Reactivity and Function of Carboxyl Groups in Bacterial and Fungal Proteinases (Subtilases): Relation to X−-Ray Models

Carboxyl groups in five proteinases from microorganisms, the bacterial mesentericopeptidase, subtilisin DY and subtilisin Carlsberg as well as the fungal proteinase K and thermitase were modified with glycinamide in the presence of water soluble carbodiimide. Computer graphic studies using crystallographic models of the investigated proteinases showed a reasonably good correlation between the chemical reactivity of the carboxyl groups on one side and the degree of their exposure to the solvent or participation in ionic interactions, on the other. Differences in the binding of a large protein substrate by proteinases from microorganisms belonging to two subgroups are discussed

Structure of the bifunctional inhibitor of trypsin and α-amylase from ragi seeds at 2.2 Å resolution

The crystal structure of a bifunctional inhibitor of α-amylase and trypsin (RATI) from ragi seeds (Indian finger millet, Eleusine coracana Gaertneri) has been determined by X-ray diffraction at 2.2Å resolution. The inhibitor consists of 122 amino acids, with five disulfide bridges, and belongs to the plant α-amylase/trypsin inhibitor family. The crystals were grown by the microdialysis method using ammonium sulfate as a precipitating agent. The structure was determined by the molecular-replacement method using as models the structures of Corn Hageman factor inhibitor (CHFI) and of RATI at 2.9 Å resolution determined previously. It has been refined to an R factor of 21.9%. The structure shows an r.m.s. deviation for C atoms of 2.0 Åcompared with its own NMR structure, whereas the corresponding value compared with CHFI is found to be 1.4 Å. The r.m.s. difference for C atoms when compared with the same protein in the structure of the complex with α-amylase is 0.7 Å. The conformations of trypsin-binding loop and the -amylase-binding N-terminal region were also found to be similar in the crystal structures of native RATI and its complex with α-amylase. These regions differed considerably in the NMR structure

Regulation of catalytic function by molecular association: structure of phospholipase A<SUB>2</SUB> from Daboia russelli pulchella (DPLA<SUB>2</SUB>) at 1.9 &#197; resolution

The crystal structure of phospholipase A2 from the venom of Daboia russelli pulchella has been refined to an R factor of 0.216 using 17 922 reflections to 1.9 &#197; resolution. The structure contains two crystallographically independent molecules in the asymmetric unit. The overall conformations of the two molecules are essentially the same except for three regions, namely the calcium-binding loop including Trp31, the &#946;-wing and the C-terminal residues 119-131. Although these differences have apparently been caused by molecular packing, they seem to have functional relevance. Particularly noteworthy is the conformation of Trp31, which is favourable for substrate binding in one molecule as it is aligned with one of the side walls of the hydrophobic channel, whereas in the other molecule it is located at the mouth of the channel, thereby blocking the entry of substrates leading to loss of activity. This feature is unique to the present structure and does not occur in the dimers and trimers of other PLA2s

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

Crystallization and preliminary X-ray crystallographic analysis of the EGF receptor ectodomain

Crystallization of the hydrophilic ectodomain of the epidermal growth factor (EGF) receptor has been accomplished in the presence of the ligand EGF. Two different crystal forms have been obtained, one of which was suitable for X-ray analysis. The space group of this form has been assigned to P21212 with unit-cell dimensions of a = 207.4, b = 113.3 and c = 120.4 Å. A native data set has been recorded and a heavy-atom search is currently under way. Diffraction from these crystals, however, is limited to low resolution and extensive trials to improve crystal quality further have all failed. To analyse the molecular shape and aggregation of the receptor protein in solution, small-angle X-ray diffraction and dynamic light-scattering techniques have been applied. Synchrotron radiation in combination with cryo-techniques is essential for data collection because of the high solvent content and radiation sensitivity

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

Mercury induced modifications in the stereochemistry of the active site through Cys-73 in a serine protease - Crystal structure of the complex of a partially modified proteinase K with mercury at 1.8 Å resolution

298-302Proteinese K(PK) isolated from Tritirachium album Limber was crystallized with HgCl2 in excess. under microgravity conditions. The intensity data were collected at 4°C to 1.8 Ǻ resolution and the final R-factor after refinement for all the reflections was 0. 164. Mercury has been found at two sites with partial occupancies (0.4 and 0.6) which are at distances of 2.48 Ǻ and 2.58 Ǻ respectively from Cys-73 Sγ. The Cys-73 in the enzyme structure is located close to the active site residue. His-69. This region is completely buri ed and is not accessible to the solvent. It is rather tightly packed. Therefore. the binding of mercury distorts the stereochemistry of the neighbouring residues including those be longing to the catalytic tri ad. As a result of this. the Oγ of Ser-224 is displaced by 0.6 Ǻ which causes the inactivation of proteinase K by increasing the H-bond distance to 3.7 Ǻ between Ser-224 Oγ and His-69 Nε2