1, 2). Whereas most EV71 infections are self-limiting, neurological and systemic complications can develop that range from aseptic meningitis to encephalitis and acute flaccid paralysis. Infection can lead to lethal pulmonary edema and heart failure (2), with mortality being especially high in young children under the age of 6 (2, 3). As seasonal outbreaks of HFMD are recurring around the world, development of a vaccine and antiviral therapies for EV71 has become an urgent concern.A member of the Picornaviridae family, EV71 has a nonenveloped, icosahedral capsid comprised of 60 copies of each of four viral structural proteins (VP1 to VP4) (4). Recent studies have solved the structures for three strains of EV71 (MY104 [5], Fuyang [6], and 1095 [7]), demonstrating that EV71 has the general features of picornavirus capsids, including the 5-fold "mesa" and the depression around the mesa called the "canyon" (5-8). Conserved residues VP1-242K and VP1-244K form positively charged patches on the 5-fold mesa (6), and this symmetry-related clustering of positive charges has been suggested as a common mechanism for heparan sulfate binding in enteroviruses (9).Several cellular receptors for EV71 have been identified: scavenger receptor B2 (SCARB2), P-selectin glycoprotein ligand-1 (PSGL-1), and heparan sulfate (HS) (10-12). SCARB2, which is expressed on a broad variety of cell types, likely binds to the virus canyon and induces the transition of the virion that is required for uncoating (13-15). PSGL-1, which is expressed exclusively on lymphocytes, binds only specific EV71 strains and supports viral replication in lymphocytes in a PSGL-1-dependent manner (11). According to recent studies, PSGL-1 and HS bind the positively charged patches on the 5-fold mesa of EV71 and provide initial attachment on the cell (12,16,17). We recently found that the PSGL-1 binding phenotype of EV71 strains is regulated b...

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Echovirus 7 (EV7) belongs to the Enterovirus genus within the family Picornaviridae. Many picornaviruses use IgG-like receptors that bind in the viral canyon and are required to initiate viral uncoating during infection. However, in addition, some of the enteroviruses use an alternative or additional receptor that binds outside the canyon. Decay-accelerating factor (DAF) has been identified as a cellular receptor for EV7. The crystal structure of EV7 has been determined to 3.1-Å resolution and used to interpret the 7.2-Å-resolution cryo-electron microscopy reconstruction of EV7 complexed with DAF. Each DAF binding site on EV7 is near a 2-fold icosahedral symmetry axis, which differs from the binding site of DAF on the surface of coxsackievirus B3, indicating that there are independent evolutionary processes by which DAF was selected as a picornavirus accessory receptor. This suggests that there is an advantage for these viruses to recognize DAF during the initial process of infection.

The biosynthesis of phospholipids and glycolipids are critical pathways for virtually all cell membranes. PatA is an essential membrane associated acyltransferase involved in the biosynthesis of mycobacterial phosphatidyl-myo-inositol mannosides (PIMs). The enzyme transfers a palmitoyl moiety from palmitoyl–CoA to the 6-position of the mannose ring linked to 2-position of inositol in PIM1/PIM2. We report here the crystal structures of PatA from Mycobacterium smegmatis in the presence of its naturally occurring acyl donor palmitate and a nonhydrolyzable palmitoyl–CoA analog. The structures reveal an α/β architecture, with the acyl chain deeply buried into a hydrophobic pocket that runs perpendicular to a long groove where the active site is located. Enzyme catalysis is mediated by an unprecedented charge relay system, which markedly diverges from the canonical HX4D motif. Our studies establish the mechanistic basis of substrate/membrane recognition and catalysis for an important family of acyltransferases, providing exciting possibilities for inhibitor design.

The coxsackievirus-adenovirus receptor (CAR) and decay-accelerating factor (DAF) have been identified as cellular receptors for coxsackievirus B3 (CVB3). The first described DAF-binding isolate was obtained during passage of the prototype strain, Nancy, on rhabdomyosarcoma (RD) cells, which express DAF but very little CAR. Here, the structure of the resulting variant, CVB3-RD, has been solved by X-ray crystallography to 2.74 Å, and a cryo-electron microscopy reconstruction of CVB3-RD complexed with DAF has been refined to 9.0 Å. This new high-resolution structure permits us to correct an error in our previous view of DAF-virus interactions, providing a new footprint of DAF that bridges two adjacent protomers. The contact sites between the virus and DAF clearly encompass CVB3-RD residues recently shown to be required for binding to DAF; these residues interact with DAF short consensus repeat 2 (SCR2), which is known to be essential for virus binding. Based on the new structure, the mode of the DAF interaction with CVB3 differs significantly from the mode reported previously for DAF binding to echoviruses. Coxsackieviruses are significant human pathogens that cause myocarditis, meningitis, and pancreatitis and have been implicated in the development of juvenile diabetes (58, 60-64). Virulence determinants have been described throughout the genome (19,20,30,51,65), including the P1 region, which encodes the structural proteins (7,10,13,25,48,49,56). The capsid surface presents a topology of structural motifs that largely dictate receptor recognition and usage, directly affecting tropism and pathogenicity.Group B coxsackieviruses (CVBs) belong to the genus Enterovirus of the family Picornaviridae. Picornaviruses are nonenveloped, positive-sense, single-stranded-RNA animal viruses with a capsid comprised of 60 protomers arranged to form an icosahedral shell ϳ300 Å in diameter with Tϭ1 (pseudo-Tϭ3) symmetry (ICTV classification) (8). In mature capsids, each protomer contains four structural proteins, VP-1, -2, -3, and -4. Structural studies have shown that capsids share common features, including a depression around the icosahedral 5-fold symmetry axes (called the "canyon") and a hydrophobic cavity located underneath the floor of the canyon (called the "pocket") (52). Biochemical and structural evidence indicates that the ligand within the pocket is a fatty acid (26, 55). For many picornaviruses, a receptor binds into the canyon and dislodges this "pocket factor," initiating conformational changes that lead to the formation of "A particles" and the subsequent uncoating of the virion (1, 12, 33, 39, 66). The major CVB receptor, the coxsackievirus-adenovirus receptor (CAR), binds within the CVB3 canyon (37) and causes the formation of A particles (16,35).A number of CVB isolates bind a second receptor, decayaccelerating factor (DAF) (CD55), a molecule that also serves as a receptor for many other enteroviruses (2,3,18,23,24,43,45). DAF, which is expressed on virtually all cell surfaces, acts to protect cells from lysis ...