Crystal structure of echicetin from Echis carinatus (Indian Saw-scaled viper) at 2.4 Å resolution

Echicetin is a heterodimeric protein from the venom of the Indian saw-scaled viper, Echis carinatus. It binds to platelet glycoprotein Ib (GPIb) and thus inhibits platelet aggregation. It has two subunits, α and β, consisting of 131 and 123 amino acid residues, respectively. The two chains are linked with a disulphide bond. The level of amino acid sequence homology between two subunits is 50%. The protein was purified from the venom of E. carinatus and crystallized using ammonium sulphate as a precipitant. The crystal structure has been determined at 2.4 Å resolution and refined to an R-factor of 0.187. Overall dimensions of the heterodimer are ˜80 Å×35 Å×35 Å. The backbone folds of the two subunits are similar. The central portions of the polypeptide chains of a and β-subunits move into each other to form a tight dimeric association. The remaining portions of the chains of both subunits fold in a manner similar to those observed in the carbohydrate-binding domains of C-type lectins. In echicetin, the Ca2+-binding sites are not present, despite being topologically equivalent to other similar Ca2+-binding proteins of the superfamily. The residues Ser41, Glu43 and Glu47 in the calcium-binding proteins of the related family are conserved but the residues Glu126/120 are replaced by lysine at the corresponding sites in the α and β-subunits

Crystal structure of a proteolytically generated functional monoferric C-lobe of bovine lactoferrin at 1.9 Å resolution

This is the first crystal structure of a proteolytically generated functional C-lobe of lactoferrin. The purified samples of iron-saturated C-lobe were crystallized in 0.1 M Mes buffer (pH 6.5) containing 25% (v/v) polyethyleneglycol monomethyl ether 550 M and 0.1 M zinc sulphate heptahydrate. The X-ray intensity data were collected with 300 mm imaging plate scanner mounted on a rotating anode generator. The structure was determined by the molecular replacement method using the coordinates of the C-terminal half of bovine lactoferrin as a search model and refined to an R-factor of 0.193 for all data to 1.9 Å resolution. The final model comprises 2593 protein atoms (residues 342-676 and 681-685), 124 carbohydrate atoms (from ten monosaccharide units, in three glycan chains), one Fe3+, one CO32-, two Zn2+ and 230 water molecules. The overall folding of the C-lobe is essentially the same as that of C-terminal half of bovine lactoferrin but differs slightly in conformations of some of the loops and reveals a number of new interactions. There are 20 Cys residues in the C-lobe forming ten disulphide links. Out of these, one involving Cys481-Cys675 provides an inter-domain link at 2.01 Å while another Cys405-Cys684 is formed between the main C-lobe 342-676 and the hydrolyzed pentapeptide 681-685 fragment. Six inter-domain hydrogen bonds have been observed in the structure whereas only four were reported in the structure of intact lactoferrin, although domain orientations have been found similar in the two structures. The good quality of electron density has also revealed all the ten oligosaccharide units in the structure. The observation of two metal ions at sites other than the iron-binding cleft is another novel feature of the present structure. These zinc ions stabilize the crystal packing. This structure is also notable for extensive inter-molecular hydrogen bonding in the crystals. Therefore, the present structure appears to be one of the best packed crystal structures among the proteins of the transferrin superfamily

Structural basis of phospholipase A<SUB>2</SUB> inhibition for the synthesis of Prostaglandins by the plant alkaloid aristolochic acid from a 1.7 &#197; crystal structure

This is the first structural observation of a plant product showing high affinity for phospholipase A2 and regulating the synthesis of arachidonic acid, an intermediate in the production of prostaglandins. The crystal structure of a complex formed between Vipera russelli phospholipase A2 and a plant alkaloid aristolochic acid has been determined and refined to 1.7 &#197; resolution. The structure contains two crystallographically independent molecules of phospholipase A2 in the form of an asymmetric dimer with one molecule of aristolochic acid bound to one of them specifically. The most significant differences introduced by asymmetric molecular association in the structures of two molecules pertain to the conformations of their calcium binding loops, B-wings, and the C-terminal regions. These differences are associated with a unique conformational behavior of Trp31. Trp31 is located at the entrance of the characteristic hydrophobic channel which works as a passage to the active site residues in the enzyme. In the case of molecule A, Trp31 is found at the interface of two molecules and it forms a number of hydrophobic interactions with the residues of molecule B. Consequently, it is pulled outwardly, leaving the mouth of the hydrophobic channel wide open. On the other hand, Trp31 in molecule B is exposed to the surface and moves inwardly due to the polar environment on the molecular surface, thus narrowing the opening of the hydrophobic channel. As a result, the aristolochic acid is bound to molecule A only while the binding site of molecule B is empty. It is noteworthy that the most critical interactions in the binding of aristolochic acid are provided by its OH group which forms two hydrogen bonds, one each with His48 and Asp49

First structural evidence of a specific inhibition of phospholipase A<SUB>2</SUB> by &#945;-Tocopherol (Vitamin E) and its implications in inflammation: crystal structure of the complex formed between phospholipase A<SUB>2</SUB> and &#945;-Tocopherol at 1.8 &#197; resolution

This is the first structural evidence of &#945;-tocopherol (&#945;-TP) as a possible candidate against inflammation, as it inhibits phospholipase A2 specifically and effectively. The crystal structure of the complex formed between Vipera russelli phospholipase A2 and &#945;-tocopherol has been determined and refined to a resolution of 1.8 &#197;. The structure contains two molecules, A and B, of phospholipase A2 in the asymmetric unit, together with one &#945;-tocopherol molecule, which is bound specifically to one of them. The phospholipase A2 molecules interact extensively with each other in the crystalline state. The two molecules were found in a stable association in the solution state as well, thus indicating their inherent tendency to remain together as a structural unit, leading to significant functional implications. In the crystal structure, the most important difference between the conformations of two molecules as a result of their association pertains to the orientation of Trp31. It may be noted that Trp31 is located at the mouth of the hydrophobic channel that forms the binding domain of the enzyme. The values of torsion angles (&#966;, &#968;, &#967;1 and &#967;2) for both the backbone as well as for the side-chain of Trp31 in molecules A and B are -94&#176;, -30&#176;, -66&#176;, 116&#176; and -128&#176;, 170&#176; -63&#176;, -81&#176;, respectively. The conformation of Trp31 in molecule A is suitable for binding, while that in B hinders the passage of the ligand to the binding site. Consequently, &#945;-tocopherol is able to bind to molecule A only, while the binding site of molecule B contains three water molecules. In the complex, the aromatic moiety of &#945;-tocopherol is placed in the large space at the active site of the enzyme, while the long hydrophobic channel in the enzyme is filled by hydrocarbon chain of &#945; -tocopherol. The critical interactions between the enzyme and &#945;-tocopherol are generated between the hydroxyl group of the six-membered ring of &#945;-tocopherol and His48 N&#948;1 and Asp49 O&#948;1 as characteristic hydrogen bonds. The remaining part of &#945;-tocopherol interacts extensively with the residues of the hydrophobic channel of the enzyme, giving rise to a number of hydrophobic interactions, resulting in the formation of a stable complex

Crystal structure of the complex formed between a group I Phospholipase A2 and a naturally occurring fatty acid at 2.7 Å resolution

This is the first evidence of a naturally bound fatty acid to a group I Phospholipase A2 (PLA2) and also to a PLA2 with Asp 49. The fatty acid identified as n-tridecanoic acid is observed at the substrate recognition site of PLA2 hydrophobic channel. The complex was isolated from the venom of Bungarus caeruleus (Common Indian Krait). The primary sequence of the PLA2 was determined using the cDNA method. Three-dimensional structure has been solved by the molecular replacement method and refined using the CNS package to a final R factor of 19.8% for the data in the resolution range from 20.0 to 2.7 Å. The final refined model is comprised of 912 protein atoms, one sodium ion, one molecule of n-tridecanoic acid, and 60 water molecules. The sodium ion is located in the calcium-binding loop with a sevenfold coordination. A characteristic extra electron density was observed in the hydrophobic channel of the enzyme, into which a molecule of n-tridecanoic acid was clearly fitted. The MALDI–TOF measurements of the crystals had earlier indicated an increase in the molecular mass of PLA2 by 212 Da over the native PLA2. A major part of the ligand fits well in the binding pocket and interacts directly with His 48 and Asp 49. Although the overall structure of PLA2 in the present complex is similar to the native structure reported earlier, it differs significantly in the folding of its calcium-binding loop

Discovery and Preclinical Profiling of 3‑[4-(Morpholin-4-yl)‑7<i>H</i>‑pyrrolo[2,3‑<i>d</i>]pyrimidin-5-yl]benzonitrile (PF-06447475), a Highly Potent, Selective, Brain Penetrant, and in Vivo Active LRRK2 Kinase Inhibitor

Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinson’s disease (PD) by genome-wide association studies (GWAS). The most common LRRK2 mutation, G2019S, which is relatively rare in the total population, gives rise to increased kinase activity. As such, LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the discovery and optimization of a novel series of potent LRRK2 inhibitors, focusing on improving kinome selectivity using a surrogate crystallography approach. This resulted in the identification of <b>14</b> (PF-06447475), a highly potent, brain penetrant and selective LRRK2 inhibitor which has been further profiled in in vivo safety and pharmacodynamic studies