Extremely Thermostabilizing Core Mutations in Coiled-Coil Mimetic Proteins of HIV-1 gp41 Produce Diverse Effects on Target Binding but Do Not Affect Their Inhibitory Activity.
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ABSTRACT:
SUBMITTER: Cano-Munoz M
PROVIDER: S-EPMC8069472 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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 (approximately 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.
| S-EPMC2635040 | biostudies-literature
Project description:A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)3 or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design.
| S-EPMC3820714 | biostudies-literature
Project description:Targeted therapy of chronic myeloid leukemia (CML) is currently based on small-molecule inhibitors that directly bind the tyrosine kinase domain of BCR-ABL1. This strategy has generally been successful, but is subject to drug resistance because of point mutations in the kinase domain. Kinase activity requires transactivation of BCR-ABL1 following an oligomerization event, which is mediated by the coiled-coil (CC) domain at the N terminus of the protein. Here, we describe a rationally engineered mutant version of the CC domain, called CC(mut3), which interferes with BCR-ABL1 oligomerization and promotes apoptosis in BCR-ABL1-expressing cells, regardless of kinase domain mutation status. CC(mut3) exhibits strong proapoptotic and antiproliferative activity in cell lines expressing native BCR-ABL1, single kinase domain mutant BCR-ABL1 (E255V and T315I) or compound-mutant BCR-ABL1 (E255V/T315I). Moreover, CC(mut3) inhibits colony formation by primary CML CD34(+) cells ex vivo, including a sample expressing the T315I mutant. These data suggest that targeting BCR-ABL1 with CC mutants may provide a novel alternative strategy for treating patients with resistance to current targeted therapies.
| S-EPMC4621806 | biostudies-literature
Project description:G protein-coupled receptor (GPCR) structures are of interest as a means to understand biological signal transduction and as tools for therapeutic discovery. The growing number of GPCR crystal structures demonstrates that the extracellular loops (EL) connecting the membrane-spanning helices show tremendous structural variability relative to the more structurally-conserved seven transmembrane ?-helical domains. The EL of the LPA(1) receptor have not yet been conclusively resolved, and bear limited sequence identity to known structures. This study involved development of a peptide to characterize the intrinsic structure of the LPA(1) GPCR second EL. The loop was embedded between two helices that assemble into a coiled-coil, which served as a receptor-mimetic folding constraint (LPA(1)-CC-EL2 peptide). The ensemble of structures from multi-dimensional NMR experiments demonstrated that a robust coiled-coil formed without noticeable deformation due to the EL2 sequence. In contrast, the EL2 sequence showed well-defined structure only near its C-terminal residues. The NMR ensemble was combined with a computational model of the LPA(1) receptor that had previously been validated. The resulting hybrid models were evaluated using docking. Nine different hybrid models interacted with LPA 18:1 as expected, based on prior mutagenesis studies, and one was additionally consistent with antagonist affinity trends.
| S-EPMC3588015 | biostudies-literature
Project description:Chlamydia trachomatis replicates in a parasitophorous membrane-bound compartment called an inclusion. The inclusions corrupt host vesicle trafficking networks to avoid the degradative endolysosomal pathway but promote fusion with each other in order to sustain higher bacterial loads in a process known as homotypic fusion. The Chlamydia protein IncA (Inclusion protein A) appears to play central roles in both these processes as it participates to homotypic fusion and inhibits endocytic SNARE-mediated membrane fusion. How IncA selectively inhibits or activates membrane fusion remains poorly understood. In this study, we analyzed the spatial and molecular determinants of IncA's fusogenic and inhibitory functions. Using a cell-free membrane fusion assay, we found that inhibition of SNARE-mediated fusion requires IncA to be on the same membrane as the endocytic SNARE proteins. IncA displays two coiled-coil domains showing high homology with SNARE proteins. Domain swap and deletion experiments revealed that although both these domains are capable of independently inhibiting SNARE-mediated fusion, these two coiled-coil domains cooperate in mediating IncA multimerization and homotypic membrane interaction. Our results support the hypothesis that Chlamydia employs SNARE-like virulence factors that positively and negatively affect membrane fusion and promote infection.
| S-EPMC3720611 | biostudies-literature
Project description:The conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 is transiently exposed during the fusion process by forming a pre-hairpin intermediate, thus representing an attractive target for the design of fusion inhibitors and neutralizing antibodies. In previous studies we reported a series of broadly neutralizing mini-antibodies derived from a synthetic naïve human combinatorial antibody library by panning against a mimetic of the trimeric N-HR coiled coil, followed by affinity maturation using targeted diversification of the CDR-H2 loop. Here we report crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Furthermore, modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5, derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop.
| S-EPMC2978731 | biostudies-literature
Project description:Coiled-coil proteins contain a characteristic seven-residue sequence repeat whose positions are designated a to g. The interacting surface between alpha-helices in a classical coiled coil is formed by interspersing nonpolar side chains at the a and d positions with hydrophilic residues at the flanking e and g positions. To explore how the chemical nature of these core amino acids dictates the overall coiled-coil architecture, we replaced all eight e and g residues in the GCN4 leucine zipper with nonpolar alanine side chains. Surprisingly, the alanine-containing mutant forms a stable alpha-helical heptamer in aqueous solution. The 1.25-A resolution crystal structure of the heptamer reveals a parallel seven-stranded coiled coil enclosing a large tubular channel with an unusual heptad register shift between adjacent staggered helices. The overall geometry comprises two interleaved hydrophobic helical screws of interacting cross-sectional a and d layers that have not been seen before. Moreover, asparagines at the a positions play an essential role in heptamer formation by participating in a set of buried interhelix hydrogen bonds. These results demonstrate that heptad repeats containing four hydrophobic positions can direct assembly of complex, higher-order coiled-coil structures with rich diversity for close packing of alpha-helices.
| S-EPMC1622844 | biostudies-literature
Project description:Peptides from the N-heptad repeat region of the HIV gp41 protein can inhibit viral fusion, but their potency is limited by a low tendency to form a trimeric coiled-coil. Accordingly, stabilization of N peptides by fusion with the stable coiled-coil IZ yields nanomolar inhibitors [Eckert, D. M. & Kim, P. S. (2001) Proc. Natl. Acad. Sci. USA 98, 11187-11192]. Because the antiviral potency of IZN17 is limited by self-association equilibrium, we covalently stabilized the peptide by using interchain disulfide bonds. The resulting covalent trimer, (CCIZN17)3, has an extraordinary thermodynamic stability that translates into unprecedented antiviral potency: (CCIZN17)3 (i) inhibits fusion in a cell-cell fusion assay (IC50 = 260 pM); (ii) is the most potent fusion inhibitor described to date (IC50 = 40-380 pM) in a single-cycle infectivity assay against HIV(HXB2), HIV(NL4-3), and HIV(MN-1); (iii) efficiently neutralizes acute viral infection in peripheral blood mononuclear cells; and (iv) displays a broad antiviral profile, being able to neutralize 100% of a large panel of HIV isolates, including R5, X4, and R5/X4 strains. In all of these assays, the potency of N-peptide inhibitor (CCIZN17)3 was equal to or more than the C-peptide inhibitor in clinical use, DP178 (also known as Enfuvirtide and Fuzeon). More importantly, we show that the two inhibitors, which have different targets in gp41, synergize when used in combination. These features make (CCIZN17)3 an attractive lead to develop as an antiviral drug, alone or in combination with DP178, as well as a promising immunogen to elicit a fusion-blocking neutralizing antibody response.
| S-EPMC1200264 | biostudies-literature
Project description:The mitotic kinesin centromere protein E (CENP-E) is an essential kinetochore component that directly contributes to the capture and stabilization of spindle microtubules by kinetochores. Although reduction in CENP-E leads to high rates of whole chromosome missegregation, neither its properties as a microtubule-dependent motor nor how it contributes to the dynamic linkage between kinetochores and microtubules is known. Using single-molecule assays, we demonstrate that CENP-E is a very slow, highly processive motor that maintains microtubule attachment for long periods. Direct visualization of full-length Xenopus laevis CENP-E reveals a highly flexible 230-nm coiled coil separating its kinetochore-binding and motor domains. We also show that full-length CENP-E is a slow plus end-directed motor whose activity is essential for metaphase chromosome alignment. We propose that the highly processive microtubule-dependent motor activity of CENP-E serves to power chromosome congression and provides a flexible, motile tether linking kinetochores to dynamic spindle microtubules.
| S-EPMC2364708 | biostudies-literature
Project description:BACKGROUND:Recombinant protein expression in bacteria often leads to the formation of intracellular insoluble protein deposits, a major bottleneck for the production of soluble and active products. However, in recent years, these bacterial protein aggregates, commonly known as inclusion bodies (IBs), have been shown to be a source of stable and active protein for biotechnological and biomedical applications. The formation of these functional IBs is usually facilitated by the fusion of aggregation-prone peptides or proteins to the protein of interest, leading to the formation of amyloid-like nanostructures, where the functional protein is embedded. RESULTS:In order to offer an alternative to the classical amyloid-like IBs, here we develop functional IBs exploiting the coiled-coil fold. An in silico analysis of coiled-coil and aggregation propensities, net charge, and hydropathicity of different potential tags identified the natural homo-dimeric and anti-parallel coiled-coil ZapB bacterial protein as an optimal candidate to form assemblies in which the native state of the fused protein is preserved. The protein itself forms supramolecular fibrillar networks exhibiting only ?-helix secondary structure. This non-amyloid self-assembly propensity allows generating innocuous IBs in which the recombinant protein of interest remains folded and functional, as demonstrated using two different fluorescent proteins. CONCLUSIONS:Here, we present a proof of concept for the use of a natural coiled-coil domain as a versatile tool for the production of functional IBs in bacteria. This ?-helix-based strategy excludes any potential toxicity drawback that might arise from the amyloid nature of ?-sheet-based IBs and renders highly active and homogeneous submicrometric particles.
| S-EPMC7268670 | biostudies-literature