Ch. Betzel

First Structural Evidence of a Specific Inhibition of Phospholipase A2 by alpha-Tocopherol (Vitamin E) and its Implications in Inflammation: Crystal Structure of the Complex Formed Between Phospholipase A2 and alpha-Tocopherol at 1.8 A Resolution

This is the first structural evidence of alpha-tocopherol (alpha-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 alpha-tocopherol has been determined and refined to a resolution of 1.8 A. The structure contains two molecules, A and B, of phospholipase A2 in the asymmetric unit, together with one alpha-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 (phi, psi, chi(1) and chi(2)) for both the backbone as well as for the side-chain of Trp31 in molecules A and B are -94 degrees, -30 degrees, -66 degrees, 116 degrees and -128 degrees, 170 degrees, -63 degrees, -81 degrees, 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, alpha-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 alpha-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 alpha-tocopherol. The critical interactions between the enzyme and alpha-tocopherol are generated between the hydroxyl group of the six-membered ring of alpha-tocopherol and His48 N(delta1) and Asp49 O(delta1) as characteristic hydrogen bonds. The remaining part of alpha-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 vipoxin at 2.0 A: an example of regulation of a toxic function generated by molecular evolution.

Vipoxin is the main toxic component in the venom of the Bulgarian snake Vipera ammodytes meridionalis, the most toxic snake in Europe. Vipoxin is a complex between a toxic phospholipase A2 (PLA2) and a non‐toxic protein inhibitor. The structure is of genetic interest due to the high degree of sequence homology (62%) between the two functionally different components. The structure shows that the formation of the complex in vipoxin is significantly different to that seen in many known structures of phospholipases and contradicts the assumptions made in earlier studies. The modulation of PLA2 activity is of great pharmacological interest, and the present structure will be a model for structure‐based drug design.

Atomic structure of the Serratia marcescens endonuclease at 1.1 A resolution and the enzyme reaction mechanism.

The three-dimensional crystal structure of Serratia marcescens endonuclease has been refined at 1.1 A resolution to an R factor of 12.9% and an R(free) of 15.6% with the use of anisotropic temperature factors. The model contains 3694 non-H atoms, 715 water molecules, four sulfate ions and two Mg(2+)-binding sites at the active sites of the homodimeric protein. It is shown that the magnesium ion linked to the active-site Asn119 of each monomer is surrounded by five water molecules and shows an octahedral coordination geometry. The temperature factors for the bound Mg(2+) ions in the A and B subunits are 7.08 and 4.60 A(2), respectively, and the average temperature factors for the surrounding water molecules are 12.13 and 10.3 A(2), respectively. In comparison with earlier structures, alternative side-chain conformations are defined for 51 residues of the dimer, including the essential active-site residue Arg57. A plausible mechanism of enzyme function is proposed based on the high-resolution S. marcescens nuclease structure, the functional characteristics of the natural and mutational forms of the enzyme and consideration of its structural analogy with homing endo-nuclease I-PpoI.

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 alpha-amylase and trypsin (RATI) from ragi seeds (Indian finger millet, Eleusine coracana Gaertneri) has been determined by X-ray diffraction at 2.2 A resolution. The inhibitor consists of 122 amino acids, with five disulfide bridges, and belongs to the plant alpha-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 A resolution determined previously. It has been refined to an R factor of 21.9%. The structure shows an r.m.s. deviation for C(alpha) atoms of 2.0 A compared with its own NMR structure, whereas the corresponding value compared with CHFI is found to be 1.4 A. The r.m.s. difference for C(alpha) atoms when compared with the same protein in the structure of the complex with alpha-amylase is 0.7 A. The conformations of trypsin-binding loop and the alpha-amylase-binding N-terminal region were also found to be similar in the crystal structures of native RATI and its complex with alpha-amylase. These regions differed considerably in the NMR structure.

Design of specific peptide inhibitors of phospholipase A2: structure of a complex formed between Russell's viper phospholipase A2 and a designed peptide Leu-Ala-Ile-Tyr-Ser (LAIYS).

Phospholipase A(2) (EC 3.1.1.4) is a key enzyme of the cascade mechanism involved in the production of proinflammatory compounds known as eicosanoids. The binding of phospholipase A(2) to membrane surfaces and the hydrolysis of phospholipids are thought to involve the formation of a hydrophobic channel into which a single substrate molecule diffuses before cleavage. In order to regulate the production of proinflammatory compounds, a specific peptide inhibitor of PLA(2), Leu-Ala-Ile-Tyr-Ser, has been designed. Phospholipase A(2) from Daboia russelli pulchella (DPLA(2)) and peptide Leu-Ala-Ile-Tyr-Ser (LAIYS) have been co-crystallized. The structure of the complex has been determined and refined to 2.0 A resolution. The structure contains two crystallographically independent molecules of DPLA(2), with one molecule of peptide specifically bound to one of them. The overall conformations of the two molecules are essentially similar except in three regions; namely, the calcium-binding loop including Trp31 (residues 25-34), the beta-wing consisting of two antiparallel beta-strands (residues 74-85) and the C-terminal region (residues 119-133). Of these, the most striking difference pertains to the orientation of Trp31 in the two molecules. The conformation of Trp31 in molecule A was suitable to allow the binding of peptide LAIYS, while that in molecule B prevented the entry of the ligand into the hydrophobic channel. The structure of the complex clearly showed that the OH group of Tyr of the inhibitor formed hydrogen bonds with both His48 N(delta1) and Asp49 O(delta1), while O(gamma)H of Ser was involved in a hydrogen bond with Trp31. Other peptide backbone atoms interact with protein through water molecules, while Leu, Ala and Ile form strong hydrophobic interactions with the residues of the hydrophobic channel.

Regulation of catalytic function by molecular association: structure of phospholipase A2 from Daboia russelli pulchella (DPLA2) at 1.9 Å resolution

The crystal structure of phospholipase A(2) from the venom of Daboia russelli pulchella has been refined to an R factor of 0.216 using 17,922 reflections to 1.9 A 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 beta-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 PLA(2)s.

Crystallisation under microgravity of mistletoe lectin I from Viscum album with adenine monophosphate and the crystal structure at 1.9 Å resolution

The crystal structure of the ribosome-inactivating protein (RIP) mistletoe lectin I (ML-I) from Viscum album in complex with adenine has been refined to 1.9 A resolution. High quality crystals of the ML-I complex were obtained by the method of vapour diffusion using the high density protein crystal growth system (HDPCG) on the international space station, mission ISS 6A. Hexagonal crystals were grown during three months under microgravity conditions. Diffraction data to 1.9A were collected applying synchrotron radiation and cryo- techniques. The structure was refined subsequently to analyse the structure of ML-I and particularly the active site conformation, complexed by adenine that mimics the RNA substrate binding.

Three-dimensional structure of neurotoxin-1 from Naja naja oxiana venom at 1.9 A resolution.

Neurotoxin‐1 from Naja naja oxiana venom (NTX‐1) has been crystallized by vapor diffusion in sitting drops. The crystals have cell dimensions of a = 25.2 Å,b = 75.6 Å, c = 35.9 Å, and are in space group P212121. Three‐dimensional data to 1.9 Å have been recorded by a Syntex P21 automatic diffractometer. The atomic structure of the toxin has been determined by molecular replacement using the α‐cobratoxin (α‐CTX) as the search model. The position of 534 non‐hydrogen protein atoms have been determined. The model contains 65 water molecules. Refinement has led to an R‐factor of 19.3% at 1.9 Å resolution. The secondary and tertiary structures of NTX‐1 have been analyzed and a comparison with structure of the α‐CTX has been made.

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 A. 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.

High resolution structure of streptavidin in complex with a novel high affinity peptide tag mimicking the biotin binding motif

A novel peptide was designed which possesses nanomolar affinity of less than 20 nM for streptavidin. Therefore it was termed Nano‐tag and has been used as an affinity tag for recombinant proteins. The minimized version of the wild type Nano‐tag is a seven‐amino acid peptide with the sequence fMDVEAWL. The three‐dimensional structure of wild type streptavidin in complex with the minimized Nano‐tag was analyzed at atomic resolution of 1.15 Å and the details of the binding motif were investigated. The peptide recognizes the same pocket of streptavidin where the natural ligand biotin is bound, but the peptide requires significantly more space than biotin. Therefore the binding loop adopts an “open” conformation in order to release additional space for the peptide. The conformation of the bound Nano‐tag corresponds to a 310 helix. However, the analysis of the intermolecular interactions of the Nano‐tag with residues of the binding pocket of streptavidin reveals astonishing similarities to the biotin binding motif. In principle the three‐dimensional conformation of the Nano‐tag mimics the binding mode of biotin. Our results explain why the use of the Nano‐tag in fusion with recombinant proteins is restricted to their N‐terminus and we describe the special significance of the fMet residue for the high affinity binding mode. Proteins 2007.