Allan M. Torres

D-amino acid residue in a defensin-like peptide from platypus venom: effect on structure and chromatographic properties.

The recent discovery that the natriuretic peptide OvCNPb (Ornithorhynchus venom C-type natriuretic peptide B) from platypus (Ornithorynchus anatinus) venom contains a D-amino acid residue suggested that other D-amino-acid-containing peptides might be present in the venom. In the present study, we show that DLP-2 (defensin-like peptide-2), a 42-amino-acid residue polypeptide in the platypus venom, also contains a D-amino acid residue, D-methionine, at position 2, while DLP-4, which has an identical amino acid sequence, has all amino acids in the L-form. These findings were supported further by the detection of isomerase activity in the platypus gland venom extract that converts DLP-4 into DLP-2. In the light of this new information, the tertiary structure of DLP-2 was recalculated using a new structural template with D-Met2. The structure of DLP-4 was also determined in order to evaluate the effect of a D-amino acid at position 2 on the structure and possibly to explain the large retention time difference observed for the two molecules in reverse-phase HPLC. The solution structures of the DLP-2 and DLP-4 are very similar to each other and to the earlier reported structure of DLP-2, which assumed that all amino acids were in the L-form. Our results suggest that the incorporation of the D-amino acid at position 2 has minimal effect on the overall fold in solution.

Defensins and the convergent evolution of platypus and reptile venom genes

When the platypus (Ornithorhynchus anatinus) was first discovered, it was thought to be a taxidermists hoax, as it has a blend of mammalian and reptilian features. It is a most remarkable mammal, not only because it lays eggs but also because it is venomous. Rather than delivering venom through a bite, as do snakes and shrews, male platypuses have venomous spurs on each hind leg. The platypus genome sequence provides a unique opportunity to unravel the evolutionary history of many of these interesting features. While searching the platypus genome for the sequences of antimicrobial defensin genes, we identified three Ornithorhynchus venom defensin-like peptide (OvDLP) genes, which produce the major components of platypus venom. We show that gene duplication and subsequent functional diversification of beta-defensins gave rise to these platypus OvDLPs. The OvDLP genes are located adjacent to the beta-defensins and share similar gene organization and peptide structures. Intriguingly, some species of snakes and lizards also produce venoms containing similar molecules called crotamines and crotamine-like peptides. This led us to trace the evolutionary origins of other components of platypus and reptile venom. Here we show that several venom components have evolved separately in the platypus and reptiles. Convergent evolution has repeatedly selected genes coding for proteins containing specific structural motifs as templates for venom molecules.

Solution structure of a defensin-like peptide from platypus venom.

Three defensin-like peptides (DLPs) were isolated from platypus venom and sequenced. One of these peptides, DLP-1, was synthesized chemically and its three-dimensional structure was determined using NMR spectroscopy. The main structural elements of this 42-residue peptide were an anti-parallel beta-sheet comprising residues 15-18 and 37-40 and a small 3(10) helix spanning residues 10-12. The overall three-dimensional fold is similar to that of beta-defensin-12, and similar to the sodium-channel neurotoxin ShI (Stichodactyla helianthus neurotoxin I). However, the side chains known to be functionally important in beta-defensin-12 and ShI are not conserved in DLP-1, suggesting that it has a different biological function. Consistent with this contention, we showed that DLP-1 possesses no anti-microbial properties and has no observable activity on rat dorsal-root-ganglion sodium-channel currents.

The HERG K+ channel: progress in understanding the molecular basis of its unusual gating kinetics

The HERG K+ channel has very unusual kinetic behaviour that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarisation as well as in preventing lethal ventricular arrhythmias. Extensive mutagenesis of the HERG K+ channel has allowed identification of which regions of the channel are important for the unusual kinetic behaviour of the channel. Furthermore, structural studies on scorpion toxins that potently inhibit HERG are beginning to provide clues as to the structural differences between HERG and other voltage-gated K+ channels.

Defensin-like peptide-2 from platypus venom: member of a class of peptides with a distinct structural fold.

The venom of the male Australian duck-billed platypus contains a family of four polypeptides of appox. 5 kDa, which are referred to as defensin-like peptides (DLPs). They are unique in that their amino acid sequences have no significant similarities to those of any known peptides; however, the tertiary structure of one of them, DLP-1, has recently been shown to be similar to beta-defensin-12 and to the sodium neurotoxin peptide ShI (Stichodactyla helianthus neurotoxin I). Although DLPs are the major peptides in the platypus venom, little is known about their biological roles. In this study, we determined the three-dimensional structure of DLP-2 by NMR spectroscopy, with the aim of gaining insights into the natural function of the DLPs in platypus venom. The DLP-2 structure was found to incorporate a short helix that spans residues 9-12, and an antiparallel beta-sheet defined by residues 15-18 and 37-40. The overall fold and cysteine-pairing pattern of DLP-2 were found to be similar to those of DLP-1, and hence beta-defensin-12; however, the sequence similarities between the three molecules are relatively small. The distinct structural fold of the DLP-1, DLP-2, and beta-defensin-12 is based upon several key residues that include six cysteines. DLP-3 and DLP-4 are also likely to be folded similarly since they have high sequence similarity with DLP-2. The DLPs, and beta-defensin-12 may thus be grouped together into a class of polypeptide molecules which have a common or very similar global fold. The fact that the DLPs did not display antimicrobial, myotoxic, or cell-growth-promoting activities implies that the nature of the side chains in this group of peptides is likely to play an important role in defining the biological function(s).

D-Amino acid residue in the C-type natriuretic peptide from the venom of the mammal, Ornithorhynchus anatinus, the Australian platypus.

The C‐type natriuretic peptide from the platypus venom (OvCNP) exists in two forms, OvCNPa and OvCNPb, whose amino acid sequences are identical. Through the use of nuclear magnetic resonance, mass spectrometry, and peptidase digestion studies, we discovered that OvCNPb incorporates a D‐amino acid at position 2 in the primary structure. Peptides containing a D‐amino acid have been found in lower forms of organism, but this report is the first for a D‐amino acid in a biologically active peptide from a mammal. The result implies the existence of a specific isomerase in the platypus that converts an L‐amino acid residue in the protein to the D‐configuration.

Identification of a novel family of proteins in snake Venoms: Purification and structural characterization of Nawaprin from Naja nigricollis snake venom

The three-dimensional structure of nawaprin has been determined by nuclear magnetic resonance spectroscopy. This 51-amino acid residue peptide was isolated from the venom of the spitting cobra, Naja nigricollis, and is the first member of a new family of snake venom proteins referred to as waprins. Nawaprin is relatively flat and disc-like in shape, characterized by a spiral backbone configuration that forms outer and inner circular segments. The two circular segments are held together by four disulfide bonds, three of which are clustered at the base of the molecule. The inner segment contains a short antiparallel β-sheet, whereas the outer segment is devoid of secondary structures except for a small turn or 310 helix. The structure of nawaprin is very similar to elafin, a human leukocyte elastase-specific inhibitor. Although substantial parts of the nawaprin molecule are well defined, the tips of the outer and inner circular segments, which are hypothesized to be critical for binding interactions, are apparently disordered, similar to that found in elafin. The amino acid residues in these important regions in nawaprin are different from those in elafin, suggesting that nawaprin is not an elastase-specific inhibitor and therefore has a different function in the snake venom.

Substrate Specificity of Platypus Venom L-to-D-Peptide Isomerase

The l-to-d-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote d-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, α-aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit l- or d-amino acid isomerization, but mutant peptides that contain residues with shorter, β-branched or long side chains with polar terminal groups, viz. Ala, α-aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for l- to d-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.

The pore domain outer helix contributes to both activation and inactivation of the HERG K+ channel

Ion flow in many voltage-gated K+ channels (VGK), including the (human ether-a-go-go-related gene) hERG channel, is regulated by reversible collapse of the selectivity filter. hERG channels, however, exhibit low sequence homology to other VGKs, particularly in the outer pore helix (S5) domain, and we hypothesize that this contributes to the unique activation and inactivation kinetics in hERG K+ channels that are so important for cardiac electrical activity. The S5 domain in hERG identified by NMR spectroscopy closely corresponded to the segment predicted by bioinformatics analysis of 676 members of the VGK superfamily. Mutations to approximately every third residue, from Phe551 to Trp563, affected steady state activation, whereas mutations to approximately every third residue on an adjacent face and spanning the entire S5 segment perturbed inactivation, suggesting that the whole span of S5 experiences a rearrangement associated with inactivation. We refined a homology model of the hERG pore domain using constraints from the mutagenesis data with residues affecting inactivation pointing in toward S6. In this model the three residues with maximum impact on activation (W563A, F559A, and F551A) face out toward the voltage sensor. In addition, the residues that when mutated to alanine, or from alanine to valine, that did not express (Ala561, His562, Ala565, Trp568, and Ile571), all point toward the pore helix and contribute to close hydrophobic packing in this region of the channel.

Mammalian l-to-d-amino-acid-residue isomerase from platypus venom

The presence of d‐amino‐acid‐containing polypeptides, defensin‐like peptide (DLP)‐2 and Ornithorhyncus venom C‐type natriuretic peptide (OvCNP)b, in platypus venom suggested the existence of a mammalian d‐amino‐acid‐residue isomerase(s) responsible for the modification of the all‐l‐amino acid precursors. We show here that this enzyme(s) is present in the venom gland extract and is responsible for the creation of DLP‐2 from DLP‐4 and OvCNPb from OvCNPa. The isomerisation reaction is freely reversible and under well defined laboratory conditions catalyses the interconversion of the DLPs to full equilibration. The isomerase is ∼50–60 kDa and is inhibited by methanol and the peptidase inhibitor amastatin. This is the first known l‐to‐d‐amino‐acid‐residue isomerase in a mammal.