Macromolecular X-Ray Crystallography

Calyxamines A (1) and B (2) are 2,2,4,6,6-pentasubstituted piperidine alkaloids possessing novel carbon skeletons isolated from the marine sponge Calyx podatypa collected in Puerto Rico. Their structures, after derivatization with trifluoroacetic acid, have been determined by a combination of X-ray and spectroscopic methods. A plausible biogenetic pathway to the calyxamines is suggested.

TIMP-3 (tissue inhibitor of metalloproteinases 3) is unique among the TIMP inhibitors, in that it effectively inhibits the TNF-α converting enzyme (TACE). In order to understand this selective capability of inhibition, we crystallized the complex formed by the catalytic domain of recombinant human TACE and the N-terminal domain of TIMP-3 (N-TIMP-3), and determined its molecular structure with X-ray data to 2.3 Å resolution. The structure reveals that TIMP-3 exhibits a fold similar to those of TIMP-1 and TIMP-2, and interacts through its functional binding edge, which consists of the N-terminal segment and other loops, with the active-site cleft of TACE in a manner similar to that of matrix metalloproteinases (MMPs). Therefore, the mechanism of TIMP-3 binding toward TACE is not fundamentally different from that previously elucidated for the MMPs. The Phe34 phenyl side chain situated at the tip of the relatively short sA-sB loop of TIMP-3 extends into a unique hydrophobic groove of the TACE surface, and two Leu residues in the adjacent sC-connector and sE-sF loops are tightly packed in the interface allowing favourable interactions, in agreement with predictions obtained by systematic mutations by Gillian Murphy's group. The combination of favourable functional epitopes together with a considerable flexibility renders TIMP-3 an efficient TACE inhibitor. This structure might provide means to design more efficient TIMP inhibitors of TACE.

Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes). They provide a fitness advantage in pathogenic strains and induce a strong pro-inflammatory response during bacteraemia. Curli formation requires a dedicated protein secretion machinery comprising the outer membrane lipoprotein CsgG and two soluble accessory proteins, CsgE and CsgF. Here we report the X-ray structure of Escherichia coli CsgG in a non-lipidated, soluble form as well as in its native membrane-extracted conformation. CsgG forms an oligomeric transport complex composed of nine anticodon-binding-domain-like units that give rise to a 36-stranded β-barrel that traverses the bilayer and is connected to a cage-like vestibule in the periplasm. The transmembrane and periplasmic domains are separated by a 0.9-nm channel constriction composed of three stacked concentric ...

Human monoclonal antibody (mAb) 447-52D neutralizes a broad spectrum of HIV-1 isolates, whereas murine mAb 0.5 , raised against gp120 of the X4 isolate HIV-1 IIIB , neutralizes this strain specifically. Two distinct gp120 V3 peptides, V3 MN and V3 IIIB , adopt alternative-hairpin conformations when bound to 447-52D and 0.5 , respectively, suggesting that the alternative conformations of this loop play a key role in determining the coreceptor specificity of HIV-1. To test this hypothesis and to better understand the molecular basis underlying an antibody's breadth of neutralization, the solution structure of the V3 IIIB peptide bound to 447-52D was determined by NMR. V3 IIIB and V3 MN peptides bound to 447-52D exhibited the same N-terminal strand conformation, while the V3 IIIB peptide revealed alternative N-terminal conformations when bound to 447-52D and 0.5. Comparison of the three known V3 structures leads to a model in which a 180°change in the orientation of the side chains and the resulting oneresidue shift in hydrogen bonding patterns in the N-terminal strand of the-hairpins markedly alter the topology of the surface that interacts with antibodies and that can potentially interact with the HIV-1 coreceptors. Predominant interactions of 447-52D with three conserved residues of the N-terminal side of the V3 loop, K312, I314, and I316, can account for its broad cross reactivity, whereas the predominant interactions of 0.5 with variable residues underlie its strain specificity. Human immunodeficiency virus type 1 (HIV-1) 1 utilizes two membrane-bound molecules to gain entry into cells: CD4 and one of several types of chemokine receptors. Most HIV strains can utilize either the receptor for CC chemokines, CCR5, or the receptor for CXC chemokines, CXCR4, and are thus termed R5-and X4-tropic viruses, respectively. A minority of HIV strains that use both of these receptors are termed dual-tropic (1). The third hypervariable region of envelope glycoprotein gp120 (V3 loop, residues 303-340) is directly involved in the binding of gp120 to the chemokine receptors (2, 3). Analysis of the HIV-1 genome demonstrated that the V3 loop contains major determinants responsible for the phenotype of the virus and its cell tropism and that the V3 sequence determines whether the virus binds to CCR5 or CXCR4 (4). Results of alanine-scanning mutagenesis led to the conclusion that Lys305, Ile307, Arg313, and Phe315 are involved in CCR5 utilization (5). [These residues are numbered Lys312, Ile314, Arg322, and Phe324 according to the numbering system used in this paper (6).] A single mutation in the V3 loop, D329R, transforms an R5 virus into an X4 virus (7). Exchange of the V3 loop of the IIIB strain (an X4 virus) with the V3 loop of an R5 virus creates an R5-like virus with infectivity that is inhibited by chemokines specific for CCR5 (8). Many HIV-1-neutralizing antibodies in infected individuals or in immunized animals are directed against the V3 loop, which was designated, accordingly, the "principal neutralizing determinant" (PND) of HIV-1 (9). HIV-neutralizing antibodies against V3 are thought to prevent the binding of gp120 to either CCR5 or CXCR4, thus abolishing viral fusion with its target cell (3, 10). HIV-1 has developed a number of highly effective mechanisms for evading the immune system and especially for escaping neutralization by anti-V3 antibodies (11, 12). Study of chimeric gp120s from R5 and X4 strains and sequence analyses revealed that neutral-† This study was supported by the NIH Grants GM 53329 (J.A.), AI36085 (S.Z.-P.), and HL 59725 (S.Z.-P.) and research funding from the U.S. Department of Veterans Affairs (S.Z.-P.). J.A. is the Dr. Joseph and Ruth Owades Professor of Chemistry. ‡ The coordinates have been deposited in the Protein Data Bank as entries 1U6U (average structure) and 1U6V (ensemble of low-energy structures).

Thiazolidinedione (TZD) compounds targeting the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) demonstrate unique benefits for the treatment of insulin resistance and type II diabetes. TZDs include rosiglitazone, pioglitazone and rivoglitazone, with the latter being the most potent. The TZDs are only marginally selective for the therapeutic target PPARγ as they also activate PPARα and PPARδ homologues to varying degrees, causing off-target effects. While crystal structures for TZD compounds in complex with PPARγ are available, minimal structural information is available for TZDs bound to PPARα and PPARδ. This paucity of structural information has hampered the determination of precise structural mechanisms involved in TZD
selectivity between PPARs. To help address these questions molecular dynamic simulations were performed of rosiglitazone, pioglitazone and rivoglitazone in complex with PPARα, PPARδ, and PPARγ in order to better understand the mechanisms of PPAR selectivity. The simulations revealed that TZD interactions with residues Tyr314 and Phe318 of PPARα and residues Phe291 and Thr253 of PPARδ as well as the omega loop, are key determinants of TZD receptor selectivity. Notably, in this study, we solve the first X-ray crystal structure of rivoglitazone bound to any PPAR. Rivoglitazone forms a unique hydrogen bond network with the residues of the PPARγ co-activator binding surface (known as AF2) and makes more extensive contacts with helix 3 and the β- sheet as compared to model TZD compounds such as rosiglitazone.

The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites ('catalophores'). As a proof-ofconcept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C-C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts.

The CD1 family is a large cluster of non-polymorphic, major histocompatibility complex (MHC) class-I-like molecules that bind distinct lipid-based antigens that are recognized by T cells. The most studied group of T cells that interact with lipid antigens are natural killer T (NKT) cells, which characteristically express a semi-invariant T-cell receptor (NKT TCR) that specifically recognizes the CD1 family member, CD1d. NKT-cell-mediated recognition of the CD1d-antigen complex has been implicated in microbial immunity, tumour immunity, autoimmunity and allergy. Here we describe the structure of a human NKT TCR in complex with CD1d bound to the potent NKT-cell agonist a-galactosylceramide, the archetypal CD1d-restricted glycolipid. In contrast to T-cell receptor-peptide-antigen-MHC complexes, the NKT TCR docked parallel to, and at the extreme end of the CD1d-binding cleft, which enables a lock-and-key type interaction with the lipid antigen. The structure provides a basis for the interaction between the highly conserved NKT TCR a-chain and the CD1d-antigen complex that is typified in innate immunity, and also indicates how variability of the NKT TCR b-chain can impact on recognition of other CD1d-antigen complexes. These findings provide direct insight into how a T-cell receptor recognizes a lipid-antigen-presenting molecule of the immune system.