Kaillathe Padmanabhan

An Ambiguous Structure of a DNA 15-mer Thrombin Complex

The structure of a complex between thrombin and a GGTTGGTGTGGTTGG DNA 15-mer has been analyzed crystallographically. The solution NMR structure of the 15-mer has two stacked G-quartets similar to that found in the previous X-ray structure determination of the 15-mer-thrombin complex [Padmanabhan, Padmanabhan, Ferrara, Sadler & Tulinsky (1993). J. Biol. Chem. 268, 17651-17654]; the strand polarity, however, is reversed from that of the crystallographic structure. The structure of the complex here has been redetermined with better diffraction data confirming the previous crystallographic structure but also indicating that the NMR solution structure fits equally well. Both 15-mer complex structures refined to an R value of about 0.16 presenting a disconcerting ambiguity. Since the two 15-mer structures associate with thrombin in different ways (through the TGT loop in the X-ray and TT loop in the NMR model), other independent lines of physical or chemical evidence are required to resolve the ambiguity.

High Resolution Structures of p-Aminobenzamidine- and Benzamidine-VIIa/Soluble Tissue Factor UNPREDICTED CONFORMATION OF THE 192-193 PEPTIDE BOND AND MAPPING OF Ca2+, Mg2+, Na+, AND Zn2+ SITES IN FACTOR VIIa

Factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like domains, and a protease domain. FVIIa binds seven Ca2+ ions in the Gla, one in the EGF1, and one in the protease domain. However, blood contains both Ca2+ and Mg2+, and the Ca2+ sites in FVIIa that could be specifically occupied by Mg2+ are unknown. Furthermore, FVIIa contains a Na+ and two Zn2+ sites, but ligands for these cations are undefined. We obtained p-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containing Ca2+, Mg2+, Na+, and Zn2+. The crystal diffracted to 1.8Å resolution, and the final structure has an R-factor of 19.8%. In this structure, the Gla domain has four Ca2+ and three bound Mg2+. The EGF1 domain contains one Ca2+ site, and the protease domain contains one Ca2+, one Na+, and two Zn2+ sites. 45Ca2+ binding in the presence/absence of Mg2+ to FVIIa, Gla-domainless FVIIa, and prothrombin fragment 1 supports the crystal data. Furthermore, unlike in other serine proteases, the amide N of Gly193 in FVIIa points away from the oxyanion hole in this structure. Importantly, the oxyanion hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87Å resolution. However, soaking benzamidine-FVIIa/sTF crystals with d-Phe-Pro-Arg-chloromethyl ketone results in benzamidine displacement, d-Phe-Pro-Arg incorporation, and oxyanion hole formation by a flip of the 192-193 peptide bond in FVIIa. Thus, it is the substrate and not the TF binding that induces oxyanion hole formation and functional active site geometry in FVIIa. Absence of oxyanion hole is unusual and has biologic implications for FVIIa macromolecular substrate specificity and catalysis.

Withanamides in Withania somnifera fruit protect PC‐12 cells from β‐amyloid responsible for Alzheimer's disease

Alzheimers disease (AD) is an irreversible neurodegenerative disorder with symptoms of confusion, memory loss, and mood swings. The β‐amyloid peptide, with 39–42 amino acid residues (BAP), plays a significant role in the development of AD. Although there is no cure for AD, it can be managed with available drugs to some degree. Several studies have revealed that natural antioxidants, such as vitamin E, vitamin C and β‐carotene, may help in scavenging free radicals generated during the initiation and progression of this disease. Therefore, there has been considerable interest in plant phytochemicals with antioxidant property as potential agents to prevent the progression of AD. Our earlier investigations of the Withania somnifera fruit afforded lipid peroxidation inhibitory withanamides that are more potent than the commercial antioxidants. In this study, we have tested two major withanamides A (WA) and C (WC) for their ability to protect the PC‐12 cells, rat neuronal cells, from β‐amyloid induced cell damage. The cell death caused by β‐amyloid was negated by withanamide treatment. Molecular modeling studies showed that withanamides A and C uniquely bind to the active motif of β‐amyloid (25–35) and suggest that withanamides have the ability to prevent the fibril formation. Further understanding of the mechanism of action and in vivo efficacy of these withanamides may facilitate its development as a prophylaxis. Copyright

Conformational rearrangements required of the V3 loop of HIV-1 gp120 for proteolytic cleavage and infection.

HIV gp120 is specifically cleaved at a single site in the V3 loop between Arg315 and Ala316 by thrombin. Previous observations by others have indicated that binding to CD4 enhances the rate of V3 loop cleavage, and that this cleavage is a prerequisite for HIV infection. Other observations also suggest that the cleavage site is in a type II β‐turn centered at Pro313‐Gly314. However, our docking experiments indicate that this conformation cannot dock to thrombin and other trypsin‐like serine proteases. Thus, based on the thrombin‐bound conformation of peptide substrates, we propose that CD4 binding, at a site remote from the V3 loop, induces and stabilizes a trans to cis isomerization of the highly conserved residue Pro313, and that this conformational shift is a prerequisite for cleavage by a ‘thrombin‐like’ cellular pro tease and subsequent infection.

Structure of the non-covalent complex of prothrombin kringle 2 with PPACK-thrombin.

Prothrombin fragment 2 (the second kringle) has been co-crystallized with PPACK (D-Phe-Pro-Arg)-thrombin and the structure of the non-covalent complex has been determined and refined (R = 0.16) at 3.2 A resolution using X-ray crystallographic methods. The kringles interact with thrombin at a site that has previously been proposed to be the heparin binding region. The latter is a highly electropositive surface near the C-terminal helix of thrombin abundant in arginine and lysine residues. These form salt bridges with acidic side chains of kringle 2. Somewhat unexpectedly, the negative groups of the kringle correspond to an enlarged anionic center of the lysine binding site of lysine binding kringles such as plasminogen K1 and K4 and TPA K2. The anionic motif is DGDEE in prothrombin kringle 2. The corresponding cationic center of the lysine binding site region has an unfavorable Arg71Phe substitution but Lys35 is conserved. However, the folding of fragment 2 is different from that of prothrombin kringle 1 and other kringles: the second outer loop possesses a distorted two-turn helix and the hairpin beta-turn of the second inner loop pivots at V64 and D70 by 60 degrees. The Lys35 is located on a turn of the helix, which causes it to project into solvent space in the fragment 2-thrombin complex, thereby devastating the cationic center of the lysine binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and Functional Studies of γ-Carboxyglutamic Acid Domains of Factor VIIa and Activated Protein C: Role of Magnesium at Physiological Calcium

Crystal structures of factor (F) VIIa/soluble tissue factor (TF), obtained under high Mg(2+) (50mM Mg(2+)/5mM Ca(2+)), have three of seven Ca(2+) sites in the γ-carboxyglutamic acid (Gla) domain replaced by Mg(2+) at positions 1, 4, and 7. We now report structures under low Mg(2+) (2.5mM Mg(2+)/5mM Ca(2+)) as well as under high Ca(2+) (5mM Mg(2+)/45 mM Ca(2+)). Under low Mg(2+), four Ca(2+) and three Mg(2+) occupy the same positions as in high-Mg(2+) structures. Conversely, under low Mg(2+), reexamination of the structure of Gla domain of activated Protein C (APC) complexed with soluble endothelial Protein C receptor (sEPCR) has position 4 occupied by Ca(2+) and positions 1 and 7 by Mg(2+). Nonetheless, in direct binding experiments, Mg(2+) replaced three Ca(2+) sites in the unliganded Protein C or APC. Further, the high-Ca(2+) condition was necessary to replace Mg4 in the FVIIa/soluble TF structure. In biological studies, Mg(2+) enhanced phospholipid binding to FVIIa and APC at physiological Ca(2+). Additionally, Mg(2+) potentiated phospholipid-dependent activations of FIX and FX by FVIIa/TF and inactivation of activated factor V by APC. Since APC and FVIIa bind to sEPCR involving similar interactions, we conclude that under the low-Mg(2+) condition, sEPCR binding to APC-Gla (or FVIIa-Gla) replaces Mg4 by Ca4 with an attendant conformational change in the Gla domain ω-loop. Moreover, since phospholipid and sEPCR bind to FVIIa or APC via the ω-loop, we predict that phospholipid binding also induces the functional Ca4 conformation in this loop. Cumulatively, the data illustrate that Mg(2+) and Ca(2+) act in concert to promote coagulation and anticoagulation.

Crystallization and preliminary diffraction data of a platelet-aggregation inhibitor from the venom of Agkistrodon piscivorus piscivorus (North American water moccasin).

Applaggin (Agkistrodon piscivorus piscivorus platelet-aggregation inhibitor) is a potent inhibitor of blood platelet aggregation derived from the venom of the North American water moccasin. The protein consists of 71 amino acids, is rich in cysteines, contains the sequence-recognition site of adhesion proteins at positions 50-52 (Arg-Gly-Asp) and shares high sequence homology with other snake-venom disintegrins such as echistatin, kistrin and trigramin. Single crystals of applaggin have been grown and X-ray diffraction data have been collected to a resolution of 3.2 A. The crystals belong to space group P4(1)2(1)2 (or its enantiomorph), with unit-cell dimensions a = b = 63.35, c = 74.18 A and two molecules per asymmetric unit. Molecular replacement using models constructed from the NMR structures of echistatin and kistrin has not been successful in producing a trial structure for applaggin.

Homology threading to generate RNA polymerase structures

Homology threading is a powerful technology for generating structural models based on homologous structures. Here we use threading to generate four complex RNA polymerase models. The models appear to be as useful as x-ray crystal structures or cryo-electron microscopy structures to support research projects.