Alan Kirkpatrick

The amino-terminal peptide of HIV-1 glycoprotein 41 interacts with human erythrocyte membranes: peptide conformation, orientation and aggregation

Structural studies assessed interactions between the amino-terminal peptide (FP-I; 23 residues 519-541) of the glycoprotein 41,000 (gp41) of Human Immunodeficiency Virus Type-1 (HIV-1) and human erythrocyte membranes and simulated membrane environments. Peptide binding was examined at sub-hemolytic (approx. less than 5 microM) and hemolytic (greater than or equal to 5 microM) doses (Mobley et al. (1992) Biochem. Biophys. Acta 1139, 251-256), using circular dichroism (CD) and Fourier-transform infrared (FTIR) measurements with FP-I, and electron spin resonance (ESR) studies employing FP-I spin-labeled at either the amino-terminal alanine (FP-II; residue 519) or methionine (FP-III; position 537). In the sub-lytic regime, FP-I binds to both erythrocyte lipids and dispersions of SDS with high alpha-helicity. Further, ESR spectra of FP-II labeled erythrocyte ghosts indicated peptide binding to both lipid and protein. In ghost lipids, FP-II was monomeric and exhibited low polarity and rapid, anisotropic motion about its long molecular axis (i.e., alpha-helical axis), with restricted motion away from this axis. The spin-label at the amino-terminal residue (Ala-519) is insensitive to the aqueous broadening agent chromium oxalate and buried within the hydrophobic core of the membrane; the angle that the alpha-helix (residues 519-536) makes to the normal of the bilayer plane is either 0 degree or 40 degrees. Contrarily, ESR spectra of ghost lipids labeled with sub-lytic doses of FP-III indicated high mobility and polarity for the reporter group (Met-537) at the aqueous-membrane interface, as well as extreme sensitivity to chromium oxalate. At lytic FP-I doses, CD and FTIR showed both alpha-helix and beta-structure for peptide in ghost lipids or detergent, while ESR spectra of high-loaded FP-II in ghost membranes indicated peptide aggregates. Membrane aggregates of FP-I may be involved in hemolysis, and models are suggested for N-terminal gp41 peptide participation in HIV-induced fusion and cytolysis.

Gly-Pro-Arg Confers Stability Similar to Gly-Pro-Hyp in the Collagen Triple-helix of Host-Guest Peptides

A set of host-guest peptides of the form Ac(Gly-Pro-Hyp)3-Gly-X-Y-(Gly-Pro-Hyp)4-Gly-Gly-NH2has been designed to evaluate the propensity of different Gly-X-Y triplets for the triple-helix conformation (Shah, N. K., Ramshaw, J. A. M., Kirkpatrick, A., Shah, C., and Brodsky, B. (1996)Biochemistry 35, 10262–10268). All Gly-X-Y guest triplets led to a decrease in melting temperature from the host (Gly-Pro-Hyp)8 peptide except for Gly-Pro-Arg. In this Gly-Pro-Hyp-rich environment, Gly-Pro-Arg was found to be as stabilizing as Gly-Pro-Hyp. Decreased stability of host-guest peptides containing Gly-Pro-Lys, Gly-Pro-homo-Arg, and Gly-Arg-Hyp compared with Gly-Pro-Arg indicated a stabilization that is optimal for Arg and specific to theY-position. Arg was found to have a similar stabilizing effect when residues other than Pro are in the X-position. Both Arg and Hyp stabilize the triple-helix preferentially in theY-position in a stereospecific manner and occupy largelyY-positions in collagen. However, contiguous Gly-Pro-Hyp units are highly stable and promote triple-helix folding, whereas incorporation of multiple Gly-Pro-Arg triplets was destabilizing and folded slowly due to charge repulsion. In collagen, Gly-Pro-Arg may contribute maximally to local triple-helix stability while also having the potential for electrostatic interactions in fibril formation and binding.

Positional preferences of ionizable residues in Gly-X-Y triplets of the collagen triple-helix

Collagens contain a high amount of charged residues involved in triple-helix stability, fibril formation, and ligand binding. The contribution of charged residues to stability was analyzed utilizing a host-guest peptide system with a single Gly-X-Y triplet embedded within Ac(Gly-Pro-Hyp)3-Gly-X-Y-(Gly-Pro-Hyp)4-Gly-Gly-NH2. The ionizable residues Arg, Lys, Glu, and Asp were incorporated into the X position of Gly-X-Hyp; in the Yposition of Gly-Pro-Y; or as pairs of oppositely charged residues occupying X and Y positions. The Gly-X-Hyp peptides had similar thermal stabilities, only marginally less stable than Gly-Pro-Hyp, whereas Gly-Pro-Ypeptides showed a wide thermal stability range (T m = 30–45 °C). The stability of peptides with oppositely charged residues in the X and Y positions appears to reflect simple additivity of the individual residues, except whenX is occupied by a basic residue and Y = Asp. The side chains of Glu, Lys, and Arg have the potential to form hydrogen bonds with available peptide backbone carbonyl groups within the triple-helix, whereas the shorter Asp side chain does not. This may relate to the unique involvement of Asp residues in energetically favorable ion pair formation. These studies clarify the dependence of triple-helix stability on the identity, position, and ionization state of charged residues.

THE INTERACTIONS OF THE N-TERMINAL FUSOGENIC PEPTIDE OF HIV-1 GP41 WITH NEUTRAL PHOSPHOLIPIDS

Abstract We have studied the interactions with neutral phospholipid bilayers of FPI, the 23-residue fusogenic N-terminal peptide of the HIV-1LAI transmembrane glycoprotein gp41, by CD, EPR, NMR, and solid state NMR (SSNMR) with the objective of understanding how it lyses and fuses cells. Using small unilamellar vesicles made from egg yolk phoshatidylcholine which were not fused or permeabilised by the peptide we obtained results suggesting that it was capable of inserting as an α-helix into neutral phospholipid bilayers but was only completely monomeric at peptide/lipid (P/L) ratios of 1/2000 or lower. Above this value, mixed populations of monomeric and multimeric forms were found with the proportion of multimer increasing proportionally to P/L, as calculated from studies on the interaction between the peptide and spin-labelled phospholipid. The CD data indicated that, at P/L between 1/200 and 1/100, approximately 68% of the peptide appeared to be in α-helical form. When P/L=1/25 the α-helical content had decreased to 41%. Measurement at a P/L of 1/100 of the spin lattice relaxation effect on the 13C nuclei of the phospholipid acyl chains of an N-terminal spin label attached to the peptide showed that most of the peptide N-termini were located in the interior hydrocarbon region of the membrane. SSNMR on multilayers of ditetradecylphosphatidyl choline at P/Ls of 1/10, 1/20 and 1/30 showed that the peptide formed multimers that affected the motion of the lipid chains and disrupted the lipid alignment. We suggest that these aggregates may be relevant to the membrane-fusing and lytic activities of FPI and that they are worthy of further study.

The effect of sequence variations and structure on the cytolytic activity of melittin peptides

The importance of various amino acid residues in melittin for cytolytic function against mammalian cells was assessed by use of a monoclonal antibody to the C-terminal region, synthesis of peptide analogues and chemical modification of specific residues. A monoclonal anti-melittin antibody directed to the basic C-terminal region inhibited cytolytic activity. Consistent with this, deletion of one of the two Lys Arg sequences at the C terminal end of the peptide reduced cytolysis 8-fold, although significant activity was still present. A similar reduction in activity was also found with a synthetic analogue which had the reverse sequence to melittin. In contrast, when the last 6 residues of the C-terminal region were transferred to the N-terminus, a peptide with markedly reduced activity was obtained. Chemical modification of lysine and arginine residues of melittin indicated that lysine was only minimally important for functional activity compared with arginine which was essential. In particular, our results demonstrate that substitution of serine for lysine 7 has no significant effect on the activity of the peptide and suggest that this residue is important only in maintaining the amphipathic helix of the peptide.

The amino-terminal peptide of HIV-1 glycoprotein 41 lyses human erythrocytes and CD4+ lymphocytes.

Functional studies assessed the cytolytic activity of the amino terminal peptide (FP-I; 23 residues 519-541) of the glycoprotein 41,000 (gp41) of the Human Immunodeficiency Virus Type-1 (HIV-1). Synthetically prepared FP-I efficiently hemolyzed human red blood cells at 37 degrees C, with 40% lysis at 32 microM. Kinetic studies indicated that FP-I induced maximal hemolysis in 30 min, probably through tight binding of the peptide with the red cell membrane. The Phe-Leu-Gly-Phe-Leu-Gly (residues 526-531) motif in FP-I apparently plays a critical role in lysis of red cells, since no hemolytic activity was observed for an amino-acid-substituted FP-I in which the unique Phe-Leu-Gly-Phe-Leu-Gly was converted to Ala-Leu-Gly-Ala-Leu-Gly. As neither smaller constituent peptides (e.g., residues 519-524 and residues 526-536) nor a N-terminal flanking peptide (e.g., residues 512-523) induced red cell hemolysis, the entire 23-residue (519-541) sequence of FP-I may be required for hemolytic activity. FP-I was also cytolytic with CD4(+)-bearing Hut-78 cells, with 40% lysis at approx. 150 microM. These results are consistent with an earlier hypothesis that the N-terminal peptide of gp41 may partially contribute to the in vivo cytopathic actions of HIV-1 infection (Gallaher, W.R. (1987) Cell 50, 327-328).

The amino-terminal peptide of HIV-1 glycoprotein 41 fuses human erythrocytes

The ability of synthetic peptides based on the amino-terminus of HIV-1 glycoprotein 41,000 (gp41) to fuse human erythrocytes was investigated. Previous site-directed mutagenesis studies have shown an important role for the N-terminal gp41 domain in HIV-fusion, in which replacement of hydrophobic amino acids with polar residues inhibits viral infection and syncytia formation. Here, a synthetic peptide (FP; 23 amino acid residues 519-541) corresponding to the N-terminus of HIV-1 gp41, and also a FP analog (FP526L/R) with Arg replacing Leu-526, were prepared with solid phase techniques. The lipid mixing and leakage of resealed ghosts triggered by these peptides were examined with fluorescence quenching techniques. Peptide-induced aggregation of human erythrocytes was studied using Coulter counter sizing and scanning electron microscopy (SEM). Using resealed erythrocyte ghosts at physiologic pH, FP induces rapid lipid mixing between red cell membranes at doses previously shown to hemolyze intact cells. FP also causes leakage from resealed ghosts, and promotes the formation of multicelled aggregates with whole erythrocytes. Contrarily, similar FP526L/R concentrations did not induce red cell lysis, lipid mixing, leakage or aggregation. Since the fusogenic potency of FP and FP526L/R parallels earlier gp41 mutagenesis studies showing that substitution of Arg for Leu-526 blocks fusion activity, these data suggest that the N-terminal gp41 domain in intact HIV participates in fusion.

New insights into heparin binding to vitronectin: studies with monoclonal antibodies

Vitronectin is a plasma glycoprotein that binds to a variety of ligands. There is considerable debate regarding the dependency of these binding interactions upon the conformational status of vitronectin, the role of multimerization and how the binding of different ligands can change vitronectins conformational state. We have developed a method of capturing vitronectin directly from fresh plasma using solid-phase monoclonal antibodies. Various biotin-labelled secondary monoclonal antibodies were used to quantify the bound vitronectin and to measure its degree of denaturation. Using these tools we demonstrated that one monoclonal antibody partially denatured vitronectin without direct multimerization. Treatment of vitronectin in plasma with soluble heparin produced a similar degree of denaturation. These results led to a proposed adaptation of the unfolding/refolding pathways for chemically denatured vitronectin originally presented by Zhuang and co-workers in 1996 [Zhuang, Blackburn and Peterson (1996) J. Biol. Chem. 271, 14323-14332 and Zhuang, Li, Williams, Wagner, Seiffert and Peterson (1996) J. Biol. Chem. 271, 14333-14343]. The adapted version allows for the production of a more stable partially unfolded intermediate, resulting from the binding of particular ligands. We also demonstrated that the avidity of heparin binding to vitronectin is governed by both the conformational state of the monomer and multimerization of the molecule.

The use of peptide-mediated electrofusion to select monoclonal antibodies directed against specific and homologous regions of the potyvirus coat protein

Whilst monoclonal antibodies (Mab) to potyviruses have been generated, it has not been possible to produce molecules with high specificity or broad reactivity to defined conserved amino acid sequences. In the current study, peptide-mediated electrofusion was used to select for high efficiency antibody-secreting hybridomas after mice were immunized with highly immunogenic viral coat protein. Mice were immunized with coat protein from either one potyvirus (potato virus Y, PVY-D) or a mixture of five distinct potyviruses. Two well-defined peptides were used for selective electrofusions. Peptide-1 was selected from the highly specific N terminal region of PVY-D and peptide-2 from the highly conserved N terminal/core junction region of Johnson grass mosaic virus (JGMV). Conventional PEG-mediated fusions using mice immunized with these peptides did not result in hybridoma formation. On the other hand, electrofusions using biotin-streptavidin to bridge peptide-specific B cells to myeloma cells produced hybridomas secreting antibodies either highly specific to PVY-D or cross-reactive with all potyviruses, depending on the peptide used.

Structure and activity of D-Pro14 melittin.

D-Pro14 melittin was synthesized to investigate the effect of increasing the angle of the bend in the hinge region between the helical segments of the molecule. Structural analysis by nuclear magnetic resonance indicated that, in methanol, the molecule consisted of two helices separated at Pro14, as in melittin. However, the two helices in D-Pro14 melittin were laterally displaced relative to each other by approximately 7 Å, and in addition, there was a small rotation of the carboxyl-terminal helix relative to the amino-terminal helix around the long axis of the molecule. The peptide had less than 5% of the cytolytic activity of melittin. Modification of Arg22 with the 2,2,5,7,8-pentamethyl-chroman-6-sulphonyl (pmc) group restored hemolytic activity to close to that of unmodified melittin. Replacement of Arg22 with Phe was less effective in restoring hemolytic activity. Electron-paramagnetic resonance studies suggest that there is a positive correlation between hemolytic activity of the peptides and interaction with phospholipid bilayers.