NH2-terminal Segment Research Articles

Structural Analysis of the fds Operon Encoding the NAD+-linked Formate Dehydrogenase of Ralstonia eutropha

The fdsGBACD operon encoding the four subunits of the NAD+-reducing formate dehydrogenase of Ralstonia eutropha H16 was cloned and sequenced. Sequence comparisons indicated a high resemblance of FdsA (alpha-subunit) to the catalytic subunits of formate dehydrogenases containing a molybdenum (or tungsten) cofactor. The NH2-terminal region (residues 1-240) of FdsA, lacking in formate dehydrogenases not linked to NAD(P)+, exhibited considerable similarity to that of NuoG of the NADH:ubiquinone oxidoreductase from Escherichia coli as well as to HoxU and the NH2-terminal segment of HndD of NAD(P)+-reducing hydrogenases. FdsB (beta-subunit) and FdsG (gamma-subunit) are closely related to NuoF and NuoE, respectively, as well as to HoxF and HndA. It is proposed that the NH2-terminal domain of FdsA together with FdsB and FdsG constitute a functional entity corresponding to the NADH dehydrogenase (diaphorase) part of NADH:ubiquinone oxidoreductase and the hydrogenases. No significant similarity to any known protein was observed for FdsD (delta-subunit). The predicted product of fdsC showed the highest resemblance to FdhD from E. coli, a protein required for the formation of active formate dehydrogenases in this organism. Transcription of the fds operon is subject to formate induction. A promoter structure resembling the consensus sequence of sigma70-dependent promoters from E. coli was identified upstream of the transcriptional start site determined by primer extension analysis.

The diphtheria toxin channel-forming T domain translocates its own NH2-terminal region across planar bilayers.

The T domain of diphtheria toxin, which extends from residue 202 to 378, causes the translocation of the catalytic A fragment (residues 1-201) across endosomal membranes and also forms ion-conducting channels in planar phospholipid bilayers. The carboxy terminal 57-amino acid segment (322-378) in the T domain is all that is required to form these channels, but its ability to do so is greatly augmented by the portion of the T domain upstream from this. In this work, we show that in association with channel formation by the T domain, its NH2 terminus, as well as some or all of the adjacent hydrophilic 63 amino acid segment, cross the lipid bilayer. The phenomenon that enabled us to demonstrate that the NH2-terminal region of the T domain was translocated across the membrane was the rapid closure of channels at cis negative voltages when the T domain contained a histidine tag at its NH2 terminus. The inhibition of this effect by trans nickel, and by trans streptavidin when the histidine tag sequence was biotinylated, clearly established that the histidine tag was present on the trans side of the membrane. Furthermore, the inhibition of rapid channel closure by trans trypsin, combined with mutagenesis to localize the trypsin site, indicated that some portion of the 63 amino acid NH2-terminal segment of the T domain was also translocated to the trans side of the membrane. If the NH2 terminus was forced to remain on the cis side, by streptavidin binding to the biotinylated histidine tag sequence, channel formation was severely disrupted. Thus, normal channel formation by the T domain requires that its NH2 terminus be translocated across the membrane from the cis to the trans side, even though the NH2 terminus is >100 residues removed from the channel-forming part of the molecule.

Interaction of caldesmon with actin subdomain-2.

The polymerization-resistant maleimidobenzoyl-G-actin (MBS-G-actin), which behaves as a functional analogue of native G-actin [Bettache, N., Bertrand, R. & Kassab, R. (1989) Proc. Natl Acad. Sci. USA 86, 6028-6032; Bettache, N., Bertrand, R. & Kassab, R. (1990) Biochemistry 29, 9085-9091) has been employed to probe the solution interaction between monomeric actin and smooth muscle caldesmon, using fluorescence measurements, limited proteolysis and covalent cross-linking reactions. MBS-G-actin associates, without polymerization, to turkey gizzard caldesmon, at about 50 mM ionic strength and 25 degrees C, with a high affinity (Kd approximately 0.04 microM) and with a 1:1 stoichiometry. However, the binding strength of the complex including caldesmon and MBS-G-actin cleaved at the subdomain-2 loop with subtilisin decreased fivefold (Kd approximately 0.20 microM). Conversely, caldesmon strongly protected subdomain-2 of MBS-G-actin from tryptic digestion at the susceptible peptide bond at positions 68-69. Furthermore, caldesmon induced the dissociation of native G-actin from its complex with DNase I, as assessed by cosedimentation assays, and increasing concentrations of the latter protein inhibited the MBS-G-actin-caldesmon interaction, suggesting mutual exclusion binding of caldesmon and DNase I to monomeric actin. MBS-G-actin was specifically coupled, via a maleimidobenzoyl group incorporated into its subdomain-2, to caldesmon, producing in high yield a 205-kDa covalent complex consisting of one actin monomer joined to Cys 580 of caldesmon. A similar conjugation process was observed with the complex of caldesmon and polymerized MBS-F-actin. MBS-G-actin could be also cross-linked to caldesmon by 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide, producing a three-band pattern identical to that of F-actin and caldesmon and previously shown to reflect the covalent union between the NH2-terminal segment of actin and the COOH-terminal actin-binding domain of caldesmon. The overall data point to a direct interaction of the latter region with actin subdomain-2 and suggest that during its binding to monomeric or filamentous actin, the caldesmon functional domain spans the entire length of a single actin and closely contacts the bottom of its subdomain-1 as well as the top portion of its subdomain-2.

Transmembrane Topography of Subunit a in theEscherichia coli F1F0 ATP Synthase

Subunit a is the least understood of the three subunits that compose the F0 sector in the Escherichia coli F0F1 ATP synthase. In this study, we have substituted Cys into predicted extramembranous loops of the protein and used chemical modification to obtain topographical information on the folding of subunit a. The extent of labeling of the substituted Cys residues by fluorescein-5'-maleimide was determined. The localization of reactive Cys residues was inferred from differences in the extent of labeling in inside out and right side out membrane vesicles. The NH2-terminal segment of subunit a was localized to the outside (periplasmic) surface and the COOH terminus to the cytoplasmic surface by these procedures. Loop residues in two periplasmic extramembranous loops and in two cytoplasmic extramembranous loops were also localized. The localization of two cytoplasmic Cys residues was confirmed by using 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid to block fluorescein-5'-maleimide labeling. From the localization of the Cys residues, a model for the topography is proposed that consists of five transmembrane segments with the NH2 terminus periplasmic and the COOH terminus cytoplasmic. The positions of second site suppressors, including several isolated here to the nonfunctional E219C and H245C substitutions, provide support for the topographical model proposed.

Structural characterization and topology of the second potential membrane anchor region in the thromboxane A2 synthase amino-terminal domain.

Thromboxane A2 synthase (TXAS) has been proposed to have two membrane-bound regions located in the NH2-terminal domain [Ruan, K.-H., Wang, L.-H., Wu, K. K., and Kulmacz, R. J. (1993) J. Biol. Chem, 268, 19483-19489; Ruan, K.-H., Li, P., Kulmacz, J. R., and Wu, K. K. (1994) J. Biol. Chem, 269, 20938-20942]. To test this hypothesis, a solution structure in membrane mimetic environments of a synthetic peptide corresponding to the second region of the NH2-terminal domain (TXAS residues 33-60) has been investigated by circular dichroism (CD), 2D nuclear magnetic resonance (NMR) spectroscopy, and peptidoliposome reconstitution. CD spectroscopy indicated that the peptide adopted a structure with significant alpha-helical content in 30% trifluoroethanol (TFE) or in dodecylphosphocholine (DPC) micelles, which mimic hydrophobic membrane environment. Through a combination of 2D NMR experiments in the presence of TFE or DPC micelles, complete 1H NMR assignments of the peptide have been obtained and the structure of the peptide has been determined. NH2-terminal segment of the peptide takes on a well-defined alpha-helical conformation; the center segment of the peptide, containing three prolines, adopts a bent conformation, and the C-terminal segment of the peptide exists in a mixture of rapidly interconverting conformations. These results provide direct structural evidence that residues 33-60 of the TXAS NH2-terminal domain contain a second membrane anchor region, with at least residues 35-46 having their helical structure expected for hydrophobic interaction with the membrane. The orientation of the peptide in DPC micelles was evaluated from the effect of incorporation of a spin-label 12-doxylstearate into the micelles. The peptide portions, found to be immersed in the micelles, include the helical segment, the bent segment, and some hydrophobic residues within the C-terminal segment. Two additional synthetic peptides, one corresponding to the NH2-terminal helical segment (TXAS residues 33-46) and the other including the bent and the C-terminal segments (TXAS residues 47-60) were analyzed for their ability to incorporate into peptidoliposomes. The helical peptide readily incorporated into liposomes; the other peptide did not. These results support the presence of a second functional membrane anchor region localized to the helical segment within TXAS residues 33-46, with passive membrane contacts in the bent and the C-terminal segments of the peptide (TXAS residues 47-60) due to immersion of the helical in the membrane.

Demonstration of Coiled-Coil Interactions within the Kinesin Neck Region Using Synthetic Peptides: IMPLICATIONS FOR MOTOR ACTIVITY

Kinesin is a dimeric motor protein that can move for several micrometers along a microtubule without dissociating. The two kinesin motor domains are thought to move processively by operating in a hand-over-hand manner, although the mechanism of such cooperativity is unknown. Recently, a approximately 50-amino acid region adjacent to the globular motor domain (termed the neck) has been shown to be sufficient for conferring dimerization and processive movement. Based upon its amino acid sequence, the neck is proposed to dimerize through a coiled-coil interaction. To determine the accuracy of this prediction and to investigate the possible function of the neck region in motor activity, we have prepared a series of synthetic peptides corresponding to different regions of the human kinesin neck (residues 316-383) and analyzed each peptide for its respective secondary structure content and stability. Results of our study show that a peptide containing residues 330-369 displays all of the characteristics of a stable, two-stranded alpha-helical coiled-coil. On the other hand, the NH2-terminal segment of the neck (residues approximately 316-330) has the capacity to adopt a beta-sheet secondary structure. The COOH-terminal residues of the neck region (residues 370-383) are not alpha-helical, nor do they contribute significantly to the overall stability of the coiled-coil, suggesting that these residues mark the beginning of a hinge located between the neck and the extended alpha-helical coiled coil stalk domain. Interestingly, the two central heptads of the coiled-coil segment in the neck contain conserved, "non-ideal" residues located within the hydrophobic core, which we show destabilize the coiled-coil interaction. These residues may enable a portion of the coiled-coil to unwind during the mechanochemical cycle, and we present a model in which such a phenomenon plays an important role in kinesin motility.

Regulation of Na,K-ATPase transport activity by protein kinase C.

Considerable evidence indicates that the renal Na+,K+-ATPase is regulated through phosphorylation/dephosphorylation reactions by kinases and phosphatases stimulated by hormones and second messengers. Recently, it has been reported that amino acids close to the NH2-terminal end of the Na+,K+-ATPase alpha-subunit are phosphorylated by protein kinase C (PKC) without apparent effect of this phosphorylation on Na+,K+-ATPase activity. To determine whether the alpha-subunit NH2-terminus is involved in the regulation of Na+, K+-ATPase activity by PKC, we have expressed the wild-type rodent Na+,K+-ATPase alpha-subunit and a mutant of this protein that lacks the first thirty-one amino acids at the NH2-terminal end in opossum kidney (OK) cells. Transfected cells expressed the ouabain-resistant phenotype characteristic of rodent kidney cells. The presence of the alpha-subunit NH2-terminal segment was not necessary to express the maximal Na+,K+-ATPase activity in cell membranes, and the sensitivity to ouabain and level of ouabain-sensitive Rb+-transport in intact cells were the same in cells transfected with the wild-type rodent alpha1 and the NH2-deletion mutant cDNAs. Activation of PKC by phorbol 12-myristate 13-acetate increased the Na+,K+-ATPase mediated Rb+-uptake and reduced the intracellular Na+ concentration of cells transfected with wild-type alpha1 cDNA. In contrast, these effects were not observed in cells expressing the NH2-deletion mutant of the alpha-subunit. Treatment with phorbol ester appears to affect specifically the Na+,K+-ATPase activity and no evidence was observed that other proteins involved in Na+-transport were affected. These results indicate that amino acid(s) located at the alpha-subunit NH2-terminus participate in the regulation of the Na+,K+-ATPase activity by PKC.

Identification of A2a Adenosine Receptor Domains Involved in Selective Coupling to GS: ANALYSIS OF CHIMERIC A1/A2a ADENOSINE RECEPTORS

Responses to adenosine are governed by selective activation of distinct G proteins by adenosine receptor (AR) subtypes. The A2aAR couples via Gs to adenylyl cyclase stimulation while the A1AR couples to Gi to inhibit adenylyl cyclase. To determine regions of the A2aAR that selectively couple to Gs, chimeric A1/A2aARs were expressed in Chinese hamster ovary cells and ligand binding and adenylyl cyclase activity analyzed. Replacement of the third intracellular loop of the A2aAR with that of the A1AR reduced maximal adenylyl cyclase stimulation and decreased agonist potency. Restricted chimeras indicated that the NH2-terminal portion of intracellular loop 3 was predominantly responsible for this impairment. Reciprocal chimeras composed primarily of A1AR sequence with limited A2aAR sequence substitution stimulated adenylyl cyclase and thus supported these findings. A lysine and glutamic acid residue were identified as necessary for efficient A2aAR-Gs coupling. Analysis of chimeric receptors in which sequence of intracellular loop 2 was substituted indicated that the nature of amino acids in this domain may indirectly modulate A2aAR-Gs coupling. Replacement of the cytoplasmic tail of the A2aAR with the A1AR tail did not affect adenylyl cyclase stimulation. Thus, selective activation of Gs is predominantly dictated by the NH2-terminal segment of the third intracellular loop of the A2aAR.

Identification of a second region of the beta-subunit involved in regulation of calcium channel inactivation.

Previous studies have shown that NH2 termini of the type 1 and 2 beta-subunits modulate the rate at which the neuronal alpha 1E calcium channel inactivates in response to voltage and that they do so independently of their common effect to stimulate activation by voltage (R. Olcese, N. Qin, T. Schneider, A. Neely, X. Wei, E. Stefani, and L. Birnbaumer, Neuron 13: 1433-1438, 1994). By constructing NH2-terminal deletions of several splice variants of beta-subunits, we have now found differences in the way they affect the rate of alpha 1E inactivation that lead us to identify a second domain that also regulates the rate of voltage-induced inactivation of the Ca2+ channel. This second domain, named segment 3, lies between two regions of high-sequence identity between all known beta-subunits and exists in two lengths (long and short), each encoded in a separate exon. Beta-Subunits with the longer 45- to 53-amino acid version cause the channel to inactivate more slowly than subunits with the shorter 7-amino acid version. As is the case for the NH2 terminus, the segment 3 does not affect the regulation of channel activation by the beta-subunit. In addition, the effect of the NH2-terminal segment prevails over that of the internal segment. This raises the possibility that phosphorylation, other types of posttranslational modification, or interaction with other auxiliary calcium channel subunits may be necessary to unmask the regulatory effect of the internal segment.

Different Regions in Activation Function-1 of the Human Estrogen Receptor Required for Antiestrogen- and Estradiol-dependent Transcription Activation

The human estrogen receptor (ER) is a ligand-inducible transcription factor that contains two transcriptional activation functions, one located in the NH2-terminal region of the protein (AF-1) and the second in the COOH-terminal region (AF-2). Antiestrogens, such as trans-hydroxytamoxifen (TOT), have partial agonistic activity in certain cell types, and studies have implied that this agonism is AF-1-dependent. We have made progressive NH2-terminal and other segment deletions and ligations in the A/B domain, and studied the transcriptional activity of these mutant ERs in ER-negative MDA-MB-231 human breast cancer and HEC-1 human endometrial cancer cells. Using several estrogens and several partial agonist/antagonist antiestrogens, we find that estrogens and antiestrogens require different regions of AF-1 for transcriptional activation. Deletion of the first 40 amino acids has no effect on receptor activity. Antiestrogen agonism is lost upon deletion to amino acid 87, while estrogen agonism is not lost until deletions progress to amino acid 109. Antiestrogen agonism has been further defined to require amino acids 41-64, as deletion of only these amino acids results in an ER that exhibits 100% activity with E2, but no longer shows an agonist response to TOT. With A/B-modified receptors in which antiestrogens lose their agonistic activity, the antiestrogens then function as pure estrogen antagonists. Our studies show that in these cellular contexts, hormone-dependent transcription utilizes a range of the amino acid sequence within the A/B domain. Furthermore, the agonist/antagonist balance and activity of antiestrogens such as TOT are determined by specific sequences within the A/B domain and thus may be influenced by differences in levels of specific factors that interact with these regions of the ER.

Binding of Fatty acid Anions to .BETA.-Conglycinin: Physical and Chemical Modification Studies on the Role of Protein Conformation and Positively Charged Groups as Binding Sites.

Binding of decanoate to soybean β-conglycinin has been studied using physically and chemically modified β-conglycinin. The role of the native conformation and the cationic groups as the binding sites and the location on the amino acid sequence have been investigated. The native conformation with a quaternary structure showed the highest number of bound ligand. None of the modified β-conglycinins binds decanoate quite as firmly as the intact β-conglycinin molecule. Competitive binding studies demonstrated that the binding of methyl orange and fatty acid to β-conglycinin occurred at the same cationic sites. The association of carboxylate anions with β-conglycinin involves electrostatic attraction of the carboxylate anions to protein cationic sites that lied close by the NH2-terminal together with the hydrophobic interactions between the fatty acid hydrocarbon tail and the nonpolar side chains of the protein. Studies with reduced and non-reduced β-conglycinins have provided additional insight into the binding mechanism. The disulfide bond formed between subunits reduces the flexibility of the β-conglycinin molecule, especially around the NH2-terminal segment, producing marked changes in the fatty acid binding characteristics of β-conglycinin. The strong fatty acid binding sites may be located in the cleft between the α and α' subunit contact regions.

Expression, Zinc-Affinity Purification, and Characterization of a Novel Metal-Binding Cluster in Troponin T: Metal-Stabilized α-Helical Structure and Effects of the NH2-Terminal Variable Region on the Conformation of Intact Troponin T and Its Association with Tropomyosin

A repeating metal-binding (Cu2+ > Ni2+ > Zn2+ approximately Co2+) sequence [HE/AEAH]4 (Tx) has been recently identified in the NH2-terminal variable region of troponin T (TnT) isoforms specifically expressed in the breast but not leg muscles of the avian orders of Galliformes and Craciformes [Jin, J.-P., & Smillie, L. B. (1994) FEBS Lett. 341, 135-140]. In the present study, two expression plasmids were constructed to produce chicken TnT1 NH2-terminal fragments of 47 (N47) or 165 (N165) amino acids containing the Tx metal-binding cluster. The recombinant protein/peptide was expressed in Escherichia coli BL21(DE3)pLysS and purified by a highly effective Zn(2+)-affinity chromatography method. Amino acid analyses, NH2-terminal peptide sequencing, mass spectrometry and immunological identification confirmed the authenticity of the genetically engineered TnT fragments. In the presence of 2,2,2-trifluoroethanol, transition metals had significant effects on the secondary structure of TnT fragment N47, as shown by circular dichroism. N165 in non-denaturing buffer demonstrated alpha-helical content comparable to previous data from rabbit fast skeletal TnT fragment T1. Zn(2+)-binding avidity of the metal-binding TnT and its fragments demonstrated tertiary relationships between the NH2-terminal variable region and the COOH-terminal segment of the intact TnT protein. Solid-phase protein-binding assays established that Zn(2+)-binding to the Tx cluster induces epitopic structure changes in this NH2-terminal segment, further affecting other epitopic structures of intact TnT as well as the function of TnT's tropomyosin binding-sites. The results demonstrate that metal ion-binding to the Tx cluster reconfigures the overall conformation of TnT through structural relationships between the NH2-terminal variable region and other domains of the intact TnT molecule. Accordingly, the developmental and/or muscle type specific NH2-terminal structure of TnT isoforms may modulate the Ca(2+)-activation of muscle contraction.

Isolation and characterization of neutralizing single-chain antibodies against Xenopus mitogen-activated protein kinase kinase from phage display libraries.

MAP kinase kinase (MAPKK) is a dual specificity protein kinase that phosphorylates and activates MAP kinase in vivo. In this study, four mouse monoclonal single-chain Fv (scFv) antibodies (Y1-6, Y1-7, Y3-6, and Y3-11) that can specifically bind to Xenopus MAPKK were isolated from combinatorial scFv-displaying phage libraries. Three scFv clones (Y1-6, Y1-7, and Y3-6) were shown to efficiently inhibit MAPKK activity in vitro. Point mutation (D98K) at VH-CDR3 of one (Y1-6) of these three clones markedly reduced its neutralizing activity. The wild-type scFv (Y1-6) inhibited the Mos-induced MAP kinase activation and germinal vesicle breakdown when injected into immature Xenopus oocytes, whereas the mutant scFv, Y1-6 (D98K), did not. The three neutralizing scFv clones (Y1-6, Y1-7, and Y3-6) were shown to bind to NH2-terminal residues 1-23 of Xenopus MAPKK, whereas the epitope of a Y3-11 clone with no neutralizing activity was shown to lie between residues 33 and 67 of MAPKK. Furthermore, a synthetic peptide (the N16 peptide) corresponding to residues 2-17 of MAPKK suppressed the neutralizing activity of the wild-type Y1-6, and a rabbit polyclonal antibody against the N16 peptide was found to possess a strong neutralizing activity against MAPKK. These results demonstrate that the neutralizing antibodies characterized here inhibit the kinase activity of MAPKK by binding to the NH2-terminal segment of MAPKK.

Effects of Subunit Mutation on the Rotational Dynamics of Fc.epsilon.RI, the High Affinity Receptor for IgE, in Transfected Cells

Erythrosin-labeled immunoglobulin E (IgE) and time-resolved phosphorescence anisotropy were used to monitor the rotational dynamics of transfected wild-type (alpha beta gamma 2) and four mutant Fc epsilon RI receptors in the monomeric and dimeric state on P815 cells. Erythrosin-IgE bound to Fc epsilon RI on cells transfected with either beta or gamma subunits with truncated COOH-terminal cytoplasmic segments exhibit faster rotational motion than when bound to Fc epsilon RI on cells transfected with wild-type subunits. Deletion of the NH2-terminal cytoplasmic segment of the beta subunit or the COOH-terminal cytoplasmic segment of the alpha subunit does not cause any significant change in the anisotropy decay. Dimers of IgE-receptor complexes formed with anti-IgE monoclonal antibody B1E3 exhibit substantially slower anisotropy decays for all the receptor constructs used, including a receptor construct that only contains the ectodomain of the alpha subunit anchored to Chinese hamster ovary (CHO) cell membranes through a lipid tail. This loss of rotational motion of dimeric IgE-Fc epsilon RI complexes may be due to nonspecific entanglement or to specific interactions involving IgE or the extracellular portion of alpha. The results suggest that the beta and gamma subunits of the tetrameric alpha beta gamma 2 receptor participate in interactions with other membrane components even in the absence of receptor aggregation. The loss of such interactions may be related to the functional impairments previously determined for these mutants.

Distinct Regions of Cu(I)·ACE1 Contact Two Spatially Resolved DNA Major Groove Sites

The interaction between the Cu(I).ACE1 (CuACE1) transcription factor and its DNA binding site in the yeast metallothionein gene was studied by systematically altering the DNA sequence through base substitution, modification, and deletions as well as by altering the protein structure through chemical modification. We show here that CuACE1 is comprised of two distinct domains that contact DNA through minor groove interactions located between two major groove interaction sites. The minor groove interactions are shown to be critical for formation of a stable CuACE1.DNA complex. The NH2-terminal segment of ACE1 is shown to contact the 5'-most distal major groove site.

Multiple crystal forms of endoglucanase CelD: Signal peptide residues modulate lattice formation

The crystal structure of Clostridium thermocellum endoglucanase Cell) revealed an extended NH 2-terminal segment (involving residues from the putative leader peptide) sticking out from the enzyme core to interact with a symmetry related molecule through an intermolecular salt bridge (LyAbstract8-Asp201). Enzymatic digestion of Cell) with various proteases emphasized the flexibility of the NH 2-segment in solution. Proteolytic removal of LyAbstract8 or the substitution of bridge-forming residues by site-directed mutagenesis promoted crystal packing arrangements that differ from that of wild type Cell). Crystals of wild-type CeID ( a = 99.3 Å c =191.8 Å) are trigonal, space group P3,21, with one molecule in the asymmetric unit (form A), whereas crystals of papain-treated CeID ( a = 100.4 Å, c = 248.7 Å), of CelD K38M ( a = 100.1 A, c = 248.4 A) and of papain-treated CeID D201A (a = 99.9 Å, c = 250.0 Å) are trigonal, space group P3,21, with two crystallographically independent molecules (form B), and crystals of chymotrypsin-treated Cell) ( a = 100.0 Å, c = 254.3 Å) and of CelD D201A ( a = 99.8 Å, c = 254.7 Å) are hexagonal, space group P6,22, with one molecule in the asymmetric unit (form C). Only chymotrypsin-treated Cell) (which preserves both LyAbstract8 and Asp201) can grow in crystal form A upon macroseeding, indicating that formation of the intermolecular salt bridge is critical for stability of this crystal form. Flexible NH 2- and COOH-terminal peptide extensions were found to influence crystal nucleation, but not crystal growth. The crystal structures of papain-treated Cell) and chymotrypsin-treated CeID, determined at 3.5 Å resolution by molecular replacement techniques, demonstrate that a small change in molecular orientation promoted by LyAbstract8 account for the differences between crystal forms B and C.

Interaction of fluorescently labeled analogues of the amino-terminal fusion peptide of Sendai virus with phospholipid membranes.

A peptide representing the NH2-terminal (33 amino acid residues) of the fusion protein (F) of Sendai virus, as well as its Gly12-->Ala12 mutant, were synthesized, fluorescently labeled, and spectroscopically and functionally characterized. Peptide-induced vesicle fusion was demonstrated by a combination of increased visible absorbance, lipid mixing assay, and electron microscopy. Both peptides, with the mutant peptide being significantly more potent, were shown to induce membrane fusion and bilayer perturbation of negatively charged phospholipid vesicles. These results are consistent with a previous study that showed that a similar mutation in the homologous NH2-terminal segment of simian virus 5 greatly enhanced syncytium formation (Horvath, C. M., and Lamb, R. A. (1992) J. Virol. 66, 2443-2455). Circular dichroism spectroscopy revealed similar high alpha-helical contents of both peptides in methanol and in trifluoroethanol. Using fluorescently labeled peptide analogues we found that (i) the peptides' membrane partition coefficients are in the range of 10(5) M-1; (ii) the NH2 terminus of the wild-type peptide is located within the lipid bilayer, whereas that of the variant peptide lies on the surface; and (iii) both peptides tend to self-associate in their membrane-bound state. The results support a model in which an alpha-helical secondary structure and self-aggregation of peptides are necessary conditions for membrane fusion. The observed differences in the peptides' fusogenic abilities are hypothesized to result from differences in the peptides' degree of penetration into the membrane, induction of membrane destabilization, and ability to cause vesicles to aggregate. The data support Sendai virus-cell fusion models in which the fusion peptide plays a crucial role in fusion induction by destabilizing the bilayer and by triggering the association of viral fusion protein molecules.

The NH2-terminal region of C5aR but not that of FPR is critical for both protein transport and ligand binding.

The N-formylated tripeptide, formylmethionylleucyl-phenylalanine (fMLP), and the 74-amino-acid long human C5a anaphylatoxin activate phagocytic cells via two structurally related G protein-coupled receptors (FPR and C5aR), which are 34% identical in amino acid sequence. C5aR chimeras were constructed in which the entire NH2-terminal extracellular sequence or part of it was replaced by the counterparts from FPR or FPRH. Although the NH2-terminal region of C5aR presents an extremely high interspecies variability, substitution of the entire NH2-terminal sequence of C5aR by that of FPR or FPRH surprisingly resulted in chimeras that were apparently retained in the endoplasmic reticulum. In contrast, when the NH2-terminal domain of FPR was replaced by the corresponding region from C5aR or FPRH normal expression to the plasma membrane and high affinity binding of N-formylated peptides were observed. Thus, the NH2-terminal region of C5aR, in contradistinction to that of FPR, seems to be required either for the translocation of C5aR through the ER membrane or for correct folding and sorting of C5aR to the plasma membrane. Replacement of the first 8 residues of C5aR by the corresponding region of FPR did not alter the cellular transport and the C5a binding capacity, whereas the exchange of the first 13 residues resulted in a chimera that was readily transported to the plasma membrane but showed no capability to bind C5a. Mutations of Asp into Asn in the NH2-terminal segment of C5aR further indicated that negative charges are required to endow the receptor with a C5a binding capacity. The residues critical for binding are either involved directly by interacting with cationic residues of C5a, or indirectly by influencing the overall structure of the ligand-binding site.

The NH2-terminal alpha-helical domain 1-18 of dermaseptin is responsible for antimicrobial activity.

Dermaseptin, a 34-amino acid residue cationic peptide, was recently shown to inhibit the growth of pathogenic fungi responsible for severe opportunistic infections accompanying immunodeficiency syndrome and the use of immunosuppressive agents. To improve our understanding of the mechanism by which dermaseptin exerts its potent antimicrobial action, a series of either NH2- or COOH-terminally truncated analogs was synthesized. These analogs were evaluated for their ability to inhibit the growth of various pathogenic agents in culture medium. Dermaseptin exerted a lytic action upon bacteria, protozoa, yeasts, and filamentous fungi at micromolar concentrations. No inhibition of proliferation was observed with human KB cells, and dermaseptin did not lyse guinea pig lymphocytes or rabbit erythrocytes at doses up to 200 micrograms/ml. Shortening the peptide chain of dermaseptin to dermaseptin-(3-34) slightly reduced the activity of the peptide, while further reduction of the chain length to residues 14-34, 16-34, 20-34, and 28-34 yielded peptide derivatives devoid of antimicrobial activity. On the other hand, lengthening the peptide chain starting from residues 1-4 to residues 1-8 and 1-18 led to a progressive recovery of the activity of the parent molecule. Whereas the central core of dermaseptin (residues 10-19) was virtually inactive, alteration of the COOH-terminal carboxylic group of dermaseptin-(1-18) to a carboxamide yielded a peptide exhibiting enhanced antimicrobial potency, yet displaying even less in vitro toxicity compared with dermaseptin. Overall, the data indicate that molecular elements responsible for the exceptional antimicrobial potency of dermaseptin are to be traced to the NH2-terminal alpha-helical amphipathic segment spanning residues 1-18 of the molecule. Dermaseptin-(1-18)-NH2 may therefore be considered as a useful and highly tractable tool for identifying key features responsible for membrane permeabilization and as a starting point for the design of new therapeutic agents.

Antigen-specific deletion of cloned T cells using peptide-toxin conjugate complexed with purified class II major histocompatibility complex antigen.

In a previous report, we showed that cloned T cells incubated with soluble, cognate major histocompatibility complex (MHC) II-peptide complex internalized the peptide moiety of the complex. Here, we report antigen-specific deletion of cloned T cells by treatment with soluble, cognate MHC II-(peptide-toxin) complexes. Toxin (doxorubicin or mycophenolic acid) was attached to synthetic AcMBP(1-14)Ala4 peptide, an analog of the natural acetylated NH2-terminal segment, AcMBP(1-14), of rat myelin basic protein (MBP). IAk-restricted, AcMBP(1-14)-Specific AJ1.2 and 4R3.9 cloned murine T cells were killed by IAk-(AcMBP(1-14)Ala4-toxin). No killing resulted from incubating AJ1.2 and 4R3.9 cells with irrelevant MHC II-(peptide-toxin) or treating IEk-restricted, pigeon cytochrome c-specific A.E7 cloned murine T cells with IAk-(AcMBP(1-14)Ala4-toxin). T cell receptor-mediated T cell uptake of the peptide-toxin moiety of relevant complex was blocked by anti-T cell receptor-alpha/beta antibody and by excess toxin-free complex. LD50 determinations revealed that cognate MHC II-(peptide-toxin) killed T cells much more effectively than did peptide-toxin conjugate alone. Finally, T cell uptake of peptide-toxin and intracellular release of toxin occurred after incubation with relevant MHC II-(peptide-toxin) containing radiolabeled toxin. These findings, which provide the first evidence that cloned T cells can be deleted with soluble, cognate MHC II-(peptide-toxin) complexes, may have significant clinical relevance for antigen-specific therapy of autoimmune or other T cell-mediated diseases.