Conotoxins and the posttranslational modification of secreted gene products.

The venoms of predatory cone snails (genus Conus) have yielded a complex library of about 50-100,000 bioactive peptides, each believed to have a specific physiological target (although peptides from different species may overlap in their target specificity). Conus has evolved the equivalent of a drug development strategy that combines the accelerated evolution of toxin sequences with an unprecedented degree of posttranslational modification. Some Conus venom peptide families are the most highly posttranslationally modified classes of gene products known. We review the variety and complexity of posttranslational modifications documented in Conus peptides so far, and explore the potential of Conus venom peptides as a model system for a more general understanding of which secreted gene products may have modified amino acids. Although the database of modified conotoxins is growing rapidly, there are far more questions raised than answers provided about possible mechanisms and functions of posttranslational modifications in Conus.

Detergent-assisted oxidative folding of delta-conotoxins.

Conotoxins comprise a diverse group of disulfide-rich peptides found in venoms of predatory Conus species. The native conformation of these peptides is marginally stable in comparison with alternative conformations, often resulting in low folding yields. The oxidative folding of hydrophobic delta-conotoxins was found to produce less than 1% of the native peptide [Bulaj, G. et al. (2001) Biochemistry 40, 13201]. In order to identify factors that might improve folding yields, we screened a number of additives including water-soluble polymers, detergents and osmolytes for their ability to increase steady-state accumulation of the native delta-conotoxin PVIA. The presence of a non-ionic detergent Tween and low temperature appeared to be the most effective factors in improving the oxidative folding. The detergent was also effective in promoting folding of other hydrophobic delta-conotoxins. Based on our findings, we discuss a possible mechanism for detergent-assisted folding and the general applicability of this mechanism to facilitating the proper folding of hydrophobic, cysteine-rich peptides.

Structure-function relationship of serine protease-protein inhibitor interaction.

We report our progress in understanding the structure-function relationship of the interaction between protein inhibitors and several serine proteases. Recently, we have determined high resolution solution structures of two inhibitors Apis mellifera chymotrypsin inhibitor-1 (AMCI-I) and Linum usitatissimum trypsin inhibitor (LUTI) in the free state and an ultra high resolution X-ray structure of BPTI. All three inhibitors, despite totally different scaffolds, contain a solvent exposed loop of similar conformation which is highly complementary to the enzyme active site. Isothermal calo- rimetry data show that the interaction between wild type BPTI and chymotrypsin is entropy driven and that the enthalpy component opposes complex formation. Our research is focused on extensive mutagenesis of the four positions from the protease binding loop of BPTI: P1, P1', P3, and P4. We mutated these residues to different amino acids and the variants were characterized by determination of the association constants, stability parameters and crystal structures of protease-inhibitor complexes. Accommodation of the P1 residue in the S1 pocket of four proteases: chymotrypsin, trypsin, neutrophil elastase and cathepsin G was probed with 18 P1 variants. High resolution X-ray structures of ten complexes between bovine trypsin and P1 variants of BPTI have been determined and compared with the cognate P1 Lys side chain. Mutations of the wild type Ala16 (P1') to larger side chains always caused a drop of the association constant. According to the crystal structure of the Leu16 BPTI-trypsin complex, introduction of the larger residue at the P1' position leads to steric conflicts in the vicinity of the mutation. Finally, mutations at the P4 site allowed an improvement of the association with several serine proteases involved in blood clotting. Conversely, introduction of Ser, Val, and Phe in place of Gly12 (P4) had invariably a destabilizing effect on the complex with these proteases.

Inhibition of six serine proteinases of the human coagulation system by mutants of bovine pancreatic trypsin inhibitor.

A series of 12 bovine pancreatic trypsin inhibitor variants mutated in the P(4) and P(3) positions of the canonical binding loop containing additional K15R and M52L mutations were used to probe the role of single amino acid substitutions on binding to bovine trypsin and to the following human proteinases involved in blood clotting: plasmin, plasma kallikrein, factors X(a) and XII(a), thrombin, and protein C. The mutants were expressed in Escherichia coli as fusion proteins with the LE1413 hydrophobic polypeptide and purified from inclusion bodies; these steps were followed by CNBr cleavage and oxidative refolding. The mutants inhibited the blood-clotting proteinases with association constants in the range of 10(3)-10(10) m(-)(1). Inhibition of plasma kallikrein, factors X(a) and XII(a), thrombin, and protein C could be improved by up to 2 orders of magnitude by the K15R substitution. The highest increase in the association constant for P(3) mutant was measured for factor XII(a); P13S substitution increased the K(a) value 58-fold. Several other substitutions at P(3) resulted in about 10-fold increase for factor X(a), thrombin, and protein C. The cumulative P(3) and P(1) effects on K(a) values for the strongest mutant compared with the wild type bovine pancreatic trypsin inhibitor were in the range of 2.2- (plasmin) to 4,000-fold (factors XII(a) and X(a)). The substitutions at the P(4) site always caused negative effects (a decrease in the range from over 1,000- to 1.3-fold) on binding to all studied enzymes, including trypsin. Thermal stability studies showed a very large decrease of the denaturation temperature (about 22 degrees C) for all P(4) mutants, suggesting that substitution of the wild type Gly-12 residue leads to a change in the binding loop conformation manifesting itself in non-optimal binding to the proteinase active site.

Inhibition of serine proteinases from human blood clotting system by squash inhibitor mutants.

A series of six CMTI I variants mutated in the P(2)-P(4)' region of the canonical binding loop were used to probe the role of single amino acid substitutions on binding to the following human proteinases involved in blood clotting: plasmin, plasma kallikrein, factors X(a) and XII(a). The mutants were expressed as fusion proteins with the LE1413 hydrophobic polypeptide in Escherichia coli, purified from inclusion bodies, followed by cyanobromide cleavage and refolding. The mutants inhibited the proteinases with the association constants in the range 10(3)-10(9) M(-1). Inhibition of plasma kallikrein and factors X(a) and XII(a) could be improved up to 30-fold by single mutations. In contrast, neither of the introduced mutations increased inhibitory properties of CMTI I against plasmin. Additionally, using two inhibitors of natural origin, CMTI I (P(1) Arg) and CPTI II (P(1) Lys), we determined the effect of Lys-->Arg on binding to four proteinases. With the exception of plasmin (no effect), P(1) Arg resulted in up to 30-fold stronger binding than P(1) Lys.

Interscaffolding additivity: binding of P1 variants of bovine pancreatic trypsin inhibitor to four serine proteases.

Different families of protein inhibitors of serine proteases share similar conformation of the enzyme-binding loop, while their scaffoldings are completely different. In the enzyme-inhibitor complex, the P1position of the loop makes numerous contacts within the S1pocket and significantly influences the energy of the interaction. Here, we determine the association energies (DeltaGavalues) for the interaction of coded P1variants of bovine pancreatic trypsin inhibitor (BPTI) with bovine beta-trypsin (BT), anionic salmon trypsin (AST), bovine alpha-chymotrypsin (BCHYM), and human neutrophil elastase (HNE). The respective DeltaGaranges are 15, 13, 9, and 8 kcal mol-1(1 cal=4.18 J). Next, through interscaffolding additivity cycles, we compare our set of DeltaGavalues determined for BCHYM and HNE with similar data sets available in the literature for three other inhibitor families. The analysis of the cycles shows that 27 to 83 % of cycles fulfil the criteria of additvity. In one particular case (comparison of associations of P1variants of BPTI and OMTKY3 with BCHYM) there is a structural basis for strongly non-additive behaviour. We argue that the interscaffolding additvity depends on sequential and conformational similarities of sites where the mutation(s) are introduced and on the particular substitution. In the second interscaffolding analysis, we compare binding of the same P1mutants to BT and AST. The high correlation coefficient shows that both trypsins recognize with comparable strength the non-cognate side-chains. However, the cognate Arg and Lys side-chains are recognized significantly more strongly by AST.