Electrostatic and steric contributions to block of the skeletal muscle sodium channel by mu-conotoxin.

Pore-blocking toxins are valuable probes of ion channels that underlie electrical signaling. To be effective inhibitors, they must show high affinity and specificity and prevent ion conduction. The 22-residue sea snail peptide, mu-conotoxin GIIIA, blocks the skeletal muscle sodium channel completely. Partially blocking peptides, derived by making single or paired amino acid substitutions in mu-conotoxin GIIIA, allow a novel analysis of blocking mechanisms. Replacement of one critical residue (Arg-13) yielded peptides that only partially blocked single-channel current. These derivatives, and others with simultaneous substitution of a second residue, were used to elucidate the structural basis of the toxin's blocking action. The charge at residue-13 was the most striking determinant. A positive charge was necessary, though not sufficient, for complete block. Blocking efficacy increased with increasing residue-13 side chain size, regardless of charge, suggesting a steric contribution to inhibition. Charges grouped on one side of the toxin molecule at positions 2, 12, and 14 had a weaker influence, whereas residue-16, on the opposite face of the toxin, was more influential. Most directly interpreted, the data suggest that one side of the toxin is masked by close apposition to a binding surface on the pore, whereas the other side, bearing Lys-16, is exposed to an aqueous cavity accessible to entering ions. Strong charge-dependent effects emanate from this toxin surface. In the native toxin, Arg-13 probably presents a strategically placed electrostatic barrier rather than effecting a complete steric occlusion of the pore. This differs from other well-described channel inhibitors such as the charybdotoxin family of potassium channel blockers and the sodium channel-blocking guanidinium toxins (tetrodotoxin and saxitoxin), which appear to occlude the narrow part of the pore.     

A mu-conotoxin-insensitive Na+ channel mutant: possible localization of a binding site at the outer vestibule.

We describe a mutation in the outer vestibule region of the adult rat skeletal muscle voltage-gated Na+ channel (microliter) that dramatically alters binding of mu-conotoxin GIIIA (mu-CTX). Mutating the glutamate at position 758 to glutamine (E758Q) decreased mu-CTX binding affinity by 48-fold. Because the mutant channel showed both low tetrodotoxin (TTX) and mu-CTX affinities, these results suggested that mu-CTX bound to the outer vestibule and implied that the TTX- and mu-CTX-binding sites partially overlapped in this region. The mutation decreased the association rate of the toxin with little effect on the dissociation rate, suggesting that Glu-758 could be involved in electrostatic guidance of mu-CTX to its binding site. We propose a mechanism for mu-CTX block of the Na+ channel based on the analogy with saxitoxin (STX) and TTX, on the requirement of mu-CTX to have an arginine in position 13 to occlude the channel, and on this experimental result suggesting that mu-CTX binds in the outer vestibule. In this model, the guanidinium group of Arg-13 of the toxin interacts with two carboxyls known to be important for selectivity (Asp-400 and Glu-755), with the association rate of the toxin increased by interaction with Glu-758 of the channel.     

Multiple, distributed interactions of μ-conotoxin PIIIA associated with broad targeting among voltage-gated sodium channels

The first μ-conotoxin studied, μCTX GIIIA, preferentially blocked voltage-gated skeletal muscle sodium channels, Na(v)1.4, while μCTX PIIIA was the first to show significant blocking action against neuronal voltage-gated sodium channels. PIIIA shares >60% sequence identity with the well-studied GIIIA, and both toxins preferentially block the skeletal muscle sodium channel isoform. Two important features of blocking by wild-type GIIIA are the toxin's high binding affinity and the completeness of block of a single channel by a bound toxin molecule. With GIIIA, neutral replacement of the critical residue, Arg-13, allows a residual single-channel current (~30% of the unblocked, unitary amplitude) when the mutant toxin is bound to the channel and reduces the binding affinity of the toxin for Na(v)1.4 (~100-fold) [Becker, S., et al. (1992) Biochemistry 31, 8229-8238]. The homologous residue in PIIIA, Arg-14, is also essential for completeness of block but less important in the toxin's binding affinity (~55% residual current and ~11-fold decrease in affinity when substituted with alanine or glutamine). The weakened dominance of this key arginine in PIIIA is also seen in the fact that there is not just one (R13 in GIIIA) but three basic residues (R12, R14, and K17) for which individual neutral replacement enables a substantial residual current through the bound channel. We suggest that, despite a high degree of sequence conservation between GIIIA and PIIIA, the weaker dependence of PIIIA's action on its key arginine and the presence of a nonconserved histidine near the C-terminus may contribute to the greater promiscuity of its interactions with different sodium channel isoforms.     

mu-conotoxin GIIIA, a peptide ligand for muscle sodium channels: chemical synthesis, radiolabeling, and receptor characterization

The peptide conotoxin GIIIA from Conus geographus L. venom, which specifically blocks sodium channels in muscle, has been synthesized by a solid-phase method. The three disulfide bridges were formed by air oxidation. After HPLC purification, the synthetic product was shown to be identical with the native conotoxin GIIIA from Conus geographus. A high specific activity, 125I derivative of mu-conotoxin was prepared and used for binding assays to the Na channel from Electrophorus electric organ. Specific binding could be abolished by competition with tetrodotoxin. The radiolabeled toxin was specifically cross-linked to the Na channel. These studies demonstrate that mu-conotoxin GIIIA can be used to define the guanidinium toxin binding site and will be a useful ligand for understanding functionally important differences between Na channel subtypes.     

Spatial requirements for location of basic residues in peptide substrates for smooth muscle myosin light chain kinase

The requirement of basic residues as substrate specificity determinants for the chicken gizzard myosin light chain kinase has been studied using synthetic peptide analogs of the local phosphorylation site sequence in the myosin light chains, Lys-Lys-Arg13-Pro-Gln-Arg16-Ala-Thr-Ser19-Asn-Val-Phe- Ala. The basic residue, Arg-16, was found to have a strong influence on the kinetics of phosphorylation similar to that reported previously for the three adjacent residues, Lys-11, Lys-12, and Lys-13 (Kemp, B. E., Pearson, R. B., and House, C. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7471-7475). The location of Arg-16 in relation to Ser-19 as well as the distance between Arg-13 and Arg-16 had a profound effect on both the kinetics and the site specificity of phosphorylation. Placement of Arg-16 at position 15 resulted in a complete switch in phosphorylation site specificity from Ser-19 to Thr-18. Increasing the number of alanine residues between Arg-13 and Arg-16 in the model peptide, Lys-Lys-Arg-(Ala)n-Arg-Ala-Thr-Ser-Asn-Val-Phe-Ala, also influenced the kinetics and site specificity of peptide phosphorylation. With two or three alanines (n = 2 or 3), the apparent Km was 7.5 and 10 microM, respectively, and 97% of the phosphate was esterified to Ser-19. Increasing or decreasing the number of alanines (n = O to n = 4) was accompanied by an increase in the apparent Km and phosphorylation of both Thr-18 and Ser-19. These results support the concept that both the presence and location of basic residues play an essential role in the substrate specificity of the smooth muscle myosin light chain kinase.     

mu-conotoxin GIIIA interactions with the voltage-gated Na(+) channel predict a clockwise arrangement of the domains

Voltage-gated Na(+) channels underlie the electrical activity of most excitable cells, and these channels are the targets of many antiarrhythmic, anticonvulsant, and local anesthetic drugs. The channel pore is formed by a single polypeptide chain, containing four different, but homologous domains that are thought to arrange themselves circumferentially to form the ion permeation pathway. Although several structural models have been proposed, there has been no agreement concerning whether the four domains are arranged in a clockwise or a counterclockwise pattern around the pore, which is a fundamental question about the tertiary structure of the channel. We have probed the local architecture of the rat adult skeletal muscle Na(+) channel (mu1) outer vestibule and selectivity filter using mu-conotoxin GIIIA (mu-CTX), a neurotoxin of known structure that binds in this region. Interactions between the pore-forming loops from three different domains and four toxin residues were distinguished by mutant cycle analysis. Three of these residues, Gln-14, Hydroxyproline-17 (Hyp-17), and Lys-16 are arranged approximately at right angles to each other in a plane above the critical Arg-13 that binds directly in the ion permeation pathway. Interaction points were identified between Hyp-17 and channel residue Met-1240 of domain III and between Lys-16 and Glu-403 of domain I and Asp-1532 of domain IV. These interactions were estimated to contribute -1.0+/-0.1, -0.9+/-0.3, and -1.4+/-0.1 kcal/mol of coupling energy to the native toxin-channel complex, respectively. mu-CTX residues Gln-14 and Arg-1, both on the same side of the toxin molecule, interacted with Thr-759 of domain II. Three analytical approaches to the pattern of interactions predict that the channel domains most probably are arranged in a clockwise configuration around the pore as viewed from the extracellular surface.     

Action of derivatives of mu-conotoxin GIIIA on sodium channels. Single amino acid substitutions in the toxin separately affect association and dissociation rates.

We have studied binding and block of sodium channels by 12 derivatives of the 22-residue peptide mu-conotoxin GIIIA (mu-CTX) in which single amino acids were substituted as follows: Arg or Lys by Gln, Gln-18 by Lys, Asp by Asn, and HO-Pro by Pro. Derivatives were synthesized as described by Becker et al. [(1989) Eur. J. Biochem. 185, 79]. Binding was measured by displacement of labeled saxitoxin from eel electroplax membranes (100 mM choline chloride, 10 mM HEPES-NaOH, pH 7.4). Blocking kinetics were evaluated from steady-state, single-channel recordings from rat skeletal muscle sodium channels incorporated into planar, neutral phospholipid/decane bilayers (200 mM NaCl, 10 mM HEPES-NaOH, pH 7.0). Blocking events generally appeared as periods of seconds to minutes in which current through the single channel was completely eliminated. A notable exception was seen for the substitution Arg-13-Gln for which the "blocked" events showed measurable conductances of about 20-40% of the open state. The substitution of Arg-13 reduced binding to electroplax membranes to undetectable levels and increased the apparent dissociation constant determined for skeletal muscle channels by greater than 80-fold compared with the native peptide. Other substitutions caused smaller decreases in affinity. The decreased potency of the toxin derivatives resulted both from increases in the rates of dissociation from the channel, and from decreases in association rates. Our data support the suggestion by Sato et al. [(1991) J. Biol. Chem. 265, 16989] that Arg-13 associates intimately with the binding site on the channel. In addition, our results suggest that certain residues affect almost exclusively the approach and docking of the toxin with its binding site, others appear to be important only to the strength of the association once binding has taken place, and yet others affect both.     

Structure-activity relationships of mu-conotoxin GIIIA: structure determination of active and inactive sodium channel blocker peptides by NMR and simulated annealing calculations.

A synthetic replacement study of the amino acid residues of mu-conotoxin GIIIA, a peptide blocker for muscle sodium channels, has recently shown that the conformation formed by three disulfide bridges and the molecular basicity, especially the one around the Arg13 residue, are important for blocking activity. In the present study, we determined the three-dimensional structure of an inactive analog, [Ala13]mu-conotoxin GIIIA, and refined that of the native toxin by NMR spectroscopy combined with simulated annealing calculations. The atomic root-mean-square difference of the mutant from the native conotoxin was 0.62 A for the backbone atoms (N, C alpha, C') of all residues except for the two terminal residues. The observation that the replacement of Arg13 by Ala13 does not significantly change the molecular conformation suggests that the loss of activity is not due to the conformational change but to the direct interaction of the essential Arg13 residue with the sodium channel molecules. In the determined structure, important residues for the activity, Arg13, Lys16, Hyp(hydroxyproline)17, and Arg19, are clustered on one side of the molecule, an observation which suggests that this face of the molecule associates with the receptor site of sodium channels. The hydroxyl group of Hyp17 is suggested to interact with the channel site with which the essential hydroxyl groups of tetrodotoxin and saxitoxin interact.     

Effects of modification at the fifth residue of mu-conotoxin GIIIA with bulky tags on the electrically stimulated contraction of the rat diaphragm.

Mu-conotoxin GIIIA, a peptide toxin from the cone snail, blocks muscle-type sodium channels. Thr-5 of mu-conotoxin GIIIA, located on the opposite side of the active site in the globular molecule, was replaced by Cys to which the bulky tags were attached. The tagged mu-conotoxin GIIIA derivatives, except for the phospholipid-tagged one, exerted the biological activity with a potency slightly weaker than natural mu-conotoxin GIIIA. When the biotinylated tags of various lengths were added, the presence of avidin suppressed the action of the biotinylated toxins of <4 nm, but not with 5 nm. The bulky biotinylated tags are useful as a caliper to measure the depth of receptor sites in the channels.     

Primary structure required for the inhibition of smooth muscle myosin light chain kinase.

Myosin light chain kinase (MLCK) contains the autoinhibitor sequence right next to the N-terminus side of the calmodulin binding region. In this paper, the structural requirement of the inhibition of MLCK activity was studied using synthetic peptide analogs. Peptides Ala-783-Lys-799 and Ala-783-Arg-798 inhibited calmodulin independent MLCK at the same potency as the peptide Ala-783-Gly-804. Deletion of Arg-797-Lys-799 or substitution of these residues to Ala markedly increased the Ki while the substitution of Lys-792 and Lys-793 to Ala and the deletion of Lys-784-Lys-785 did not affect the inhibitory activity of the peptides. The results suggest that Arg-797-Arg-798 are especially important for the inhibitory activity among other basic residues in the autoinhibitory region.     

Importance of Arg-599 of β-galactosidase (Escherichia coli) as an anchor for the open conformations of Phe-601 and the active-site loop

Structural and kinetic data show that Arg-599 of β-galactosidase plays an important role in anchoring the "open" conformations of both Phe-601 and an active-site loop (residues 794-803). When alanine was substituted for Arg-599, the conformations of Phe-601 and the loop shifted towards the "closed" positions because interactions with the guanidinium side chain were lost. Also, Phe-601, the loop, and Na+, which is ligated by the backbone carbonyl of Phe-601, lost structural order, as indicated by large B-factors. IPTG, a substrate analog, restored the conformations of Phe-601 and the loop of R599A-β-galactosidase to the open state found with IPTG-complexed native enzyme and partially reinstated order. ?-Galactonolactone, a transition state analog, restored the closed conformations of R599A-β-galactosidase to those found with ?-galactonolactone-complexed native enzyme and completely re-established the order. Substrates and substrate analogs bound R599A-β-galactosidase with less affinity because the closed conformation does not allow substrate binding and extra energy is required for Phe-601 and the loop to open. In contrast, transition state analog binding, which occurs best when the loop is closed, was several-fold better. The higher energy level of the enzyme?substrate complex and the lower energy level of the first transition state means that less activation energy is needed to form the first transition state and thus the rate of the first catalytic step (k2) increased substantially. The rate of the second catalytic step (k3) decreased, likely because the covalent form is more stabilized than the second transition state when Phe-601 and the loop are closed. The importance of the guanidinium group of Arg-599 was confirmed by restoration of conformation, order, and activity by guanidinium ions.     

Structure-function relationships and site of action of apamin, a neurotoxic polypeptide of bee venom with an action on the central nervous system.

Specific chemical modifications of apamin have been used to study the residues involved in its toxic action. Transformation of Lys4 into homoarginine did not affect toxicity. Modification of the alpha-amino group of Cys1 and of the epsilon-amino group of Lys4 by acetic anhydride or fluorescamine decreased toxicity only by a factor of 2.5-2.8. Modification of the gamma-carboxylate of Glu7 with glycine ethyl ester in the presence of a soluble carbodiimide decreased toxicity by a factor of 2. Diethyl pyrocarbonate treated of the imidazole side chain of His18 decreased toxicity by a factor of 2.6. Thus none of these residues is essential for toxicity. However, combined modification of amino groups and of the imidazole side chain of His-18 completely abolished biological activity. Complete loss of toxicity also resulted from reduction and alkylation of both disulfide bridges, from chemical modification with cyclohexanedione of Arg-13 and Arg-14, and from removal of Arg-14 of acetylated apamin by digestion with trypsin. Incorporation of radioactive acetyl groups on both amino groups of apamin gave an active labeled toxin which has been used to localize the site of action of apamin in the spinal cord, principally in the lumbar part of the neuraxis.     

Roles of Hyp Residues in the Folding and Activity of μ-Conotoxin GIIIA,a Peptide Blocker of Muscle Sodium Channels

Attempts were made to synthesize seven analogs of µ-conotoxin GIIIA, a specific blocker of muscle sodium channels, by replacing the three Hyp residues (Hyp⁶, Hyp⁷, and Hyp¹⁷) with various amino acids. Replacement with Ala residue at these positions resulted in a very low isolation yield, suggesting that these three Hyp residues are essential for the folding of the molecule. CD spectra of the synthesized analogs suggest that, once synthesized, the replacement did not affect the three dimensional structure. The inhibitory effects on the twitch contractions of the rat diaphragm showed that the hydroxyl group at side chains of Hyp residues are not essential for the activity.     

Solution structure of BmKK4, the first member of subfamily alpha-KTx 17 of scorpion toxins

BmKK4 is a 30 amino acid peptide purified from the venom of the Chinese scorpion Buthus martensi Karsch. It has been classified as the first member of scorpion toxin subfamily alpha-KTx 17. The 3D structure of BmKK4 in solution has been determined by 2D NMR spectroscopy. This toxin adopts a common alpha/beta-motif, but shows a distinctive local conformation. The most novel feature is that the regular arrangements of the side chains of the residues involved in the beta-sheet of BmKK4 are distorted by a classic beta-bulge structure, which involves two residues (Asp18 and Arg19) in the first strand opposite a single residue (Tyr26) in the second strand. The bulge produces two main changes in the structure of the antiparallel beta-sheet: (1) It disrupts the normal alteration of the side chain direction; the side chain of Asp18 turns over to form a salt bridge with that of Arg19. (2) It accentuates the twist of the sheet, and alters the direction of the antiparallel beta-sheet. The unusual structural feature of the toxin is attributed to the shorter peptide segment (Leu15-Arg19) between the third and fourth Cys residues and two unique residues (Asp18 and Arg19) at the position preceding the fourth Cys. In addition, the lower affinity of the peptide for the Kv channel is correlated to the structural features: residue Arg19 instead of a Lys residue at the critical position for binding and the salt bridge formed between residues Arg19 and Asp18.     

Refined crystal structure of cytoplasmic malate dehydrogenase at 2.5-A resolution

The molecular structure of cytoplasmic malate dehydrogenase from pig heart has been refined by alternating rounds of restrained least-squares methods and model readjustment on an interactive graphics system. The resulting structure contains 333 amino acids in each of the two subunits, 2 NAD molecules, 471 solvent molecules, and 2 large noncovalently bound molecules that are assumed to be sulfate ions. The crystallographic study was done on one entire dimer without symmetry restraints. Analysis of the relative position of the two subunits shows that the dimer does not obey exact 2-fold rotational symmetry; instead, the subunits are related by a 173 degrees rotation. The structure results in a R factor of 16.7% for diffraction data between 6.0 and 2.5 A, and the rms deviations from ideal bond lengths and angles are 0.017 A and 2.57 degrees, respectively. The bound coenzyme in addition to hydrophobic interactions makes numerous hydrogen bonds that either are directly between NAD and the enzyme or are with solvent molecules, some of which in turn are hydrogen bonded to the enzyme. The carboxamide group of NAD is hydrogen bonded to the side chain of Asn-130 and via a water molecule to the backbone nitrogens of Leu-157 and Asp-158 and to the carbonyl oxygen of Leu-154. Asn-130 is one of the corner residues in a beta-turn that contains the lone cis peptide bond in cytoplasmic malate dehydrogenase, situated between Asn-130 and Pro-131. The active site histidine, His-186, is hydrogen bonded from nitrogen ND1 to the carboxylate of Asp-158 and from its nitrogen NE2 to the sulfate ion bound in the putative substrate binding site. In addition to interacting with the active site histidine, this sulfate ion is also hydrogen bonded to the guanidinium group of Arg-161, to the carboxamide group of Asn-140, and to the hydroxyl group of Ser-241. It is speculated that the substrate, malate or oxaloacetate, is bound in the sulfate binding site with the substrate 1-carboxyl hydrogen bonded to the guanidinium group of Arg-161.     

Determination of the carboxyl termini of the alpha and beta subunits of yeast K1 killer toxin. Requirement of a carboxypeptidase B-like activity for maturation

The carboxyl-terminal sequences of the two polypeptide chains of the Saccharomyces cerevisiae K1 killer toxin were determined by protein sequencing and amino acid analysis of peptide fragments generated from the mature, secreted toxin. The COOH-terminal amino acid of the beta chain is histidine 316, the final residue encoded by the precursor gene. The COOH terminus of the alpha chain is at alanine 147 of the preprotoxin. Amino acid composition data for the purified toxin are consistent with that predicted from the gene sequence of the preprotoxin where the alpha and beta subunits consist of amino acid residues 45-147 and 234-316, respectively. The molecular weight of the mature alpha beta dimer is about 20,658. The COOH-terminal sequence determination completes the location of the toxin subunits in the precursor, and its configuration may be represented as prepropeptide-Pro-Arg-alpha-Arg-Arg-gamma-Lys-Arg-beta, where gamma represents the interstitial glycosylated peptide. The COOH terminal side of the paired basic residues (Arg-148 Arg-149 and Lys-232 Arg-233 of preprotoxin) are endoproteolytic processing sites for the product of the KEX2 gene (Julius, D., Brake, A., Blair, L., Kunisawa, R., and Thorner, J. (1984) Cell 37, 1075-1089), and thus maturation of the alpha subunit of killer toxin apparently requires a carboxypeptidase B-like activity. A possible candidate for this activity is the product of the KEX1 gene (Dmochowska, A., Dignard, D., Henning, D., Thomas, D.Y., and Bussey, H. (1987) Cell, in press).     

Generation of polyclonal antibody against mu-conotoxin GIIIA using an immunogen of [Cys(5)]mu-conotoxin GIIIA site-specifically conjugated with bovine serum albumin.

mu-Conotoxin GIIIA, one of the strong peptide toxins in the cone shell, preferentially blocks the skeletal muscle-type sodium channels in vertebrates. The toxicity of mu-conotoxin GIIIA is nearly equal to that of tetrodotoxin. The generation of an antibody for the native toxins is analytically useful, but practically difficult due to its high toxicity to animals. In this study, we generated the polyclonal antibody for mu-conotoxin GIIIA using a specific conjugation method in which the immunogen was detoxified while retaining the active-site structure for the sodium channels. ELISA analysis showed that the generated antibody recognized the native toxin folded with three disulfide bridges, but not the linear one. Furthermore, the physiologically active mutants of GIIIA were recognized while the inactive mutants were not, suggesting that the newly generated antibody can selectively recognize the physiologically active toxins. These methods for generating an antibody against peptide toxins will be applicable to other peptide toxins.     

Chemical modification of arginine residues in alpha-bungarotoxin.

The reaction of alpha-bungarotoxin (alpha-BuTX) with 1,2-cyclohexanedione resulted in the modification of only Arg-72 but arginine at position 36 or 72, as well as both were modified by reaction of the toxin with p-hydroxyphenylglyoxal. No derivative modified at Arg-25 was obtained, indicating that this residue may be located in the interior region of alpha-BuTX molecule. Monoderivative at Arg-72 showed about 50% of the lethal toxicity and binding activity of alpha-BuTX to nicotinic acetylcholine receptor (AChR), while the activity was decreased to one-third when the invariant Arg-36 was modified, indicating that the latter residue is more closely related to the interaction of the toxin with AChR. Approx. 13% of the residual activity was observed when both arginine residues at 36 and 72 were modified. The antigenicity of alpha-BuTX was still retained essentially intact after Arg-36 or -72 was modified, whereas it decreased to 50% when both these arginine residues were modified. The present study indicates that Arg-36 and -72 in alpha-BuTX may be involved in the multipoint contact between the toxin and AChR, but neither is absolutely essential for the binding.     

Swapping the substrate specificities of the neuropeptidases neurolysin and thimet oligopeptidase

Thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are closely related zinc-dependent metallopeptidases that metabolize small bioactive peptides. They cleave many substrates at the same sites, but they recognize different positions on others, including neurotensin, a 13-residue peptide involved in modulation of dopaminergic circuits, pain perception, and thermoregulation. On the basis of crystal structures and previous mapping studies, four sites (Glu-469/Arg-470, Met-490/Arg-491, His-495/Asn-496, and Arg-498/Thr-499; thimet oligopeptidase residues listed first) in their substrate-binding channels appear positioned to account for differences in specificity. Thimet oligopeptidase mutated so that neurolysin residues are at all four positions cleaves neurotensin at the neurolysin site, and the reverse mutations in neurolysin switch hydrolysis to the thimet oligopeptidase site. Using a series of constructs mutated at just three of the sites, it was determined that mutations at only two (Glu-469/Arg-470 and Arg-498/Thr-499) are required to swap specificity, a result that was confirmed by testing the two-mutant constructs. If only either one of the two sites is mutated in thimet oligopeptidase, then the enzyme cleaves almost equally at the two hydrolysis positions. Crystal structures of both two-mutant constructs show that the mutations do not perturb local structure, but side chain conformations at the Arg-498/Thr-499 position differ from those of the mimicked enzyme. A model for differential recognition of neurotensin based on differences in surface charge distribution in the substrate binding sites is proposed. The model is supported by the finding that reducing the positive charge on the peptide results in cleavage at both hydrolysis sites.     

Characterization of the binding site on the formyl peptide receptor using three receptor mutants and analogs of Met-Leu-Phe and Met-Met-Trp-Leu-Leu

The formyl peptide receptor (FPR) is a chemotactic G protein-coupled receptor found on the surface of phagocytes. We have previously shown that the formyl peptide binding site maps to the membrane-spanning region (Miettinen, H. M., Mills, J. S., Gripentrog, J. M., Dratz, E. A., Granger, B. L., and Jesaitis, A. J. (1997) J. Immunol. 159, 4045-4054). Recent reports have indicated that non-formylated peptides, such as MMWLL can also activate this receptor (Chen, J., Bernstein, H. S., Chen, M., Wang, L., Ishi, M., Turck, C. W., and Coughlin, S. R. (1995) J. Biol. Chem. 270, 23398-23401.) Here we show that the selectivity for the binding of different NH(2)-terminal analogs of MMWLL or MLF can be markedly altered by mutating Asp-106 to asparagine or Arg-201 to alanine. Both D106N and R201A produced a similar change in ligand specificity, including an enhanced ability to bind the HIV-1 peptide DP178. In contrast, the mutation R205A exhibited altered specificity at the COOH terminus of fMLF, with R205A binding fMLF-O-butyl > fMLF-O-methyl > fMLF, whereas wt FPR bound fMLF > fMLF-O-methyl approximately fMLF-O-butyl. These data, taken together with our previous finding that the leucine side chain of fMLF is probably bound to FPR near FPR (93)VRK(95) (Mills, J. S., Miettinen, H. M., Barnidge, D., Vlases, M. J., Wimer-Mackin, S., Dratz, E. A., and Jesaitis, A. J. (1998) J. Biol. Chem. 273, 10428-10435.), indicate that the most likely positioning of fMLF in the binding pocket of FPR is approximately parallel to the fifth transmembrane helix with the formamide group of fMLF hydrogen-bonded to both Asp-106 and Arg-201, the leucine side chain pointing toward the second transmembrane region, and the COOH-terminal carboxyl group of fMLF ion-paired with Arg-205.