Α-helix Dipole Research Articles

Molecular mechanism of Mad1 kinetochore targeting by phosphorylated Bub1.

During metaphase, in response to improper kinetochore‐microtubule attachments, the spindle assembly checkpoint (SAC) activates the mitotic checkpoint complex (MCC), an inhibitor of the anaphase‐promoting complex/cyclosome (APC/C). This process is orchestrated by the kinase Mps1, which initiates the assembly of the MCC onto kinetochores through a sequential phosphorylation‐dependent signalling cascade. The Mad1‐Mad2 complex, which is required to catalyse MCC formation, is targeted to kinetochores through a direct interaction with the phosphorylated conserved domain 1 (CD1) of Bub1. Here, we present the crystal structure of the C‐terminal domain of Mad1 (Mad1CTD) bound to two phosphorylated Bub1CD1 peptides at 1.75 Å resolution. This interaction is mediated by phosphorylated Bub1 Thr461, which not only directly interacts with Arg617 of the Mad1 RLK (Arg‐Leu‐Lys) motif, but also directly acts as an N‐terminal cap to the CD1 α‐helix dipole. Surprisingly, only one Bub1CD1 peptide binds to the Mad1 homodimer in solution. We suggest that this stoichiometry is due to inherent asymmetry in the coiled‐coil of Mad1CTD and has implications for how the Mad1‐Bub1 complex at kinetochores promotes efficient MCC assembly.

Effects of aligned α-helix peptide dipoles on experimental electrostatic potentials.

Aligned protein α-helix dipoles have been implicated in protein function and structure. The recent breakthroughs in high-resolution electron microscopy (EM) of macromolecules makes it possible to explore fundamental aspects of structural biology at the detailed molecular level. The electrostatic potential (ESP) generated by aligned protein α-helix dipole should be observable in high-resolution EM maps despite the fact that the effect may be partially screened by induced electric fields. Here, we show that aligned backbone dipoles in protein α-helices account for long-range features in the protein ESP functions. Our results are consistent with experimental EM maps and density functional theory calculations, including direct Fourier summation for proper calculation of the ESP due to the nonlocal nature of the ESP function from aligned dipoles and other partial atomic charges.

Proline Primed Helix Length as a Modulator of the Nuclear Receptor–Coactivator Interaction

Nuclear receptor binding to coactivator proteins is an obligate first step in the regulation of gene transcription. Nuclear receptors preferentially bind to an LXXLL peptide motif which is highly conserved throughout the 300 or so natural coactivator proteins. This knowledge has shaped current understanding of this fundamental protein-protein interaction, and continues to inspire the search for new drug therapies. However, sequence specificity beyond the LXXLL motif and the molecular functioning of flanking residues still requires urgent addressing. Here, ribosome display has been used to reassess the estrogen receptor for new and enlarged peptide recognition motifs, leading to the discovery of a potent and highly evolved PXLXXLLXXP binding consensus. Molecular modeling and X-ray crystallography studies have provided the molecular insights on the role of the flanking prolines in priming the length of the α-helix and enabling optimal interactions of the α-helix dipole and its surrounding amino acids with the surface charge clamp and the receptor activation function 2. These findings represent new structural parameters for modulating the nuclear receptor-coactivator interaction based on linear sequences of proteinogenic amino acids and for the design of chemically modified inhibitors.

Hydroxynitrile lyases with α/β-hydrolase fold: two enzymes with almost identical 3D structures but opposite enantioselectivities and different reaction mechanisms.

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins to yield hydrocyanic acid (HCN) and the respective carbonyl compound and are key enzymes in the process of cyanogenesis in plants. In organic syntheses, HNLs are used as biocatalysts for the formation of enantiopure cyanohydrins. We determined the structure of the recently identified, R-selective HNL from Arabidopsis thaliana (AtHNL) at a crystallographic resolution of 2.5 Å. The structure exhibits an α/β-hydrolase fold, very similar to the homologous, but S-selective, HNL from Hevea brasiliensis (HbHNL). The similarities also extend to the active sites of these enzymes, with a Ser-His-Asp catalytic triad present in all three cases. In order to elucidate the mode of substrate binding and to understand the unexpected opposite enantioselectivity of AtHNL, complexes of the enzyme with both (R)- and (S)-mandelonitrile were modeled using molecular docking simulations. Compared to the complex of HbHNL with (S)-mandelonitrile, the calculations produced an approximate mirror image binding mode of the substrate with the phenyl rings located at very similar positions, but with the cyano groups pointing in opposite directions. A catalytic mechanism for AtHNL is proposed, in which His236 from the catalytic triad acts as a general base and the emerging negative charge on the cyano group is stabilized by main-chain amide groups and an α-helix dipole very similar to α/β-hydrolases. This mechanistic proposal is additionally supported by mutagenesis studies.

Effect of pulsed electric field on the secondary structure and thermal properties of soy protein isolate

Changes of structure and thermal stability of soy protein isolate after pulsed electric field treatment were analyzed by Fourier transform infrared spectroscopy and differential scanning calorimetry (DSC). When the applied pulsed electric field (PEF) treatment intensity was over 35 kV/cm, the amino acid side chain, anti-parallel β-sheets, β-turn as well as β-sheets in soy protein isolate (SPI) secondary structure were significantly changed, which suggested that PEF treatment might be a new processing method for SPI; the dipole moments of some bonds, such as C=O, C–O, and C–O–C were partially polarized, accompanying with complete denaturation of β-conglycinin and glycinin obtained from DSC. Furthermore, self-reassembly from β-turn to α-helix in SPI structure abruptly happened under intense PEF treatment condition, which suggested that the PEF treatment had induced change of the orientation of α-helix dipole moment to stabilize α-helix. This observation implies that PEF treatment technique may be a novel method for preparation of protein nanotube.

A systematic study of fundamentals in α-helical coiled coil mimicry by alternating sequences of β- and γ-amino acids

Aimed at understanding the crucially important structural features for the integrity of α-helical mimicry by βγ-sequences, an α-amino acid sequence in a native peptide was substituted by differently arranged βγ-sequences. The self- and hetero-assembly of a series of αβγ-chimeric sequences based on a 33-residue GCN4-derived peptide was investigated by means of molecular dynamics, circular dichroism, and a disulfide exchange assay. Despite the native-like behavior of βγ alternating sequences such as retention of α-helix dipole and the formation of 13-membered α-helix turns, the αβγ-chimeras with different βγ substitution patterns do not equally mimic the structural behavior of the native parent peptide in solution. The preservation of the key residue contacts such as van der Waals interactions and intrahelical H-bonding, which can be met only by particular substitution patterns, thermodynamically favor the adoption of coiled coil folding motif. In this study, we show how successfully the destabilizing structural consequences of α→βγ modification can be harnessed by reducing the solvent-exposed hydrophobic surface area and placing of suitably long and bulky helix-forming side chains at the hydrophobic core. The pairing of αβγ-chimeric sequences with the native wild-type are thermodynamically allowed in the case of ideal arrangement of β- and γ-residues. This indicates a similarity in local side chain packing of β- and γ-amino acids at the helical interface of αβγ-chimeras and the native α-peptide. Consequently, the backbone extended residues are able to participate in classical "knob-into-hole" packing with native α-peptide.

Using Soft X-Rays for a Detailed Picture of Divalent Metal Binding in the Nucleosome

Divalent metals associate with DNA in a site-selective manner, which can influence nucleosome positioning, mobility, compaction, and recognition by nuclear factors. We previously characterized divalent metal binding in the nucleosome core using hard (short-wavelength) X-rays allowing high-resolution crystallographic determination of the strongest affinity sites, which revealed that Mn(2+) associates with the DNA major groove in a sequence- and conformation-dependent manner. In this study, we obtained diffraction data with soft X-rays at the Mn(2+) absorption edge for a core particle crystal in the presence of 10 mM MnSO(4), mimicking prevailing Mg(2+) concentration in the nucleus. This provides an exceptional view of counterion binding in the nucleosome through identification of 45 divalent metal binding sites. In addition to that at the well-characterized major interparticle interface, only one other histone-divalent metal binding site is found, which corresponds to a symmetry-related counterpart on the 'free' H2B alpha1 helix C-terminus. This emphasizes the importance of the alpha-helix dipole in ion binding and suggests that the H2B motif may serve as a nucleation site in nucleosome compaction. The 43 sites associated with the DNA are characterized by (1) high-affinity direct coordination at the most electrostatically favorable major groove locations, (2) metal hydrate binding to the major groove, (3) direct coordination to phosphate groups at sites of high charge density, (4) metal hydrate binding in the minor groove, or (5) metal hydrate-divalent anion pairing. Metal hydrates are found within the minor groove only at locations displaying a narrow range of high-intermediate width and to which histone N-terminal tails are not associated or proximal. This indicates that divalent metals and histone tails can both collaborate and compete in minor groove association, which sheds light on nucleosome solubility and chromatin compaction behavior.

Mechanism of Selective Urea Permeation through a Bacterial Urea Transporter

In addition to its function as an intermediate in nitrogen metabolism, the small molecule urea plays an important role in the homeostasis of osmolarity and fluid volume in many organisms, including mammals, which concentrate urea in the kidney to produce the osmotic gradient necessary for water re-absorption. Because urea is highly polar and consequently poorly permeable through lipid bilayers, specialized urea transporters have evolved to increase its rate of diffusion across cell membranes. To better understand how urea transporters achieve the rapid and selective transport of urea, we have solved the 2.3 A structure of a urea transporter from the bacterium Desulfovibrio vulgaris (dvUT), which has significant homology to mammalian urea transporters. The dvUT fold contains two homologous domains related by a two-fold pseudosymmetry axis perpendicular to the plane of the membrane. Each protomer contains a continuous membrane-spanning pore, suggesting that the protein operates by a channel-like rather than transporter-like mechanism. The constricted selectivity filter at the center of pore can accommodate dehydrated urea molecules passing in single file. Urea is stabilized by backbone and side chain oxygen atoms that provide continuous coordination as it progresses through the filter, and by well-positioned α-helix dipoles. We are now using a variety of functional assays to probe the physical and chemical interactions involved in the transport mechanism, as well as interactions between dvUT and various high-affinity UT blockers.

Relationship between the Stability of Hen Egg-White Lysozymes Mutated at Sites Designed to Interact with α-Helix Dipoles and Their Secretion Amounts in Yeast

The positively charged lysine at the C-terminals of three long alpha-helices (5-15, 25-35, and 88-99) was replaced with alanine (K13A, K33A, K97A) or aspartic acid (K13D, K33D, K97D) in hen lysozyme by genetic engineering. The denaturation transition point (Tm) and Gibbs energy change Delta G of the mutant lysozymes decreased remarkably, suggesting that the positive charge at the C-terminals of helices is involved in the stabilization of the helix dipole. On the other hand, the non-charged asparagine at the N-terminal of the long alpha-helices (25-35 and 88-99) was replaced with negatively charged aspartic acid (N27D and N93D). The Tm and Delta G of N27D increased, suggesting that the dipole moment of the N-terminal of the helices is diminished by replacement with negatively charged amino acid strengthening the stability of the helices. The stabilities of those hen egg white lysozymes mutated at the N- or C-terminal sites of the three long alpha-helices were related with their secretion amounts in yeast (Pichia pastoris). The secretion amounts of these mutant lysozymes in yeast were closely correlated with their stability.

Structural basis for ion conduction and gating in ClC chloride channels

Members of the ClC family of voltage-gated chloride channels are found from bacteria to mammals with a considerable degree of conservation in the membrane-inserted, pore-forming region. The crystal structures of the ClC channels of Escherichia coli and Salmonella typhimurium provide a structural framework for the entire family. The ClC channels are homodimeric proteins with an overall rhombus-like shape. Each ClC dimer has two pores each contained within a single subunit. The ClC subunit consists of two roughly repeated halves that span the membrane with opposite orientations. This antiparallel architecture defines a chloride selectivity filter within the 15-Å neck of a hourglass-shaped pore. Three Cl− binding sites within the selectivity filter stabilize ions by interactions with α-helix dipoles and by chemical interactions with nitrogen atoms and hydroxyl groups of residues in the protein. The Cl− binding site nearest the extracellular solution can be occupied either by a Cl− ion or by a glutamate carboxyl group. Mutations of this glutamate residue in Torpedo ray ClC channels alter gating in electrophysiological assays. These findings reveal a form of gating in which the glutamate carboxyl group closes the pore by mimicking a Cl− ion.

Electrostatic Interactions in Leucine Zippers: Thermodynamic Analysis of the Contributions of Glu and His Residues and the Effect of Mutating Salt Bridges

Electrostatic interactions play a complex role in stabilizing proteins. Here, we present a rigorous thermodynamic analysis of the contribution of individual Glu and His residues to the relative pH-dependent stability of the designed disulfide-linked leucine zipper AB SS. The contribution of an ionized side-chain to the pH-dependent stability is related to the shift of the p K a induced by folding of the coiled coil structure. p K a F values of ten Glu and two His side-chains in folded AB SS and the corresponding p K a U values in unfolded peptides with partial sequences of AB SS were determined by 1H NMR spectroscopy: of four Glu residues not involved in ion pairing, two are destabilizing (−5.6 kJ mol −1) and two are interacting with the positive α-helix dipoles and are thus stabilizing (+3.8 kJ mol −1) in charged form. The two His residues positioned in the C-terminal moiety of AB SS interact with the negative α-helix dipoles resulting in net stabilization of the coiled coil conformation carrying charged His (−2.6 kJ mol −1). Of the six Glu residues involved in inter-helical salt bridges, three are destabilizing and three are stabilizing in charged form, the net contribution of salt-bridged Glu side-chains being destabilizing (−1.1 kJ mol −1). The sum of the individual contributions of protonated Glu and His to the higher stability of AB SS at acidic pH (−5.4 kJ mol −1) agrees with the difference in stability determined by thermal unfolding at pH 8 and pH 2 (−5.3 kJ mol −1). To confirm salt bridge formation, the positive charge of the basic partner residue of one stabilizing and one destabilizing Glu was removed by isosteric mutations (Lys→norleucine, Arg→norvaline). Both mutations destabilize the coiled coil conformation at neutral pH and increase the p K a of the formerly ion-paired Glu side-chain, verifying the formation of a salt bridge even in the case where a charged side-chain is destabilizing. Because removing charges by a double mutation cycle mainly discloses the immediate charge–charge effect, mutational analysis tends to overestimate the overall energetic contribution of salt bridges to protein stability.

The 1.8 Å crystal structure and active-site architecture of β-ketoacyl-acyl carrier protein synthase III (FabH) from Escherichia coli

Background: β-Ketoacyl-acyl carrier protein synthase III (FabH) initiates elongation in type II fatty acid synthase systems found in bacteria and plants. FabH is a ubiquitous component of the type II system and is positioned ideally in the pathway to control the production of fatty acids. The elucidation of the structure of FabH is important for the understanding of its regulation by feedback inhibition and its interaction with drugs. Although the structures of two related condensing enzymes are known, the roles of the active-site residues have not been experimentally tested. Results: The 1.8 Å crystal structure of FabH was determined using a 12-site selenium multiwavelength anomalous dispersion experiment. The active site (Cys112, His244 and Asn274) is formed by the convergence of two α helices and is accessed via a narrow hydrophobic tunnel. Hydrogen-bonding networks that include two tightly bound water molecules fix the positions of His244 and Asn274, which are critical for the decarboxylation and condensation reactions. Surprisingly, the His244→Ala mutation does not affect the transacylation reaction suggesting that His244 has only a minor influence on the nucleophilicity of Cys112. Conclusions: The histidine and asparagine active-site residues are both required for the decarboxylation step in the condensation reaction. The nucleophilicity of the active-site cysteine is enhanced by the α-helix dipole effect, and an oxyanion hole promotes the formation of the tetrahedral transition state.

Refined structure of Cro repressor protein from bacteriophage λ suggests both flexibility and plasticity

The structure of the Cro repressor protein from phage λ has been refined to a crystallographic R-value of 19.3% at 2.3 Å resolution. The re fined model supports the structure as originally described in 1981 and provides a basis for comparison with the Cro-operator complex described in the accompanying paper. Changes in structure seen in different crystal forms and modifications of Cro suggest that the individual subunits are somewhat plastic in nature. In addition, the dimer of Cro suggests a high degree of flexibility, which may be important in forming the Cro-DNA complex. The structure of the Cro subunit as determined by NMR agrees reasonably well with that in the crystals (root-mean-square discrepancy of about 2 Å for all atoms). There are, however, only a limited number of intersubunit distance constraints and, presumably for this reason, the different NMR models for the dimer vary substantially among themselves (discrepancies of 1.3 to 5.5 Å). Because of this variation it is not possible to say whether the range of discrepancies between the X-ray and NMR Cro dimers (2.9 to 7.5 Å) represent a significant difference between the X-ray and solution structures. It has previously been proposed that substitutions of Tyr26 in Cro increase thermal stability by the “reverse hydrophobic effect”, i.e. by exposing 40% more hydrophobic surface to solvent in the folded form than in the unfolded state. The refined structure, however, suggests that Tyr26 is equally solvent exposed in the folded and unfolded states. The most stabilizing substitution is Tyr26 → Asp and in this case it appears that interaction with an α-helix dipole is at least partly responsible for the enhanced stability.

Channels formed by the transmembrane helix of phospholamban: a simulation study

Phospholamban is a small membrane protein which can form cation selective ion channels in lipid bilayers. Each subunit contains a single, largely hydrophobic transmembrane helix. The helices are thought to assemble as a pentameric and approximately parallel bundle surrounding a central pore. A model of this assembly (PDB code IPSL) has been used as the starting point for molecular dynamics (MD) simulations of a system consisting of the pentameric helix bundle, plus 217 water molecules located within and at either mouth of the pore. Interhelix distance restraints were employed to maintain the integrity of the helix bundle during a 500 ps MD simulation. Water molecules within the pore exhibited reduced diffusional and rotational mobility. Interactions between the α-helix dipoles and the water dipoles, the latter aligned anti-parallel to the former, contribute to the stability of the system. Analysis of the potential energy of interaction of a K + ion as it was moved through the pore suggested that unfavourable interactions of the cation with the aligned helix dipoles at the N-terminal mouth were overcome by favourable ion-water interactions. Comparable analysis for a Cl ion revealed that the ion-(pore + water) interactions were unfavourable along the whole of the pore, increasingly so from the N- to the C-terminal mouth. Overall, the interaction energy profiles were consistent with a pore selective for cations over anions. Pore radius profiles were used to predict a channel conductance of 50 to 70 ps in 0.2 M KCl, which compares well with an experimental value of 100 ps.

Ionisation of Cysteine Residues at the Termini of Model α-Helical Peptides. Relevance to Unusual Thiol p KaValues in Proteins of the Thioredoxin Family

The physical basis of the unusually lowp K avalues of an active site cysteine thiol group in proteins with the thioredoxin fold is unknown.The electrostatic field associated with an α-helixpointing with its N terminus towards the cysteine residue has been implicated to lower the thiol p K avalue by up to 5 pH units in glutaredoxin and DsbA. Here, the influence of the presence of an α-helical conformation on the ionisation of a cysteine thiol group located at or near the helix terminus is investigated in highly helical synthetic peptides with the generic sequence Ac- AAAAAAAAARAAAARAAAARAA-(NH 2). The thiol p K avalues have been determined by monitoring the pH dependence of the absorbance at 240 nm, of the α-helix content measured by the mean residue ellipticity at 222 nm, and of the chemical shifts of protons close to the sulphur atom of the cysteine residue. The favourable interaction between the thiolate anion at the N terminus and the α-helix decreases the thiol p K avalue by up to 1.6 pH units when compared to a normal thiol p K avalue measured in an unfolded control peptide, corresponding to a stabilisation energy of 2.1 kcal/mol. At the C terminus, the thiol p K avalue is increased, but by only 0.2 pH units. The observations are consistent with an interaction of the α-helix dipole with the cysteine thiolate anion, involving both its charge and hydrogen-bonding. Subtle conformational effects in different model peptides appear to influence the ionisation of the thiol group significantly, with an N terminal Cys-Pro sequence having the most favourable interaction with the α-helix.

Electrostatic forces in two lysozymes: calculations and measurements of histidine pKa values.

In order to examine the electrostatic forces in globular proteins, pKa values and their ionic strength dependence of His residues of hen egg white lysozyme (HEWL) and human lysozyme (HUML) were measured, and they were compared with those calculated numerically. pKa values of His residues in HEWL, HUML, and short oligopeptides were determined from chemical shift changes of His side chains by 1H-nmr measurements. The associated changes in pKa values in HEWL and HUML were calculated by solving the Poisson-Boltzmann equations numerically for macroscopic dielectric models. The calculated pKa changes and their ionic strength dependence agreed fairly well with the observed ones. The contribution from each residue of each alpha-helix dipole to the pKa values and their ionic strength dependence was analyzed using Green's reciprocity theorem. The results indicate that (1) the pKa of His residues are largely affected by surrounding ionized and polar groups; (2) the ionic strength dependence of the pKa values is determined by the overall charge distributions and their accessibilities to solvent; and (3) alpha-helix dipoles make a significant contribution to the pKa, when the His residue is close to the helix terminus and not fully exposed to the solvent.

Prediction of the histidine-95 pK a perturbation in triosephosphate isomerase using an electrostatically trained neural network (SONNIC)

A backpropagation neural-network trained with AM1 SCF-MO derived molecular electrostatic potentials (MEPs) for a set of 30 substituted imidazoles of known ionisation constant can be used to predict the pKa of imidazole under the influence of the α-helix dipole found for histidine-95 in triosephosphate isomerase; the results agree quantitatively with experimental evidence that this residue has an anomalous value such that its catalytic nature is unconventional.

Resonance Raman and absorption spectroscopic characterization of the chemically engineered enzyme thiolsubtilisin; comparison with a natural thiolenzyme

Abstract The serine protease subtilisin Carlsberg has been converted to the cysteine protease thiolsubtilisin by chemically converting the active site Ser-221 to cysteine. The present experiments have been designed to explore the differences in the active site properties of thiolsubtilisin and papain - a natural cysteine protease. Resonance Raman and electronic absorption studies for the enzyme—substrate intermediates involving the acyl groups 5-methylthienylacryloyl and furylacryloyl have been used to probe the electrical effects of thiolsubtilisin's active site on the groups' delocalised π-electron systems. Unlike papain, thiolsubtilisin does not give rise to highly polarized π-electron systems in the bound substrates, characterized by low frequency νCC modes and red shifted λmax's. However, for the 5-methylthienylacryloyl intermediate there is evidence for a minor photoinduced population which does have a polarized π-electron system. The results can be explained by differential binding of the substrate with respect to the α-helix dipole found in the active sites of thiolsubtilisin and papain.

Enhanced protein thermostability from designed mutations that interact with alpha-helix dipoles.

Two different genetically engineered amino-acid substitutions designed to interact with alpha-helix dipoles in T4 lysozyme are shown to increase the thermal stability of the protein. Crystallographic analyses of the mutant lysozyme structures suggest that the stabilization is due to electrostatic interaction and does not require precise hydrogen bonding between the substituted amino acid and the end of the alpha-helix.

Stabilization of protein structure by interaction of alpha-helix dipole with a charged side chain.

The alpha-helix in proteins has a dipole moment resulting from the alignment of dipoles of the peptide bond which can perturb the pKas of ionizing groups. One of the two histidine residues (His18) in barnase, the small ribonuclease from Bacillus amyloliquefaciens, is located at the negatively charged end (C-terminal) of an alpha-helix. From NMR titrations of wild-type and engineered mutants we find that the pKa of His18 is 7.9 in wild-type enzyme, 1.6 units above the value in the urea-denatured enzyme and in model peptides. This implies that there is a favourable interaction between the protonated form of His18 and the alpha-helix that should stabilize the native structure at neutral pH by 2.1 kcal mol-1. Denaturation at various values of pH of wild-type and muant enzymes engineered at position 18 shows that this is so. The increase in stability of the enzyme as the pH changes from 8.5 to 6.3 is attributable to this interaction, and the pH-stability curve fits pKa values for His18 in native and urea-denatured enzymes that are consistent with the NMR data.