Interactions of HIV-1 Inhibitory Peptide T20 with the gp41 N-HR Coiled Coil

Cellular entry of human immunodeficiency virus type 1 (HIV-1) involves fusion of viral and cellular membranes and is mediated by structural transitions in viral glycoprotein gp41. The antiviral C-peptide T20 targets the gp41 N-terminal heptad repeat region (N-HR), blocking gp41 conformational changes essential for the entry process. To probe the T20 structure-activity relationship, we engineered a molecular mimic of the entire gp41 N-HR coiled coil using the 5-Helix design strategy. T20 bound this artificial protein (denoted 5H-ex) with nanomolar affinity (K_D = 30 nm), close to its IC₅₀ concentration (∼3 nm) but much weaker than the affinity of a related inhibitory C-peptide C37 (K_D = 0.0007 nm). T20/C37 competitive binding assays confirmed that T20 interacts with the hydrophobic groove on the surface of the N-HR coiled coil outside of a deep pocket region crucial for C37 binding. We used 5H-ex to investigate how the T20 N and C termini contributed to the inhibitor binding activity. Mutating three aromatic residues at the T20 C terminus (WNWF → ANAA) had no effect on affinity, suggesting that these amino acids do not participate in T20 binding to the gp41 N-HR. The results support recent evidence pointing to a different role for these residues in T20 inhibition (Peisajovich, S. G., Gallo, S. A., Blumenthal, R., and Shai, Y. (2003) J. Biol. Chem. 278, 21012–21017; Liu, S., Jing, W., Cheung, B., Lu, H., Sun, J., Yan, X., Niu, J., Farmar, J., Wu, S., and Jiang, S. (2007) J. Biol. Chem. 282, 9612–9620). By contrast, mutations near the T20 N terminus substantially influenced inhibitor binding strength. When Ile was substituted for Thr in the second T20 position, a 40-fold increase in binding affinity was measured (K_D = 0.75 nm). The effect of this affinity enhancement on T20 inhibitory potency varied among different viral strains. The original T20 and the higher affinity T20 variant had similar potency against wild type HIV-1. However, the higher affinity T20 variant was significantly more potent against T20-resistant virus. The findings suggest that other factors in addition to binding affinity play a role in limiting T20 potency. As a mimetic of the complete gp41 N-HR coiled coil region, 5H-ex will be a useful tool to further elucidate mechanistic profiles of C-peptide inhibitors.The HIV-1² surface glycoprotein Env promotes viral entry through the fusion of viral and cellular membranes (3). Env consists of three gp120 surface subunits and three gp41 transmembrane subunits arranged as a trimer-of-heterodimers on the virion surface. In the current model of HIV-1 entry, cellular receptor binding to gp120 initiates a series of coordinated structural transformations that stimulate gp41 to extend and insert its N-terminal fusion peptide into target cell membranes (see Fig. 1A) (4, 5). This high energy extended intermediate structure ultimately collapses into a trimer-of-hairpins conformation that juxtaposes the gp41 fusion peptide and transmembrane domain. Because the fusion peptide and transmembrane domain are inserted in target cell and viral membranes, formation of the trimer-of-hairpins is proposed to bring these membranes into the close proximity required for efficient fusion.Open in a separate windowFIGURE 1.HIV-1 gp41 and its role in viral membrane fusion. A, a model of HIV-1 entry (46). In native Env prior to receptor activation, gp41 is held in a metastable conformation by a canopy of gp120 proteins (green). Receptor binding to gp120 stimulates gp41 to extend and insert its fusion peptide segment (red) into the target cell membrane. The N-HR (gray) and C-HR (blue) regions of the gp41 ectodomain are transiently exposed in this prehairpin state. Subsequently, gp41 collapses into the trimer-of-hairpins conformation that brings the gp41 fusion peptides, transmembrane regions (purple), and their associated membranes into the close proximity for membrane fusion. The actual disposition of gp120 in both the prehairpin and trimer-of-hairpins states is uncertain; for clarity, the protein is omitted in the schematic of the trimer-of-hairpins conformation. B, a diagram of HIV-1 gp41 identifying its fusion peptide (FP), N-HR, C-HR, MPER (MP), transmembrane (TM), and cytoplasmic (cyto) domains. Amino acid sequences above and below the diagram are derived from the N-HR and C-HR/MPER regions of Env_HXB2; all but the MPER sequence WNWF (magenta) were used in the design of 5H-ex. The N-HR and C-HR segments found in the original 5-Helix are boxed in gray and blue, respectively, whereas the sequences of C37 and T20 are denoted by lines. The side chains of the C-HR amino acids marked with + pack into the hydrophobic pocket at the C terminus of the N-HR coiled coil.The core of the trimer-of-hairpins is a bundle of six α-helices formed by two hydrophobic heptad repeat sequences in the N- and C-terminal regions of the gp41 ectodomain (N-HR and C-HR, respectively) (6, 7). In the trimer-of-hairpins, the N-HR segments from three gp41 ectodomains form a central trimeric coiled coil, around which the three C-HR segments pack as antiparallel helices into hydrophobic grooves (8–11). In the prehairpin extended conformation, the N-HR and C-HR segments are unassociated and transiently accessible to inhibitors of HIV-1 entry (5, 12). Several such inhibitors are formed from the peptide sequence of the C-HR and adjacent gp41 regions (4, 6, 13, 14). Denoted C-peptides, they work in a dominant negative fashion by binding to the exposed N-HR coiled coil, thereby blocking trimer-of-hairpins formation and inhibiting viral membrane fusion (4, 15–21). One C-peptide, T20 (also called enfuvirtide), has shown antiviral activity in vivo and has been approved for use in the treatment of HIV-1 infection (22, 23).T20 is a 36-amino acid peptide extending from Tyr⁶³⁸ in the middle of the C-HR to Phe⁶⁷³ in the Trp-rich membrane proximal external region (MPER) that precedes the gp41 transmembrane domain (residue numbering is according to the Env_HXB2 sequence; see Fig. 1B) (13). In T20, these C-terminal MPER-derived residues are critical for inhibitory activity, although their structure and function in the gp41-bound state are currently unknown (1, 24, 25). A second class of similarly potent C-peptides includes C34 (residues 628–661) and the slightly larger C37 (residues 625–661) (4, 6, 26, 27). These peptides are derived entirely from the C-HR sequence and thus are shifted in the N-terminal direction compared with T20 (Fig. 1B). The interactions of C34 and C37 with gp41 are greatly stabilized by residues Trp⁶²⁸, Trp⁶³¹, and Ile⁶³⁵ near the C-HR N terminus (4). Their bulky hydrophobic side chains pack into a deep hydrophobic pocket on the surface of the N-HR coiled coil. T20 lacks these pocket binding residues and their stabilizing effect. However, T20 does contain bulky hydrophobic residues (Trp⁶⁷⁰, Trp⁶⁷², and Phe⁶⁷³) at its C terminus that might pack into a similar pocket at the other end of the N-HR coiled coil.High resolution structures of the gp41 trimer-of-hairpins have aided our understanding of the mechanism of C-peptide inhibition. These structures have enabled the design of polypeptides that mimic the gp41 N-HR coiled coil and bind C34/C37, thereby providing a tool to probe the structure-activity relationships of the inhibitors (26, 28–30). No similar tool is available for investigating T20 inhibition in detail. The structures of the gp41 trimer-of-hairpins do not include the T20 C terminus (9 residues) nor the gp41 N-terminal segments that putatively interact with it. Furthermore, gp41 N-HR-derived peptides predicted to interact with T20 are poorly soluble and difficult to use in solution phase interaction assays (6). Here we describe the design of a soluble protein (denoted 5H-ex) that mimics the putative T20-binding site on the N-HR coiled coil. 5H-ex interacts with T20 with an equilibrium dissociation constant (K_D) of 30 nm, close to the T20 50% inhibitory concentration (IC₅₀) of 3 nm. Using this protein, we explored the extent to which the N and C termini of T20 contribute to its binding activity. First, we showed that the MPER-derived residues at the peptide C terminus do not stabilize the 5H-ex/T20 interaction. Second, we identified an N-terminal substitution that significantly enhanced T20 binding affinity and improved peptide inhibitory activity against T20-resistant HIV-1. The results suggest that T20 binding to the N-HR coiled coil is stabilized primarily by residues derived from the C-HR and not the MPER. 5H-ex is likely to be a useful tool in probing the structure-activity relationship of T20.     

The Cytoplasmic Tail of GM3 Synthase Defines Its Subcellular Localization,Stability, and In Vivo Activity

GM3 synthase (SAT-I) is the primary glycosyltransferase responsible for the biosynthesis of ganglio-series gangliosides. In this study, we identify three isoforms of mouse SAT-I proteins, named M1-SAT-I, M2-SAT-I, and M3-SAT-I, which possess distinct lengths in their NH₂-terminal cytoplasmic tails. These isoforms are produced by leaky scanning from mRNA variants of mSAT-Ia and mSAT-Ib. M2-SAT-I and M3-SAT-I were found to be localized in the Golgi apparatus, as expected, whereas M1-SAT-I was exclusively found in the endoplasmic reticulum (ER). Specific multiple arginines (R) arranged in an R-based motif, RRXXXXR necessary for ER targeting, were found in the cytoplasmic tail of M1-SAT-I, and in vivo GM3 biosynthesis by M1-SAT-I was very low because of restricted transport to the Golgi apparatus. In addition, M1-SAT-I and M3-SAT-I had a long half-life relative to M2-SAT-I. This is the first report demonstrating the presence of an ER-targeting R-based motif in the cytoplasmic tail of a protein in the mammalian glycosyltransferase family of enzymes. The system, which produces SAT-I isoforms having distinct characteristics, is likely to be of critical importance for the regulation of GM3 biosynthesis under various pathological and physiological conditions.     

Expression of the autofluorescent protein, DsRed2, in the recombinants of the ectomycorrhizal basidiomycete, Suillus grevillei, generated by Agrobacterium-mediated transformation

Recombinants were generated from the ectomycorrhizal basidiomycete, Suillus grevillei, through agroinfection using a binary vector carrying the hygromycin B resistance and the autofluorescent protein, DsRed2, markers. DsRed2 was driven by a cis-regulatory region of the glyceraldeyde-3-phosphate dehydrogenase gene (gpd) from the wood-rotting basidiomycete, Coriolus hirsutus, which contains promoters and 5′ gpd sequences with first through fourth exons and expressed for the first time in Suillus spp. The transformation system and recombinants expressing an autofluorescent protein may be useful in genetic analysis of the symbiosis.     

Cloning of Lentinus edodes mitochondrial DNA fragment capable of autonomous replication in Saccharomyces cerevisiae

Mitochondrial (mt) DNA of the higher basidiomycetes Lentinus edodes with a molecular weight of about 69 kb was partially digested with Sau3AI, cloned with plasmid YIp32 (a hybrid of pBR322 and the yeast leu2 gene) and analyzed for sequences capable of autonomous replication (ARSs) in the eukaryote Saccharomyces cerevisiae. One recombinant plasmid was isolated which contained 3.2 kb fragment of the mtDNA with ARS activity. This plasmid (named pSK52) exhibited a high-frequency yeast transformation and was found to be maintained within the cell as an extrachromosomal element. The stability and copy number properties of pSK52 were similar to those of the recombinant plasmid of YIp32 and S. cerevisiae mt-ARS constructed as a reference. Subcloning experiments were carried out to assess the localization of ARS on the above 3.2 kb fragment, revealing that the fragment contains at least two ARSs.