Cryptic association of B7‐2 molecules and its implication for clustering

T‐cell co‐stimulation through CD28/CTLA4:B7‐1/B7‐2 axis is one of the extensively studied pathways that resulted in the discovery of several FDA‐approved drugs for autoimmunity and cancer. However, many aspects of the signaling mechanism remain elusive, including oligomeric association and clustering of B7‐2 on the cell surface. Here, we describe the structure of the IgV domain of B7‐2 and its cryptic association into 1D arrays that appear to represent the pre‐signaling state of B7‐2 on the cell membrane. Super‐resolution microscopy experiments on heterologous cells expressing B7‐2 and B7‐1 suggest, B7‐2 form relatively elongated and larger clusters compared to B7‐1. The sequence and structural comparison of other B7 family members, B7‐1:CTLA4 and B7‐2:CTLA‐4 complex structures, support our view that the observed B7‐2 1D zipper array is physiologically important. This observed 1D zipper‐like array also provides an explanation for its clustering, and upright orientation on the cell surface, and avoidance of spurious signaling.     

Structural basis of the anionic interface preference and kcat* activation of pancreatic phospholipase A2

Pancreatic phospholipase A(2) (PLA2) shows a strong preference for the binding to the anionic interface and a consequent allosteric activation. In this paper, we show that virtually all the preference is mediated through 3 (Lys-53, -56, and -120) of the 12 cationic residues of bovine pancreatic PLA2. The lysine-to-methionine substitution enhances the binding of the enzyme to the zwitterionic interface, and for the K53,56,120M triple mutant at the zwitterionic interface is comparable to that for the wild type (WT) at the anionic interface. In the isomorphous crystal structure, the backbone folding of K53,56M K120,121A and WT are virtually identical, yet a significant change in the side chains of certain residues, away from the site of substitution, mostly at the putative contact site with the interface (i-face), is discernible. Such reciprocity, also supported by the spectroscopic results for the free and bound forms of the enzyme, is expected because a distal structural change that perturbs the interfacial binding could also affect the i-face. The results show that lysine-to-methionine substitution induces a structural change that promotes the binding of PLA2 to the interface as well as the substrate binding to the enzyme at the interface. The kinetic results are consistent with a model in which the interfacial Michaelis complex exists in two forms, and the complex that undergoes the chemical step is formed by the charge compensation of Lys-53 and -56. Analysis of the incremental changes in the kinetic parameters shows that the charge compensation of Lys-53 and -56 contributes to the activation and that of Lys-120 contributes only to the structural change that promotes the stability of the Michaelis complex at the interface. The charge compensation effects on these three residues also account for the differences in the anionic interface preference of the evolutionarily divergent secreted PLA2.     

Structural Basis of CD160:HVEM Recognition

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Protein synthesis in a maize callus exposed to NaCl and mannitol

A maize (Zea mays, L) callus was exposed to media containing different levels of NaCl (0 to 3%) and mannitol (0 to 18.2%) for a period of 4 weeks, and the changes in growth and protein synthesis were determined. Cells are able to tolerate and grow in NaCl up to 1% (0.17 M) or mannitol up to 9.1% (0.5 M), but the relative overall growth rates are about 1/6 and 1/8 of the control, respectively. Protein synthesis, as assessed by pulselabeling of the cells with ³⁵S-methionine after exposure to the stress reagents at various times of incubation, suggests that the relative rates of amino acid uptake and its incorporation into proteins are inhibited as early as 4 h after exposure, and the extent of inhibition does not increase appreciably until after 1 week. Severe inhibition of uptake and protein synthesis results from prolonged exposures at growth-inhibitory concentrations of NaCl and mannitol. In general, the overall mean inhibition of cellular uptake and protein synthesis in the first 2-week period are approximately 50% and 35% for the NaCl (1%) and mannitol (7.3 %) treatments, respectively. No detectable differences are apparent in the abundant, steady state protein population as revealed by SDS-PAGE and on staining with Coomassie blue or silver, but random losses of individual proteins occur after 2 weeks at 2% and 3% NaCl and at 18.2% mannitol. Of the newly-synthesized proteins, discernible changes are found in 7 and 4 polypeptides in NaCl and mannitol treatments, respectively. Apparently three new proteins (74 kd, 28.5 kd, and 26.2 kd) are induced de novo under both treatments. Other proteins (39.5 kd, 39.0 kd, 30 kd, and 16.5 kd) show an increased or decreased level of synthesis. NaCl levels above 0.5% or mannitol levels above 3.6% do not after the pattern of newly-synthesized proteins. This altered expression of newly-made proteins in the maize callus occurs only after a week of exposure to salinity or osmotic stress and coincides with the cell growth phase.     

A structural basis for the role of nucleotide specifying residues in regulating the oligomerization of the Rv1625c adenylyl cyclase from M. tuberculosis

The Rv1625c Class III adenylyl cyclase from Mycobacterium tuberculosis is a homodimeric enzyme with two catalytic centers at the dimer interface, and shows sequence similarity with the mammalian adenylyl and guanylyl cyclases. Mutation of the substrate-specifying residues in the catalytic domain of Rv1625c, either independently or together, to those present in guanylyl cyclases not only failed to confer guanylyl cyclase activity to the protein, but also severely abrogated the adenylyl cyclase activity of the enzyme. Biochemical analysis revealed alterations in the behavior of the mutants on ion-exchange chromatography, indicating differences in the surface-exposed charge upon mutation of substrate-specifying residues. The mutant proteins showed alterations in oligomeric status as compared to the wild-type enzyme, and differing abilities to heterodimerize with the wild-type protein. The crystal structure of a mutant has been solved to a resolution of 2.7A. On the basis of the structure, and additional biochemical studies, we provide possible reasons for the altered properties of the mutant proteins, as well as highlight unique structural features of the Rv1625c adenylyl cyclase.     

Decoy strategies: the structure of TL1A:DcR3 complex

Decoy Receptor 3 (DcR3), a secreted member of the Tumor Necrosis Factor (TNF) receptor superfamily, neutralizes three different TNF ligands: FasL, LIGHT, and TL1A. Each of these ligands engages unique signaling receptors which direct distinct and critical immune responses. We report the crystal structures of the unliganded DcR3 ectodomain and its complex with TL1A, as well as complementary mutagenesis and biochemical studies. These analyses demonstrate that DcR3 interacts with invariant backbone and side-chain atoms in the membrane-proximal half of TL1A which supports recognition of its three distinct TNF ligands. Additional features serve as antideterminants that preclude interaction with other members of the TNF superfamily. This mode of interaction is unique among characterized TNF:TNFR family members and provides a mechanistic basis for the broadened specificity required to support the decoy function of DcR3, as well as for the rational manipulation of specificity and affinity of DcR3 and its ligands.