Electrically conductive pili from Geobacter species, termed bacterial nanowires, are intensely studied for their biological significance and potential in the development of new materials. Using cryo-electron microscopy, we have characterized nanowires from conductive G. sulfurreducens pili preparations that are composed solely of head-to-tail stacked monomers of the six-heme C-type cytochrome OmcS. The unique fold of OmcSclosely wrapped around a continuous stack of hemes that can serve as an uninterrupted path for electron transportgenerates a scaffold that supports the unbranched chain of hemes along the central axis of the filament. We present here, at 3.4 Å resolution, the structure of this cytochrome-based filament and discuss its possible role in long-range biological electron transport.

P2X receptors (P2XRs) are ligand-gated ion channels activated by extracellular ATP. Although the crystal structure of the zebrafish P2X4R has been solved, the exact mode of ATP binding and the conformational changes governing channel opening and desensitization remain unknown. Here, we used voltage clamp fluorometry to investigate movements in the cysteine-rich head domain of the rat P2X1R (A118-I125) that projects over the proposed ATP binding site. On substitution with cysteine residues, six of these residues (N120-I125) were specifically labeled by tetramethyl-rhodaminemaleimide and showed significant changes in the emission of the fluorescence probe on application of the agonists ATP and benzoylbenzoyl-ATP. Mutants N120C and G123C showed fast fluorescence decreases with similar kinetics as the current increases. In contrast, mutants P121C and I125C showed slow fluorescence increases that seemed to correlate with the current decline during desensitization. Mutant E122C showed a slow fluorescence increase and fast decrease with ATP and benzoyl-benzoyl-ATP, respectively. Application of the competitive antagonist 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) resulted in large fluorescence changes with the N120C, E122C, and G123C mutants and minor or no changes with the other mutants. Likewise, TNP-ATP-induced changes in control mutants distant from the proposed ATP binding site were comparably small or absent. Combined with molecular modeling studies, our data confirm the proposed ATP binding site and provide evidence that ATP orients in its binding site with the ribose moiety facing the solution. We also conclude that P2XR activation and desensitization involve movements of the cysteine-rich head domain. P 2X receptors (P2XRs) represent a family of nonselective cation channels gated by extracellular ATP. They are widely distributed in mammalian tissues and have been shown to be involved in diverse physiological functions (1). The seven known subunits all contain two transmembrane domains linked by a large extracellular loop. Functional receptors are homo-or heteromeric trimers (2, 3).Based on mutagenesis studies, it has been suggested that conserved positively charged and aromatic residues are crucial for ATP binding, presumably by interacting with the negatively charged phosphate chain of ATP (4-6) and its adenine ring (6), respectively. We have previously shown that replacement of two of these residues, K68 and F291, by cysteine residues allows disulfide cross-linking between neighboring P2X1 subunits and that this reaction is prevented in the presence of ATP. Based on these data, we concluded that the ATP binding sites are located at the subunit interfaces (7,8). This conclusion is in good agreement with the positions of the relevant amino acids in the crystal structure of the unliganded P2X4R from zebrafish (2). This zP2X4 structure revealed an ion channel architecture that resembles a dolphin, with the transmembrane helices and the extracellular region forming the fluke and the upper body, respectively. Att...

We describe the successful design of a tetrahedral His3Cys Zn(II)-binding site in a small protein of known structure: the B1 domain of Streptococcal protein G. The B1 variants containing the novel metal-binding site were characterized using a combination of optical absorption, circular dichroism and NMR spectroscopies. The results indicate that the designed proteins bind Zn(II) with high affinity and tetrahedral coordination geometry, and that the overall secondary and tertiary structure of the B1 domain is maintained.

Autologous T cells genetically modified with a chimeric antigen receptor (CAR) redirected at CD19 have potent activity in the treatment of B cell leukemia and B cell non-Hodgkin's lymphoma (B-NHL). Immunotherapies to treat multiple myeloma (MM) targeted the B cell maturation antigen (BCMA), which is expressed in most cases of MM. We developed a humanized CAR with specificity for BCMA based on our previously generated anti-BCMA monoclonal antibody. The targeting single-chain variable fragment (scFv) domain exhibited a binding affinity in the low nanomolar range, conferring T cells with high functional avidity. Redirecting T cells by this CAR allowed us to explore BCMA as an alternative target for mature B-NHLs. We validated BCMA expression in diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia. BCMA CAR T cells triggered target cell lysis with an activation threshold in the range of 100 BCMA molecules, which allowed for an efficient eradication of B-NHL cells in vitro and in vivo. Our data corroborate BCMA is a suitable target in B cell tumors beyond MM, providing a novel therapeutic option for patients where BCMA is expressed at low abundance or where anti-CD19 immunotherapies have failed due to antigen loss.

GTPases of immunity-associated proteins (GIMAPs) are regulators of lymphocyte survival and homeostasis. We previously determined the structural basis of GTP-dependent GIMAP2 scaffold formation on lipid droplets. To understand how its GTP hydrolysis is activated, we screened for other GIMAPs on lipid droplets and identified GIMAP7. In contrast to GIMAP2, GIMAP7 displayed dimerization-stimulated GTP hydrolysis. The crystal structure of GTP-bound GIMAP7 showed a homodimer that assembled via the G domains, with the helical extensions protruding in opposite directions. We identified a catalytic arginine that is supplied to the opposing monomer to stimulate GTP hydrolysis. GIMAP7 also stimulated GTP hydrolysis by GIMAP2 via an analogous mechanism. Finally, we found GIMAP2 and GIMAP7 expression differentially regulated in several human T cell lymphoma lines. Our findings suggest that GTPase activity in the GIMAP family is controlled by homo- and heterodimerization. This may have implications for the differential roles of some GIMAPs in lymphocyte survival.

Proteinase 3 is a serine protease found in neutrophil granules and on the extracellular neutrophil membrane (mPR3). mPR3 is a major antigen for anti-neutrophil cytoplasmic antibodies (PR3-ANCAs), autoantibodies causing fatal autoimmune diseases. In most individuals, a subpopulation of neutrophils also produce CD177, proposed to present additional PR3 on the surface, resulting in CD177neg/mPR3low and CD177pos/mPR3high neutrophil subsets. A positive correlation has been shown between mPR3 abundance, disease incidence, and clinical outcome. We present here a detailed investigation of the PR3:CD177 complex, verifying the interaction, demonstrating the effect of binding on PR3 proteolytic activity and explaining the accessibility of major PR3-ANCA epitopes. We observed high affinity PR3:CD177 complex formation by surface plasmon resonance. Using flow cytometry and a PR3-specific FRET assay, we found that CD177 binding reduced the proteolytic activity of PR3 in vitro using purified proteins, in neutrophil degranulation supernatants containing wtPR3 and directly on mPR3high neutrophils and PR3-loaded HEK cells. Finally, CD177pos/mPR3high neutrophils showed no migration advantage in vitro or in vivo when migrating from the blood into the oral cavity. We illuminate details of the PR3:CD177 interaction explaining mPR3 membrane orientation and proteolytic activity with relevance to ANCA activation of the distinct mPR3 neutrophil populations.

The intended effects of secondary ligand addition on metal affinity were observed in each mutant and demonstrated to be additive. Addition of metal also stabilized the protein's structure, partially offsetting the destabilizing effect of the mutations. These results represent a successful first attempt at designing an extended metal-binding site environment and illustrate the importance of including secondary interactions in the design of metal-binding sites.

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