Structure, activity and interactions of the cysteine deleted analog of tachyplesin-1 with lipopolysaccharide micelle: Mechanistic insights into outer-membrane permeabilization and endotoxin neutralization.

Tachyplesin-1, a disulfide stabilized beta-hairpin antimicrobial peptide, can be found at the hemocytes of horse shoe crab Tachypleus tridentatus. A cysteine deleted linear analog of tachyplesin-1 or CDT (KWFRVYRGIYRRR-NH2) contains a broad spectrum of bactericidal activity with a reduced hemolytic property. The bactericidal activity of CDT stems from selective interactions with the negatively charged lipids including LPS. In this work, CDT-LPS interactions were investigated using NMR spectroscopy, optical spectroscopy and functional assays. We found that CDT neutralized LPS and disrupted permeability barrier of the outer membrane. Zeta potential and ITC studies demonstrated charge compensation and hydrophobic interactions of CDT with the LPS-outer membrane, respectively. Secondary structure of the peptide was probed by CD and FT-IR experiments indicating beta-strands and/or beta-turn conformations in the LPS micelle. An ensemble of structures, determined in LPS micelle by NMR, revealed a beta-hairpin like topology of the CDT peptide that was typified by an extended cationic surface and a relatively shorter segment of hydrophobic region. Interestingly, at the non-polar face, residue R11 was found to be in a close proximity to the indole ring of W2, suggesting a cation-n type interactions. Further, saturation transfer difference (STD) NMR studies established intimate contacts among the aromatic and cationic residues of CDT with the LPS micelle. Fluorescence and dynamic light scattering experiments demonstrated that CDT imparted structural destabilization to the aggregated states of LPS. Collectively, atomic resolution structure and interactions of CDT with the outer membrane-LPS could be exploited for developing potent broad spectrum antimicrobial and anti-sepsis agents.

NMR structure, localization, and vesicle fusion of Chikungunya virus fusion peptide.

The virus-host cell fusion process is mediated by a membrane anchored viral fusion protein that inserts its hydrophobic fusion peptide into the plasma membrane of the host cell, initiating the fusion reaction. Therefore, fusion peptides are an important functional constituent of the fusion proteins of enveloped viruses. In this work, we characterize the fusion peptide or VT18 (V(84)YPFMWGGAYCFCDAENT(101)) of Chikungunya virus (CHIKV) using NMR and fluorescence spectroscopy in zwitterionic lipid environments. Our results demonstrate that the VT18 peptide is able to induce liposome fusions in a pH independent manner and interacts with the zwitterionic lipid vesicles. The NMR derived three-dimensional structure of VT18, in solution of dodecylphosphocholine (DPC) micelles, is typified by extended or ß-type conformations for most of the residues, whereby residues M88-W89-G90-G91 adopt a type I ß-turn conformation. Strikingly, the aromatic side chains of residues Y85, F87, Y93, and F95 in the VT18 structure are found to be well-packed forming an aromatic core. In particular, residue F87 is situated at the center of the aromatic core establishing a close proximity with other aromatic side chains. Further, the aromatic core residues are also involved in packing interactions with the side chains of residues M88, C94. Paramagnetic relaxation enhancement NMR, using spin labeled doxyl lipids, indicated that the aromatic core residues of VT18 are well inserted into the micelles, whereas the polar residues at the C-terminus may be surface localized. The atomic resolution structure and lipid interactions of CHIKV fusion peptide presented here will aid to uncover the fusion mechanism by the type II viral fusion proteins.

NMR structure of pardaxin, a pore-forming antimicrobial peptide, in lipopolysaccharide micelles: mechanism of outer membrane permeabilization.

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is an important element against permeability of bactericidal agents, including antimicrobial peptides. However, structural determinants of antimicrobial peptides for LPS recognition are not clearly understood. Pardaxins (Pa1, Pa2, Pa3, and Pa4) are a group of pore-forming bactericidal peptides found in the mucous glands of sole fishes. Despite having a low net positive charge, pardaxins contain a broad spectrum of antibacterial activities. To elucidate the structural basis of LPS interactions of pardaxins, herein, we report the first three-dimensional structure of Pa4 bound to LPS micelles. The binding kinetics of Pa4 with LPS is estimated using [(15)N-Leu-19] relaxation dispersion NMR experiments. LPS/Pa4 interactions are further characterized by a number of biophysical methods, including isothermal titration calorimetry, (31)P NMR, saturation transfer difference NMR, dynamic light scattering, and IR spectroscopy. In the LPS-Pa4 complex, Pa4 adopts a unique helix-turn-helix conformation resembling a "horseshoe." Interestingly, the LPS-bound structure of Pa4 shows striking differences with the structures determined in lipid micelles or organic solvents. Saturation transfer difference NMR identifies residues of Pa4 that are intimately associated with LPS micelles. Collectively, our results provide mechanistic insights into the outer membrane permeabilization by pardaxin.

Structure, interactions, and antibacterial activities of MSI-594 derived mutant peptide MSI-594F5A in lipopolysaccharide micelles: role of the helical hairpin conformation in outer-membrane permeabilization.

Lipopolysaccharide (LPS) provides a well-organized permeability barrier at the outer membrane of Gram-negative bacteria. Host defense cationic antimicrobial peptides (AMPs) need to disrupt the outer membrane before gaining access to the inner cytoplasmic membrane or intracellular targets. Several AMPs are largely inactive against Gram-negative pathogens due to the restricted permeation through the LPS layer of the outer membrane. MSI-594 (GIGKFLKKAKKGIGAVLKVLTTG) is a highly active AMP with a broad-spectrum of activities against bacteria, fungi, and virus. In the context of LPS, MSI-594 assumes a hairpin helical structure dictated by packing interactions between two helical segments. Residue Phe5 of MSI-594 has been found to be engaged in important interhelical interactions. In order to understand plausible structural and functional inter-relationship of the helical hairpin structure of MSI-594 with outer membrane permeabilization, a mutant peptide, termed MSI-594F5A, containing a replacement of Phe5 with Ala has been prepared. We have compared antibacterial activities, outer and inner membrane permeabilizations, LPS binding affinity, perturbation of LPS micelles structures by MSI-594 and MSI-594F5A peptides. Our results demonstrated that the MSI-594F5A has lower activities against Gram-negative bacteria, due to limited permeabilization through the LPS layer, however, retains Gram-positive activity, akin to MSI-594. The atomic-resolution structure of MSI-594F5A has been determined in LPS micelles by NMR spectroscopy showing an amphipathic curved helix without any packing interactions. The 3D structures, interactions, and activities of MSI-594 and its mutant MSI-594F5A in LPS provide important mechanistic insights toward the requirements of LPS specific conformations and outer membrane permeabilization by broad-spectrum antimicrobial peptides.

Functional and structural characterization of the talin F0F1 domain.

The globular head domain of talin, a large multi-domain cytoplasmic protein, is required for inside-out activation of the integrins, a family of heterodimeric transmembrane cell adhesion molecules. Talin head contains a FERM domain that is composed of F1, F2, and F3 subdomains. A F0 subdomain is located N-terminus to F1. The F3 contains a canonical phosphotyrosine binding (PTB) fold that directly interacts with the membrane proximal NPxY/F motif in the integrin beta cytoplasmic tail. This interaction is stabilized by the F2 that interacts with the lipid head-groups of the plasma membrane. In comparison to F2 and F3, the properties of the F0F1 remains poorly characterized. Here, we showed that F0F1 is essential for talin-induced activation of integrin alphaLbeta2 (LFA-1). F0F1 has a high content of beta-sheet secondary structure, and it tends to homodimerize that may provide stability against proteolysis and chaotrope induced unfolding.

NMR structural studies of the Ste11 SAM domain in the dodecyl phosphocholine micelle.

The sterile alpha-motif (SAM), a relatively small ( approximately 70 amino acids) interaction domain, is found in a variety of proteins involved in cell signaling, transcription regulation, and scaffolding. The Ste11 protein kinase from the mitogen activated protein kinase (MAPK) signaling cascades of the budding yeast is regulated by a SAM domain located at the N-terminus of full-length protein. In solution, the Ste11 SAM domain exists as a well-folded dimeric structure that is involved in interaction with the cognate SAM domain from an adaptor protein Ste50. In this work, we show that the Ste11 SAM domain has an intrinsic affinity towards the lipid membranes. The solution conformation of the Ste11 SAM determined in perdeuterated DPC micelle, using NMR spectroscopy, is defined by five helices of different lengths connected by a number of loops. In the micelle bound state, the non-polar and aromatic residues of the Ste11 SAM lack a native-like packing and are presumably engaged in interactions with the micelle. Using two different paramagnetic doxyl-lipids; we have mapped out localization of Ste11 SAM residues at the micelle surface. Most of the residues appear to localize at the interfacial region of the micelle. However, a number of non-polar residues from the central region of the domain are found to be located inside the core of the micelle including residues from the helix 4 and a loop between helix 2 and helix 3. Isothermal titration calorimetry studies demonstrate that a facile insertion of the Ste11 SAM into the DPC micelle is primarily driven by a large change in enthalpy, -50 kcal/mol with an apparent equilibrium association constant (Ka) of 7.86 x 10(6) M(-1). Interestingly, an interfacial mutant L60R of the Ste11 SAM lacking the dimeric structure does not show detectable interactions with the lipid micelle. The micelle-bound structure of the Ste11 SAM domain described in this work may have potential implications in the regulation of MAPK signaling whereby positioning of the Ste11 protein in close proximity to the membrane may facilitate efficient phosphorylation of the Ste11 kinase by the membrane attached upstream Ste20/pak kinase.

Structural and thermodynamic analyses of the interaction between melittin and lipopolysaccharide.

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is the very first site of interactions with the antimicrobial peptides. In this work, we have determined a solution conformation of melittin, a well-known membrane active amphiphilic peptide from honey bee venom, by transferred nuclear Overhauser effect (Tr-NOE) spectroscopy in its bound state with lipopolysaccharide. The LPS bound conformation of melittin is characterized by a helical structure restricted only to the C-terminus region (residues A15-R24) of the molecule. Saturation transfer difference (STD) NMR studies reveal that several C-terminal residues of melittin including Trp19 are in close proximity with LPS. Isothermal titration calorimetry (ITC) data demonstrates that melittin binding to LPS or lipid A is an endothermic process. The interaction between melittin and lipid A is further characterized by an equilibrium association constant (Ka) of 2.85 x 10(6) M(-1) and a stoichiometry of 0.80, melittin/lipid A. The estimated free energy of binding (delta G0), -8.8 kcal mol(-1), obtained from ITC experiments correlates well with a partial helical structure of melittin in complex with LPS. Moreover, a synthetic peptide fragment, residues L13-Q26 or mel-C, derived from the C-terminus of melittin has been found to contain comparable outer membrane permeabilizing activity against Escherichia coli cells. Intrinsic tryptophan fluorescence experiments of melittin and mel-C demonstrate very similar emission maxima and quenching in presence of LPS micelles. The Red Edge Excitation Shift (REES) studies of tryptophan residue indicate that both peptides are located in very similar environment in complex with LPS. Collectively, these results suggest that a helical conformation of melittin, at its C-terminus, could be an important element in recognition of LPS in the outer membrane.

Interactions of a designed peptide with lipopolysaccharide: Bound conformation and anti-endotoxic activity.

Designed peptides that would selectively interact with lipopolysaccharide (LPS) or endotoxin and fold into specific conformations could serve as important scaffolds toward the development of antisepsis compounds. Here, we describe solution structure of a designed amphipathic peptide, H(2)N-YVKLWRMIKFIR-CONH(2) (YW12D) in complex with endotoxin as determined by transferred nuclear Overhauser effect spectroscopy. The conformation of the isolated peptide is highly flexible, but undergoes a dramatic structural stabilization in the presence of LPS. Structure calculations reveal that the peptide presents two amphipathic surfaces in its bound state to LPS whereby each surface is characterized by two positive charges and a number of aromatic and/or aliphatic residues. ITC data suggests that peptide interacts with two molecules of lipid A. In activity assays, YW12D exhibits neutralization of LPS toxicity with very little hemolysis of red blood cells. Structural and functional properties of YW12D would be applicable in designing low molecular weight non-toxic antisepsis molecules.

Designed beta-boomerang antiendotoxic and antimicrobial peptides: structures and activities in lipopolysaccharide.

Lipopolysaccharide (LPS), an integral part of the outer membrane of Gram-negative bacteria, is involved in a variety of biological processes including inflammation, septic shock, and resistance to host-defense molecules. LPS also provides an environment for folding of outer membrane proteins. In this work, we describe the structure-activity correlation of a series of 12-residue peptides in LPS. NMR structures of the peptides derived in complex with LPS reveal boomerang-like beta-strand conformations that are stabilized by intimate packing between the two aromatic residues located at the 4 and 9 positions. This structural feature renders these peptides with a high ability to neutralize endotoxicity, >80% at 10 nM concentration, of LPS. Replacements of these aromatic residues either with Ala or with Leu destabilizes the boomerang structure with the concomitant loss of antiendotoxic and antimicrobial activities. Furthermore, the aromatic packing stabilizing the beta-boomerang structure in LPS is found to be maintained even in a truncated octapeptide, defining a structured LPS binding motif. The mode of action of the active designed peptides correlates well with their ability to perturb LPS micelle structures. Fourier transform infrared spectroscopy studies of the peptides delineate beta-type conformations and immobilization of phosphate head groups of LPS. Trp fluorescence studies demonstrated selective interactions with LPS and the depth of insertion into the LPS bilayer. Our results demonstrate the requirement of LPS-specific structures of peptides for endotoxin neutralizations. In addition, we propose that structures of these peptides may be employed to design proteins for the outer membrane.

Berberine targets assembly of Escherichia coli cell division protein FtsZ.

The ever increasing problem of antibiotic resistance necessitates a search for new drug molecules that would target novel proteins in the prokaryotic system. FtsZ is one such target protein involved in the bacterial cell division machinery. In this study, we have shown that berberine, a natural plant alkaloid, targets Escherichia coli FtsZ, inhibits the assembly kinetics of the Z-ring, and perturbs cytokinesis. It also destabilizes FtsZ protofilaments and inhibits the FtsZ GTPase activity. Saturation transfer difference NMR spectroscopy of the FtsZ-berberine complex revealed that the dimethoxy groups, isoquinoline nucleus, and benzodioxolo ring of berberine are intimately involved in the interaction with FtsZ. Berberine perturbs the Z-ring morphology by disturbing its typical midcell localization and reduces the frequency of Z-rings per unit cell length to half. Berberine binds FtsZ with high affinity ( K D approximately 0.023 microM) and displaces bis-ANS, suggesting that it may bind FtsZ in a hydrophobic pocket. Isothermal titration calorimetry suggests that the FtsZ-berberine interaction occurs spontaneously and is enthalpy/entropy-driven. In silico molecular modeling suggests that the rearrangement of the side chains of the hydrophobic residues in the GTP binding pocket may facilitate the binding of the berberine to FtsZ and lead to inhibition of the association between FtsZ monomers. Together, these results clearly indicate the inhibitory role of berberine on the assembly function of FtsZ, establishing it as a novel FtsZ inhibitor that halts the first stage in bacterial cell division.

Equilibrium unfolding of the dimeric SAM domain of MAPKKK Ste11 from the budding yeast: role of the interfacial residues in structural stability and binding.

The sterile alpha motifs or SAM domains are small ( approximately 70 amino acids) protein-protein interaction modules that are involved in diverse functions ranging from cell signaling, transcription regulation, and scaffolding. The Ste11 protein kinase in the mitogen-activated protein kinase (MAPK) signaling cascades of the budding yeast is regulated by a SAM domain located at the N-terminus of the full-length protein. The Ste11 SAM domain forms a symmetrical dimeric structure with an interface stabilized presumably by hydrophobic and ionic interactions. Here, we investigated urea-induced unfolding, using NMR and other optical spectroscopic methods, of the dimeric Ste11 SAM domain and two of the variants, namely, L57R and L60R, each containing a point mutation at the interfacial region. Our results demonstrate that the residue-specific or global unfolding of the Ste11 SAM is highly cooperative without any evidence for folded monomeric or partially folded species. However, replacement of hydrophobic residues with basic residues in the interface caused considerable changes in the stability and folding of the Ste11 SAM domain. The native dimeric structure of the L60R mutant protein is severely affected as indicated by a high propensity toward aggregation. On the other hand, the L57R mutant, although retaining the native structure, shows a dramatic decrease in the conformational stability as revealed by urea-induced denaturation and amide proton exchange studies. Furthermore, isothermal titration calorimetry and intrinsic tryptophan fluorescence experiments demonstrate that the L57R interacts with the cognate SAM domain from Ste50 with reduced affinity, while the L60R protein is devoid of any detectable binding activity. These results demonstrate that the interfacial residues of the dimeric SAM domain of Ste11 are critically involved in its structural stability and binding to the Ste50 SAM domain.

High-resolution solution structure of a designed peptide bound to lipopolysaccharide: transferred nuclear Overhauser effects, micelle selectivity, and anti-endotoxic activity.

Designing peptides that would interact with lipopolysaccharides (LPS) and acquire a specific folded conformation can generate useful structural insights toward the development of anti-sepsis agents. In this work, we have constructed a 12-residue linear peptide, YW12, rich in aromatic and aliphatic amino acid residues with a centrally located stretch of four consecutive positively charged (KRKR) residues. In absence of LPS, YW12 is predominantly unstructured in aqueous solution. Using transferred nuclear Overhauser effect (Tr-NOE) spectroscopy, we demonstrate that YW12 adopts a well-folded structure as a complex with LPS. Structure calculations reveal that YW12 assumes an extended conformation at the N-terminus followed by two consecutive beta-turns at its C-terminus. A hydrophobic core is formed by extensive packing between number of aromatic and nonpolar residues, whereas the positively charged residues are segregated out to a separate region essentially stabilizing an amphipathic structure. In an in vitro LPS neutralization assay using NF-kappaB induction as the readout, YW12 shows moderate activity with an IC50 value of approximately 10 microM. As would be expected, tryptophan fluorescence studies demonstrate that YW12 shows selective interactions only with the negatively charged lipid micelles including sodium dodecyl sulfate (SDS), 1-palmitoyl-2-oleoylphosphatidyl-dl-glycerol (POPG), and LPS, and no significant interactions are detected with zwitterionic lipid micelles such as dodecyl-phosphocholine (DPC). Far-UV CD studies indicate the presence of beta-turns or beta-sheet-like conformations of the peptide in negatively charged micelles, whereas no structural transitions are apparent in DPC micelles. These results suggest that structural features of YW12 could be utilized to develop nontoxic antisepsis compounds.