NMR structure and molecular dynamics of the in-plane membrane anchor of nonstructural protein 5A from bovine viral diarrhea virus

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a monotopic membrane protein anchored to the membrane by an N-terminal in-plane amphipathic alpha-helix. This membrane anchor is essential for the assembly of a functional viral replication complex. Although amino acid sequences differ considerably, putative membrane anchors with amphipathic features were predicted in NS5A from related Flaviviridae family members, in particular bovine viral diarrhea virus (BVDV), the prototype representative of the genus Pestivirus. We report here the NMR structure of the membrane anchor 1-28 of NS5A from BVDV in the presence of different membrane mimetic media. This anchor includes a long amphipathic alpha-helix of 21 residues interacting in-plane with the membrane interface and including a putative flexible region. Molecular dynamic simulation at a water-dodecane interface used to mimic the surface separating a lipid bilayer and an aqueous medium demonstrated the stability of the helix orientation and the location at the hydrophobic-hydrophilic interface. The flexible region of the helix appears to be required to allow the most favorable interaction of hydrophobic and hydrophilic side chain residues with their respective environment at the membrane interface. Despite the lack of amino acid sequence similarity, this amphipathic helix shares common structural features with that of the HCV counterpart, including a stable, hydrophobic N-terminal segment separated from the more hydrophilic C-terminal segment by a local, flexible region. These structural conservations point toward conserved roles of the N-terminal in-plane membrane anchors of NS5A in replication complex formation of HCV, BVDV, and other related viruses.     

Novel lactoferrampin antimicrobial peptides derived from human lactoferrin

Human lactoferrampin is a novel antimicrobial peptide found in the cationic N-terminal lobe of the iron-binding human lactoferrin protein. The amino acid sequence that directly corresponds to the previously characterized bovine lactoferrin-derived lactoferrampin peptide is inactive on its own (WNLLRQAQEKFGKDKSP, residues 269-285). However, by increasing the net positive charge near the C-terminal end of human lactoferrampin, a significant increase in its antibacterial and Candidacidal activity was obtained. Conversely, the addition of an N-terminal helix cap (sequence DAI) did not have any appreciable effect on the antibacterial or antifungal activity of human lactoferrampin peptides, even though it markedly influenced that of bovine lactoferrampin. The solution structure of five human lactoferrampin variants was determined in SDS micelles and all of the structures display a well-defined amphipathic N-terminal helix and a flexible cationic C-terminus. Differential scanning calorimetry studies indicate that this peptide is capable of inserting into the hydrophobic core of a membrane, while fluorescence spectroscopy results suggest that a hydrophobic patch encompassing the single Trp and Phe residues as well as Leu, Ile and Ala side chains mediates the interaction between the peptide and the hydrophobic core of a phospholipid bilayer.     

Isolation, gene expression and solution structure of a novel moricin analogue, antibacterial peptide from a lepidopteran insect, Spodoptera litura

An antibacterial peptide was isolated from a lepidopteran insect, Spodoptera litura. The molecular mass of this peptide was determined to be 4489.55 by matrix assisted laser desorption/ionization-time of flight mass (MALDI-TOF MS) spectrometry. The peptide consists of 42 amino acids and the sequence has 69-98% identity to those of moricin-related peptides, antibacterial peptides from lepidopetran insects. Thus, the peptide was designated S. litura (Sl) moricin. Sl moricin showed a broad antibacterial spectrum against Gram-positive and negative bacteria. Sl moricin gene was inducible by bacterial injection and expressed tissue-specifically in the fat body and hemocytes. Furthermore, the solution structure of Sl moricin was determined by two-dimensional (2D) 1H-nuclear magnetic resonance (NMR) spectroscopy and hybrid distance geometry-simulated annealing calculation. The tertiary structure revealed a long alpha-helix containing eight turns along nearly the full length of the peptide like that of moricin, confirming that Sl moricin is a new moricin-like antibacterial peptide. These results suggest that moricin is present not only in B. mori but also in other lepidopteran insects forming a gene family.     

NMR solution structure of the mitochondrial F1beta presequence from Nicotiana plumbaginifolia

We have isolated, characterized and determined the three-dimensional NMR solution structure of the presequence of ATPsynthase F1beta subunit from Nicotiana plumbaginifolia. A general method for purification of presequences is presented. The method is based on overexpression of a mutant precursor containing a methionine residue introduced at the processing site, followed by CNBr-cleavage and purification of the presequence on a cation-exchange column. The F1beta presequence, 53 amino acid residues long, retained its native properties as evidenced by inhibition of in vitro mitochondrial import and processing at micromolar concentrations. CD spectroscopy revealed that the F1beta presequence formed an alpha-helical structure in membrane mimetic environments such as SDS and DPC micelles (approximately 50% alpha-helix), and in acidic phospholipid bicelles (approximately 60% alpha-helix). The NMR solution structure of the F1beta presequence in SDS micelles was determined on the basis of 518 distance and 21 torsion angle constraints. The structure was found to contain two helices, an N-terminal amphipathic alpha-helix (residues 4-15) and a C-terminal alpha-helix (residues 43-53), separated by a largely unstructured 27 residue long internal domain. The N-terminal amphipathic alpha-helix forms the putative Tom20 receptor binding site, whereas the C-terminal alpha-helix is located upstream of the mitochondrial processing peptidase cleavage site.     

The solution structure of frenatin 3, a neuronal nitric oxide synthase inhibitor from the giant tree frog, Litoria infrafrenata

The peptide frenatin 3 is a major component of the skin secretion of the Australian giant tree frog, Litoria infrafrenata. Frenatin 3 is 22 amino acids in length, and shows neither antimicrobial nor anticancer activity. It inhibits the production of nitric oxide by the enzyme neuronal nitric oxide synthase at a micromolar concentration by binding to its regulatory protein, Ca2+ calmodulin, a protein known to recognize and bind amphipathic alpha-helices. The solution structure of frenatin 3 has been investigated using NMR spectroscopy and restrained molecular dynamics calculations. In trifluoroethanol/water mixtures, the peptide forms an amphipathic alpha-helix over residues 1-14 while the C-terminal eight residues are more flexible and less structured. The flexible region may be responsible for the lack of antimicrobial activity. In water, frenatin 3 exhibits some alpha-helical character in its N-terminal region.     

Characterization of a synthetic peptide corresponding to a receptor binding domain of mouse interferon gamma.

A receptor binding region of mouse interferon gamma (IFN gamma) has previously been localized to the N-terminal 39 amino acids of the molecule by use of synthetic peptides and monoclonal antibodies. In this report, a detailed analysis of the synthetic peptide corresponding to this region, IFN gamma (1-39), is presented. Circular dichroism (CD) spectroscopy indicated that the peptide has stable secondary structure under aqueous conditions and adopts a combination of alpha-helical and random structure. A peptide lacking two N-terminal amino acids, IFN gamma (3-39), had similar secondary structure and equivalent ability to compete for receptor binding, while peptides lacking four or more N-terminal residues had reduced alpha-helical structure and did not inhibit 125I-IFN gamma binding. Substitution of proline, a helix-destabilizing amino acid, for leucine (residue 8) of a predicted amphipathic alpha-helix (residues 3-12), IFN gamma (1-39) [Pro]8, resulted in a substantial reduction in the helical content of the peptide, supporting the presence of helical structure in this region. However, destabilization of the helix did not reduce the competitive ability of the peptide. A peptide lacking eight C-terminal residues, IFN gamma (1-31), did not block 125I-IFN gamma binding and had no detectable alpha-helical structure, suggesting a requirement of the predicted second alpha-helix (residues 20-34) for receptor interaction and helix stabilization. Substitution of phenylalanine for tyrosine at position 14, IFN gamma (1-39) [Phe]14, a central location of a predicted omega-loop structure, did not affect the secondary structure associated with the region yet resulted in a 30-fold increase in receptor competition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural requirements of tropomodulin for tropomyosin binding and actin filament capping

Regulation of actin filament dynamics underlies many cellular functions. Tropomodulin together with tropomyosin can cap the pointed, slowly polymerizing, filament end, inhibiting addition or loss of actin monomers. Tropomodulin has an unstructured N-terminal region that binds tropomyosin and a folded C-terminal domain with six leucine-rich repeats. Of tropomodulin 1's 359 amino acids, an N-terminal fragment (Tmod1(1)(-)(92)) suffices for in vitro function, even though the C-terminal domain can weakly cap filaments independent of tropomyosin. Except for one short alpha-helix with coiled coil propensity (residues 24-35), the Tmod1(1)(-)(92) solution structure shows that the fragment is disordered and highly flexible. On the basis of the solution structure and predicted secondary structure, we have introduced a series of mutations to determine the structural requirements for tropomyosin binding (using native gels and CD) and filament capping (by measuring actin polymerization using pyrene fluorescence). Tmod1(1)(-)(92) fragments with mutations of an interface hydrophobic residue, L27G and L27E, designed to destroy the alpha-helix or coiled coil propensity, lost binding ability to tropomyosin but retained partial capping function in the presence of tropomyosin. Replacement of a flexible region with alpha-helical residues (residues 59-61 mutated to Ala) had no effect on tropomyosin binding but inhibited the capping function. A mutation in a region predicted to be an amphipathic helix (residues 65-75), L71D, destroyed the capping function. The results suggest that molecular flexibility and binding to actin via an amphipathic helix are both required for tropomyosin-dependent capping of the pointed end of the actin filament.     

Molecular basis of the interaction between the flagellar export proteins FliI and FliH from Helicobacter pylori

Bacterial flagellar protein export requires an ATPase, FliI, and presumptive inhibitor, FliH. We have explored the molecular basis for FliI/FliH interaction in the human gastric pathogen Helicobacter pylori. By using bioinformatic and biochemical analyses, we showed that residues 1-18 of FliI very likely form an amphipathic alpha-helix upon interaction with FliH, and that residues 21-91 of FliI resemble the N-terminal oligomerization domain of the F1-ATPase catalytic subunits. A truncated FliI-(2-91) protein was shown to be folded, although the N-terminal 18 residues were likely unstructured. Deletion and scanning mutagenesis showed that residues 1-18 of FliI were essential for the FliI/FliH interaction. Scanning mutation of amino acids in the N-terminal 10 residues of FliI indicated that a cluster of hydrophobic residues in this segment was critical for the interaction with FliH. The interaction between FliI and FliH has similarities to the interaction between the N-terminal alpha-helix of the F1-ATPase alpha-subunit and the globular domain of the F1-ATPase delta-subunit, respectively. This similarity suggests that FliH may function as a molecular stator.     

Structural elucidation of the protein- and membrane-binding properties of the N-terminal tail domain of human annexin II

The conformational preferences and the solution structure of AnxII(N31), a peptide corresponding to the full-length sequence (residues 1-31) of the human annexin II N-terminal tail domain, were investigated by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. CD results showed that AnxII(N31) adopts a mainly alpha-helical conformation in hydrophobic or membrane-mimetic environments, while a predominantly random structure is adopted in aqueous buffer. In contrast to previous results of the annexin I N-terminal domain peptide [Yoon et al. (2000) FEBS Lett. 484, 241-245], calcium ions showed no effect on the structure of AnxII(N31). The NMR-derived structure of AnxII(N31) in 50% TFE/water mixture showed a horseshoe-like fold comprising the N-terminal amphipathic alpha-helix, the following loop, and the C-terminal helical region. Together, the results establish the first detailed structural data on the N-terminal tail domain of annexin II, and suggest the possibility of the domain to undergo Ca(2+)-independent membrane-binding.     

Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain

The glucagon-like peptide-1 receptor (GLP-1R) belongs to Family B1 of the seven-transmembrane G protein-coupled receptors, and its natural agonist ligand is the peptide hormone glucagon-like peptide-1 (GLP-1). GLP-1 is involved in glucose homeostasis, and activation of GLP-1R in the plasma membrane of pancreatic beta-cells potentiates glucose-dependent insulin secretion. The N-terminal extracellular domain (nGLP-1R) is an important ligand binding domain that binds GLP-1 and the homologous peptide Exendin-4 with differential affinity. Exendin-4 has a C-terminal extension of nine amino acid residues known as the "Trp cage", which is absent in GLP-1. The Trp cage was believed to interact with nGLP-1R and thereby explain the superior affinity of Exendin-4. However, the molecular details that govern ligand binding and specificity of nGLP-1R remain undefined. Here we report the crystal structure of human nGLP-1R in complex with the antagonist Exendin-4(9-39) solved by the multiwavelength anomalous dispersion method to 2.2A resolution. The structure reveals that Exendin-4(9-39) is an amphipathic alpha-helix forming both hydrophobic and hydrophilic interactions with nGLP-1R. The Trp cage of Exendin-4 is not involved in binding to nGLP-1R. The hydrophobic binding site of nGLP-1R is defined by discontinuous segments including primarily a well defined alpha-helix in the N terminus of nGLP-1R and a loop between two antiparallel beta-strands. The structure provides for the first time detailed molecular insight into ligand binding of the human GLP-1 receptor, an established target for treatment of type 2 diabetes.     

NMR structures of the C-terminal segment of surfactant protein B in detergent micelles and hexafluoro-2-propanol

Although the membrane-associated surfactant protein B (SP-B) is an essential component of lung surfactant, which is itself essential for life, the molecular basis for its activity is not understood. SP-B's biophysical functions can be partially mimicked by subfragments of the protein, including the C-terminus. We have used NMR to determine the structure of a C-terminal fragment of human SP-B that includes residues 63-78. Structure determination was performed both in the fluorinated alcohol hexafluoro-2-propanol (HFIP) and in sodium dodecyl sulfate (SDS) micelles. In both solvents, residues 68-78 take on an amphipathic helical structure, in agreement with predictions made by comparison to homologous saposin family proteins. In HFIP, the five N-terminal residues of the peptide are largely unstructured, while in SDS micelles, these residues take on a well-defined compact conformation. Differences in helical residue side chain positioning between the two solvents were also found, with better agreement between the structures for the hydrophobic face than the hydrophilic face. A paramagnetic probe was used to investigate the position of the peptide within the SDS micelles and indicated that the peptide is located at the water interface with the hydrophobic face of the helix oriented inward, the hydrophilic face of the helix oriented outward, and the N-terminal residues even farther from the micelle center than those on the hydrophilic face of the alpha-helix. Interactions of basic residues of SP-B with anionic lipid headgroups are known to have an impact on function, and these studies demonstrate structural ramifications of such interactions via the differences observed between the peptide structures determined in HFIP and SDS.     

Structural similarities of micelle-bound peptide YY (PYY) and neuropeptide Y (NPY) are related to their affinity profiles at the Y receptors

Here, we investigate the structure of porcine peptide YY (pPYY) both when unligated in solution at pH 4.2 and when bound to dodecylphosphocholine (DPC) micelles at pH 5.5. pPYY in solution displays the PP-fold, with the N-terminal segment being back-folded onto the C-terminal alpha-helix, which extends from residue 17 to 31. In contrast to the solution structure of Keire et al. published in the year 2000 the C-terminal helix does not display a kink around residue 23-25. The root mean square deviation (RMSD) for backbone atoms of the NMR ensemble of conformers to the mean structure is 0.99(+/-0.35) Angstrom for residues 14-31. The back-fold is supported by values of 0.60+/-0.1 for the (15)N(1)H-NOE and by generalized order parameters S(2) of 0.74+/-0.1 for residues 5-31 which indicate that the peptide is folded in that segment. We have additionally used DPC micelles as a membrane model and determined the structure of pPYY when bound to it. Therein, an alpha-helix occurs in the segment comprising residues 17-31 and the N terminus freely diffuses in solution. The hydrophobic side of the amphipathic helix forms the micelle-binding interface and hydrophobic side-chains extend into the micelle interior. A significant stabilization of helical conformation occurs in the C-terminal pentapeptide, which is important for receptor binding. The latter is supported by positive values of the heteronuclear NOE in that segment (0.52+/-0.1 compared to 0.08+/-0.4 for the unligated form) and by values of S(2) of 0.6+/-0.2 (versus 0.38+/-0.2 for the unligated form). The structures of micelle-bound pPYY and pNPY are much more similar than those of pPYY and bPP with pairwise RMSDs of 1.23(+/-0.21)A or 3.21(+/-0.39) Angstrom, respectively. In contrast to the conformational similarities in the DPC-bound state their structures in solution are very different. In fact pPYY is more similar to bPP, which with its strong preference for the Y(4) receptor displays a completely different binding profile. Considering the high degree of sequence homology of pNPY and pPYY (>80%) and the fact, that their binding affinities at all receptor subtypes are high and, more importantly, rather similar, it is much more likely that PYY and NPY are recognized by the Y receptors from the membrane-bound state. As a consequence of the latter the PP-fold is not important for recognition of PYY or NPY at the Y receptors. To our knowledge this work provides for the first time strong arguments derived from structural data that support a membrane-bound receptor recognition pathway.     

Helical structure of dermaseptin B2 in a membrane-mimetic environment

Dermaseptins are antimicrobial peptides from frog skin that have high membrane-lytic activity against a broad spectrum of microorganisms. The structure of dermaseptin B2 in aqueous solution, in TFE/water mixtures, and in micellar and nonmicellar SDS was analyzed by CD, FTIR, fluorescence, and NMR spectroscopy combined with molecular dynamics calculations. Dermaseptin B2 is unstructured in water, but helical conformations, mostly in segment 3-18, are stabilized by addition of TFE. SDS titration showed that dermaseptin B2 assumes nonhelical structures at SDS concentrations far below the critical micellar concentration and helical structures at micellar concentrations. Dermaseptin B2 bound to SDS micelles (0.4 mM peptide, 80 mM SDS) adopts a well-defined amphipathic helix between residues 11-31 connected to a more flexible helical segment spanning residues 1-8 by a flexible hinge region around Val9 and Gly10. Experiments using paramagnetic probes showed that dermaseptin B2 lies near the surface of SDS micelles and that residue Trp3 is buried in the SDS micelle, but close to the surface. A slow exchange equilibrium occurs at higher peptide/SDS ratios (2 mM peptide, 80 mM SDS) between forms having distinct sets of resonances in the N-terminal 1-11 segment. This equilibrium could reflect different oligomeric states of dermaseptin B2 interacting with SDS micelles. Structure-activity studies on dermaseptin B2 analogues showed that the N-terminal 1-11 segment is an absolute requirement for antibacterial activity, while the C-terminal 10-33 region is also important for full antibiotic activity.     

RGS4 binds to membranes through an amphipathic alpha -helix

RGS4, a mammalian GTPase-activating protein for G protein alpha subunits, requires its N-terminal 33 amino acids for plasma membrane localization and biological activity (Srinivasa, S. P., Bernstein, L. S., Blumer, K. J., and Linder, M. E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5584-5589). In this study, we tested the hypothesis that the N-terminal domain mediates membrane binding by forming an amphipathic alpha-helix. RGS4 bound to liposomes containing anionic phospholipids in a manner dependent on the first 33 amino acids. Circular dichroism spectroscopy of a peptide corresponding to amino acids 1-31 of RGS4 revealed that the peptide adopted an alpha-helical conformation in the presence of anionic phospholipids. Point mutations that either neutralized positive charges on the hydrophilic face or substituted polar residues on the hydrophobic face of the model helix disrupted plasma membrane targeting and biological activity of RGS4 expressed in yeast. Recombinant mutant proteins were active as GTPase-activating proteins in solution but exhibited diminished binding to anionic liposomes. Peptides corresponding to mutants with the most pronounced phenotypes were also defective in forming an alpha-helix as measured by circular dichroism spectroscopy. These results support a model for direct interaction of RGS4 with membranes through hydrophobic and electrostatic interactions of an N-terminal alpha-helix.     

The structure of the mammalian antibacterial peptide cecropin P1 in solution, determined by proton-NMR.

Cecropins are peptides with antibacterial activity originally found in insects. Recently a cecropin-type peptide was isolated from pig intestine. This peptide, porcine cecropin P1, which has 31 amino acid residues and is not amidated in the C-terminus, has been synthesized, purified, and investigated by CD and two-dimensional 1H-NMR at pH 5.0 in aqueous solution with 30% (by vol.) 1,1,1,3,3,3-hexafluoro-2-propanol. All proton resonances have been assigned except for the N-terminal serine. Using constraints derived from NOE connectivities and 3JNH alpha-coupling constants, three-dimensional structures have been calculated by means of a distance-geometry program. Some of these structures have been refined by energy minimization and restrained molecular dynamics. The structures reveal an alpha-helix of approximately seven turns along nearly the full length of the peptide. The central part of the helix is very well defined by the NMR constraints. Also the chemical shifts of the alpha protons and the results of CD measurements are in accord with this structure, which is different from the helix-hinge-helix structure earlier found in cecropin A and related peptides. In the alpha-helix of cecropin P1 there is a long amphipathic section, of 4-5 turns, and a short hydrophobic section of one to two turns, with an intervening Glu-Gly sequence, which is a potential bend-forming section. The helix can easily span a lipid membrane.     

Surface binding of alamethicin stabilizes its helical structure: molecular dynamics simulations.

Alamethicin is an amphipathic alpha-helical peptide that forms ion channels. An early event in channel formation is believed to be the binding of alamethicin to the surface of a lipid bilayer. Molecular dynamics simulations are used to compare the structural and dynamic properties of alamethicin in water and alamethicin bound to the surface of a phosphatidylcholine bilayer. The bilayer surface simulation corresponded to a loosely bound alamethicin molecule that interacted with lipid headgroups but did not penetrate the hydrophobic core of the bilayer. Both simulations started with the peptide molecule in an alpha-helical conformation and lasted 2 ns. In water, the helix started to unfold after approximately 300 ps and by the end of the simulation only the N-terminal region of the peptide remained alpha-helical and the molecule had collapsed into a more compact form. At the surface of the bilayer, loss of helicity was restricted to the C-terminal third of the molecule and the rod-shaped structure of the peptide was retained. In the surface simulation about 10% of the peptide/water H-bonds were replaced by peptide/lipid H-bonds. These simulations suggest that some degree of stabilization of an amphipathic alpha-helix occurs at a bilayer surface even without interactions between hydrophobic side chains and the acyl chain core of the bilayer.     

NMR solution structure of the glucagon antagonist [desHis1, desPhe6, Glu9]glucagon amide in the presence of perdeuterated dodecylphosphocholine micelles

Glucagon, a 29-residue peptide hormone, plays an important role in glucose homeostasis and in diabetes mellitus. Several glucagon antagonists and agonists have been developed, but limited structural information is available to clarify the basis of their biological activity. The solution structure of the potent glucagon antagonist, [desHis1, desPhe6, Glu9]glucagon amide, was determined by homonuclear 2D NMR spectroscopy at pH 6.0 and 37 degrees C in perdeuterated dodecylphosphocholine micelles. The overall backbone root-mean-square deviation (rmsd) for the structured portion (residues 7-29, glucagon numbering) of the micelle-bound 27-residue peptide is 1.36 A for the 15 lowest-energy structures, after restrained molecular dynamics simulation. The structure consists of four regions (segment backbone rmsd in A): an unstructured N-terminal segment between residues 2 and 5 (1.68), an irregular helix between residues 7 and 14 (0.79), a hinge region between residues 15 and 18 (0.54), and a well-defined alpha-helix between residues 19 and 29 (0.33). The two helices form an L-shaped structure with an angle of about 90 degrees between the helix axes. There is an extended hydrophobic cluster, which runs along the inner surface of the L-structure and incorporates the side chains of the hydrophobic residues of each of the amphipathic helices. The outer surface contains the hydrophilic side chains, with two salt bridges (D15-R18 and R17-D21) implied from close approach of the charged groups. This result is the first clear indication of an overall tertiary fold for a glucagon analogue in the micelle-bound state. The relationship of the two helical structural elements may have important implications for the biological activity of the glucagon antagonist.     

Structure-activity relationship of HP (2-20) analog peptide: enhanced antimicrobial activity by N-terminal random coil region deletion

HP (2-20) (AKKVFKRLEKLFSKIQNDK) is a 19-aa antimicrobial peptide derived from N-terminus of Helicobacter pylori Ribosomal protein L1 (RpL1). In the previous study, several analogs with amino acid substitutions were designed to increase or decrease only the net hydrophobicity. In particular, substitutions of Gln(16) and Asp(18) with Trp (Anal 3) for hydrophobic amino acid caused a dramatic increase in antibiotic activity without a hemolytic effect. HP-A3 is a potent antimicrobial peptide that forms, in a hydrophobic medium, an amphipathic structure consisting of an N-terminal random coil region (residues 2-5) and extended C-terminal regular alpha-helical region (residues 6-20). To obtain the short and potent alpha-helical antimicrobial peptide, we synthesized a N-terminal random coil deleted HP-A3 (A3-NT) and examined their antimicrobial activity and mechanism of action. The resulting 15mer peptide showed increased antibacterial and antifungal activity to 2- and 4-fold, respectively, without hemolysis. Confocal fluorescence microscopy studies showed that A3-NT was accumulated in the plasma membrane. Flow cytometric analysis revealed that A3-NT acted in salt- and energy-independent manner. Furthermore, A3-NT causes significant morphological alterations of the bacterial surfaces as shown by scanning electron microscopy. Circular dichroism (CD) analysis revealed that A3-NT showed higher alpha-helical contents than the HP-A3 peptide in 50% TFE solution. Therefore, the cell-lytic efficiency of HP-A3, which depended on the alpha-helical content of peptide, correlated linearly with their antimicrobial potency.     

Structure of the human anti-apoptotic protein survivin reveals a dimeric arrangement

Survivin is a 16.5 kDa protein that is expressed during the G2/M phase of the cell cycle and is hypothesized to inhibit a default apoptotic cascade initiated in mitosis. This inhibitory function is coupled to survivin's localization to the mitotic spindle. To begin to address the structural basis of survivin's function, we report the X-ray crystal structure of a recombinant form of full length survivin to 2.58 A resolution. Survivin consists of two defined domains including an N-terminal Zn2+-binding BIR domain linked to a 65 A amphipathic C-terminal alpha-helix. The crystal structure reveals an extensive dimerization interface along a hydrophobic surface on the BIR domain of each survivin monomer. A basic patch acting as a sulfate/phosphate-binding module, an acidic cluster projecting off the BIR domain, and a solvent-accessible hydrophobic surface residing on the C-terminal amphipathic helix, are suggestive of functional protein-protein interaction surfaces.