Solution structure of P01, a natural scorpion peptide structurally analogous to scorpion toxins specific for apamin-sensitive potassium channel

The venom of the North African scorpion Androctonus mauretanicus mauretanicus possesses numerous highly active neurotoxins that specifically bind to various ion channels. One of these, P05, has been found to bind specifically to calcium-activated potassium channels and also to compete with apamin, a toxin extracted from bee venom. Besides the highly potent ones, several of these peptides (including that of P01) have been purified and been found to possess only a very weak, although significant, activity in competition with apamin. The amino acid sequence of P01 shows that it is shorter than P05 by two residues. This deletion occurs within an α-helix stretch (residues 5–12). This α-helix has been shown to be involved in the interaction of P05 with its receptor via two arginine residues. These two arginines are absent in the P01 sequence. Furthermore, a proline residue in position 7 of the P01 sequence may act as an α-helix breaker. We have determined the solution structure of P01 by conventional two-dimensional ¹H nuclear magnetic resonance and show that 1) the proline residue does not disturb the α-helix running from residues 5 to 12; 2) the two arginines are topologically replaced by two acidic residues, which explains the drop in activity; 3) the residual binding activity may be due to the histidine residue in position 9; and 4) the overall secondary structure is conserved, i.e., an α-helix running from residues 5 to 12, two antiparallel stretches of β-sheet (residues 15–20 and 23–27) connected by a type I′ β-turn, and three disulfide bridges connecting the α-helix to the β-sheet.     

Spectroscopic investigations of peptide 401 from bee venom

The circular dichroism (CD) and ¹H-nmr properties of peptide 401, a bee venom component with 22 amino acid residues and two disulfide bridges, have been studied under a variety of conditions and compared with those of the structurally related octadecapeptide apamin. The major component of the relatively intense CD signal in the 200–230-nm region in both cases probably arises from the rigid asymmetric ring structures of the disulfide bridges. CD spectra are practically unaffected by pH (in the region 1–7), solvent (water, trifluoroethanol, dioxane/water mixtures), concentration of peptide, or additions of salt (guanidinium chloride, KCl). Temperature changes (in the range 20–59°C) have only a modest influence. For both apamin and peptide 401, reduction of the two disulfide bridges results in a dramatic change of the CD spectrum, which acquires the characteristic form of a random coil. Preliminary ¹H-nmr data are presented for both the reduced and the oxidized form. Several resonance peaks could be assigned on the basis of the theoretical random-coil spectrum. In the oxidized forms, six slowly exchangeable amide protons could be found in a spectrum taken at low pH, which are ascribed to intramolecular hydrogen bonds. Each of the four protons of the two histidine residues of peptide 401 appears as two distinct resonance peaks in the oxidized form but not in the reduced form. This is interpreted as arising from conformational heterogeneity of peptide 401.     

The asparagine-stabilized beta-turn of apamin: contribution to structural stability from dynamics simulation and amide hydrogen exchange analysis

Molecular dynamics simulations of bee venom apamin, and an analogue having an Asn to Ala substitution at residue 2 (apamin-N2A), were analyzed to explore the contribution of hydrogen bonds involving Asn2 to local (beta-turn residues N2, C3, K4, A5) and global stability. The wild-type peptide retained a stable conformation during 2.4 ns of simulation at 67 degrees C, with high beta-turn stability characterized by backbone-side chain hydrogen bonds involving beta-turn residues K4 and A5, with the N2 side chain amide carbonyl. The loss of stabilizing interactions involving the N2 side chain resulted in the loss of the beta-turn conformation in the apamin N2A simulations (27 or 67 degrees C). This loss of beta-turn stability propagates throughout the peptide structure, with destabilization of the C-terminal helix connected to the N-terminal region by two disulfide bonds. Backbone stability in a synthetic peptide analogue (apamin-N2A) was characterized by NMR and amide hydrogen exchange measurements. Consistent with the simulations, loss of hydrogen bonds involving the N2 side chain resulted in destabilization of both the N-terminal beta-turn and the C-terminal helix. Amide exchange protection factors in the C-terminal helix were reduced by 9-11-fold in apamin N2A as compared with apamin, corresponding to free energy (deltaDeltaG(uf)) of around 1.5 kcal M(-1) at 20 degrees C. This is equivalent to the contribution of hydrogen bond interactions involving the N2 side chain to the stability of the beta-turn. Together with additional measures of exchange protection factors, the three main contributions to backbone stability in apamin that account for virtually the full thermodynamic stability of the peptide have been quantitated.     

Spatial structure of apamin in solution]

The calculation of the complete spatial structure of the bee venom peptide neurotoxin apamin has been carried out by means of a method elaborated earlier. It is based on the joint utilization of the molecular mechanics algorithms and NMR spectroscopy data. It was established that the molecule backbone conformation in solution may be represented as the combination of the beta-turn III (residues 2-5) and alpha-helical segment (9-18) both separated by the non-standard bend IV (5-8). The most probable system of the intramolecular hydrogen bonds in the apamin polypeptide backbone was proposed. Certain amino acid residues have been shown to be characterized by the lack of strict determination of the conformations of their side chains which may be realized in a few states providing approximately equal stabilization of the same form of the main chain. The conformational parameters of the proposed apamin structural model are appropriate to the NMR spectroscopy data derived from the literature and used in the calculations and are not contradictory to other experimental information.     

Partial activation of antithrombin without heparin through deletion of a unique sequence on the reactive site loop of the serpin.

Native antithrombin (AT) has an inactive reactive site loop conformation unless it is activated by a unique pentasaccharide fragment of heparin (H(5)). Structural data suggests that this may be due to preinsertion of two N-terminal residues of the reactive site loop of the serpin into the A-beta-sheet of the molecule. Relative to alpha(1)-antitrypsin, the reactive site loop of AT has three additional residues, Arg(399), Val(400), and Thr(401), at the C-terminal P' end of the loop. To determine whether a longer reactive site loop of AT is responsible for loop preinsertion in the native conformation, mutants of the serpin were expressed in which these residues were individually or in combination deleted. Kinetic analysis suggested that deletion of two residues, Val(400) and Thr(401), changed the solution equilibrium of the serpin in favor of the active conformation, thereby enhancing the inhibition of factor Xa by an order of magnitude independent of H(5). Interestingly, the reactivity of this mutant with thrombin was impaired by the same order of magnitude in the absence, but not in the presence of H(5). These results suggest that a longer reactive site loop in AT is responsible for its inactive native conformation toward factor Xa, while at same time AT requires this feature to regulate the activity of thrombin.     

The solution structure of a chimeric LEKTI domain reveals a chameleon sequence

The conversion of an alpha-helical to a beta-strand conformation and the presence of chameleon sequences are fascinating from the perspective that such structural features are implicated in the induction of amyloid-related fatal diseases. In this study, we have determined the solution structure of a chimeric domain (Dom1PI) from the multidomain Kazal-type serine proteinase inhibitor LEKTI using multidimensional NMR spectroscopy. This chimeric protein was constructed to investigate the reasons for differences in the folds of the homologous LEKTI domains 1 and 6 [Lauber, T., et al. (2003) J. Mol. Biol. 328, 205-219]. In Dom1PI, two adjacent phenylalanine residues (F28 and F29) of domain 1 were substituted with proline and isoleucine, respectively, as found in the corresponding P4' and P5' positions of domain 6. The three-dimensional structure of Dom1PI is significantly different from the structure of domain 1 and closely resembles the structure of domain 6, despite the sequence being identical to that of domain 1 except for the two substituted phenylalanine residues and being only 31% identical to the sequence of domain 6. The mutation converted a short 3(10)-helix into an extended loop conformation and parts of the long COOH-terminal alpha-helix of domain 1 into a beta-hairpin structure. The latter conformational change occurs in a sequence stretch distinct from the region containing the substituted residues. Therefore, this switch from an alpha-helical structure to a beta-hairpin structure indicates a chameleon sequence of seven residues. We conclude that the secondary structure of Dom1PI is determined not only by the local protein sequence but also by nonlocal interactions.     

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 cis-trans 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 α-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 α-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.© 1997 John Wiley & Sons, Inc. Biopoly 41 : 451–460, 1997     

Beta-alanine containing peptides: a novel molecular tool for the design of gamma-turns.

In the present paper we describe the synthesis, purification, single crystal x-ray analysis, and solution conformational characterization of the cyclic tetrapeptide cyclo-(L-Pro-beta-Ala-L-Pro-beta-Ala). This peptide was synthesized by classical solution methods and the cyclization of the free tetrapeptide was accomplished in good yields in diluted methylene chloride solution using N,N-dicyclohexyl-carbodiimide (DCCI). The compound crystallizes in the orthorombic space group P2(1)2(1)2(1) from ethyl acetate. All peptide bonds are trans. The molecular conformation is stabilized by two intramolecular hydrogen bonds between the CO and NH groups of the two beta-alanine residues. These hydrogen bonds take place in a C7 structure in which both proline residues occupy the 2 position of an inverse gamma-turn. The two beta-alanine residues have a typical folded conformation (around the C alpha-C beta bond) observed in other cyclic peptides containing this residue. A detailed 1H-nmr analysis in CD3CN solution has been carried out. The molecule assumes a twofold symmetry in solution with a molecular conformation consistent with that observed in the solid state.     

Structural and mechanistic characterization of leukocyte-type core 2 β1,6-N-acetylglucosaminyltransferase: a metal-ion-independent GT-A glycosyltransferase

Leukocyte-type core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT-L) is an inverting, metal-ion-independent glycosyltransferase that catalyzes the formation of mucin-type core 2 O-glycans. C2GnT-L belongs to the GT-A fold, yet it lacks the metal ion binding DXD motif characteristic of other nucleoside disphosphate GT-A fold glycosyltransferases. To shed light on the basis for its metal ion independence, we have solved the X-ray crystal structure (2.3 Å resolution) of a mutant form of C2GnT-L (C217S) in complex with the nucleotide sugar product UDP and, using site-directed mutagenesis, examined the roles of R378 and K401 in both substrate binding and catalysis. The structure shows that C2GnT-L exists in an “open” conformation and a “closed” conformation and that, in the latter, R378 and K401 interact with the β-phosphate moiety of the bound UDP. The two conformations are likely to be important in catalysis, but the conformational changes that lead to their interconversion do not resemble the nucleotide-sugar-mediated loop ordering observed in other GT-A glycosyltransferases. R378 and K401 were found to be important in substrate binding and/or catalysis, an observation consistent with the suggestion that they serve the same role played by metal ion in all of the other GT-A glycosyltransferases studied to date. Notably, R378 and K401 appear to function in a manner similar to that of the arginine and lysine residues contained in the RX_4-5K motif found in the retaining GT-B glycosyltransferases.     

Crystal structure of bisphosphorylated IGF-1 receptor kinase: insight into domain movements upon kinase activation

BACKGROUND: The insulin-like growth-factor-1 (IGF-1) receptor, which is widely expressed in cells that have undergone oncogenic transformation, is emerging as a novel target in cancer therapy. IGF-1-induced receptor activation results in autophosphorylation of cytoplasmic kinase domains and enhances their capability to phosphorylate downstream substrates. Structures of the homologous insulin receptor kinase (IRK) exist in an open, unphosphorylated form and a closed, trisphosphorylated form. RESULTS: We have determined the 2.1 A crystal structure of the IGF-1 receptor protein tyrosine kinase domain phosphorylated at two tyrosine residues within the activation loop (IGF-1RK2P) and bound to an ATP analog. The ligand is not in a conformation compatible with phosphoryl transfer, and the activation loop is partially disordered. Compared to the homologous insulin receptor kinase, IGF-1RK2P is trapped in a half-closed, previously unobserved conformation. Observed domain movements can be dissected into two orthogonal rotational components. CONCLUSIONS: Conformational changes upon kinase activation are triggered by the degree of phosphorylation and are crucially dependent on the conformation of the proximal end of the kinase activation loop. This IGF-1RK structure will provide a molecular basis for the design of selective antioncogenic therapeutic agents.     

Two parts of the T3S4 domain of the hook-length control protein FliK are essential for the substrate specificity switching of the flagellar type III export apparatus

The switch in export specificity of the type III flagellar protein export apparatus from rod/hook type to filament type is believed to occur upon completion of hook assembly by way of an interaction of the type III secretion substrate specificity switch (T3S4) domain of the hook-length control protein FliK, with the integral membrane export apparatus component FlhB. The T3S4 domain of FliK (FliKT3S4) consisting of amino acid residues 265-405 has an unstable and flexible conformation in its last 35 residues (FliKCT). To investigate the role of FliKT3S4 in substrate specificity switching, we studied the effect of deletions and point mutations within this domain and characterized suppressor mutations. Deletions of ten amino acid residues within the region of residues 301-350 and five amino acids of residues 401-405 abolished switching of export specificity. Site directed mutagenesis showed that highly conserved residues, Val302, Ile304, Leu335, Val401 and Ala405, are essential, and that the five C terminal residues (401-405) are restricted in conformation for the switching process. Suppressor mutant analysis of the fliK(S319Y) mutant, which produces extended hooks with filaments attached due to delayed switching, suggested that FliKT3S4 interacts with the C terminal half of the cytoplasmic domain of FlhB (FlhBC). We propose a two step binding model of FliKT3S4 and FlhBC, in which residues 301-350 of FliK bind to FlhBC upon hook assembly completion at about 55 nm, and then unfolded FliKCT binds to FlhBC to trigger the switch in substrate specificity.     

Altered dark- and photoconversion of phytochrome B mediate extreme light sensitivity and loss of photoreversibility of the phyB-401 mutant

The phyB-401 mutant is 10(3) fold more sensitive to red light than its wild-type analogue and shows loss of photoreversibility of hypocotyl growth inhibition. The phyB-401 photoreceptor displays normal spectral properties and shows almost no dark reversion when expressed in yeast cells. To gain insight into the molecular mechanism underlying this complex phenotype, we generated transgenic lines expressing the mutant and wild-type phyB in phyB-9 background. Analysis of these transgenic lines demonstrated that the mutant photoreceptor displays a reduced rate of dark-reversion but normal P(fr) to P(r) photoconversion in vivo and shows an altered pattern of association/dissociation with nuclear bodies compared to wild-type phyB. In addition we show (i) an enhanced responsiveness to far-red light for hypocotyl growth inhibition and CAB2 expression and (ii) that far-red light mediated photoreversibility of red light induced responses, including inhibition of hypocotyl growth, formation of nuclear bodies and induction of CAB2 expression is reduced in these transgenic lines. We hypothesize that the incomplete photoreversibility of signalling is due to the fact that far-red light induced photoconversion of the chromophore is at least partially uncoupled from the P(fr) to P(r) conformation change of the protein. It follows that the phyB-401 photoreceptor retains a P(fr)-like structure (P(r) (*)) for a few hours after the far-red light treatment. The greatly reduced rate of dark reversion and the formation of a biologically active P(r) (*) conformer satisfactorily explain the complex phenotype of the phyB-401 mutant and suggest that amino acid residues surrounding the position 564 G play an important role in fine-tuning phyB signalling.     

A 1H-NMR study of the solution conformation of secretin. Resonance assignment and secondary structure

The solution conformation of the 27 residue polypeptide hormone secretin has been investigated by 1H-NMR spectroscopy under conditions where it adopts a fully ordered structure as judged by circular dichroism spectroscopy, namely in an aqueous solution of 40% (v/v) trifluoroethanol. Using a combination of two-dimensional NMR techniques the 1H-NMR spectrum of secretin is completely assigned and its secondary structure is determined from a qualitative interpretation of the nuclear Overhauser enhancement data. It is shown that under these conditions secretin adopts a conformation consisting of an N-terminal irregular strand (residues 1-6) followed by two helices (residues 7-13 and 17-25) connected by a 'half-turn' (residues 14-16); the last two residues (26 and 27) are again irregular. This conformation is shown to be very similar to that of glucagon in perdeuterated dodecylphosphocholine micelles and to that of the active 1-29 fragment of growth hormone releasing factor in 30% (v/v) trifluoroethanol:     

Introducing Wilson disease mutations into the zinc-transporting P-type ATPase of Escherichia coli. The mutation P634L in the 'hinge' motif (GDGXNDXP) perturbs the formation of the E2P state.

ZntA, a bacterial zinc-transporting P-type ATPase, is homologous to two human ATPases mutated in Menkes and Wilson diseases. To explore the roles of the bacterial ATPase residues homologous to those involved in the human diseases, we have introduced several point mutations into ZntA. The mutants P401L, D628A and P634L correspond to the Wilson disease mutations P992L, D1267A and P1273L, respectively. The mutations D628A and P634L are located in the C-terminal part of the phosphorylation domain in the so-called hinge motif conserved in all P-type ATPases. P401L resides near the N-terminal portion of the phosphorylation domain whereas the mutations H475Q and P476L affect the heavy metal ATPase-specific HP motif in the nucleotide binding domain. All mutants show reduced ATPase activity corresponding 0-37% of the wild-type activity. The mutants P401L, H475Q and P476L are poorly phosphorylated by both ATP and P(i). Their dephosphorylation rates are slow. The D628A mutant is inactive and cannot be phosphorylated at all. In contrast, the mutant P634L six residues apart in the same domain shows normal phosphorylation by ATP. However, phosphorylation by P(i) is almost absent. In the absence of added ADP the P634L mutant dephosphorylates much more slowly than the wild-type, whereas in the presence of ADP the dephosphorylation rate is faster than that of the wild-type. We conclude that the mutation P634L affects the conversion between the states E1P and E2P so that the mutant favors the E1 or E1P state.     

Role of Asn(2) and Glu(7) residues in the oxidative folding and on the conformation of the N-terminal loop of apamin

The X-ray structure of [N-acetyl]-apamin has been solved at 0.95 A resolution. It consists of an 1-7 N-terminal loop stabilized by an Asn-beta-turn motif (2-5 residues) and a helical structure spanning the 9-18 residues tightly linked together by two disulfide bonds. However, neither this accurate X-ray nor the available solution structures allowed us to rationally explain the unusual downfield shifts observed for the Asn(2) and Glu(7) amide signals upon Glu(7) carboxylic group ionization. Thus, apamin and its [N-acetyl], [Glu(7)Gln], [Glu(7)Asp], and [Asn(2)Abu] analogues and submitted to NMR structural studies as a function of pH. We first demonstrated that the Glu(7) carboxylate group is responsible for the large downfield shifts of the Asn(2) and Glu(7) amide signals. Then, molecular dynamics (MD) simulations suggested unexpected interactions between the carboxylate group and the Asn(2) and Glu(7) amide protons as well as the N-terminal alpha-amino group, through subtle conformational changes that do not alter the global fold of apamin. In addition, a structural study of the [Asn(2)Abu] analogue, revealed an essential role of Asn(2) in the beta-turn stability and the cis/trans isomerization of the Ala(5)-Pro(6) amide bond. Interestingly, this proline isomerization was shown to also depend on the ionization state of the Glu(7) carboxyl group. However, neither destabilization of the beta-turn nor proline isomerization drastically altered the helical structure that contains the residues essential for binding. Altogether, the Asn(2) and Glu(7) residues appeared essential for the N-terminal loop conformation and thus for the selective formation of the native disulfide bonds but not for the activity.     

Crystal Structure of P13K SH3 Domain at 2.0 Å Resolution

The P13K SH3 domain, residues 1 to 85 of the P1 – 3 kinase p85 subunit, has been characterized by X-ray diffraction. Crystals belonging to space groupP4₃2₁2 diffract to 2.0 Å resolution and the structure was phased by single isomorphous replacement and anomalous scattering (SIRAS). As expected, the domain is a compact β barrel with an over-all conformation very similar to the independently determined NMR structures. The X-ray structure illuminates a discrepancy between the two NMR structures on the conformation of the loop region unique to P13K SH3. Furthermore, the ligand binding pockets of P13K SH3 domain are occupied by amino acid residues from symmetry-related P13K SH3 molecules: the C-terminal residues I(82) SPP of one and R18 of another. The interaction modes clearly resemble those observed for the P13K SH3 domain complexed with the synthetic peptide RLP1, a class 1 ligand, although there are significant differences. The solid-state interactions suggest a model of protein – protein aggregation that could be mediated by SH3 domains.     

Processing by accessory cells for presentation to murine T cells of apamin, a disulfide-bonded 18 amino acid peptide

Apamin, an 18 amino acid peptide with two disulfide bonds, elicits specific T cell proliferative responses in H-2d and H-2b mouse strains. We evaluated the processing requirement of this compact peptide by accessory cells for presentation to apamin-reactive T hybridoma cells (THC) by analyzing the IL-2 responses of 16 THC from apamin-primed BALB/c or C57BL/6 mice, to various forms of either native or chemically synthesized apamin analogs. These included: unfolded peptides (whose four sulfhydryl groups were blocked by acetamidomethyl residues), N-and/or C-truncated peptides, and an analog with a single amino acid substitution at position 10. Assessment of the Ag-specific THC responses in the presence of either live or formaldehyde-prefixed APC indicated the following: 1) all THC stringently required Ag processing; 2) in 8 of 16 cases, the simple unfolding of apamin was sufficient to eliminate the need for Ag processing, or even induced increased THC IL-2 responses (other cells required further antigenic alterations in addition to unfolding, or rare processing steps dependent on the integrity of the two disulfide bonds); and 3) for most THC, the Leu10 and the N terminus arm of apamin were shown to be critical for expression of the epitopes involved in T cell recognition. These data indicate that apamin, a natural peptide having an appropriate size for T cell triggering, acquires its antigenic conformation after a processing by APC which primarily involves an alteration of a disulfide bond-dependent peptide folding.     

Preparation of a pure monoiodo derivative of the bee venom neurotoxin apamin and its binding properties to rat brain synaptosomes

The preparation and purification of an active monoiodo derivative of apamin is described. Radiolabeled monoiodoapamin (2000 Ci/mmol) binds specifically to rat brain synaptosomes at 0 degrees C and pH 7.5 with a second order rate constant of association (ka = 2.6 x 10(7) M-1 s-1) and a first order rate constant of dissociation (kd = 3.8 x 10(-4) s-1). The maximal binding capacity is 12.5 fmol/mg of protein and the dissociation constant is 15-25 pM for the monoiodo derivative and 10 pM for the native toxin. The apamin receptor is destroyed by proteases suggesting that it is of a proteic nature. Neurotensin and its COOH-terminal partial sequences are the only molecules unrelated to apamin that are able to displace monoiodoapamin from its receptor at low concentrations. Half-displacement occurs at 170 nM neurotensin. This property is due to the presence in the COOH-terminal sequence of neurotensin of two contiguous arginine residues, a structure analogous to that of the apamin active site. The binding of monoiodoapamin to its receptor is sensitive to cations. Increasing K+ or Rb+ concentrations from 10 microM to 5 mM selectively enhances the binding by a factor of 1.8. Increasing the concentration of any cation from 1 to 100 mM completely inhibits iodoapamin binding. Both effects are due to a cation-induced modulation of the affinity of monoidoapamin for its receptor without any change of the maximal toxin binding capacity of synaptosomes. Guanidinium and molecules containing a guanidinium group are better inhibitors of iodoapamin binding than other inorganic cations or positively charged organic molecules.     

Synthesis and properties of chemotactic peptide analogs. II. HCO-Met-Leu-Phe-OMe analogs containing cyclic alpha,alpha-disubstituted amino acids as Met and Phe mimicking residues.

As a part of a research program aimed at studying structure activity relationship in the field of chemotactic peptides, modified analogs of the potent chemoattractant HCO-Met-Leu-Phe-OH (fMLP) of the general formula HCO-Xaa-Leu-Yaa-OMe are examined. 4-Aminotetrahydrothiopyran-4-carboxylic acid (Thp) and 2-aminoindane-2-carboxylic acid (Ain) have been chosen as achiral, conformationally restricted amino acids suitable to mimick the external Met and Phe residues of fMLP-OMe. Studies on a first model, namely [Ain3]fMLP-OMe 1, have already been reported (12). Here the two remaining analogs [Thp1, Ain3] 2 and [Thp1] 3 have been synthesized. The conformation in the crystal of the disubstituted analog 2 has been determined and compared with those adopted by the parent fMLP-OMe and by previously studied models. The backbone conformation of 2 is characterized by helical folding centred at each of the three residues with the central Leu presenting helical handedness opposite to those of the two adjacent achiral residues. This conformation presents strong similarities with that adopted in the crystal by fMLP-OMe and resembles the conformation of fMLP bound to immunoglobulin (Bence-Jones dimer). The conformationally restricted analogs 2 and 3 are more active than the parent in the stimulation of directed mobility of human neutrophils but are practically inactive in the superoxide production. Crystals of 2 are orthorhombic, s.g. P2(1)2(1)2(1), with a = 21.934 (8), b = 10.856 (2), c = 10.380 (2) A. The structure has been refined to R = 0.071 for 2301 independent reflections with I greater than 1.5 sigma.