Gingipains are cysteine proteinases acting as key virulence factors of the bacterium Porphyromonas gingivalis, the major pathogen in periodontal disease. The 1.5 and 2.0 Å crystal structures of free and D-Phe-Phe-Arg-chloromethylketone-inhibited gingipain R reveal a 435-residue, single-polypeptide chain organized into a catalytic and an immunoglobulin-like domain. The catalytic domain is subdivided into two subdomains comprising four-and six-stranded β-sheets sandwiched by α-helices. Each subdomain bears topological similarities to the p20-p10 heterodimer of caspase-1. The second subdomain harbours the Cys-His catalytic diad and a nearby Glu arranged around the S1 specificity pocket, which carries an Asp residue to enforce preference for Arg-P1 residues. This gingipain R structure is an excellent template for the rational design of drugs with a potential to cure and prevent periodontitis. Here we show the binding mode of an arginine-containing inhibitor in the active-site, thus identifying major interaction sites defining a suitable pharmacophor.

Most known archaeal DNA polymerases belong to the type B family, which also includes the DNA replication polymerases of eukaryotes, but maintain high fidelity at extreme conditions. We describe here the 2.5 Å resolution crystal structure of a DNA polymerase from the Archaea Thermococcus gorgonarius and identify structural features of the fold and the active site that are likely responsible for its thermostable function. Comparison with the mesophilic B type DNA polymerase gp43 of the bacteriophage RB69 highlights thermophilic adaptations, which include the presence of two disulfide bonds and an enhanced electrostatic complementarity at the DNA-protein interface. In contrast to gp43, several loops in the exonuclease and thumb domains are more closely packed; this apparently blocks primer binding to the exonuclease active site. A physiological role of this ''closed'' conformation is unknown but may represent a polymerase mode, in contrast to an editing mode with an open exonuclease site. This archaeal B DNA polymerase structure provides a starting point for structure-based design of polymerases or ligands with applications in biotechnology and the development of antiviral or anticancer agents.Propagation of cells requires faithful DNA replication. This is performed in vivo by DNA polymerases (pols), which attach the appropriate dNTP to the nascent DNA primer strand to match its paired template. Different families of pols are involved in different DNA polymerization processes including not only DNA replication (1, 2) but also repair and recombination (3, 4), a heterogeneity also reflected by varying polypeptide structures and͞or subunit compositions (3, 5). Some pols complement polymerase activity with 3Ј 3 5Ј exonuclease activity (editing activity) and͞or 5Ј 3 3Ј ''structure-specific endonuclease'' activity, often located in separate structural domains on the same polypeptide chain (4-8).Crystal structures are available for most known polymerase families, including the A family DNA polymerases (9-14), pol ␤ (15-17), HIV reverse transcriptase (18)(19)(20), and recently, the B family pol gp43 from bacteriophage RB69 (21). All share a functional polymerase structure, which resembles a right hand built by the palm, fingers and thumb domains (see ref. 7 for review). Although the fingers and thumb domains are highly diverse among the different families, the palm domains, which contain the conserved catalytic aspartate residues, show a similar topology among all families except pol ␤. The polymerase nucleotidyl transfer was studied in detail for the A family polymerases, HIV reverse transcriptase, and pol ␤, and was shown to involve two metal ions (summarized in ref. 7).Considerably less is known for the family of type B pols, which are replicative enzymes in eukaryotes and most likely also Archaea (22,23). The structure of gp43 from bacteriophage RB69 (21) provided an excellent first insight into this family. In addition to the three polymerase domains, gp43 contains an 3Ј 3 5Ј exonuclease domain and an N-termin...

Human apolipoprotein D (ApoD) occurs in plasma associated with high density lipoprotein. Apart from the involvement in lipid metabolism, its binding activity for progesterone and arachidonic acid plays a role in cancer development and neurological diseases. The crystal structures of free ApoD and its complex with progesterone were determined at 1.8 Å resolution and reveal a lipocalin fold. The narrow, mainly uncharged pocket within the typical ␤-barrel accommodates progesterone with its acetyl side chain oriented toward the bottom. The cavity adopts essentially the same shape in the absence of progesterone and allows complexation of arachidonic acid as another cognate ligand. Three of the four extended loops at the open end of the ␤-barrel expose hydrophobic side chains, which is an unusual feature for lipocalins and probably effects association with the high density lipoprotein particle by mediating insertion into the lipid phase. This mechanism is in line with an unpaired Cys residue in the same surface region that can form a disulfide cross-link with apolipoprotein A-II.Human ApoD 2 (1, 2) is a glycoprotein of 169 amino acids, which was discovered in plasma (3) as an atypical lipoprotein. There, ApoD is peripherally associated with HDL via disulfide bond with ApoA-II (4), which itself forms an amphipathic ␣-helical belt that wraps around the lipid disc (5).ApoD was also isolated as a progesterone-binding protein abundant in mammary gross cystic disease fluid (6), and it was found as a monomeric protein in apocrine secretion, where it seems to bind odorants (7). ApoD mRNA is expressed in a variety of organs (8) as well as in certain human cancers. Indeed, ApoD has received attention as prognostic marker for various, often steroid-responsive tumors, including prostate cancer (9), breast carcinoma, and cutaneous malignant melanoma (10).In addition, ApoD is synthesized by astrocytes in the central nervous system (11), and there, it seems to be involved in arachidonic acid transport, metabolism, and signaling (12). Notably, ApoD plays a well documented pathophysiological role in several psychiatric disorders (12), especially in schizophrenia (13).ApoD has been prepared as a soluble recombinant protein via secretion into the periplasm of Escherichia coli, and its binding activity for progesterone and arachidonic acid, both with K D values around 1 M, was demonstrated (14). However, recombinant ApoD reveals a pronounced tendency to adsorb to surfaces and to form aggregates upon storage, which may be explained by hydrophobic surface properties that enable ApoD to interact with HDL and/or with lipid membranes in its physiological environment. Using systematic substitution of presumably exposed hydrophobic residues, we were able to construct a "solubilized" mutant of ApoD that retained native ligand binding function and appeared to be suitable for structural analysis (15). EXPERIMENTAL PROCEDURESProtein Production and Crystallization-An engineered version of ApoD was produced in E. coli via periplasmic secretion essent...

Protein flexibility and variable water structures are essential elements in protein-ligand interactions. Ligand design strategies that fail to take this into account may overlook or underestimate the potential of lead structures. Further, the significance of 'weak' interactions must be considered both in crystallographic refinement and in analysis of binding mechanisms.

Photolabeling of the benzodiazepine receptor, which to date has been done with benzodiazepine agonists such as flunitrazepam, can also be achieved with Ro 15-4513, a partial inverse agonist of the benzodiazepine receptor. [3H]Ro 15-4513 specifically and irreversibly labeled a protein with an apparent molecular weight of 51,000 (P51) in cerebellum and at least two proteins with apparent molecular weights of 51,000 (P51) and 55,000 (P55) in hippocampus. Photolabeling was inhibited by 10 microM diazepam but not by 10 microM Ro 5-4864. The BZ1 receptor-selective ligands CL 218872 and beta-carboline-3-carboxylate ethyl ester preferentially inhibited irreversible binding of [3H]Ro 15-4513 to protein P51. Not only these biochemical results but also the distribution and density of [3H]Ro 15-4513 binding sites in rat brain sections were similar to the findings with [3H]flunitrazepam. Thus, the binding sites for agonists and inverse agonists appear to be located on the same proteins. In contrast, whereas [3H]flunitrazepam is known to label only 25% of the benzodiazepine binding sites in brain membranes, all binding sites are photolabeled by [3H]Ro 15-4513. Thus, all benzodiazepine receptor sites are associated with photolabeled proteins with apparent molecular weights of 51,000 and/or 55,000. In cerebellum, an additional protein (MW 57,000) unrelated to the benzodiazepine receptor was labeled by [3H]Ro 15-4513 but not by [3H]flunitrazepam. In brain sections, this component contributed to higher labeling by [3H]Ro 15-4513 in the granular than the molecular layer.