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The antiviral and antiproliferative activities of human type I interferons (IFNs) are mediated by two transmembrane receptor subunits, IFNAR1 and IFNAR2. To elucidate the role of IFNAR1 in IFN binding and the establishment of biological activity, specific residues of IFNAR1 were mutated. Residues (62)FSSLKLNVY(70) of the S5-S6 loop of the N-terminal subdomain of IFNAR1 and tryptophan-129 of the second subdomain of IFNAR1 were shown to be crucial for IFN-alpha binding and signaling and establishment of biological activity. Mutagenesis of peptide (278)LRV in the third subdomain shows that these residues are critical for IFN-alpha-induced biological activity but not for ligand binding. These data, together with the sequence homology of IFNAR1 with cytokine receptors of known structure and the recently resolved NMR structure of IFNAR2, led to the establishment of a three-dimensional model of the human IFN-alpha/IFNAR1/IFNAR2 complex. This model predicts that following binding of IFN to IFNAR1 and IFNAR2 the receptor complex assumes a "closed form", in which the N-terminal domain of IFNAR1 acts as a lid, resulting in the activation of intracellular kinases. Differences in the primary sequence of individual IFN-alpha subtypes and resulting differences in binding affinity, duration of ligand/receptor association, or both would explain differences in intracellular signal intensities and biological activity observed for individual IFN-alpha subtypes.

A simple method for predicting residues involved in protein interaction sites is proposed. In the absence of any structural report, the procedure identifies linear stretches of sequences as "receptor-binding domains" (RBDs) by analysing hydrophobicity distribution. The sequences of two databases of non-homologous interaction sites eliciting various biological activities were tested; 59-80 % were detected as RBDs. A statistical analysis of amino acid frequencies was carried out in known interaction sites and in predicted RBDs. RBDs were predicted from the 80,000 sequences of the Swissprot database. In both cases, arginine is the most frequently occurring residue. The RBD procedure can also detect residues involved in specific interaction sites such as the DNA-binding (95 % detected) and Ca-binding domains (83 % detected). We report two recent analyses; from the prediction of RBDs in sequences to the experimental demonstration of the functional activities. The examples concern a retroviral Gag protein and a penicillin-binding protein. We support that this method is a quick way to predict protein interaction sites from sequences and is helpful for guiding experiments such as site-specific mutageneses, two-hybrid systems or the synthesis of inhibitors.

The class B M1-V577 penicillin-binding protein (PBP) 3 of Escherichia coli consists of a M1-L39 membrane anchor (bearing a cytosolic tail) that is linked via a G40-S70 intervening peptide to an R71-I236 non-catalytic module (containing the conserved motifs 1-3) itself linked via motif 4 to a D237-V577 catalytic module (containing the conserved motifs 5-7 of the penicilloyl serine transferases superfamily). It has been proposed that during cell septation the peptidoglycan crosslinking activity of the acyl transferase module of PBP3 is regulated by the associated M1-I236 polypeptide itself in interaction with other components of the divisome. The fold adopted by the R71-V577 polypeptide of PBP3 has been modelled by reference to the corresponding R76-S634 polypeptide of the class B Streptococcus pneumoniae PBP2x. Based on these data and the results of site-directed mutagenesis of motifs 1-3 and of peptide segments of high amphiphilicity (identified from hydrophobic moment plots), the M1-I236 polypeptide of PBP3 appears to be precisely designed to work in the way proposed. The membrane anchor and the G40-S70 sequence (containing the G57-Q66 peptide segment) upstream from the non-catalytic module have the information ensuring that PBP3 undergoes proper insertion within the divisome at the cell septation site. Motif 1 and the I74-L82 overlapping peptide segment, motif 2 and the H160-G172 overlapping peptide segment, and the G188-D197 motif 3 are located at or close to the intermodule junction. They contain the information ensuring that PBP3 folds correctly and the acyl transferase catalytic centre adopts the active configuration. The E206-V217 peptide segment is exposed at the surface of the non-catalytic module. It has the information ensuring that PBP3 fulfils its cell septation activity within the fully complemented divisome.

Monoclonal antibodies (mAbs) were produced against gastric H,K-ATPase using a theoretical and experimental strategy based on prediction of linear epitopes by molecular modelling followed by production of anti-peptide antibodies. By analysing the alpha subunit sequence, we predicted several epitopes corresponding to amino acids K519-L533, E543-Y553 and S786-L798 and produced monoclonal antibodies HK519, HK543 and HK786. All three react against gastric H,K-ATPase in RaLISA, immunohistochemistry and Western blots demonstrating that they recognize the native and the SDS-denatured ionic pump and that the epitopes are located at the surface of the native ATPase. Antibody Kd are in the range 6-10x10(-8) M. Monoclonal antibody HK519 is a competitive inhibitor of ATP, in agreement with ATP binding to K519. Neither mAb 543, nor mAb 786 inhibit the ATPase activity. Monoclonal antibody 95111, whose epitope is mapped between residues C529 and E561, competes with mAb HK543 but not with the other two. We suggest that the 95111 epitope is overlapping or very close to the HK543-553 sequence. Induction of E1 conformer by binding FITC to K519 increases the number of mAb 95111 and mAb HK543 epitopes but not that of mAb 786, supporting the fact that the fragment E543-Y553 changes accessibility, maybe during the E1-E2 transconformation.

International audience; 2-D and 3-D NMR techniques were used to investigate the conformations in solution of several peptides and proteins for which crystalline structures are not available yet. Insect defensin A is a small (40 aa) antibiotic protein exhibiting a characteristic 'loop-helix-beta-sheet' structure. A striking analogy was found with charybdotoxin, a scorpion toxin in which a CSH (cysteine stabilized alpha-helix) motif is also present. Wheat phospholipid transfer protein (PLTP) (90 aa) has a 3-D structure resulting from the packing of four helices and of a C-terminal less well-defined fragment. Preliminary results show that PLTP forms a complex with lyso-PC and that such an interaction results in a conformational change affecting principally the C-terminal half of the protein. A last example is given with surfactin, a lipopeptide biosurfactant from bacterial origin. Its protonated form shows a very compact structure in which the two acidic residues located on the top of a 'horse saddle' topology face each other, whereas the ionized form could adopt a more extended conformation. A common property of these compounds is their capacity to interact with lipids. The present structural data open the way for a further establishment of structure-activity relationships.

International audience; A 500 MHz 2D 1H NMR study of recombinant insect defensin A is reported. This defense protein of 40 residues contains 3 disulfide bridges, is positively charged and exhibits antibacterial properties. 2D NMR maps of recombinant defensin A were fully assigned and secondary structure elements were localized. The set of NOE connectivities, 3JNH-alpha H coupling constants as well as 1H/2H exchange rates and delta delta/delta T temperature coefficients of NH protons strongly support the existence of an alpha-helix (residues 14-24) and of an antiparallel beta-sheet (residues 27-40). Models of the backbone folding were generated by using the DISMAN program and energy refined by using the AMBER program. This was done on the basis of: (i) 133 selected NOEs, (ii) 21 dihedral restraints from 3JNH-alpha H coupling constants, (iii) 12 hydrogen bonds mostly deduced from 1H/2H exchange rates or temperature coefficients, in addition to 9 initial disulfide bridge covalent constraints. The two secondary structure elements and the two bends connecting them involve approximately 70% of the total number of residues, which impose some stability in the C-terminal part of the molecule. The remaining N-terminal fragment forms a less well defined loop. This spatial organization, in which a beta-sheet is linked to an alpha-helix by two disulfide bridges and to a large loop by a third disulfide bridge, is rather similar to that found in scorpion charybdotoxin and seems to be partly present in several invertebrate toxins.

Butenafine ( N -4- tert -butylbenzyl- N -methyl-1-naphtalenemethylamine hydrochloride) is an antifungal agent of the benzylamine class that has excellent therapeutic efficacy and a remarkably long duration of action when applied topically to treat various mycoses. Given the lipophilic nature of the molecule, efficacy may be related to an interaction with cell membrane phospholipids and permeabilization of the fungal cell wall. Similarly, high lipophilicity could account for the long duration of action, since fixation to lipids in cutaneous tissues might allow them to act as local depots for slow release of the drug. We have therefore used computer-assisted conformational analysis to investigate the interaction of butenafine with lipids and extended these observations with experimental studies in vitro using liposomes. Conformational analysis of mixed monolayers of phospholipids with the neutral and protonated forms of butenafine highlighted a possible interaction with both the hydrophilic and hydrophobic domains of membrane phospholipids. Studies using liposomes demonstrated that butenafine increases membrane fluidity [assessed by fluorescence polarization of 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene and 1,6-diphenylhexatriene] and membrane permeability (studied by release of calcein from liposomes). The results show, therefore, that butenafine readily interacts with lipids and is incorporated into membrane phospholipids. These findings may help explain the excellent antifungal efficacy and long duration of action of this drug when it is used as a topical antifungal agent in humans.

Penicillin-binding protein 4a (PBP4a) from Bacillus subtilis was overproduced and purified to homogeneity. It clearly exhibits dd -carboxypeptidase and thiolesterase activities in vitro. Although highly isologous to the Actinomadura sp. strain R39 DD-peptidase (B. Granier, C. Duez, S. Lepage, S. Englebert, J. Dusart, O. Dideberg, J. van Beeumen, J. M. Frère, and J. M. Ghuysen, Biochem. J. 282:781–788, 1992), which is rapidly inactivated by many β-lactams, PBP4a is only moderately sensitive to these compounds. The second-order rate constant ( k 2 / K ) for the acylation of the essential serine by benzylpenicillin is 300,000 M −1 s −1 for the Actinomadura sp. strain R39 peptidase, 1,400 M −1 s −1 for B. subtilis PBP4a, and 7,000 M −1 s −1 for Escherichia coli PBP4, the third member of this class of PBPs. Cephaloridine, however, efficiently inactivates PBP4a ( k 2 / K = 46,000 M −1 s −1 ). PBP4a is also much more thermostable than the R39 enzyme.

Dipeptidyl peptidase IV (DPP IV/CD26) belongs to a non-classical subfamily of serine-proteases. Sequence comparisons have identified Asp 599 , Ser 624 , Asp 657 , Asp 702 , and His 734 as highly conserved residues of mouse DPP IV. We previously reported the identification of Ser 624 , Asp 702 and His 734 as the catalytic triad of mouse DPP IV (David, F., Bernard, A. M., Pierres, M., and Marguet, D. (1993) J. Biol. Chem. 268, 17247–17252). Using site-directed mutagenesis, we have shown here that substitution of Asp 599 for Ala (D599A) specifically decreases the cell-surface expression of DPP IV in stably transfected mouse fibroblasts. The D599A mutant remained as a high mannose immature glycoprotein and was rapidly degraded. This retention/degradation process correlates with the generation of a β strand in the C-terminal region of DPP IV as shown by three dimensional computer modeling. Our results suggest that conserved residue Asp 599 is important for the proper folding, glycosylation and transport of mouse DPP IV.

Antibodies are powerful tools for studying the in situ localization and physiology of proteins. The prediction of epitopes by molecular modelling has been used successfully for the papilloma virus, and valuable antibodies have been raised [Muller et al. (1990) J. Gen. Virol., 71, 2709-2717]. We have improved the modelling approach to allow us to predict epitopes from the primary sequences of the cystic fibrosis transmembrane conductance regulator. The procedure involves searching for fragments of primary sequences likely to make amphipathic secondary structures, which are hydrophilic enough to be at the surface of the folded protein and thus accessible to antibodies. Amphipathic helices were predicted using the methods of Berzofsky, Eisenberg and Jahnig. Their hydrophobic-hydrophilic interface was calculated and drawn, and used to predict the orientation of the helices at the surface of the native protein. Amino acids involved in turns were selected using the algorithm of Eisenberg. Tertiary structures were calculated using 'FOLDING', a software developed by R. Brasseur for the prediction of small protein structures [Brasseur (1995) J. Mol. Graphics, in press]. We selected sequences that folded as turns with at least five protruding polar residues. One important property of antibodies is selectivity. To optimize the selectivity of the raised antibodies, each sequence was screened for similarity (FASTA) to the protein sequence from several databanks. Ubiquitous sequences were discarded. This approach led to the identification of 13 potential epitopes in the cystic fibrosis transmembrane conductance regulator: seven helices and six loops.

Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the N-terminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably the length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity.

Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the N-terminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably the length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity.