Ding-Kwo Chang

Membrane interaction and structure of the transmembrane domain of influenza hemagglutinin and its fusion peptide complex

BackgroundTo study the organization and interaction with the fusion domain (or fusion peptide, FP) of the transmembrane domain (TMD) of influenza virus envelope glycoprotein for its role in membrane fusion which is also essential in the cellular trafficking of biomolecules and sperm-egg fusion.ResultsThe fluorescence and gel electrophoresis experiments revealed a tight self-assembly of TMD in the model membrane. A weak but non-random interaction between TMD and FP in the membrane was found. In the complex, the central TMD oligomer was packed by FP in an antiparallel fashion. FP insertion into the membrane was altered by binding to TMD. An infrared study exhibited an enhanced membrane perturbation by the complex formation. A model was built to illustrate the role of TMD in the late stages of influenza virus-mediated membrane fusion reaction.ConclusionThe TMD oligomer anchors the fusion protein in the membrane with minimal destabilization to the membrane. Upon associating with FP, the complex exerts a synergistic effect on the membrane perturbation. This effect is likely to contribute to the complete membrane fusion during the late phase of fusion protein-induced fusion cascade. The results presented in the work characterize the nature of the interaction of TMD with the membrane and TMD in a complex with FP in the steps leading to pore initiation and dilation during virus-induced fusion. Our data and proposed fusion model highlight the key role of TMD-FP interaction and have implications on the fusion reaction mediated by other type I viral fusion proteins. Understanding the molecular mechanism of membrane fusion may assist in the design of anti-viral drugs.

Biophysical Characterization of the Structure of the Amino-terminal Region of gp41 of HIV-1 IMPLICATIONS ON VIRAL FUSION MECHANISM

A peptide of 51 amino acids corresponding to the NH2-terminal region (5–55) of the glycoprotein gp41 of human immunodeficiency virus type 1 was synthesized to study its conformation and assembly. Nuclear magnetic resonance experiments indicated the sequence NH2-terminal to the leucine zipper-like domain of gp41 was induced into helix in the micellar solution, in agreement with circular dichroism data. Light scattering experiment showed that the peptide molecules self-assembled in water into trimeric structure on average. That the peptide molecules oligomerize in aqueous solution was supported by gel filtration and diffusion coefficient experiments. Molecular dynamics simulation based on the NMR data revealed a flexible region adjacent to the hydrophobic NH2 terminus of gp41. The biological significance of the present findings on the conformational flexibility and the propensity of oligomerization of the peptide may be envisioned by a proposed model for the interaction of gp41 with membranes during fusion process.

Nuclear Overhauser effect and computational characterization of the beta-spiral of the polypentapeptide of elastin.

The structure of the elastin polypentapeptide, poly(VPGVG), was studied by nuclear Overhauser effect experiments using perdeuterated Val1 and Val4 samples under the condition where intermolecular interactions are absent. More extensive interaction was found between the Val1 gamma CH and Pro2 beta CH protons than between the Val4 gamma CH and Pro2 beta CH protons. The Val1 gamma CH3-Pro2 beta CH interaction does not occur within the same pentamer as previously shown experimentally and as expected from steric considerations. The results are incompatible with the presence of a random chain network in poly(VPGVG) at room temperature but are readily explicable in terms of interturn interactions in a beta-spiral structure. More specifically, the results indicate that the beta-spiral conformation with 2.9 pentamers/turn is more prevalent than that with 2.7 pentamers/turn. Using conformations developed by molecular mechanics calculations, molecular dynamics simulations were carried out to compare the relative energies of these two variants of this class of beta-spiral structures. It was found in vacuo that the structure with 2.9 pentamers/turn is indeed more stable than that of 2.7 pentamers/turn by approximately 1 kcal/mole-pentamer.

EVENTS IN THE KINETIC FOLDING PATHWAY OF A SMALL, ALL BETA -SHEET PROTEIN

The folding of cardiotoxin analogue III (CTX III), a small (60 amino acids), all β-sheet protein from the venom of the Taiwan Cobra (Naja naja atra) is here investigated. The folding kinetics is monitored by using a variety of techniques such as NMR, fluorescence, and circular dichroism spectroscopy. The folding of the protein is complete within a time scale of 200 ms. The earliest detectable event in the folding pathway of CTX III is the formation of a hydrophobic cluster, which possess strong affinity to bind to nonpolar dye such as 1-anilino-8-napthalene-sulfonic acid. Quenched-flow deuterium-hydrogen exchange experiments indicate that the segment spanning residues 51–55 along with Lys23, Ile39, Val49, Tyr51 and Val52 could constitute the “hydrophobic cluster.” Folding kinetics of CTX III based on the amide-protection data reveals that the triple-stranded, antiparallel β-sheet segment, which is located in the central core of the molecule, appears to fold faster than the double-stranded β-sheet segment.

Identification of the LWYIK Motif Located in the Human Immunodeficiency Virus Type 1 Transmembrane gp41 Protein as a Distinct Determinant for Viral Infection

ABSTRACT The highly conserved LWYIK motif located immediately proximal to the membrane-spanning domain of the gp41 transmembrane protein of human immunodeficiency virus type 1 has been proposed as being important for the surface envelope (Env) glycoproteins association with lipid rafts and gp41-mediated membrane fusion. Here we employed substitution and deletion mutagenesis to understand the role of this motif in the virus life cycle. None of the mutants examined affected the synthesis, precursor processing, CD4 binding, oligomerization, or cell surface expression of the Env, nor did they alter Env incorporation into the virus. All of the mutants, particularly the ΔYI, ΔIK, and ΔLWYIK mutants, in which the indicated residues were deleted, exhibited greatly reduced one-cycle viral replication and the Env trans-complementation ability. All of these deletion mutant proteins were still localized in the lipid rafts. With the exception of the Trp-to-Ala (WA) mutant, which exhibited reduced viral infectivity albeit with normal membrane fusion, all mutants displayed loss of some or almost all of the membrane fusion ability. Although these deletion mutants partially inhibited in trans wild-type (WT) Env-mediated fusion, they were more effective in dominantly interfering with WT Env-mediated viral entry when coexpressed with the WT Env, implying a role of this motif in postfusion events as well. Both T20 and L43L peptides derived from the two gp41 extracellular C- and N-terminal α-helical heptad repeats, respectively, inhibited WT and ΔLWYIK Env-mediated viral entry with comparable efficacies. Biotin-tagged T20 effectively captured both the fusion-active, prehairpin intermediates of WT and mutant gp41 upon CD4 activation. Env without the deletion of the LWYIK motif still effectively mediated lipid mixing but inhibited content mixing. Our study demonstrates that the immediate membrane-proximal LWYIK motif acts as a unique and distinct determinant located in the gp41 C-terminal ectodomain by promoting enlargement of fusion pores and postfusion activities.

Molecular dynamics calculations on relaxed and extended states of the polypentapeptide of elastin

Abstract Reported are the first molecular dynamics calculations on the elastomeric polypentapeptide of elastin as (VPGVG)7. The salient points are that (1) there is little change in internal energy on extension; (2) a trajectory of 50 ps is insufficient to reflect the primary structural periodicity in rms displacements of torsion angles, but does show librational processes and their damping on extension; and (3) the recurring β-turn structure is retained.

pK Shift of functional group in mechanochemical coupling due to hydrophobic effect: Evidence for an apolar-polar repulsion free energy in water

In the sequential polypeptide poly[4(VPGVG),(VPGEG)] and its more hydrophobic analog poly[4(IPGVG),(IPGEG)] when the material is gamma-irradiation cross-linked to form an elastomeric matrix, mechanochemical coupling occurs on changing the pH, that is, motion and mechanical work are achieved by a change in proton chemical potential. The temperature dependence of aggregation at different pH values in phosphate buffered saline demonstrates the pK to be shifted approximately one pH unit higher for the more hydrophobic sequential polypeptide. The pH dependence of contraction or relaxation for each elastomer shows a similar shift. Data are reviewed and 2D-NMR data are presented which argue that the pK shift is not due to different conformations of the polypentapeptides. Specifically it is proposed that there exist a competition between carboxylates and hydrophobic side chains for mutually incompatible water structures; this results in an apolar-polar repulsion free energy in water with the difference in free energy reflecting the difference in the lle and Val hydrophobicities.

The function of coreceptor as a basis for the kinetic dissection of HIV type 1 envelope protein-mediated cell fusion

The function of HIV‐1 HXB2 envelope (Env) glycoprotein (gp) was investigated by surface plasmon resonance and fluorescence imaging techniques. Strikingly, it was found that gp120 shedding requires the presence of the X4 coreceptor. A similar coreceptor requirement was observed for the membrane mixing and the Env recruitment on the cell surface. However, exposure and membrane penetration of the fusion peptide do not require X4 and occur within the first minute after incubation of Env with CD4 and/or X4. Analogously X4 was not required but enhanced binding of the fusion inhibitor. In contrast, bundle formation of the gp41 ectodomain, as monitored by NC‐1, was accelerated by the presence of X4. The kinetics of these key post‐Env binding events as determined in real time by fluorescence microscopic imaging, coupled with the differential coreceptor requirement, led to the proposition that gp120 shedding, which takes place from 1 to 10 min after engagement of receptor and coreceptor to Env, is a primary function of the coreceptor. The shedding of the surface subunits is needed for the subsequent processes including hemi‐fusion, full fusion, and Env recruitment. The temporal order of these fusogenic steps allows construction of a refined model on the Env‐mediated cell fusion event. Chien M.‐P., Jiang, S., Chang D.‐K. The function of coreceptor as a basis for the kinetic dissection of HIV type 1 Env‐mediated cell fusion. FASEB J. 22, 1179–1192 (2008)

Fluorescence evidence for a loose self-assembly of the fusion peptide of influenza virus HA2 in the lipid bilayer.

Steady state fluorescence experiments were performed on a 25-mer synthetic peptide incorporated in the phospholipid vesicle to study the role of oligomerization of the fusion peptide in membrane fusion. It was found from fluorescence resonance energy transfer (FRET) that the extent of lipid mixing and the initial mixing rate varied with the fusion peptide concentration in a higher than linear fashion, indicating that the peptide promoted membrane mixing as oligomers. Results of self-quenching of the Rhodamine (Rho) in Rho-labelled peptide incorporated in the phospholipid bilayer indicated that the peptide molecules assembled in the bilayer with an order higher than dimer. The data also revealed that the peptides were not tightly packed in the membrane. Binding affinity measurement monitored by the NBD fluorescence intensity on the fluorophore-labelled fusion peptide supports the notion of self-association of the peptide in the vesicular dispersion. In the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments, a diffuse band with apparent molecular mass close to a dimeric species of the wild type fusion peptide suggested that the fusion peptides formed loose oligomers under the influence of SDS detergent in the electric field. The result is in contrast to a less fusion-active variant which appears to exhibit less propensity for self-association.

Effects of alterations of the amino-terminal glycine of influenza hemagglutinin fusion peptide on its structure, organization and membrane interactions

Mutations of the glycine residue at the amino terminus of HA2 have been shown to have a large effect on the fusion activity of HA2, the extent of which apparently correlates with the side chain bulkiness of the substituting amino acids. To investigate into the cause of abrogation in fusogenicity and virus-promoted fusion mechanism, we synthesized several peptides in which this glycine was substituted by serine, glutamic acid, or lysine. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl sn-glycero-3-phosphoglycerol (DMPG) were used as model membranes in the fluorescence, circular dichroism (CD), and FTIR measurements while sodium dodecyl sulfate was used in NMR studies. We found that, for the less active variants, affinity to membrane, degree of solvent dehydration, lipid perturbation, depth of insertion, and helicity were less. Comparison of affinity to membrane bilayer among these analogs revealed that binding of the fusion peptide is determined largely by the hydrophobic effect. Additionally, the orientation is closer to the membrane normal for the wild-type fusion peptide in the helix form while the inactive analogs inserted more parallel to the membrane surface.