The efficiency of HIV infection is greatly enhanced when the virus is delivered at conjugates between CD4 + T cells and virus-bearing antigen-presenting cells such as macrophages or dendritic cells via specialized structures known as virological synapses. Using ion abrasion SEM, electron tomography, and superresolution light microscopy, we have analyzed the spatial architecture of cell-cell contacts and distribution of HIV virions at virological synapses formed between mature dendritic cells and T cells. We demonstrate the striking envelopment of T cells by sheet-like membrane extensions derived from mature dendritic cells, resulting in a shielded region for formation of virological synapses. Within the synapse, filopodial extensions emanating from CD4 + T cells make contact with HIV virions sequestered deep within a 3D network of surface-accessible compartments in the dendritic cell. Viruses are detected at the membrane surfaces of both dendritic cells and T cells, but virions are not released passively at the synapse; instead, virus transfer requires the engagement of T-cell CD4 receptors. The relative seclusion of T cells from the extracellular milieu, the burial of the site of HIV transfer, and the receptor-dependent initiation of virion transfer by T cells highlight unique aspects of cell-cell HIV transmission.

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AcpA is a respiratory burst-inhibiting acid phosphatase from the Centers for Disease Control and Prevention Category A bioterrorism agent Francisella tularensis and prototype of a superfamily of acid phosphatases and phospholipases C. We report the 1.75-Å resolution crystal structure of AcpA complexed with the inhibitor orthovanadate, which is the first structure of any F. tularensis protein and the first for any member of this superfamily. The core domain is a twisted 8-stranded ␤-sheet flanked by three ␣-helices on either side, with the active site located above the carboxyl-terminal edge of the ␤-sheet. This architecture is unique among acid phosphatases and resembles that of alkaline phosphatase. Unexpectedly, the active site features a serine nucleophile and metal ion with octahedral coordination. Structure-based sequence analysis of the AcpA superfamily predicts that the hydroxyl nucleophile and metal center are also present in AcpA-like phospholipases C. These results imply a phospholipase C catalytic mechanism that is radically different from that of zinc metallophospholipases.

SummaryHistidine acid phosphatases catalyze the transfer of a phosphoryl group from phosphomonoesters to water at acidic pH using an active site histidine. The histidine acid phosphatase from the category A pathogen Francisella tularensis (FtHAP) has been implicated in intramacrophage survival and virulence, motivating interest in understanding the structure and mechanism of this enzyme. Here we report a structure-based study of ligand recognition by FtHAP. The 1.70 Å resolution structure of FtHAP complexed with the competitive inhibitor L(+)-tartrate was solved using single-wavelength anomalous phasing. Structures of the ligand-free enzyme and the complex with inorganic phosphate were determined at resolutions of 1.85 Å and 1.70 Å, respectively. To gain insight into substrate recognition, the structure of the Asp261Ala mutant enzyme complexed with the substrate 3′-AMP was determined at 1.50 Å resolution. FtHAP exhibits a two-domain fold similar to that of human prostatic acid phosphatase, consisting of an α/β core domain and a smaller domain that caps the core domain. The structures show that the core domain supplies the phosphoryl binding site, catalytic histidine (His17), and an aspartic acid residue (Asp261) that protonates the leaving group, while the cap domain contributes residues that enforce substrate preference. FtHAP and human prostatic acid phosphatase differ in the orientation of the crucial first helix of the cap domain, implying differences in the substrate preferences of the two enzymes. 3′-AMP binds in one end of a 15-Å long tunnel, with the adenine clamped between Phe23 and Tyr135, and the ribose 2′-hydroxyl interacting with Gln132. The importance of the clamp is confirmed with site-directed mutagenesis; mutation of Phe23 and Tyr135 individually to Ala increases K m by factors of seven and ten, respectively. The structural data are consistent with a role for FtHAP in scavenging phosphate from small molecules present in host macrophage cells.

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Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was ϳ31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3 and 5 nucleoside monophosphates at pH 6. Calculated K m s ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 mol of P i /s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class.Clostridium perfringens is a ubiquitous gram-positive, endospore-forming anaerobic bacterium. While found in soil, the organism is often a constituent of the intestinal flora and is capable of causing severe gastrointestinal and histotoxic infections in humans and animals. As a species, C. perfringens is a very heterogeneous group of organisms with respect to metabolic by-products produced in vitro and pathogenic potential (25). Although the literature is replete with descriptions of the immunological, bio-and physicochemical attributes of C. perfringens-encoded toxins, as well as molecular and morphological details of sporulation, detailed analysis of other constituent enzymes, including those involved in acquisition and regulation of inorganic phosphate, has received relatively little attention. This lack of information is notable. A growing cadre of investigators (1, 7) has recognized the clinical utility of acid phosphatase as a biochemical marker for the identification of C. perfringens isolated from water and other sources and its use in the differentiation of C. perfringens from ϳ95% of all currently recognized clostridial species in the genus (42).Acid phosphatases (EC 3.1.3.2) are ubiquitous and catalyze, at an acidic pH, the transfer of phosphoryl groups from phosphomonoesters to water (48). These enzymes play essential roles in the generation, acquisition, and mobilization of inorganic phosphate and critical roles in phosphoryl relay systems intimately involved in signal transduction pathways in both prokaryotes and eukaryotes. A subgroup of phosphatases has been designated nonspecific acid phosphatases (NSAPs) by Thaller and coworkers (45) and includes those bacterial polyspecific phosphatases that are secreted across the cytoplasmic membrane and exhibit optimum catalytic activity at an acidic pH (41). NSAPs are recognized as a distinct group of phosphatases and categorized into three classes (46). The class C enzyme...

Lipoprotein e (P4) from Haemophilus influenzae belongs to the "DDDD" superfamily of phosphohydrolases and is the prototype of class C nonspecific acid phosphatases. P4 is also a component of a H. influenzae vaccine. We report the crystal structures of recombinant P4 in the ligand-free and tungstate-inhibited forms, which are the first structures of a class C phosphatase. P4 has a two-domain architecture consisting of a core alpha/beta domain and a smaller alpha domain. The core domain features a five-stranded beta-sheet flanked by helices on both sides that is reminiscent of the haloacid dehalogenase superfamily. The alpha domain appears to be unique and plays roles in substrate binding and dimerization. The active site is solvent accessible and located in a cleft between the two domains. The structure shows that P4 is a metalloenzyme and that magnesium is the most likely metal ion in the crystalline recombinant enzyme. The ligands of the metal ion are the carboxyl groups of the first and third Asp residues of the DDDD motif, the backbone carbonyl of the second Asp of the DDDD motif, and two water molecules. The structure of the tungstate-bound enzyme suggests that Asp64 is the nucleophile that attacks the substrate P atom. Dimerization appears to be important for catalysis because intersubunit contacts stabilize the active site. Analysis of the structural context of mutations engineered for vaccine studies shows that the most promising mutations are located in the dimer interface. This observation suggests a structure-based vaccine design strategy in which the dimer interface is disrupted in order to expose epitopes that are buried in dimeric P4.

Cloning, expression, purification and crystallization studies of a recombinant class C acid phosphatase from the Category A pathogen Bacillus anthracis are reported. Large diffraction-quality crystals were grown in the presence of HEPES and Jeffamine ED-2001 at pH 7.0. The crystals belong to space group P2 1 2 1 2 1 , with unit-cell parameters a = 53.4, b = 90.1, c = 104.2 Å . The asymmetric unit is predicted to contain two protein molecules with a solvent content of 38%. Two native data sets were collected from the same crystal before and after flashannealing. The first data set had a mosaicity of 1.6 and a high-resolution limit of 1.8 Å . After flash-annealing, the apparent mosaicity decreased to 0.9 and the high-resolution limit of usable data increased to 1.6 Å . This crystal form is currently being used to determine the structure of B. anthracis class C acid phosphatase with experimental phasing techniques.