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Local anesthetics are able to induce pain relief by binding to the sodium channel of excitable membranes, blocking the influx of sodium ions and the propagation of the nervous impulse. Benzocaine (BZC) is a local anesthetic whose low water-solubility limits its application to topical formulations. The present work focuses on the characterization of inclusion complexes of BZC in beta-cyclodextrin (beta-CD). Differential scanning calorimetry and electron microscopy gave evidences of the formation and the morphology of the complex. Fluorescence spectroscopy showed a BZC/beta-CD 1:1 stoichiometry. Phase-solubility diagrams allowed the determination of the association constants between BZC and beta-CD (549 M(-1)) and revealed that a three-fold increase in BZC solubility can be reached upon complexation with beta-CD. The details of BZC/beta-CD molecular interaction were analyzed by 1H 2D NMR allowing the proposition of an inclusion model for BZC into beta-CD where the aromatic ring of the anesthetic is located near the head of the beta-CD cavity. Moreover, in preliminary toxicity studies, the complex seems to be less toxic than BZC alone, since it induced a decrease in the in vitro oxidation of human hemoglobin. These results suggest that the BZC/beta-CD complex represents an effective novel formulation to enhance BZC solubility in water, turning it promising for use outside its traditional application, i.e., in infiltrative anesthesia.

Trypanosoma cruzi expresses a set of glycoproteins encoded by the gp85/trans-sialidase gene superfamily. In this report a structure model is proposed for a cloned member of the superfamily, the Tc85-11 protein. The structure consists of an N-terminus beta-propeller and a C-terminus beta-sandwich interconnected by an alpha-helix. The recombinant protein, corresponding to the N-domain (Tc85-N), binds to laminin in a selective manner. Six synthetic 20-mer peptides from the N-domain adhere onto the surface of LLC-MK(2) cells and two of these peptides specifically inhibit the Tc85-N/laminin interaction, indicating that they are the laminin-binding sites of the molecule. Thus, Tc85-11 and other related members of the family appear to be good candidates to play an important role in T. cruzi infection via a laminin mediated host-parasite interaction.

Fibroblast growth factors (FGFs) are regulatory proteins associated with a number of physiological and pathological states. On the basis of data suggesting a functional role for specific regions of human acidic FGF (aFGF), a linear peptide encompassing residues 99-108 (peptide1) and its cyclic analogue (peptide 2) were synthesized and their functional and structural features were investigated. While peptide 1 is inactive on Balb/c 3T3 fibroblasts, peptide 2 is mitogenic with ED(50) of approximately 50 microM. Moreover, peptide 1 is not able to inhibit the binding of human aFGF to cellular receptors whereas peptide 2 exhibits significant inhibitory activity. The NMR-derived solution conformers indicated the presence, only in peptide 2, of structural elements that we believe are related to its ability to emulate the biological activity of the native protein. These results suggest that the expression of mitogenic activity in short peptides, besides the presence of specific amino acids, requires the existence of stable structural features. In addition, they indicate that the introduction of chemical restraints in peptides can provide novel possibilities for the development of receptor agonists or antagonists.

Molecular modeling has allowed us to propose that one main contact surface of the Fibroblast Growth Factor Receptor -1 (FGFR-1) to the ligand FGF-1 is formed by a 16 amino acid sequence comprised by the C-terminal region of the domain II (DII) plus the hinge linking DII and DIII domains and the N-terminal region of domain III (DIII). Therefore, this sequence was used to design the following three peptides: Ac-YQLDVVERS-NH2 (R1); Ac-YQLDVVERSPHRPILQ-NH2 (R2) and Ac-RSPHRPILQ-NH2 (R3). The synthetic peptides were tested in their ability to inhibit the mitogenic activity of FGF-1 and FGF-2 in cultured Balb/c 3T3 fibroblasts. The results showed that R1 and R2 inhibited the activity of FGF-1 (ID50 = 40 -50 μM) but not that of FGF-2. Molecular modeling studies of R1 and its docking to FGF-1 suggested that this peptide could assume a conformation very similar to that found in the corresponding segment of FGFR-1. All these results support our hypothesis that the C-terminal residues of the DII domain, represented by peptide R1, are part of a surface responsible for the binding of FGF-1 to FGFR-1 but not of FGF-2. Also, they indicate that peptide R1 may be useful for the development of small selective peptide inhibitors of the FGF-1 biological activities.

α-KTx toxin Tc32, from the Amazonian scorpion Tityus cambridgei, lacks the dyad motif, including Lys27, characteristic of the family and generally associated with channel blockage. The toxin has been cloned and expressed for the first time. Electrophysiological experiments, by showing that the recombinant form blocks Kv1.3 channels of olfactory bulb periglomerular cells like the natural Tc32 toxin, when tested on the Kv1.3 channel of human T lymphocytes, confirmed it is in an active fold. The nuclear magnetic resonance-derived structure revealed it exhibits an α/β scaffold typical of the members of the α-KTx family. TdK2 and TdK3, all belonging to the same α-KTx 18 subfamily, share significant sequence identity with Tc32 but diverse selectivity and affinity for Kv1.3 and Kv1.1 channels. To gain insight into the structural features that may justify those differences, we used the recombinant Tc32 nuclear magnetic resonance-derived structure to model the other two toxins, for which no experimental structure is available. Their interaction with Kv1.3 and Kv1.1 has been investigated by means of docking simulations. The results suggest that differences in the electrostatic features of the toxins and channels, in their contact surfaces, and in their total dipole moment orientations govern the affinity and selectivity of toxins. In addition, we found that, regardless of whether the dyad motif is present, it is always a Lys side chain that physically blocks the channels, irrespective of its position in the toxin sequence.

The hemoglobins of three snake species: Liophis miliaris, Bothrops alternatus and Boa constrictor present a single ATP binding site per tetramer. The ATP association constant values for the deoxyhemoglobins at pH 7.5 were about KD ≅ 106 M−1 (107 M−1 for B. contrictor), three to four orders of magnitude higher than the respective values for oxyhemoglobin of about KO ≅ 102 M−1. The deoxyhemoglobin constant values markedly decrease as a function of pH, becoming, at pH 8.5, about KD ≅ 103 M−1 whereas for the oxyhemoglobin the constants remain of about the same, KO ≅ 102 M−1, at the pH range studied. The high ATP binding affinity constants, compared to those of human hemoglobin A, were explained from a molecular structural standpoint, considering L. miliaris hemoglobin, whose complete primary sequence is known. Two distinct amino acid residue differences were found in the β-chain, one being Trp (NA3) (more hydrophobic) in the snake hemoglobin which substitutes the Leu (NA3) in human hemoglobin, and the second being Val 101 β (G3) instead of Glu 101 β (G3). The substitutions could provide an un-neutralized, positively charged, residue Lys-104β and, taking into account its high pK value, the pH dependence of ATP binding affinity for the snake hemoglobin would originate from pH-dependent ionization of phosphate groups of the allosteric effector. The physiological implications of the high ATP binding constant, as well as the possible protective role of the nucleotide binding against the effect of high environmental temperatures on the oxygen dissociation curves, are discussed.

Eukaryotic MPN domain proteins are components of the complexes proteasome lid, COP9-signalosome (CSN), and translation initiation factor 3 (eIF3). The proteasome lid Rpn11 and COP9-signalosome Csn5 subunits, which contain the conserved JAMM motif involved in zinc ion coordination, show catalytic isopeptidase activity. Homology modeling indicates that the MPN domain of Mov34 cannot coordinate a zinc ion in the same manner as catalytically active MPN domains. In this work, we show that the MPN domain of Mov34 is highly resistant to proteolysis and the major product comprises residues 9–186, which includes the conserved MPN domain. Two clones containing the MPN domain region (MPN1–177 and MPN1–186) including the eight N-terminal residues show a less pronounced band in the 220 nm region of the CD, indicating lower α-helical content relative to the clones lacking these residues (MPN9–177 and MPN9–186). However, clones lacking residues 1–8 show lower expression levels and thermal stability, indicating that residues 1–8 are required for proper folding and stability of this particular MPN domain.

Trialysin is a pore-forming protein found in the saliva of Triatoma infestans (Hemiptera, Reduviidae), the insect vector of Chagas' disease. The protein is active against a broad range of cell types from bacteria to eukaryotic cells. Recognizing that the N-terminus of trialysin harbors the lytic motif [Amino, R., Martins, R. M., Procopio, J., Hirata, I. Y., Juliano, M. A., and Schenkman, S. (2002) J. Biol. Chem. 277, 6207-6213], we designed a set of peptides scanning this region to investigate the structural basis of its biological function. Peptides encompassing residues 1-32 (P6), 1-27 (P7), and 6-32 (P5) efficiently induced lysis of the protozoan parasite Trypanosoma cruzi and Escherichia coli in the 0.4-9.0 microM range, while much higher concentrations were required to cause hemolysis. Other more internal peptides, including peptide P2 (residues 21-47) and others up to residue 52, were less effective. P6 turned out to be the most active of all. P7 has a significantly higher activity than P5 against E. coli, while P5 has a hemolytic activity comparable to that of P6. CD spectroscopy showed that all tested peptides acquire a comparable helical content in solvent mixtures or in detergent micelles. The solution structure of P2 and P5-P7 was determined in a 30% trifluoroethanol/water mixture by nuclear magnetic resonance. All peptides exhibit a structure characterized by a central helical fold, and except for P2, which does not show a continuous hydrophobic surface, they are amphipathic. The structural models show that P5 and P7 extend their structural similarities with the most active peptide, P6, in either the C-terminus or the N-terminus. Amino acid substitutions in the N-terminus of P6 improved hemolysis but did not change the activity against T. cruzi. These results suggest that while amphipathicity is essential for the lytic activity, the selectivity of the active peptides for specific organisms appears to be associated with the structural features of their N- and C-termini.

The peptide pIV/S4-S5 encompasses the cytoplasmic linker between helices S4-S5 in domain IV of the voltage-gated Na+ channel, residues 1644-1664. The interaction of two local anesthetics (LA), lidocaine and benzocaine, with pIV/S4-S5 has been studied by DOSY, heteronuclear NMR 1H-15N-HSQC spectroscopy and computational methods. DOSY indicates that benzocaine, a neutral ester, exhibits stronger interaction with pIV/S4-S5 than lidocaine, a charged amine-amide. Weighted average chemical shifts, Deltadelta(1H-15N), show that benzocaine affects residues L1653, M1655 and S1656 while lidocaine slightly perturbs residues I1646, L1649 and A1659, L1660, near the N- and C-terminus, respectively. Computational methods confirmed the stability of the benzocaine binding and the existence of two binding sites for lidocaine. Even considering that the approach of studying the peptide in the presence of a co-solvent (TFE/H2O, 30%/70% v/v) has an inherently limited implication, our data strongly support the existence of multiple LA binding sites in the IV/S4-S5 linker, as suggested in the literature. In addition, we consider that LA can bind to the S4-S5 linker with diverse binding modes and strength since this linker is part of the receptor for the "inactivation gate particle". Conditions for devising new functional studies, aiming to better understand Na+ channel functionality as well as the various facets of LA pharmacological activity are proposed in this work.

Ki-1/57 is a 57-kDa cytoplasmic and nuclear protein associated with protein kinase activity and is hyper-phosphorylated on Ser/Thr residues upon cellular activation. In previous studies we identified the receptor of activated kinase-1 (RACK1), a signaling adaptor protein that binds activated PKC, as a protein that interacts with Ki-1/57. Here we demonstrate that the far-UV circular dichroism spectrum of the WD repeat-containing RACK1 protein shows an unusual positive ellipticity at 229 nm, which in other proteins of the WD family has been attributed to surface tryptophans that are quenchable by N-bromosuccinimide (NBS). As well as NBS, in vitro binding of 6xHis-Ki-1/57(122-413) and 6xHis-Ki-1/57(264-413) can also quench the positive ellipticity of the RACK1 spectrum. We generated a model of RACK1 by homology modeling using a G protein beta subunit as template. Our model suggests the family-typical seven-bladed beta-propeller, with an aromatic cluster around the central tunnel that contains four Trp residues (17, 83, 150, 170), which are likely involved in the interaction with Ki-1/57.

Hemoglobin is known to be a source of peptides involved in several functions. The peptide FLSFPTTKTYFPHFDLSHGSAQVKGHGAK (Hb33-61) is a proteolytic product of the bovine hemoglobin alpha-chain found in the gut content of the cattle tick, Boophilus microplus, and it possesses antimicrobial activity. Since in the past we showed that the amidated form of Hb33-61, Hb33-61a, is active against a few Gram-positive bacteria and fungi strains at micromolar concentration [Fogaca et al. (1999) J. Biol. Chem. 274, 25330-25334], we have been prompted to shed more light on its functional and structural features. Here we show that the peptide is able to disrupt the bacterial membrane ofMicrococcus luteus A270. As for its structure, it has a random conformation in water, and it does not interact with zwitterionic micelles. On the other hand, it binds to negatively charged micelles acquiring a finite structural organization. The 3D structure of Hb33-61a bound to SDS micelles exhibits a nonconventional conformation for an antimicrobial peptide. The backbone is characterized by the presence of a beta-turn in the N-terminus and by a beta-turn followed by a alpha-helical stretch in the C-terminus. A hinge, whose spatial organization is stabilized by side-chain-side-chain interactions, joins these two regions. Interestingly, it preserves structural features present in the corresponding segment of the bovine hemoglobin alpha-chain. Hb33-61a does not possess a well-defined amphipathic nature, and H/D exchange experiments show that while the C-terminal region is embedded in the SDS micelle, one face of the N-terminal half is partly exposed to the solvent.

Animal venoms are constantly being studied in search of new pharmacologically active molecules that can be used for therapeutic purposes. In this respect, scorpion venom is an incredible resource of molecules, most of which are peptides and small proteins characterized by a variety of biological activities. A good number of them are able to bind specifically to ion channels, producing mainly neurotoxic effects. On the other hand, it is worth remembering that from the point of view of scorpions, spiders, and snakes, these toxins are essential tools for their survival. As a result, they must be quite robust in order to reach their targets and exert their functions. In fact, they have to resist and overcome the defensive systems of the hosts such as, for instance, proteases and/or significant local pH variations that may result from inflammation states (Steen et al. 1996; Waldmann and Lasdunski 1998) induced by the animal bite itself. Potassium channels are widely spread in the living kingdom and are part of a large variety of biological processes. In addition, it is now well recognized that they are involved in an increasing number of human pathologies (Ashcroft 2000; Shieh et al. 2000). Thus, the perception that potassium channels are important therapeutic targets explains the constantly growing efforts to comprehend their three-dimensional structure and mechanism of action. Scorpion toxins selective for K+ channels (KTx) are short-chain peptides (23–43 amino acids) cross-linked by three or four disulfide bridges. They have been classified into α-, β-, and γ- toxins (Tytgat et al. 1999; Corona et al. 2002). The α-KTx family is the best studied to date. At first, it was divided into 12 subfamilies comprising a total of 49 members (Tytgat et al. 1999). More recently, six new subfamilies have been added (Batista et al. 2002; Goudet et al. 2002). Figure 1A ▶ shows the sequence alignment of a selected number of α-KTx toxins. Their global fold displays the same α/β scaffold formed by an α-helix and a two- or three-stranded β-sheet linked by two disulfide bridges (Tenenholz et al. 2000). This α/β fold, however, is shared by a variety of polypeptides with diverse functions, including toxins active on Na+ channels (Cohen et al. 2004), some antimicrobial peptides such as defensins (Bauer et al. 2001; Sawai et al. 2001), γ-thionins (Bloch et al. 1998), and antifungal proteins (Fant et al. 1998), thus suggesting that the α/β fold does not correlate directly with a specific target. Figure 1. (A) Sequence alignment of selected members of the α-KTx family carried out using CLUSTAL W (Thompson et al. 1994) followed by a manual adjustment. Identical and highly conserved residues are indicated by stars and by colons, respectively. (B) ... Overall, despite their similar architecture, α-KTx toxins exhibit distinct selectivity and affinity for a variety of K+ channels (Rodriguez de la Vega et al. 2003), thus posing interesting questions concerning the molecular factors that influence their diversified K+ channel blocker activity. α-KTx12.1 is a four disulfide-bridged neurotoxin (Fig. 1B ▶), first isolated from the venom of the Brazilian scorpion Tityus serrulatus. Due to the lack of sequence homology with any other toxin belonging to the α-KTx family, it has been considered the first member of the subfamily 12 (Tytgat et al. 1999). Originally, it was named TsTX-IV (Tityus toxin IV) (Arantes et al. 1989) and described as a 41-residue protein (Novello et al. 1999). However, according to the results reported here and in agreement with data recently published by another group (Pimenta et al. 2003), it turns out that the polypeptide sequence lacks the residue Asn41. Based on these findings, it follows that α-KTx12.1 and the 40-residue-long butantoxin (BuTX) originally isolated from the venom of Tityus serrulatus and subsequently from T. bahiensis and T. stigmurus (Holaday et al. 2000) are the same molecule. Finally, a peptide recently purified from the venom of the Argentinean scorpion Tityus trivittatus, despite presenting the same primary sequence as α-KTx12.1, has been classified as α-KTx12.2 because of the different geographic origin (Coronas et al. 2003). As for its biological activity, α-KTx12.1, at nM concentration, was shown to block the high-conductance Ca2+-activated K+ channels in Leydig cells (Novello et al. 1999). Furthermore, it was reported that α-KTx12.1, while it does not compete with 125I-apamin, a very specific ligand of the low-conductance activated K+ channels, is able to inhibit the binding of 125I-kaliotoxin to its receptor in rat brain synaptosomal preparations, with an IC50 of 46 nM, thus suggesting an affinity also for the Kv1.3 K+ channel (Pimenta et al. 2003). Finally, α-KTx12.2 was shown to block, reversibly, the Shaker B K+-channels, with a Kd of ~660 nM (Coronas et al. 2003). In summary, this toxin appears to be able to interact with a variety of K+ channels, exhibiting a variable affinity for each of them. Such behavior suggests that, despite the four disulfide bridges, the toxin may be characterized by a conformational flexibility that allows its structure to fit into the pore region of diverse K+ channels. The current models describing the functionality of toxins assume that the type and position of the amino acids exposed at the molecular surface are critical in determining the biological functions and binding properties of each particular toxin. In fact, these models suggest that the inhibition of the K+ conductance results from the ability of the toxin to occlude the pore of the channel at the extracellular opening. As for the binding process, it is described as a reversible, bimolecular reaction which is governed by electrostatic interactions between negatively charged residues in the channel and positively charged residues in the toxin. Consequently, the binding is expected to be affected by both the pH and the ionic strength of the external media (Ellis et al. 2001; Garcia et al. 2001). In this work, aiming to disclose how α-KTx12.1 succeeds to actively interact with various types of K+ channels and knowing that the binding is influenced by the structural features the toxin possesses just before interacting with the channel, we investigated the conformational properties of the toxin at that particular step. Recognizing that the toxin may experience environments where the pH varies significantly, and that the protein ionization state can affect its structural stability and flexibility (Fersht 1998; Kipping et al. 2001; Hu et al. 2003), we combined NMR spectroscopy and molecular dynamics (MD) simulations to probe the structural and dynamic features of α-KTx12.1 at both acidic and neutral pH.

Inflammatory peptides display different types of post-transcriptional modifications, such as C-terminal amidation, that alter their biological activity. Here we describe the structural and molecular dynamics features of the mast cell degranulating peptide, eumenine mastoparan-AF (EMP-AF-NH(2)), found in the venom of the solitary wasp, and of its carboxyl-free C-terminal form (EMP-AF-COO(-)) characterized by a reduced activity. Circular dichroism indicates that both peptides switch from a random coil conformation in water to a helical structure in TFE and SDS micelles. NMR data, in 30% TFE, reveal that the two peptides fold into an alpha-helix spanning most of their length, while they differ in terms of molecular rigidity. To understand the origins of the conformational flexibility observed in the case of EMP-AF-COO(-), a 5 ns MD simulation was carried out for each peptide, in an explicit water/TFE environment. The results show that the two peptides differ in an H-bond between Leu14 NH(2) and the backbone carbonyl of Ile11. The loss of that H-bond in EMP-AF-COO(-) leads to a significant modification of its structural dynamics. In fact, as evidenced by essential dynamics analysis, while EMP-AF-NH(2) exists mainly as a rigid structure, EMP-AF-COO(-) presents two helical stretches that fluctuate in some sort of independent fashion. We conclude that the diverse biological activity of the two peptides is not simply due to the reduction of the net positive charge, as generally suggested, but also to a structural perturbation of the amphipathic alpha-helix that affects their ability to perturb the cell membrane.

Linear synthetic peptides related to the human Fibroblast Growth Factor-1 (hFGF-1) segment [112-147] were tested for their capacity of mimicking FGF mitogenic activity, binding to heparin-Sepharose columns, stimulating DNA synthesis and competing with hFGF-1 for the cellular receptors. The results obtained indicated that the activity of these compounds is dependent on the presence of the sequence WFVGLK in their structures. The affinity for the cellular receptors increased when this sequence was elongated in order to incorporate amino acid residues that are important for FGF-heparin binding.