NMR structure of a parallel homotrimeric coiled coil

The solution structure of the oligomerization domain of cartilage matrix protein (also known as matrilin-1) has been determined by heteronuclear NMR spectroscopy. The domain folds into a parallel, disulfide-linked, three-stranded, alpha-helical coiled coil, spanning five heptad repeats in the amino acid sequence. The sequence of the first two heptad repeats shows some deviations from the consensus of hydrophobic and hydrophilic residue preferences. While the corresponding region of the coiled coil has a higher intrinsic flexibility, backbone alpha-helix and superhelix parameters are consistent with a regular coiled coil structure.     

Chronic neurologic effects of pesticide overexposure

To investigate the molecular mechanisms involved in paramyxovirus-induced cell fusion, the function and structure of a peptide with a 20-amino-acid sequence from the leucine zipper region (heptad repeat region 2) of the Newcastle disease virus fusion protein (F) were characterized. A peptide with the sequence ALDKLEESNSKLDKVNVKLT (amino acids 478-497 of the F protein) was found to inhibit syncytia formation after virus infection and after transfection of Cos cells with the HN (hemagglutinin-neuraminidase) and F protein cDNAs. Using an F protein gene that requires addition of exogenous trypsin for cleavage, it was shown that the peptide exerted its inhibitory effect prior to cleavage. The three-dimensional conformation of the peptide in aqueous solution was determined through the use of NMR and molecular modeling. Results showed that the peptide formed a helix with properties between an alpha-helix and a 3(10)-helix and that leucine residues aligned along one face of the helix. Side chain salt bridges and hydrogen bonds likely contributed to the stability of the peptide secondary structure. Analysis of the aqueous solution conformation of the peptide suggested mechanisms for specificity of interaction with the intact F protein.     

Crystal structure of the simian immunodeficiency virus (SIV) gp41 core: conserved helical interactions underlie the broad inhibitory activity of gp41 peptides

The gp41 subunit of the envelope protein complex from human and simian immunodeficiency viruses (HIV and SIV) mediates membrane fusion during viral entry. The crystal structure of the HIV-1 gp41 ectodomain core in its proposed fusion-active state is a six-helix bundle. Here we have reconstituted the core of the SIV gp41 ectodomain with two synthetic peptides called SIV N36 and SIV C34, which form a highly helical trimer of heterodimers. The 2.2 A resolution crystal structure of this SIV N36/C34 complex is very similar to the analogous structure in HIV-1 gp41. In both structures, three N36 helices form a central trimeric coiled coil. Three C34 helices pack in an antiparallel orientation into highly conserved, hydrophobic grooves along the surface of this coiled coil. The conserved nature of the N36-C34 interface suggests that the HIV-1 and SIV peptides are functionally interchangeable. Indeed, a heterotypic complex between HIV-1 N36 and SIV C34 peptides is highly helical and stable. Moreover, as with HIV-1 C34, the SIV C34 peptide is a potent inhibitor of HIV-1 infection. These results identify conserved packing interactions between the N and C helices of gp41 and have implications for the development of C peptide analogs with broad inhibitory activity.     

The course of childhood bronchiectasis: a case report and considerations of hospital use

Using an inhibitory synthetic peptide (DP-178) from HIV-1 gp41, we have trapped HIV-1 envelope glycoprotein (Env) undergoing conformational changes during virus entry. Our data show that DP-178 binds gp41 and inhibits Env-mediated membrane fusion after gp120 interacts with cellular receptors, indicating that conformational changes involving the coiled coil domain of gp41 are required for entry. Capture of this fusion-active conformation of Env provides insights into the early events leading to Env-mediated membrane fusion.     

Managed mental health care: problems and possibilities

To investigate the molecular mechanisms involved in paramyxovirus-induced cell fusion, the function and structure of synthetic peptide analogs of the sequence from the leucine zipper region (heptad repeat region 2) of the Newcastle disease virus fusion protein (F) were characterized. As previously reported (Young et al., Virology, 238, 291), a peptide with the sequence ALDKLEESNSKLDKVNVKLT (amino acids 478-497 of the F protein) inhibited syncytia formation after transfection of Cos cells with the hemagglutinin-neuraminidase and F protein cDNAs. A peptide analog which had an alanine residue in place of the first leucine residue in the zipper motif (ALDKAEESNSKLDKVNVKLT) retained inhibitory activity but less than the original peptide. Further loss in activity was observed in a peptide in which two of the leucine residues were replaced with alanine (ALDKAEESNSKADKVNVKLT), and a peptide which had all leucine residues in the zipper motif replaced with alanine (ALDKAEESNSKADKVNVKLT) had no inhibitory activity. The three-dimensional conformations of these peptides in aqueous solution were determined through the use of nuclear magnetic spectroscopy and molecular modeling. Results showed that while the wild-type peptide formed a helix with properties between an alpha-helix and a 3(10) helix with leucine residues aligned along one face of the helix, progressive substitution of leucine residues with alanine resulted in the progressive loss of helical structure. The results suggest that alterations of leucine residues in the zipper motif disrupt secondary structure of the peptide and that this structure is critical to the inhibitory activity of the peptide.     

The YXXL sequences of a transmembrane protein of bovine leukemia virus are required for viral entry and incorporation of viral envelope protein into virions

The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)2 motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.     

Role of autologous bone marrow transplantation as consolidation therapy in acute promyelocytic leukemia patients in complete remission

Recent studies in the field of de novo protein design have focused on the construction of native-like structures. Here we describe the design and characterization of an isoleucine zipper peptide intended to form a parallel triple-stranded coiled coil. To obtain the native-like structural uniqueness, the hydrophobic interface of the peptide consists of beta-branched Ile residues for complementary side chain packing. The peptide forms a stable triple-stranded coiled coil, as determined by circular dichroism and sedimentation equilibrium analyses. A fluorescence quenching assay after the incorporation of acridine revealed a parallel orientation of the peptides. The structural uniqueness of the coiled coil was confirmed by proton-deuterium amide hydrogen exchange and hydrophobic dye binding. The peptide contains amide protons with hydrogen exchange rates that are approximately an order of magnitude slower than those expected if the exchange occurred via global unfolding. A hydrophobic dye does not bind to the peptide. These results strongly suggest that the peptide folds into a well-packed structure that is very similar to the native state of a natural protein. Thus, Ile residues in the hydrophobic interface can improve the side chain packing, which can impart native-like structural uniqueness to the designed coiled coil.     

Structural studies on membrane-embedded influenza hemagglutinin and its fragments

The mechanism of influenza virus hemagglutinin (HA)-mediated membrane fusion has been inferred in part from studies examining pH-induced structural changes in soluble HA derivatives lacking the viral membrane anchor and, sometimes, the fusion peptide (the C- and N-terminal residues of the HA2 chain, respectively). To reconcile structure-based mechanisms of HA-mediated membrane fusion with structural implications of functional studies performed on membrane-embedded HA, we have undertaken attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic analyses of membrane-embedded HA (strain X:31) and its fragments reconstituted into supported lipid bilayers. The fragments correspond to proteolytic products with the majority of the HA1 chain and, in some cases, the fusion peptide removed (THA2 and THA2F-, respectively). In combination with R18 fluorescence dequenching to monitor the functional implications of HA1 subunit removal, we have assessed the influence of pH and target membrane presentation on the secondary structures, orientations relative to the membrane, and dynamics of these molecules. We find that X:31 HA is more tilted towards the plane of the membrane under fusion than under resting conditions, that the fitting of HA depends on the presence of the HA1 chain, that the residues connecting the membrane-inserted fusion peptide with the crystallographically determined coiled coil probably adopt an alpha-helical conformation, and that several changes in the secondary structure and the amide H/D exchange kinetics occur as a result of acidification and target membrane presentation, which can be interpreted as small changes and a release of strain in the static and dynamic structure of membrane-bound HA. THA2 mediatcs fusion, but less efficiently and with less pH-selectivity than HA.     

An effective peptide vaccine to eliminate bovine leukaemia virus (BLV) infected cells in carrier sheep

Protective effects of the gp51 of bovine leukaemia virus (BLV) expressed by a recombinant baculovirus (rgp51) and synthetic multiple antigenic peptides (MAP) of T-helper, T-cytotoxic, and B-cell epitopes of gp51 were investigated against BLV challenge. Two and three sheep were immunized with rgp51 and a mixture of peptides with Freund's complete adjuvant, respectively. BLV was detected from all the immunized sheep at 2 weeks and showed peak levels at 4 weeks after the challenge. However, in two sheep immunized with the mixed peptides, the titer of BLV gradually decreased and one sheep eliminated BLV completely at 28 weeks after the challenge. These two sheep showed higher lymphocyte proliferative responses against the immunized peptides than the other sheep. One of the sheep also showed the specific cytotoxic lymphocyte activity against the BLV gp51-expressing target in vitro. These results suggest the possibility of the peptide vaccine for elimination of BLV in carrier animals in vivo.     

Macrophages infected with bovine leukaemia virus (BLV) induce humoral response in rabbits

BLV is a lymphotropic retrovirus which infects mainly B-cells. However, the possible infection of cells of the monocyte/macrophage lineage (M/M) might explain some aspects of the disease such as latency or disease progression. We infected sheep M/M with BLV either by culturing M/M with supernatant containing virus, or coculturing M/M with persistently infected cell lines. These BLV-infected M/M were inoculated into rabbits and the serological response was followed for two years. ELISA results using adsorbed sera showed a persistent production of specific antibodies from as early as the first week post inoculation. Two tests were used to detect the response against envelope glycoprotein gp51: Agar gel immunodiffusion (AGID) and a virus neutralization test read as syncytia inhibition (SI). Sera were positive by AGID after the second or third inoculation. Neutralizing titres (SI) were higher than those seen in control rabbits inoculated with persistently infected cell lines, suggesting that the virus may be expressed better in M/M. Gag-related proteins were analyzed by Western Blot (WB). Sera from rabbits inoculated with BLV-infected M/M recognized as many viral proteins as sera from BLV immunized control rabbits or infected cows, and this profile did not change with repeated inoculations. All these results suggest that BLV may infect M/M, where viral proteins are actively expressed to the point that they induce a humoral immune response in animals, and that animals get persistently infected.     

Identification of residues in the drug-binding site of human P-glycoprotein using a thiol-reactive substrate

We identified a thiol-reactive compound, dibromobimane (dBBn), that was a potent stimulator (8.2-fold) of the ATPase activity of Cys-less P-glycoprotein. We then used this compound together with cysteine-scanning mutagenesis to identify residues in transmembrane segment (TM) 6 and TM12 that are important for function. TM6 and TM12 lie close to each other in the tertiary structure and are postulated to be important for drug-protein interactions. The majority of P-glycoprotein mutants containing a single cysteine residue retained substantial amounts of drug-stimulated ATPase activity and were not inhibited by dBBn. The ATPase activities of mutants L339C, A342C, L975C, V982C, and A985C, however, were markedly inhibited (>60%) by dBBn. The drug substrates verapamil, vinblastine, and colchicine protected these mutants against inhibition by dBBn, suggesting that these residues are important for interaction of substrates with P-glycoprotein. We previously showed that residues Leu339, Ala342, Leu975, Val982, and Ala985 lie along the point of contact between helices TM6 and TM12, when both are aligned in a left-handed coiled coil (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 20986-20989). Taken together, these results suggest that the interface between TM6 and TM12 likely forms part of the potential drug-binding pocket in P-glycoprotein.     

HtrI is a dimer whose interface is sensitive to receptor photoactivation and His-166 replacements in sensory rhodopsin I

Single cysteine substitutions were introduced into three positions of otherwise cysteineless HtrI, a phototaxis transducer found in Halobacterium salinarum that transmits signals from the photoreceptor sensory rhodopsin I (SRI) to a cytoplasmic pathway controlling the cell's motility. Oxidative cross-linking of the monocysteine HtrI mutants in membrane suspensions resulted in dimer forms evident in SDS-polyacrylamide gels. The rate of cross-linking of I64C on the cytoplasmic side of HtrI was accelerated by SRI binding in the dark and further increased by SRI photoactivation. Several residue replacements of His-166 in SRI accelerated the cross-linking rate of I64C in the dark and His-166 mutants that exhibit "inverted signaling" (mediating repellent instead of the normally attractant response to orange light) inverted the light effect on the cross-linking rate of I64C. Secondary structure prediction of HtrI indicates a coiled coil structure in the cytoplasmic region following TM2, a dimerization domain found in a diverse group of proteins. We conclude that 1) HtrI exists as a dimer both in the absence of SRI and in the SRI-HtrI complex, 2) binding of SRI in the dark increases reactivity of the two cysteines at position 64 in the dimer by increasing their proximity or mobility, 3) light activation of wild-type SRI further increases their reactivity, 4) His-166 replacements in the SRI receptor have conformational effects on the structure of HtrI at position 64, and 5) inverted signaling by His-166 mutants likely results from an inverted conformational change at this region induced by SRI photoactivation.     

Mutational analysis of the candidate internal fusion peptide of the avian leukosis and sarcoma virus subgroup A envelope glycoprotein

The transmembrane subunit (TM) of the avian leukosis and sarcoma virus (ALSV) envelope glycoprotein (Env) contains a stretch of conserved hydrophobic amino acids internal to its amino terminus (residues 21 to 42). By analogy with similar sequences in other viral envelope glycoproteins, this region has been proposed to be a fusion peptide. We investigated the role of this region by changing each of three hydrophobic residues (Ile-21, Val-30, and Ile-39) to glutamatic acid and lysine in the ALSV subgroup A Env. Like wild-type (wt) Env, all six mutant Env proteins were proteolytically processed, oligomerized, and expressed at the cell surface in a form that bound Tva, the ALSV subgroup A receptor. Like wt Env, Ile21Glu, Ile21Lys, Va30Glu, and Val30Lys changed conformation upon binding Tva, as assayed by sensitivity to thermolysin. Ile39Glu and Ile39Lys were cleaved by thermolysin in both the absence and presence of Tva. Although incorporated into virus particles at approximately equal levels, all mutant Envs were compromised in their ability to support infection. The mutants at residues 21 and 30 showed levels of infection 2 to 3 orders of magnitude lower than that of wt Env. The mutants at residue 39 were noninfectious. Furthermore, none of the mutants displayed activity in a cell-cell fusion assay. Our results support the contention that residues 21 to 42 of ALSV subgroup A Env constitute its fusion peptide.     

Subdomain folding of the coiled coil leucine zipper from the bZIP transcriptional activator GCN4

One popular model for protein folding, the framework model, postulates initial formation of secondary structure elements, which then assemble into the native conformation. However, short peptides that correspond to secondary structure elements in proteins are often only marginally stable in isolation. A 33-residue peptide (GCN4-p1) corresponding to the GCN4 leucine zipper folds as a parallel, two-stranded coiled coil [O'Shea, E.K., Klemm, J.D., Kim, P.S., & Alber, T.A. (1991) Science 254, 539-544]. Deletion of the first residue (Arg 1) results in local, N-terminal unfolding of the coiled coil, suggesting that a stable subdomain of GCN4-p1 can form. N- and C-terminal deletion studies result in a 23-residue peptide, corresponding to residues 8-30 of GCN4-p1, that folds as a parallel, two-stranded coil with substantial stability (the melting temperature of a 1 mM solution is 43 degrees C at pH 7). In contrast, a closely related 23-residue peptide (residues 11-33 of GCN4-p1) is predominantly unfolded, even at 0 degrees C, as observed previously for many isolated peptides of similar length. Thus, specific tertiary packing interactions between two short units of secondary structure can be energetically more important in stabilizing folded structure than secondary structure propensities. These results provide strong support for the notion that stable, cooperatively folded subdomains are the important determinants of protein folding.     

Conversion of a beta-strand to an alpha-helix induced by a single-site mutation observed in the crystal structure of Fis mutant Pro26Ala

The conversion from an alpha-helix to a beta-strand has received extensive attention since this structural change may induce many amyloidogenic proteins to self-assemble into fibrils and cause fatal diseases. Here we report the conversion of a peptide segment from a beta-strand to an alpha-helix by a single-site mutation as observed in the crystal structure of Fis mutant Pro26Ala determined at 2.0 A resolution. Pro26 in Fis occurs at the point where a flexible extended beta-hairpin arm leaves the core structure. Thus it can be classified as a "hinge proline" located at the C-terminal end of the beta2-strand and the N-terminal cap of the A alpha-helix. The replacement of Pro26 to alanine extends the A alpha-helix for two additional turns in one of the dimeric subunits; therefore, the structure of the peptide from residues 22 to 26 is converted from a beta-strand to an alpha-helix. This result confirms the structural importance of the proline residue located at the hinge region and may explain the mutant's reduced ability to activate Hin-catalyzed DNA inversion. The peptide (residues 20 to 26) in the second monomer subunit presumably retains its beta-strand conformation in the crystal; therefore, this peptide shows a "chameleon-like" character since it can adopt either an alpha-helix or a beta-strand structure in different environments. The structure of Pro26Ala provides an additional example where not only the protein sequence, but also non-local interactions determine the secondary structure of proteins.     

Mutational analysis of residues in the coiled-coil domain of human immunodeficiency virus type 1 transmembrane protein gp41

The envelope glycoprotein (Env) of human immunodeficiency virus mediates virus entry into cells by undergoing conformational changes that lead to fusion between viral and cellular membranes. A six-helix bundle in gp41, consisting of an interior trimeric coiled-coil core with three exterior helices packed in the grooves (core structure), has been proposed to be part of a fusion-active structure of Env (D. C. Chan, D. Fass, J. M. Berger, and P. S. Kim, Cell 89:263-273, 1997; W. Weissenhorn, A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley, Nature 387:426-430, 1997; and K. Tan, J. Liu, J. Wang, S. Shen, and M. Lu, Proc. Natl. Acad. Sci. USA 94:12303, 1997). We analyzed the effects of amino acid substitutions of arginine or glutamic acid in residues in the coiled-coil (heptad repeat) domain that line the interface between the helices in the gp41 core structure. We found that mutations of leucine to arginine or glutamic acid in position 556 and of alanine to arginine in position 558 resulted in undetectable levels of Env expression. Seven other mutations in six positions completely abolished fusion activity despite incorporation of the mutant Env into virions and normal gp160 processing. Single-residue substitutions of glutamic acid at position 570 or 577 resulted in the only viable mutants among the 16 mutants studied, although both viable mutants exhibited impaired fusion activity compared to that of the wild type. The glutamic acid 577 mutant was more sensitive than the wild type to inhibition by a gp41 coiled-coil peptide (DP-107) but not to that by another peptide corresponding to the C helix in the gp41 core structure (DP-178). These results provide insight into the gp41 fusion mechanism and suggest that the DP-107 peptide may inhibit fusion by binding to the homologous region in gp41, probably by forming a peptide-gp41 coiled-coil structure.     

A mutational analysis identifies three functional regions of the spindle pole component Spc110p in Saccharomyces cerevisiae

The central coiled coil of the essential spindle pole component Spc110p spans the distance between the central and inner plaques of the Saccharomyces cerevisiae spindle pole body (SPB). The carboxy terminus of Spc110p, which binds calmodulin, resides at the central plaque, and the amino terminus resides at the inner plaque from which nuclear microtubules originate. To dissect the functions of Spc110p, we created temperature-sensitive mutations in the amino and carboxy termini. Analysis of the temperature-sensitive spc110 mutations and intragenic complementation analysis of the spc110 alleles defined three functional regions of Spc110p. Region I is located at the amino terminus. Region II is located at the carboxy-terminal end of the coiled coil, and region III is the previously defined calmodulin-binding site. Overexpression of SPC98 suppresses the temperature sensitivity conferred by mutations in region I but not the phenotypes conferred by mutations in the other two regions, suggesting that the amino terminus of Spc110p is involved in an interaction with the gamma-tubulin complex composed of Spc97p, Spc98p, and Tub4p. Mutations in region II lead to loss of SPB integrity during mitosis, suggesting that this region is required for the stable attachment of Spc110p to the central plaque. Our results strongly argue that Spc110p links the gamma-tubulin complex to the central plaque of the SPB.     

A conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type 1 gp41 ectodomain is important for Env-mediated fusion and virus infectivity

Mutations were introduced into the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane envelope glycoprotein, gp41, within a region immediately adjacent to the membrane-spanning domain. This region, which is predicted to form an alpha-helix, contains highly conserved hydrophobic residues and is unusually rich in tryptophan residues. In addition, this domain overlaps the epitope of a neutralizing monoclonal antibody, 2F5, as well as the sequence corresponding to a peptide, DP-178, shown to potently neutralize virus. Site-directed mutagenesis was used to create deletions, substitutions, and insertions centered around a stretch of 17 hydrophobic and uncharged amino acids (residues 666 to 682 of the HXB2 strain of HIV-1) in order to determine the role of this region in the maturation and function of the envelope glycoprotein. Deletion of the entire stretch of 17 amino acids abrogated the ability of the envelope glycoprotein to mediate both cell-cell fusion and virus entry without affecting the normal maturation, transport, or CD4-binding ability of the protein. This phenotype was also demonstrated by substituting alanine residues for three of the five tryptophan residues within this sequence. Smaller deletions, as well as multiple amino acid substitutions, were also found to inhibit but not block cell-cell fusion. These results demonstrate the crucial role of a tryptophan-rich motif in gp41 during a post-CD4-binding step of glycoprotein-mediated fusion. The basis for the invariant nature of the tryptophans, however, appears to be at the level of glycoprotein incorporation into virions. Even the substitution of phenylalanine for a single tryptophan residue was sufficient to reduce Env incorporation and drop the efficiency of virus entry approximately 10-fold, despite the fact that the same mutation had no significant effect on syncytium formation.