The Australian elapids are some of the most deadly snakes in the world. Their venoms contains a cocktail of chemicals including peptides and proteins with a broad range of biological activities including pre and post synaptic neurotoxins, anti and pro coagulants, cardiotoxins, cytotoxins, hemorrhagic toxins and myotoxins. To date, however, a systematic approach aimed at the annotation and characterization of all components of all Australian snake venoms has not been undertaken. Therefore, the overall aim of our “Venomics” group is to isolate and evaluate a large number of venom proteins from Australian snakes with a view to finding biologically active molecules that could ultimately have application in a therapeutic setting. A key component of the overall effort is the understanding of structure function relationships for many of these molecules. This thesis focuses on the structure determination and analysis of two proteins, textilinin-1 from the Australian Common Brown snake, Pseudonaja textilis, and ACII-4 a phospholipase from the King Brown snake, Pseudechis australis.
Textilinin-1 is a small protein of 59 amino acids. A BLAST analysis showed this molecule has a sequence identity of 45% to the well known Kunitz inhibitor, aprotinin (bovine pancreatic trypsin inhibitor). The most interesting property of textilinin-1 is that it has potent anti-fibrinolytic activity, most likely because it is a nM inhibitor of plasmin. For this reason, textilinin-1 is being considered as an alternative to aprotinin in surgery as an anti-bleeding agent. In this study, I determined the three-dimensional structure of recombinant textilinin-1 as the free inhibitor to 1.63 Å resolution, and its structure in complex with trypsin and microplasmin (the catalytic domain of plasmin) to 1.64 Å and 2.78 Å resolution, respectively. An unusual feature of the structure of free textilinin-1 is that its canonical loop, the region which predominantly interacts with and inhibits the protease, can adopt multiple conformations. In one snapshot taken from the crystal structure this loop is inverted such that the critical protease binding residue Val18 becomes partially buried.In complex with trypsin or microplasmin, textilinin-1 binds such that its P1 residue, Arg17, inserts into the specificity pocket.In these complexes the catalytic serine hydroxyl oxygen of the protease is in closer-than-van der Waals contact with the carbonyl carbon of the scissile peptide bond of textilinin-1. In the microplasmin complex, His603 adopts an altered non-catalytically competent position such that its side-chain has swiveled around its χ1 bond and out of its classical catalytic triad location. To compensate, a water molecule is observed bridging the serine and aspartate side-chains. The reason for this appears to be due to the close approach of Val18 from textilinin-1 forcing the histidine to move. An interesting observation from the trypsin-textilinin-1 complex when compared to the structure of trypsin in complex with aprotinin is that the relative docking angles of these two inhibitors differs by ∼25°.The reason for this difference is the variations in the sequences of the two inhibitors.At the P1' and P3' sites, two bulkier amino acids, valine and phenylalanine are observed in textilinin-1 as compared to alanine and isoleucine which are present in aprotinin. As a result the textilinin-1 molecule is unable to dock into trypsin in the same orientation as aprotinin.
ACII-4 has a number of important biological activities including its ability to increase prothrombin time in citrated plasma and to inhibit the conversion of prothrombin to thrombin.ACII-4 has also been shown to inhibit platelet aggregation by collagen, arachionic acid and ADP, and inhibit the conversion of factor X to factor Xa. As a first step to understanding structure-function relationships of this molecule its X-ray crystal structure has been determined to 1.56 Å resolution.ACII-4 has a characteristic PLA2 fold stabilized by seven disulfide bonds. In the crystal structure, a Ca2+ ion is observed bound to the polypeptide, coordinated to seven ligands Tyr28O, Gly30O, Gly32O, the side-chain oxygens of Asp49, and two water molecules. The active site consisting of the catalytic triad of His48, Asp92, and a water molecule are visible in the structure. A polyethylene glycol molecule from the crystallization buffer is positioned in the active site where the fatty acid substrate would be found.The side-chain of Trp31 is situated in the opening to the active site and forms a hydrophobic contact with a second polyethylene glycol molecule. Two possible dimer formations of ACII-4 can be deduced from the crystal lattice contacts. One is a hydrophilic interface with a buried surface of 1141 Å2 and the other is a hydrophobic interface with a buried surface of 686 Å2. The implications of the structures determined in this thesis for the functions of textilinin-1 and ACII-4 as venom components and as potential pharmaceuticals are discussed.