Critical Main-Chain Length for Conformational Conversion From 310-Helix to α-Helix in Polypeptides

Abstract

To assess the minimal peptide length required for the stabilization of the a-helix relative to the 3₁₀-helix in Aib-rich peptides, we have solved the X-ray diffraction structures of the terminally blocked sequential hexa- and octapeptides with the general formula -(Aib-L-Ala)_n-(n = 3 and 4, respectively). The hexapeptide molecules are completely 3₁₀-helical with four 1 ← 4 intramolecular N-H … O=C H-bonds. On the other hand, the octapeptide molecules are essentially α-helical with four 1 ← 5 H-bonds; however, the helix is elongated at the N-terminus, with two 1 ← 4 H-bonds, giving these molecules a mixed α/3₁₀-helical character. In both compounds the right-handed screw sense of the helix is dictated by the presence of the Ala residues of L-configuration. This study represents the first experimental proof for a 3₁₀ →α-helix conversion in the crystal state induced by peptide backbone lengthening only.     

α-Helix mimetics: Outwards and upwards

α-Helices are common secondary structural elements forming key parts of the large, generally featureless interfacial regions of many therapeutically-relevant protein–protein interactions (PPIs). The rational design of helix mimetics is an appealing small-molecule strategy for the mediation of aberrant PPIs, however the first generation of scaffolds presented a relatively small number of residues on a single recognition surface. Increasingly, helices involved in PPIs are found to have more complex binding modes, utilizing two or three recognition surfaces, or binding with extended points of contact. To address these unmet needs the design and synthesis of new generations of multi-sided, extended, and supersecondary structures are underway.     

Theoretical Studies on α-Helix—DNA Interactions

Abstract

The ¹H NMR relaxation effects produced by paramagnetic Cr(III) complexes on nucleoside 5′-mono- and -triphosphates in D₂O solution at Ph′=3 were measured. The paramagnetic probes were [Cr(III)(H₂O) ₆]³⁺, [Cr(III)(H₂O)₃ (HATP)], [Cr(III)(H₂O)₃(HCTP)] and [Cr(III) (H₂O)₃(UTP)^?, while the matrix nucleotides (0.1 M) were H₂AMP, HIMP^?, and H₂ATP^2-. For the aromatic base protons, the ratios of the transverse to longitudinal paramagnetic relaxation rates (R_2p/R_1p) for the [Cr(III)(H₂O)₆]³⁺/H₂ATP^2-, [Cr(III)(H₂O)₃(HATP)]/H₂ATP^2-, [Cr(III)(H₂O)₃(HCTP)]/H₂ATP² and [Cr(III)(H₂O)₃(UTP)]^?/H₂ATP² systems were below 2.33 so the dipolar term predominates. For a given nucleotide, R_1p for the purine H(8) signal was larger than for the H(2) signal with the [Cr(III)(H₂O)₆]³⁺ probe, while R_1p for the H(2) signal was larger with all the other Cr(III) probes. Molecular mechanics computations on the [Cr(III)(H₂O)₄(HPP)(α,β)], [Cr(III)(NH₃)₄(HPP)(α,β)], [Co(III)(NH₃)₃(H₂PPP)(α,βγ)] and [Co(III)(NH₃)₄(HPP)(α,β)] complexes gave calculated energy-minimized geometries in good agreement with those reported in crystal structures. The molecular mechanics force constants found were then used to calculate the geometry of the inner sphere [Cr(III)(H₂O)₆]³⁺ and [Cr(III)(H₂O)₃(HATP)(α,βγ)] complexes as well as the structures of the outer sphere [Cr(III) (H₂O)₆]³⁺-(H₂AMP) and [Cr(III)(H₂O)₆]-(HIMP)^? species. The gas-phase structure of the [Cr(III)(H₂O)₃(HATP)(α,βγ)] complex shows the existence of a hydrogen bond interaction between a water ligand and the adenine N(7) (O…N = 2.82 Å). The structure is also stabilized by intramolecular hydrogen bonds involving the -O(2′)H group and the adenine N(3) (O…N = 2.80 Å) as well as phosphate oxygen atoms and a water molecule (O…O = 2.47 Å). The metal center has an almost regular octahedral coordination geometry.

The structures of the two outer-sphere species reveal that the phosphate group interacts strongly with the hexa-aquochromium probe. In both complexes, the nucleotides have a similar “anti” conformation around the N(9)-C(l′) glycosidic bond. However, a very important difference characterizes the two structures. For the (HIMP)^? complex, strong hydrogen bond interactions exist between one and two water ligands and the inosine N(7) and O(6) atoms, respectively (O…O = 2.63 Å O…N = 2.72, 2.70 Å). For the H₂AMP complex, the [Cr(III) (H₂O)c]³⁺ cation does not interact with N(7) since it is far from the purine system. Hydrogen bonds occur between water ligands and phosphate oxygens. The Cr-H(8) and Cr-H(2) distances revealed by the energy-minimized geometries for the two outer sphere species were used to calculate the R_1p values for the H(8) and H(2) signals for comparison with the observed R_1p values: 0.92(c), 1.04(ob) (H(8)) and 0.06(c), 0.35(ob) (H(2)) for H₂AMP; and 3.76(c), 4.53(ob) (H(8)) and 0.16(c), 0.77(ob) s^?1 (H(2)) for HIMP^?. These results suggest that the dynamic relaxation effects can be only partially understood with molecular mechanics computations, although the success of the geometry calculations suggests that future efforts in the development of computational methods are justified.     

Cotranslational Folding of a Pentarepeat β-Helix Protein

It is becoming increasingly clear that many proteins start to fold cotranslationally before the entire polypeptide chain has been synthesized on the ribosome. One class of proteins that a priori would seem particularly prone to cotranslational folding is repeat proteins, that is, proteins that are built from an array of nearly identical sequence repeats. However, while the folding of repeat proteins has been studied extensively in vitro with purified proteins, only a handful of studies have addressed the issue of cotranslational folding of repeat proteins. Here, we have determined the structure and studied the cotranslational folding of a β-helix pentarepeat protein from the human pathogen Clostridium botulinum—a homolog of the fluoroquinolone resistance protein MfpA—using an assay in which the SecM translational arrest peptide serves as a force sensor to detect folding events. We find that cotranslational folding of a segment corresponding to the first four of the eight β-helix coils in the protein produces enough force to release ribosome stalling and that folding starts when this unit is ~ 35 residues away from the P-site, near the distal end of the ribosome exit tunnel. An additional folding transition is seen when the whole PENT moiety emerges from the exit tunnel. The early cotranslational formation of a folded unit may be important to avoid misfolding events in vivo and may reflect the minimal size of a stable β-helix since it is structurally homologous to the smallest known β-helix protein, a four-coil protein that is stable in solution.     

Dynamic ��-Helix Structure of Micelle-bound Human Amylin

Amylin is an endocrine hormone that regulates metabolism. In patients afflicted with type 2 diabetes, amylin is found in fibrillar deposits in the pancreas. Membranes are thought to facilitate the aggregation of amylin, and membrane-bound oligomers may be responsible for the islet β-cell toxicity that develops during type 2 diabetes. To better understand the structural basis for the interactions between amylin and membranes, we determined the NMR structure of human amylin bound to SDS micelles. The first four residues in the structure are constrained to form a hairpin loop by the single disulfide bond in amylin. The last nine residues near the C terminus are unfolded. The core of the structure is an α-helix that runs from about residues 5–28. A distortion or kink near residues 18–22 introduces pliancy in the angle between the N- and C-terminal segments of the α-helix. Mobility, as determined by ¹⁵N relaxation experiments, increases from the N to the C terminus and is strongly correlated with the accessibility of the polypeptide to spin probes in the solution phase. The spin probe data suggest that the segment between residues 5 and 17 is positioned within the hydrophobic lipid environment, whereas the amyloidogenic segment between residues 20 and 29 is at the interface between the lipid and solvent. This orientation may direct the aggregation of amylin on membranes, whereas coupling between the two segments may mediate the transition to a toxic structure.Type 2 diabetes affects over 100 million people worldwide (1) and is thought to cost upward of $130 billion dollars a year to treat in the United States alone (2). The endocrine hormone amylin (also known as islet amyloid polypeptide) appears to have key roles in diabetes pathology (3–5). The normal functions of amylin include the inhibition of glucagon secretion, slowing down the emptying of the stomach, and inducing a feeling of satiety through the actions of the hormone on neurons of the hypothalamus in the brain (5). The effects of amylin are exerted in concert with those of insulin and reduce the level of glucose in the blood (3, 5). Circulating amylin levels increase in a number of pathological conditions, including obesity, syndrome X, pancreatic cancer, and renal failure (3). Amylin levels together with insulin are raised initially in type 2 diabetes but fall as the disease progresses to a stage where the pancreatic islets of Langerhans β-cells that synthesize amylin no longer function (3).One of the hallmarks of type 2 diabetes, found in 90% of patients, is the formation of extracellular amyloid aggregates composed of amylin (3–5). The amyloid deposits accumulate in the interstitial fluid between islet cells and are usually juxtaposed with the β-cell membranes (3). Aggregates of amylin are toxic when added to cultures of β-cells, so that the amyloid found in situ may be responsible for β-cell death as type 2 diabetes progresses (6, 7). Genetic evidence that amylin is directly involved in pathology includes a familial S20G mutation that leads to early onset of the disease (8) and produces an amylin variant that aggregates more readily (9).As with all amyloids it is unclear whether fibrillar structures or soluble oligomers are responsible for pathology. A recurrent theme for amyloidogenic proteins is that toxicity appears to be exerted through membrane-bound oligomers that form pores and disrupt ion balance across membranes (4, 10–13). Experimental evidence for such oligomers has been found for the amyloid-β (Aβ)² peptides (14), which cause Alzheimer disease, and for α-synuclein (αS), the protein involved in Parkinson disease (15), a particular interest of our laboratory. The similar toxic effects exerted by these amyloidogenic molecules may have a common structural and physical basis. Detailed structural models are available for Aβ (16) and αS (17) bound to SDS micelle mimetics of membranes. For amylin there are models of peptide fragments 1–19 (18), 20–29 (19), and 17–29 (20) bound to micelles but as of yet no model of the complete hormone. This turns out to be particularly important as the interplay between structure and dynamics in amylin only comes to light when considering the whole molecule.Here we report the solution structure of human amylin bound to SDS micelles. We complement the structure with information on dynamics and on the immersion of amylin into micelles.     

The Role of Arrestin α-Helix I in Receptor Binding

Arrestins rapidly bind phosphorylated activated forms of their cognate G protein-coupled receptors, thereby preventing G protein coupling and often switching signaling to other pathways. Amphipathic α-helix I (residues 100-111) has been implicated in receptor binding, but the mechanism of its action has not been determined yet. Here we show that several mutations in the helix itself and in adjacent hydrophobic residues in the body of the N-domain reduce arrestin1 binding to light-activated phosphorylated rhodopsin (P-Rh^?). On the background of phosphorylation-independent mutants that bind with high affinity to both P-Rh^? and light-activated unphosphorylated rhodopsin, these mutations reduce the stability of the arrestin complex with P-Rh^?, but not with light-activated unphosphorylated rhodopsin. Using site-directed spin labeling, we found that the local structure around α-helix I changes upon binding to rhodopsin. However, the intramolecular distances between α-helix I and adjacent β-strand I (or the rest of the N-domain), measured using double electron-electron resonance, do not change, ruling out relocation of the helix due to receptor binding. Collectively, these data demonstrate that α-helix I plays an indirect role in receptor binding, likely keeping β-strand I, which carries several phosphate-binding residues, in a position favorable for its interaction with receptor-attached phosphates.     

Conformational Similarity Indices Between Different Residues in Proteins and α-Helix Propensities

Abstract

Various amino acid similarity matrices have been derived using data on physicochemical properties and molecular evolution. Conformational similarity indices, CS_XX′, between different residues are computed here using the distribution of the main-chain and side-chain torsion angles and the values have been used to cluster amino acids in proteins. A subset of these parameters, CS_AX′ indicates the extent of similarity in the main-chain and side-chain conformations (φ ψ and χ₁) of different residues (X) with Ala (A) and is found to have strong correlation with α-helix propensities. However, no subset of CS_XX′ provides any linear relationship with β-sheet propensities, suggesting that the conformational feature favouring the location of a residue in an a-helix is different from the one favouring the β-sheet. Conformationally similar residues (close CS_AX values) have similar steric framework of the side-chain (linear/branched, aliphatic/aromatic), irrespective of the polarity or hydrophobicity. Cooperative nucleation of helix may be facile for a contiguous stretch of residues with high overall CS_AX values.     

Alcohol-Induced Conformational Transitions in Ervatamin C. An α-Helix to β-Sheet Switchover

Alcohol-induced conformational transitions of erv C, a highly stable cysteine protease, were followed by CD, fluorescence, and activity. At acidic pH, the addition of different alcohols caused two types of conformational transitions. Increasing the concentration of nonfluorinated alkyl alcohols induced a conformational switch from α-helix to β-sheet. Under these conditions, the protein lost its proteolytic activity and tertiary structure. The switch was a sudden one, observed in 50% methanol, 45% ethanol, and 40% propanol. Under similar conditions of pH and concentration, however, glycerol and TFE enhanced the α-helicity of the protein. Methanol-induced denaturation was observed to occur in two stages; the first is the β-sheet state stabilized at low alcohol concentrations, and the other is the β-sheet state with enhanced ellipticity stabilized at high alcohol concentrations. This β-sheet conformation can be attained from the native as well as 6 M GuHCl-denatured state by addition of methanol and exhibits properties different from the native or unfolded state. This state shows loss of tertiary structure and activity, enhanced nonnative secondary structure, noncooperative temperature unfolding, and higher stability toward denaturants as compared to the native state, which are characteristic of the molten globule-like state or O-state, and thus this state may be functioning as an intermediate in the folding pathway of erv C.     

A Comparative Analysis of Interhelical Polar Interactions of Various α-Helix Packings in Proteins

One hundred twenty globular proteins and forty five leucine zippers representing all types of packing of long -helices were studied in terms of revealing and comparing their interhelical hydrogen and salt bonds. Many previous studies of leucine zippers and their analogs showed that interhelical interactions between polar groups could impart specificity to packing of an -helix. The current comparison demonstrated that basically, globular proteins and leucine zippers had similar interhelical polar interactions with presumably a similar structural role. However, depending on the packing of -helices, the networks of interhelical polar bonds were shown to be distinct and determined both by physicochemical properties of involved amino acid residues and by the relative positions of hydrophobic and hydrophilic residues on the surface of -helices. The revealed distinction is probably crucial for selecting the unique packing of an -helix.     

Accessory α-Helix of Complexin I Can Displace VAMP2 Locally in the Complexin-SNARE Quaternary Complex

The calcium-triggered neurotransmitter release requires three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins: synaptobrevin 2 (or vesicle-associated membrane protein 2) on the synaptic vesicle and syntaxin 1 and SNAP-25 (synaptosome-associated protein of 25 kDa) at the presynaptic plasma membrane. This minimal fusion machinery is believed to drive fusion of the vesicle to the presynaptic membrane. Complexin, also known as synaphin, is a neuronal cytosolic protein that acts as a major regulator of synaptic vesicle exocytosis. Stimulatory and inhibitory effects of complexin have both been reported, suggesting the duality of its function. To shed light on the molecular basis of the complexin's dual function, we have performed an EPR investigation of the complexin-SNARE quaternary complex. We found that the accessory α-helix (amino acids 27-48) by itself has the capacity to replace the C-terminus of the SNARE motif of vesicle-associated membrane protein 2 in the four-helix bundle and makes the SNARE complex weaker when the N-terminal region of complexin I (amino acids 1-26) is removed. However, the accessory α-helix remains detached from the SNARE core when the N-terminal region of complexin I is present. Thus, our data show the possibility that the balance between the activities of the accessory α-helix and the N-terminal domain might determine the final outcome of the complexin function, either stimulatory or inhibitory.     

The N Terminus of Connexin37 Contains an ��-Helix That Is Required for Channel Function

The cytoplasmic N-terminal domain of connexins has been implicated in multiple aspects of gap junction function, including connexin trafficking/assembly and channel gating. A synthetic peptide corresponding to the first 23 amino acids of human connexin37 was prepared, and circular dichroism and nuclear magnetic resonance studies showed that this N-terminal peptide was predominantly α-helical between glycine 5 and glutamate 16. The importance of this structure for localization of the protein at appositional membranes and channel function was tested by expression of site-directed mutants of connexin37 in which amino acids leucine 10 and glutamine 15 were replaced with prolines or alanines. Wild type connexin37 and both substitution mutants localized to appositional membranes between transfected HeLa cells. The proline mutant did not allow intercellular transfer of microinjected neurobiotin; the alanine mutant allowed transfer, but less extensively than wild type connexin37. When expressed alone in Xenopus oocytes, wild type connexin37 produced hemichannel currents, but neither of the double substitution mutants produced detectable currents. The proline mutant (but not the alanine mutant) inhibited co-expressed wild type connexin37. Taken together, our data suggest that the α-helical structure of the connexin37 N terminus may be dispensable for protein localization, but it is required for channel and hemichannel function.Gap junction channels allow intercellular passage of ions and small molecules up to 1000 Da. They are oligomeric assemblies of members of a family of related proteins called connexins (CX)² (reviewed in Ref. 1). Six connexin monomers assemble to form a hemichannel or connexon (Fig. 1, top panel), which, in turn, forms a complete gap junction channel by docking with a hemichannel from an adjacent cell. Based on sequence similarities, connexins have been separated into subfamilies designated by Greek characters (2, 3). The majority of connexins are members of the α- and β-subfamilies. Connexin polypeptides span the plasma membrane four times and have three cytoplasmic regions: the N terminus (NT), a cytoplasmic loop between the second and third transmembrane domains, and the C terminus (Fig. 1, middle panel). Structural studies of gap junctions have revealed that each hemichannel contains a ring of 24 transmembrane spanning helices (4, 5). Most topological models suggest that the NT of α-subfamily connexins contains 23 amino acids (illustrated for connexin37, CX37, in Fig. 1, bottom panel) and that of β-subfamily connexins contains 22 amino acids.Open in a separate windowFIGURE 1.Diagrams depicting the relationships between a gap junction hemichannel (top), the connexin polypeptide (middle), and the amino acid sequence of the CX37 N-terminal domain (bottom). Thick vertical lines represent the boundaries of the plasma membrane; the intracellular and extracellular spaces are indicated. The transmembrane (M1–M4), extracellular (E1 and E2), and cytoplasmic (NT, N terminus; CL, cytoplasmic loop; and CT, C terminus) domains within a connexin are indicated.The importance of the connexin NT has been emphasized by the identification of a number of connexin mutants that cause amino acid substitutions within this region and are linked to diseases including sensorineural deafness (CX26, CX30, and CX31), Charcot-Marie-Tooth disease (CX32), oculodentodigital dysplasia (CX43), and congenital cataracts (CX46 and CX50). Among the disease-linked mutants that have been studied, some show impaired protein trafficking to the cell surface, whereas others traffic properly, but show loss or alterations of channel function (6–16). Heterologous expression of site-directed mutants and chimeric connexins has demonstrated the influence of NT amino acids upon channel properties, including transjunctional voltage (V_j)-dependent gating, unitary conductance, permeability, and sensitivity to regulation by polyamines (17–22). Lagree et al. (23) have provided evidence that the NT influences the compatibility of connexin hetero-oligomerization.The structure of the NT domain of a β-group connexin, Cx26, has been investigated through circular dichroism (CD) and nuclear magnetic resonance (NMR) of a synthetic peptide corresponding to part of the predicted CX26NT (24, 25). Based on their data, Purnick et al. (24) proposed a model for the NT of CX26 with an α-helix extending from position 1 to 10 and a critical bend at positions 11 and 12 that was suggested to act as a “hinge” allowing the first 10 amino acids to swing into the pore and block the channel. Oshima et al. (5) have published structural studies of a “permeability” mutant (M34A) of CX26 (26) showing a density within the pore of the channel that they suggested might represent a bundle of N termini acting as a “plug” to close the channel.We have been studying CX37, an α-group connexin that is expressed in endothelial cells (27), which may be important for development of atherosclerotic disease (28) and that can form large conductance channels and hemichannels (27, 29). We have shown that as much as half the length of the CX37NT can be deleted without affecting formation of gap junction plaques, but a full-length N terminus is required for hemichannel gating and intercellular communication (30). These observations suggested that the CX37NT may have a structure that is required for function. Therefore, the present experiments were designed to determine the structure of the NT of CX37 and the importance of that structure for protein localization and formation of functional channels and hemichannels. Differences between our data and those previously reported in studies of CX26 suggest that the structure of the NT in α-group connexins may differ from that in β-group connexins.     

α-Helix peptides designed from EBV-gH protein display higher antigenicity and induction of monocyte apoptosis than the native peptide

We tested the hypothesis that stabilizing α-helix of Epstein–Barr virus gH-derived peptide 11438 used for binding human cells will increase its biological activity. Non-stable α-helix of peptide 11438 was unfolded in an entropy-driven process, despite the opposing effect of the enthalpy factor. Adding and/or changing amino acids in peptide 11438 allowed the designing of peptides 33207, 33208 and 33210; peptides 33208 and 33210 displayed higher helical content due to a decreased unfolding entropy change as was determined by AGADIR, molecular dynamics and circular dichroism analysis. Peptides 33207, 33208 and 33210 inhibited EBV invasion of peripheral blood mononuclear cells and displayed epitopes more similar to native protein than peptide 11438; these peptides could be useful for detecting antibodies induced by native gH protein since they displayed high reactivity with anti-EBV antibodies. Anti-peptide 33207 antibodies showed higher reactivity with EBV than anti-peptide 11438 antibodies being useful for inducing antibodies against EBV. Anti-peptide 33210 antibodies inhibit EBV invasion of epithelial cells better than anti-peptide 11438 antibodies. Peptide 33210 bound to normal T lymphocytes and Raji cells stronger than peptide 11438 and also induced apoptosis of monocytes and Raji cells but not of normal T cells in a similar way to EBV-gH. Peptide 33210 inhibited the monocytes’ development toward dendritic cells better than EBV and peptide 11438. In conclusion, stabilizing the α-helix in peptides 33208 and 33210 designed from peptide 11438 increased the antigenicity and the ability of the antibodies induced by peptides of inhibiting EBV invasion of host cells.     

The Cancer Mutation D83V Induces an α-Helix to β-Strand Conformation Switch in MEF2B

MEF2B is a major target of somatic mutations in non-Hodgkin lymphoma. Most of these mutations are non-synonymous substitutions of surface residues in the MADS-box/MEF2 domain. Among them, D83V is the most frequent mutation found in tumor cells. The link between this hotspot mutation and cancer is not well understood. Here we show that the D83V mutation induces a dramatic α-helix to β-strand switch in the MEF2 domain. Located in an α-helix region rich in β-branched residues, the D83V mutation not only removes the extensive helix stabilization interactions but also introduces an additional β-branched residue that further shifts the conformation equilibrium from α-helix to β-strand. Cross-database analyses of cancer mutations and chameleon sequences revealed a number of well-known cancer targets harboring β-strand favoring mutations in chameleon α-helices, suggesting a commonality of such conformational switch in certain cancers and a new factor to consider when stratifying the rapidly expanding cancer mutation data.     

The α-Helix of Ribonuclease T1as an Independent Stability Unit: Direct Comparison of Peptide and Protein Stability

Measurements of the change in conformational stability, Δ(ΔG), upon mutation of two acidic residues at the C terminus of the helix of ribonuclease T₁have recently been reported. Here, we investigate peptides based on the sequence of the helix with the same mutations: Glu28 replaced with Gln, Asp29 replaced with Asn, and the double mutant. In addition, the mutant Lys25 to Gln was studied. Changes in helix content of the peptides with pH confirm the conclusion found in the intact protein, that the charged forms of the acidic residues destabilize the protein by destabilizing the helix. The pH-dependence of the change in confor mational free energy for the peptides and mutant proteins show fair correspondence for D29N and the double mutant. The mutants E28Q and K25Q, on the other hand, give striking agreement between the protein and peptide systems. This agreement suggests that the helix of ribonuclease T₁behaves as an independently stabilized structural unit of the intact protein and that stabilization of the helical form of the peptide is mirrored in the protein.     

A Spiroligomer α-Helix Mimic That Binds HDM2, Penetrates Human Cells and Stabilizes HDM2 in Cell Culture

We demonstrate functionalized spiroligomers that mimic the HDM2-bound conformation of the p53 activation domain. Spiroligomers are stereochemically defined, functionalized, spirocyclic monomers coupled through pairs of amide bonds to create spiro-ladder oligomers [1]. Two series of spiroligomers were synthesized, one of structural analogs and one of stereochemical analogs, from which we identified compound 1, that binds HDM2 with a Kd value of 400 nM. The spiroligomer 1 penetrates human liver cancer cells through passive diffusion and in a dose-dependent and time-dependent manner increases the levels of HDM2 more than 30-fold in Huh7 cells in which the p53/HDM2 negative feed-back loop is inoperative. This is a biological effect that is not seen with the HDM2 ligand nutlin-3a. We propose that compound 1 modulates the levels of HDM2 by stabilizing it to proteolysis, allowing it to accumulate in the absence of a p53/HDM2 feedback loop.     

A Non-Native α-Helix Is Formed in the β-Sheet Region of the Molten Globule State of Canine Milk Lysozyme

The native and the molten globule states (N and MG states, respectively) of canine milk lysozyme (CML) were examined by CD spectroscopy and AGADIR algorithm, a helix-coil transition program. It revealed that the helical content of the MG state was higher than that of the N-state, suggesting that non-native alpha-helix is formed in the MG state of CML. Using AGADIR, it indicated the possibility of alpha-helix formation in the third beta-strand region in the MG state. To investigate this possibility, we designed a mutant, Q58P, in which the helical propensity of the MG state was significantly decreased around the third beta-strand region. It appeared that the absolute ellipticity value at 222 nm of the mutant in the MG state was smaller than that of the wild-type protein. It could be assumed that the non-native alpha-helix is formed around the third beta-strand region of wild-type CML in the MG state.     

The M4 Transmembrane α-Helix Contributes Differently to Both the Maturation and Function of Two Prokaryotic Pentameric Ligand-gated Ion Channels

The role of the outermost transmembrane α-helix in both the maturation and function of the prokaryotic pentameric ligand-gated ion channels, GLIC and ELIC, was examined by Ala scanning mutagenesis, deletion mutations, and mutant cycle analyses. Ala mutations at the M4-M1/M3 interface lead to loss-of-function phenotypes in GLIC, with the largest negative effects occurring near the M4 C terminus. In particular, two aromatic residues at the M4 C terminus form a network of π-π and/or cation-π interactions with residues on M3 and the β6-β7 loop that is essential for both maturation and function. M4-M1/M3 interactions appear to be optimized in GLIC with even subtle structural changes at this interface leading to detrimental effects. In contrast, mutations along the M4-M1/M3 interface of ELIC typically lead to gain-of-function phenotypes, suggesting that these interactions in ELIC are not optimized for channel function. In addition, no cluster of interacting residues involving the M4 C terminus, M3, and the β6-β7 loop was found, suggesting that the M4 C terminus plays little role in ELIC maturation or function. This study shows that M4 makes distinct contributions to the maturation and gating of these two closely related homologs, suggesting that GLIC and ELIC exhibit divergent features of channel function.     

Inclusion of Many-Body Effects in the Additive CHARMM Protein CMAP Potential Results in Enhanced Cooperativity of α-Helix and β-Hairpin Formation

Folding simulations on peptides and proteins using empirical force fields have demonstrated the sensitivity of the results to details of the backbone potential. A recently revised version of the additive CHARMM protein force field, which includes optimization of the backbone CMAP potential to achieve good balance between different types of secondary structure, correcting the α-helical bias present in the former CHARMM22/CMAP energy function, is shown to result in improved cooperativity for the helix-coil transition. This is due to retention of the empirical corrections introduced in the original CMAP to reproduce folded protein structures—corrections that capture many-body effects missing from an energy surface fitted to gas phase calculations on dipeptides. The experimental temperature dependence of helix formation in (AAQAA)₃ and parameters for helix nucleation and elongation are in much better agreement with experiment than those obtained with other recent force fields. In contrast, CMAP parameters derived by fitting to a vacuum quantum mechanical surface for the alanine dipeptide do not reproduce the enhanced cooperativity, showing that the empirical backbone corrections, and not some other feature of the force field, are responsible. We also find that the cooperativity of β-hairpin formation is much improved relative to other force fields we have studied. Comparison with (ϕ,ψ) distributions from the Protein Data Bank further justifies the inclusion of many-body effects in the CMAP. These results suggest that the revised energy function will be suitable for both simulations of unfolded or intrinsically disordered proteins and for investigating protein-folding mechanisms.     

The C-Terminal α-Helix Domain of Apolipoprotein E Is Required for Interaction with Nonstructural Protein 5A and Assembly of Hepatitis C Virus

We have recently demonstrated that human apolipoprotein E (apoE) is required for the infectivity and assembly of hepatitis C virus (HCV) (K. S. Chang, J. Jiang, Z. Cai, and G. Luo, J. Virol. 81:13783-13793, 2007; J. Jiang and G. Luo, J. Virol. 83:12680-12691, 2009). In the present study, we have determined the molecular basis underlying the importance of apoE in HCV assembly. Results derived from mammalian two-hybrid studies demonstrate a specific interaction between apoE and HCV nonstructural protein 5A (NS5A). The C-terminal third of apoE per se is sufficient for interaction with NS5A. Progressive deletion mutagenesis analysis identified that the C-terminal α-helix domain of apoE is important for NS5A binding. The N-terminal receptor-binding domain and the C-terminal 20 amino acids of apoE are dispensable for the apoE-NS5A interaction. The NS5A-binding domain of apoE was mapped to the middle of the C-terminal α-helix domain between amino acids 205 and 280. Likewise, deletion mutations disrupting the apoE-NS5A interaction resulted in blockade of HCV production. These findings demonstrate that the specific apoE-NS5A interaction is required for assembly of infectious HCV. Additionally, we have determined that using different major isoforms of apoE (E2, E3, and E4) made no significant difference in the apoE-NS5A interaction. Likewise, these three major isoforms of apoE are equally compatible with infectivity and assembly of infectious HCV, suggesting that apoE isoforms do not differentially modulate the infectivity and/or assembly of HCV in cell culture.Hepatitis C virus (HCV) remains a major global health problem, chronically infecting approximately 170 million people worldwide, with severe consequences such as hepatitis, fibrosis/cirrhosis, and hepatocellular carcinoma (HCC) (2, 57). The current standard therapy for hepatitis C is pegylated alpha interferon in combination with ribavirin. However, this anti-HCV regimen has limited efficacy (<50% sustained antiviral response for the dominant genotype 1 HCV) and causes severe side effects (17, 39). Recent clinical studies on the HCV protease- and polymerase-specific inhibitors showed promising results but also found that drug-resistant HCV mutants emerged rapidly (3, 27), undermining the efficacy of specific antiviral therapy for hepatitis C. Therefore, future antiviral therapies for hepatitis C likely require a combination of several safer and more efficacious antiviral drugs that target different steps of the HCV life cycle. The lack of knowledge about the molecular details of the HCV life cycle has significantly impeded the discovery of antiviral drugs and development of HCV vaccines.HCV is a small enveloped RNA virus classified as a member of the Hepacivirus genus in the family Flaviviridae (46, 47). It contains a single positive-sense RNA genome that encodes a large viral polypeptide, which is proteolytically processed by cellular peptidases and viral proteases into different structural and nonstructural proteins in the order of C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (30, 31). Other novel viral proteins derived from the C-coding region have also been discovered (11, 13, 55, 59). The nucleotides at both the 5′ and 3′ untranslated regions (UTR) are highly conserved and contain cis-acting RNA elements important for internal ribosome entry site (IRES)-mediated initiation of protein translation and viral RNA replication (15, 16, 33, 56, 60).The success in the development of HCV replicon replication systems has made enormous contributions to the determination of the roles of the conserved RNA sequences/structures and viral NS proteins in HCV RNA replication (4, 5, 7, 32). However, the molecular mechanisms of HCV assembly, morphogenesis, and egression have not been well understood. A breakthrough advance has been the development of robust cell culture systems for HCV infection and propagation, which allow us to determine the roles of viral and cellular proteins in the HCV infectious cycle (9, 29, 54, 63). We have recently demonstrated that infectious HCV particles are enriched in apolipoprotein E (apoE) and that apoE is required for HCV infection and assembly (10, 23). apoE-specific monoclonal antibodies efficiently neutralized HCV infectivity. The knockdown of endogenous apoE expression by a specific small interfering RNA (siRNA) and the blockade of apoE secretion by microsomal triglyceride transfer protein (MTP) inhibitors remarkably suppressed HCV assembly (10, 23). More importantly, apoE was found to interact with the HCV NS5A in the cell and purified HCV particles, as determined by yeast two-hybrid and coimmunoprecipitation (co-IP) studies (6, 23). These findings suggest that apoE has dual functions in HCV infection and assembly via distinct interactions with cell surface receptors and HCV NS5A. To further understand the molecular mechanism of apoE in HCV assembly, we carried out a mutagenesis analysis of apoE and determined the importance of the apoE-NS5A interaction in HCV assembly. Progressive deletion mutagenesis analysis has mapped the NS5A-binding domain of apoE to the C-terminal α-helix region between amino acid residues 205 and 280. Mutations disrupting the apoE-NS5A interaction also blocked HCV production. Additionally, we have determined the effects of three major isoforms of apoE on HCV infection and assembly. Our results demonstrate that apoE isoforms do not determine the infectivity and assembly of infectious HCV in cell culture.