Walter R. P. Scott

Curling of flap tips in HIV-1 protease as a mechanism for substrate entry and tolerance of drug resistance

BACKGROUND The human immunodeficiency virus type 1 (HIV-1) protease is an essential viral protein that is a major drug target in the fight against Acquired Immune Deficiency Syndrome (AIDS). Access to the active site of this homodimeric enzyme is gained when two large flaps, one from each monomer, open. The flap movements are therefore central to the function of the enzyme, yet determining how these flaps move at an atomic level has not been experimentally possible. RESULTS In the present study, we observe the flaps of HIV-1 protease completely opening during a 10 ns solvated molecular dynamics simulation starting from the unliganded crystal structure. This movement is on the time scale observed by Nuclear Magnetic Resonance (NMR) relaxation data. The highly flexible tips of the flaps, with the sequence Gly-Gly-Ile-Gly-Gly, are seen curling back into the protein and thereby burying many hydrophobic residues. CONCLUSIONS This curled-in conformational change has never been previously described. Previous models of this movement, with the flaps as rigid levers, are not consistent with the experimental data. The residues that participate in this hydrophobic cluster as a result of the conformational change are highly sensitive to mutation and often contribute to drug resistance when they do change. However, several of these residues are not part of the active site cavity, and their essential role in causing drug resistance could possibly be rationalized if this conformational change actually occurs. Trapping HIV-1 protease in this inactive conformation would provide a unique opportunity for future drug design.

Structural Studies of a Peptide with Immune Modulating and Direct Antimicrobial Activity

The structure and function of the synthetic innate defense regulator peptide 1018 was investigated. This 12 residue synthetic peptide derived by substantial modification of the bovine cathelicidin bactenecin has enhanced innate immune regulatory and moderate direct antibacterial activities. The solution state NMR structure of 1018 in zwitterionic dodecyl phosphocholine (DPC) micelles indicated an α-helical conformation, while secondary structures, based on circular dichroism measurements, in anionic sodium dodecyl sulfate (SDS) and phospholipid vesicles (POPC/PG in a 1:1 molar ratio) and simulations revealed that 1018 can adopt a variety of folds, tailored to its different functions. The structural data are discussed in light of the ability of 1018 to potently induce chemokine responses, suppress the LPS-induced TNF-α response, and directly kill both Gram-positive and Gram-negative bacteria.

Effect of divalent cations on the structure of the antibiotic daptomycin

Daptomycin, a cyclic anionic lipopeptide antibiotic, whose three-dimensional structure was recently solved using solution state NMR (Ball et al. 2004; Jung et al. 2004; Rotondi and Gierasch 2005), requires calcium for function. To date, the exact nature of the interaction between divalent cations, such as Ca2+ or Mg2+, has not been fully characterized. It has, however, been suggested that addition of Ca2+ to daptomycin in a 1:1 molar ratio induces aggregation. Moreover, it has been suggested that certain residues, e.g. Asp3 and Asp7, which are essential for activity (Grunewald et al. 2004; Kopp et al. 2006), may also be important for Ca2+ binding (Jung et al. 2004). In this work, we have tried: (1) to further pinpoint how Ca2+ affects daptomycin structure/oligomerization using analytical ultracentrifugation; and (2) to determine whether a specific calcium binding site exists, based on one-dimensional 13C NMR spectra and molecular dynamics (MD) simulations. The centrifugation results indicated that daptomycin formed micelles of between 14 and 16 monomers in the presence of a 1:1 molar ratio of Ca2+ and daptomycin. The 13C NMR data indicated that addition of calcium had a significant effect on the Trp1 and Kyn13 residues, indicating that either calcium binds in this region or that these residues may be important for oligomerization. Finally, the molecular dynamics simulation results indicated that the conformational change of daptomycin upon calcium binding might not be as significant as originally proposed. Similar studies on the divalent cation Mg2+ are also presented. The implication of these results for the biological function of daptomycin is discussed.

Mechanism of Action and Limited Cross-Resistance of New Lipopeptide MX-2401

ABSTRACT MX-2401 is a semisynthetic calcium-dependent lipopeptide antibiotic (analogue of amphomycin) in preclinical development for the treatment of serious Gram-positive infections. In vitro and in vivo, MX-2401 demonstrates broad-spectrum bactericidal activity against Gram-positive organisms, including antibiotic-resistant strains. The objective of this study was to investigate the mechanism of action of MX-2401 and compare it with that of the lipopeptide daptomycin. The results indicated that although both daptomycin and MX-2401 are in the structural class of Ca2+-dependent lipopeptide antibiotics, the latter has a different mechanism of action. Specifically, MX-2401 inhibits peptidoglycan synthesis by binding to the substrate undecaprenylphosphate (C55-P), the universal carbohydrate carrier involved in several biosynthetic pathways. This interaction resulted in inhibition, in a dose-dependent manner, of the biosynthesis of the cell wall precursors lipids I and II and the wall teichoic acid precursor lipid III, while daptomycin had no significant effect on these processes. MX-2401 induced very slow membrane depolarization that was observed only at high concentrations. Unlike daptomycin, membrane depolarization by MX-2401 did not correlate with its bactericidal activity and did not affect general membrane permeability. In contrast to daptomycin, MX-2401 had no effect on lipid flip-flop, calcein release, or membrane fusion with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (POPG) liposomes. MX-2401 adopts a more defined structure than daptomycin, presumably to facilitate interaction with C55-P. Mutants resistant to MX-2401 demonstrated low cross-resistance to other antibiotics. Overall, these results provided strong evidence that the mode of action of MX-2401 is unique and different from that of any of the approved antibiotics, including daptomycin.

Cooperative fluctuations of unliganded and substrate-bound HIV-1 protease: a structure-based analysis on a variety of conformations from crystallography and molecular dynamics simulations

The dynamics of HIV‐1 protease, both in unliganded and substrate‐bound forms have been analyzed by using an analytical method, Gaussian network model (GNM). The method is applied to different conformations accessible to the protein backbone in the native state, observed in crystal structures and snapshots from fully atomistic molecular dynamics (MD) simulation trajectories. The modes of motion obtained from GNM on different conformations of HIV‐1 protease are conserved throughout the MD simulations. The flaps and 40s loop of the unliganded HIV‐1 protease structure are identified as the most mobile regions. However, in the liganded structure these flaps lose mobility, and terminal regions of the monomers become more flexible. Analysis of the fast modes shows that residues important for stability are in the same regions of all the structures examined. Among these, Gly86 appears to be a key residue for stability. The contribution of residues in the active site region and flaps to the stability is more pronounced in the substrate‐bound form than in the unliganded form. The convergence of modes in GNM to similar regions of HIV‐1 protease, regardless of the conformation of the protein, supports the robustness of GNM as a potentially useful and predictive tool. Proteins 2003;51:409–422.

Assessing the effects of time and spatial averaging in 15N chemical shift/15N-1H dipolar correlation solid state NMR experiments.

The effect of time and spatial averaging on 15N chemical shift/1H-15N dipolar correlation spectra, i.e., PISEMA spectra, of α-helical membrane peptides and proteins is investigated. Three types of motion are considered: (a) Librational motion of the peptide planes in the α-helix; (b) rotation of the helix about its long axis; and (c) wobble of the helix about a nominal tilt angle. A 2ns molecular dynamics simulation of helix D of bacteriorhodopsin is used to determine the effect of librational motion on the spectral parameters. For the time averaging, the rotation and wobble of this same helix are modelled by assuming either Gaussian motion about the respective angles or a uniform distribution of a given width. For the spatial averaging, regions of possible 15N chemical shift/1H-15N dipolar splittings are computed for a distribution of rotations and/or tilt angles of the helix. The computed spectra show that under certain motional modes the 15N chemical shift/1H-15N dipolar pairs for each of the residues do not form patterns which mimic helical wheel patterns. As a result, the unambiguous identification of helix tilt and helix rotation without any resonance assignments or on the basis of a single assignment may be difficult.

On the structures of filamentous bacteriophage Ff (fd, f1, M13)

The filamentous bacteriophage (Inovirus) strain Ff (fd, f1, M13) is widely used in molecular biophysics as a simple model system. A low resolution molecular model of the fd protein coat has been reported, derived from iterative helical real space reconstruction of cryo-electron micrographs (cryoEM). This model is significantly different from the model previously derived from X-ray fibre diffraction and solid-state NMR. We show that the cryoEM model agrees neither with solid-state NMR data nor with X-ray fibre diffraction data of fd, and has some puzzling structural features, for instance nanometre holes through the protein coat. We refine the cryoEM model against the X-ray data, and find that the model after refinement closely approximates the model derived directly from X-ray fibre diffraction and solid-state NMR data. We suggest possible reasons for the differences between the models derived from cryoEM and X-ray diffraction.

On using time-averaging restraints in molecular dynamics simulation

Introducing experimental values as restraints into molecular dynamics (MD) simulations to bias the values of particular molecular properties, such as nuclear Overhauser effect intensities or distances, 3J coupling constants, chemical shifts or crystallographic structure factors, towards experimental values is a widely used structure refinement method. To account for the averaging of experimentally derived quantities inherent in the experimental techniques, time-averaging restraining methods may be used. In the case of structure refinement using 3J coupling constants from NMR experiments, time-averaging methods previously proposed can suffer from large artificially induced structural fluctuations. A modified time-averaged restraining potential energy function is proposed which overcomes this problem. The different possible approaches are compared using stochastic dynamics simulations of antamanide, a cyclic peptide of ten residues.

Protein structure prediction force fields: Parametrization with quasi‐newtonian dynamics

We present an unusual method for parametrizing low‐resolution force fields of the type used for protein structure prediction. Force field parameters were‐determined by assigning each a fictitious mass and using a quasi‐molecular dynamics algorithm in parameter space. The quasi‐energy term favored folded native structures and specifically penalized folded nonnative structures. The force field was generated after optimizing less than 70 adjustable parameters, but shows a strong ability to discriminate between native structures and compact misfolded‐alternatives. The functional form of the force field was chosen as in molecular mechanics and is not table‐driven. It is continuous with continuous derivatives and is thus suitable for use with algorithms such as energy minimization or newtonian dynamics. Proteins 27:367–384, 1997.

Synthetic Fusion Peptides of Tick-Borne Encephalitis Virus as Models for Membrane Fusion

The fusion peptide of TBEV is a short segment of the envelope protein that mediates viral and host cell membrane fusion at acidic pH. Previous studies on the E protein have shown that mutations at L107 have an effect on fusogenic activity. Structural studies have also suggested that during the fusion process the E protein rearranges to form a trimer. In the present study, a number of short peptides were synthesized, and their structure/activity was examined: (1) monomers consisting of residues 93-113 of the wild-type E protein with Leu at position 107 (WT) and two mutants, namely, L107F and L107T; (2) a monomer consisting of residues 93-113 of the E protein with a C105A mutation (TFPmn); (3) a trimer consisting of three monomers described in (2), linked at the C-terminus via 1 Lys (TFPtr); (4) a monomer consisting of residues 93-113 of the E protein plus six additional Lys at the C-terminus; and (5) a trimer consisting of three monomers described in (3), linked via the side chain of the sixth lysine. The secondary structure content of all peptides was investigated using circular dichroism (CD). Approximately seven of the residues were in beta-strand conformation, in the presence of POPC/POPE/cholesterol. The structures did not depend on pH significantly. The fusogenicity of the peptides was measured by FRET and photon correlation spectroscopy. The data suggest that TFPtr is the most fusogenic at acidic pH and that the mutation from L107 to T reduces activity. Molecular dynamics simulations of WT, L107T, and L107F suggest that this reduction in activity may be related to the fact that the mutations disrupt trimer stability. Finally, tryptophan fluorescence experiments were used to localize the peptides in the membrane. It was found that WT, L107F, TFPmn, and TFPtr could penetrate better into the acyl chain region of the lipids than the other peptides tested. The implications of these results on the fusion mechanism of TBEV E protein will be presented.