Computational design, synthesis, and evaluation of miniproteins as androgen receptor coactivator mimics.

Insertion of 3 to 4 mutations, based on in silico modelling, in a diverse set of natural miniproteins generates potent androgen receptor (AR) binders and a clear insight into the structure-activity relationship of such coactivator mimics concerning helix length.

Novel families of toxin-like peptides in insects and mammals: a computational approach.

Most animal toxins are short proteins that appear in venom and vary in sequence, structure and function. A common characteristic of many such toxins is their apparent structural stability. Sporadic instances of endogenous toxin-like proteins that function in non-venom context have been reported. We have utilized machine learning methodology, based on sequence-derived features and guided by the notion of structural stability, in order to conduct a large-scale search for toxin and toxin-like proteins. Application of the method to insect and mammalian sequences revealed novel families of toxin-like proteins. One of these proteins shows significant similarity to ion channel inhibitors that are expressed in cone snail and assassin bug venom, and is surprisingly expressed in the bee brain. A toxicity assay in which the protein was injected to fish induced a strong yet reversible paralytic effect. We suggest that the protein may function as an endogenous modulator of voltage-gated Ca(2+) channels. Additionally, we have identified a novel mammalian cluster of toxin-like proteins that are expressed in the testis. We suggest that these proteins might be involved in regulation of nicotinic acetylcholine receptors that affect the acrosome reaction and sperm motility. Finally, we highlight a possible evolutionary link between venom toxins and antibacterial proteins. We expect our methodology to enhance the discovery of additional novel protein families.

Characterization of the outer pore region of the apamin-sensitive Ca2+-activated K+ channel rSK2.

We have studied the interaction between the SK2 channel and different scorpion toxins in order to find similarity and differences to other K+ channels. Beside apamin, ScTX is a high affinity blocker of the SK2 channel, whereas CTX is unable to block current through SK2. In order to prove that the ScTX affinity can be explained by the character of the different residues in the outer pore of the SK channels we introduced point mutations that render SK2 K+ channel SK1 and SK3 like. Directed by the results of the toxin receptor on the ShakerK+ channel, we changed single amino acids of the SK2 K+ channel that should render it sensitive to other peptide toxins like CTX a blocker of the IK channel, or KTX a blocker of the voltage-dependent channel Kv1.1 and Kv1.3. Amino acids V342G, S344E, and G384D of SK2 were changed to amino acids known from ShakerK+ channel to improve Shaker K+ channel CTX sensitivity. Interestingly SK2 V342G became CTX sensitive with a Kd of 19 nM and was also KTX sensitive Kd=97 nM. SK2 S344E (KdCTX = 105 nM,KdKTX = 144 nM) and G348D (KdCTX = 31 nM,Kd KTX = 89 nM) became also CTX and KTX sensitive with a lower affinity. The mutant channels SK V342G, SK2 S344E and SK2 G348D showed reduced ScTX sensitivity (Kd = 6 nM,Kd = 48 nM, and Kd = 12 nM). Because the exchange of a single residue could create a new high affinity binding site for CTX and KTX we concluded that the outer vestibule around position V342, S344, and G348 of the SK2 K+ channel pore is very similar to those of voltage-gated K+ channels such as the Shaker K+ channel, Kv1.1 and Kv1.3 channels and also to the prokaryotic KcsA channel. From mutant cycle analysis of KTX position H34 and SK2 position V342G, S344E, and G348D we could deduce that KTX binds in a similar way to SK2 channel mutant pore than to the Kv1.1 pore. We have studied the interaction between the SK2 channel and different scorpion toxins in order to find similarity and differences to other K+ channels. Beside apamin, ScTX is a high affinity blocker of the SK2 channel, whereas CTX is unable to block current through SK2. In order to prove that the ScTX affinity can be explained by the character of the different residues in the outer pore of the SK channels we introduced point mutations that render SK2 K+ channel SK1 and SK3 like. Directed by the results of the toxin receptor on the ShakerK+ channel, we changed single amino acids of the SK2 K+ channel that should render it sensitive to other peptide toxins like CTX a blocker of the IK channel, or KTX a blocker of the voltage-dependent channel Kv1.1 and Kv1.3. Amino acids V342G, S344E, and G384D of SK2 were changed to amino acids known from ShakerK+ channel to improve Shaker K+ channel CTX sensitivity. Interestingly SK2 V342G became CTX sensitive with a Kd of 19 nM and was also KTX sensitive Kd = 97 nM. SK2 S344E (KdCTX = 105 nM,KdKTX = 144 nM) and G348D (KdCTX = 31 nM,Kd KTX = 89 nM) became also CTX and KTX sensitive with a lower affinity. The mutant channels SK V342G, SK2 S344E and SK2 G348D showed reduced ScTX sensitivity (Kd = 6 nM,Kd = 48 nM, and Kd = 12 nM). Because the exchange of a single residue could create a new high affinity binding site for CTX and KTX we concluded that the outer vestibule around position V342, S344, and G348 of the SK2 K+ channel pore is very similar to those of voltage-gated K+ channels such as the Shaker K+ channel, Kv1.1 and Kv1.3 channels and also to the prokaryotic KcsA channel. From mutant cycle analysis of KTX position H34 and SK2 position V342G, S344E, and G348D we could deduce that KTX binds in a similar way to SK2 channel mutant pore than to the Kv1.1 pore.

Deletion of a single amino acid changes the folding of an apamin hybrid sequence peptide to that of endothelin.

The solution conformations of a hybrid sequence peptide related to the bee venom peptide apamin have been determined using two-dimensional 1H-nmr. Apamin is an 18 amino acid peptide containing a C-terminal helix that is stabilized by two disulfide bonds. The deletion of one residue (K4) of the N-terminal "scaffold" region of the apamin sequence results in a helical peptide, but with a change in the pairing of cysteines to form the disulfide cross links. The new disulfide arrangement is analogous to that of the vasoconstrictor peptide endothelin. Two sets of nmr resonances were observed for the apamin-deletion (AD) peptide, due to cistrans isomerism at the A4-P5 peptide bond. The cis isomer of the AD peptide contains a tight turn in residues 3-6, which is required for formation of the alpha-helix in residues 7-15. Nuclear Overhauser effects observed for the trans AD peptide are not consistent with any single unique fold, indicating the presence of conformational averaging when the peptide adopts the trans form. Distance geometry calculations on the cis AD peptide reveal an alpha-helical structure that appears to be more like that of apamin than the crystal structure of human endothelin, despite the reversal of the disulfide pattern in the AD peptide from that of apamin to that of endothelin.

Recombinant and chemical derivatives of apamin. Implication of post-transcriptional C-terminal amidation of apamin in biological activity.

The use of the colicin A lysis protein to direct the extracellular release of a fusion protein from Escherichia coli was investigated as an approach for the preparation of recombinant animal toxins. Apamin, a bee venom neurotoxin, was used as the model toxin. It is reticulated by two disulfide bridges and interacts with small conductance Ca(2+)-activated K+ channels. Substantial amounts of free recombinant apamin were obtained by CNBr cleavage of the fusion protein [col-(1-171)-apa] and HPLC purification. It was recognized by conformation-dependent monoclonal antibodies with a K0.5 value close to that for natural apamin, indicating that folding was correct. In toxicity and binding experiments, the recombinant apamin displayed low activity. The recombinant and natural molecules differed by the amidation of the C-terminal histidine residue. Previous structure/activity relationship studies do not implicate this C-terminal residue in activity but the role of its amidation was not investigated. An apamin analog with a non-amidated C-terminal residue was then chemically synthesized. The biological properties of both recombinant and chemical molecules were determined. Amidation of the C-terminal alpha-carboxyl of apamin appears to be essential for full expression of its biological activity.

A genetic algorithm that seeks native states of peptides and proteins.

We describe a computer algorithm to predict native structures of proteins and peptides from their primary sequences, their known native radii of gyration, and their known disulfide bonding patterns, starting from random conformations. Proteins are represented as simplified real-space main chains with single-bead side chains. Nonlocal interactions are taken from structural database-derived statistical potentials, as in an earlier treatment. Local interactions are taken from simulations of (phi, psi) energy surfaces for each amino acid generated using the Biosym Discover program. Conformational searching is done by a genetic algorithm-based method. Reasonable structures are obtained for melittin (a 26-mer), avian pancreatic polypeptide inhibitor (a 36-mer), crambin (a 46-mer), apamin (an 18-mer), tachyplesin (a 17-mer), C-peptide of ribonuclease A (a 13-mer), and four different designed helical peptides. A hydrogen bond interaction was tested and found to be generally unnecessary for helical peptides, but it helps fold some sheet regions in these structures. For the few longer chains we tested, the method appears not to converge. In those cases, it appears to recover native-like secondary structures, but gets incorrect tertiary folds.

The precursors of the bee venom constituents apamin and MCD peptide are encoded by two genes in tandem which share the same 3'-exon.

From a cDNA library prepared from venom glands of worker bees, clones encoding the precursors of apamin and MCD peptide have been isolated. The cDNAs are similar at the 5'-ends and identical in their 3'-regions. Analysis of the corresponding genes has revealed the existence of six exons separated by introns rich in A + T. Starting from the 5'-end, these exons are arranged in the following order: three exons of the mast cell-degranulating (MCD) peptide precursor, two exons of the gene for the apamin precursor, and finally a 3'-exon present in both cDNAs. This suggests that the bulk of the apamin gene resides in the third intron of the MCD peptide gene. Using inverse polymerase chain reaction, a segment of genomic DNA upstream of the first exon of the MCD precursor gene was obtained. The sequence of this segment shows 81% identity to the DNA sequence preceding the first exon of the apamin gene and both contain a putative TATA box. We thus propose that the mRNA encoding the apamin precursor originates from a primary transcript which starts in the third intron of the MCD peptide gene. Both cDNAs encode unusually small precursors comprising only 46 amino acids in case of apamin and 50 in the case of the MCD peptide.