Relative Orientation Of Helices Research Articles

Domain Swapping in Abiotic Foldamers.

Foldamer sequences that adopt tertiary helix-turn-helix folds mediated by helix-helix hydrogen bonding in organic solvents have been previously reported. In an attempt to create genuine abiotic quaternary structures, i.e. assemblies of tertiary structures, new sequences were prepared that possess additional hydrogen bond donors at positions that may promote an association between the tertiary folds. However, a solid state structure and extensive solution state investigations by Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) show that, instead of forming a quaternary structure, the tertiary folds assemble into stable domain-swapped dimer motifs. Domain swapping entails a complete reorganization of the arrays of hydrogen bonds and changes in relative helix orientation and handedness that can all be rationalized.

Domain Swapping in Abiotic Foldamers

AbstractFoldamer sequences that adopt tertiary helix‐turn‐helix folds mediated by helix‐helix hydrogen bonding in organic solvents have been previously reported. In an attempt to create genuine abiotic quaternary structures, i.e. assemblies of tertiary structures, new sequences were prepared that possess additional hydrogen bond donors at positions that may promote an association between the tertiary folds. However, a solid state structure and extensive solution state investigations by Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) show that, instead of forming a quaternary structure, the tertiary folds assemble into stable domain‐swapped dimer motifs. Domain swapping entails a complete reorganization of the arrays of hydrogen bonds and changes in relative helix orientation and handedness that can all be rationalized.

Synthesis and Crystallographic Characterization of Helical Hairpin Oligourea Foldamers.

Oligomers designed to form a helix-turn-helix super-secondary structure have been prepared by covalently bridging aliphatic oligourea foldamer helices with either rigid aromatic or more flexible aliphatic spacers. The relative helix orientation in these dimers has been investigated at high resolution using X-ray diffraction analysis. In several cases, racemic crystallography was used to facilitate crystallization and structure determination. All structures were solved by direct methods. Well-defined parallel helical hairpin motifs were observed in all cases when 4,4'-methylene diphenyl diisocyanate was employed as a dimerizing agent, irrespective of primary sequence and chain length.

Designing cooperatively folded abiotic uni- and multimolecular helix bundles.

Abiotic foldamers, that is foldamers that have backbones chemically remote from peptidic and nucleotidic skeletons, may give access to shapes and functions different to those of peptides and nucleotides. However, design methodologies towards abiotic tertiary and quaternary structures are yet to be developed. Here we report rationally designed interactional patterns to guide the folding and assembly of abiotic helix bundles. Computational design facilitated the introduction of hydrogen-bonding functionalities at defined locations on the aromatic amide backbones that promote cooperative folding into helix-turn-helix motifs in organic solvents. The hydrogen-bond-directed aggregation of helices not linked by a turn unit produced several thermodynamically and kinetically stable homochiral dimeric and trimeric bundles with structures that are distinct from the designed helix-turn-helix. Relative helix orientation within the bundles may be changed from parallel to tilted on subtle solvent variations. Altogether, these results prefigure the richness and uniqueness of abiotic tertiary structure behaviour.

Structural effect of the L16Q, K50E, and R53P mutations on homeodomain of pituitary homeobox protein 2

The transcription factor pituitary homeobox protein 2 (PITX2) is involved in genetic control of development. Mutations in PITX2, most in the homeodomain, cause the autosomal-dominant disorder Rieger syndrome. The mutants L16Q, K50E and R53P destabilize the structure and disrupt DNA-binding activity. The biological functions of these mutants have been characterized but not the structural basis behind the loss of DNA-binding activity. We performed multiple molecular dynamics simulations at 37°C to investigate the structural and dynamic effects of the 3 PITX2 homeodomain mutants. Compared with the wild type (WT), the L16Q mutant induces a kink in the α3 helix, which is stabilized by the hydrogen bond of Q21-R59. The disruption in backbone hydrogen bonds of V47-N51 and W48-R52 leads to a kink formation in the α3 helix of K50E. The R53P mutant alters the relative orientation of helices, which is apparently stabilized by the formation of new hydrogen bonds of T38-Q11, T38-Q12, T38-R2, N39-R2, L40-Q1, L40-R2, and T41-Q4. The hydrophobic core residues F8, L13, L40 and V45 change their positions in all mutants to break the hydrophobic core. Thus, changes in helical orientations and hydrophobic core cause rearrangement of the DNA-binding surface and disrupt DNA-binding activity in the mutants. The structural and molecular dynamics properties of 3 PITX2 homeodomain mutants differ from those of the WT, especially in formation of a kink in the recognition helix, change in the packing of helices and disruption of the hydrophobic core. This structural basis for the loss of DNA-binding activity for these polymorphisms may help in understanding the effect of mutations on other homeodomains with other diseases.

3D maps of RNA interhelical junctions

More than 50% of RNA secondary structure is estimated to be A-form helices, which are linked together by various junctions. Here we describe a protocol for computing three interhelical Euler angles describing the relative orientation of helices across RNA junctions. 5' and 3' helices, H1 and H2, respectively, are assigned based on the junction topology. A reference canonical helix is constructed using an appropriate molecular builder software consisting of two continuous idealized A-form helices (iH1 and iH2) with helix axis oriented along the molecular Z-direction running toward the positive direction from iH1 to iH2. The phosphate groups and the carbon and oxygen atoms of the sugars are used to superimpose helix H1 of a target interhelical junction onto the corresponding iH1 of the reference helix. A copy of iH2 is then superimposed onto the resulting H2 helix to generate iH2'. A rotation matrix R is computed, which rotates iH2' into iH2 and expresses the rotation parameters in terms of three Euler angles α(h), β(h) and γ(h). The angles are processed to resolve a twofold degeneracy and to select an overall rotation around the axis of the reference helix. The three interhelical Euler angles define clockwise rotations around the 5' (-γ(h)) and 3' (α(h)) helices and an interhelical bend angle (β(h)). The angles can be depicted graphically to provide a 'Ramachandran'-type view of RNA global structure that can be used to identify unusual conformations as well as to understand variations due to changes in sequence, junction topology and other parameters.

Topology Links RNA Secondary Structure with Global Conformation, Dynamics, and Adaptation

Thermodynamic rules that link RNA sequences to secondary structure are well established, but the link between secondary structure and three-dimensional global conformation remains poorly understood. We constructed comprehensive three-dimensional maps depicting the orientation of A-form helices across RNA junctions in the Protein Data Bank and rationalized our findings with modeling and nuclear magnetic resonance spectroscopy. We show that the secondary structures of junctions encode readily computable topological constraints that accurately predict the three-dimensional orientation of helices across all two-way junctions. Our results suggest that RNA global conformation is largely defined by topological constraints encoded at the secondary structural level and that tertiary contacts and intermolecular interactions serve to stabilize specific conformers within the topologically allowed ensemble.

Structure and Dynamics of Human Apolipoprotein CIII

Human apolipoprotein CIII (apoCIII) is a surface component of chylomicrons, very low density lipoproteins, and high density lipoproteins. ApoCIII inhibits lipoprotein lipase as well as binding of lipoproteins to cell surface heparan sulfate proteoglycans and receptors. High levels of apoCIII are often correlated with elevated levels of blood lipids (hypertriglyceridemia). Here, we report the three-dimensional NMR structure and dynamics of human apo-CIII in complex with SDS micelles, mimicking its natural lipid-bound state. Thanks to residual dipolar coupling data, the first detailed view is obtained of the structure and dynamics of an intact apolipoprotein in its lipid-bound state. ApoCIII wraps around the micelle surface as a necklace of six approximately 10-residue amphipathic helices, which are curved and connected via semiflexible hinges. Three positively charged (Lys) residues line the polar faces of helices 1 and 2. Interestingly, their three-dimensional conformation is similar to that of the low density lipoprotein receptor binding motifs of apoE/B and the receptor-associated protein. At the C-terminal side of apoCIII, an array of negatively charged residues lines the polar faces of helices 4 and 5 and the adjacent flexible loop. Sequence comparison shows that this asymmetric charge distribution along the solvent-exposed face of apoCIII as well as other structural features are conserved among mammals. This structure provides a template for exploration of molecular mechanisms by which human apoCIII inhibits lipoprotein lipase and receptor binding.

Characterizing the relative orientation and dynamics of RNA A-form helices using NMR residual dipolar couplings

We present a protocol for determining the relative orientation and dynamics of A-form helices in 13C/15N isotopically enriched RNA samples using NMR residual dipolar couplings (RDCs). Non-terminal Watson-Crick base pairs in helical stems are experimentally identified using NOE and trans-hydrogen bond connectivity and modeled using the idealized A-form helix geometry. RDCs measured in the partially aligned RNA are used to compute order tensors describing average alignment of each helix relative to the applied magnetic field. The order tensors are translated into Euler angles defining the average relative orientation of helices and order parameters describing the amplitude and asymmetry of interhelix motions. The protocol does not require complete resonance assignments and therefore can be implemented rapidly to RNAs much larger than those for which complete high-resolution NMR structure determination is feasible. The protocol is particularly valuable for exploring adaptive changes in RNA conformation that occur in response to biologically relevant signals. Following resonance assignments, the procedure is expected to take no more than 2 weeks of acquisition and data analysis time.

Importance of Potential Interhelical Salt-bridges Involving Interior Residues for Coiled-coil Stability and Quaternary Structure

Coiled coils are formed by two or more α-helices that align in a parallel or an antiparallel relative orientation. Polar interactions involving residues at the interior a and d positions are important for determining the quaternary structure of coiled coils. In the model heterodimeric coiled-coil Acid-a1-Base-a1, a buried a– d′ Asn–Asn interaction is sufficient to specify both a dimeric structure and an antiparallel relative helix orientation. Although the equivalent a– a′ interaction is found in parallel coiled coils, there is no example of an a– d′ Asn–Asn interaction in structurally characterized, naturally occurring antiparallel coiled coils. Instead, interior charged residues form interhelical salt-bridges with residues at the adjacent e or g positions. Using a model coiled-coil heterodimer, we have explored the role of a potential interhelical interaction between an Arg at an interior d position and a Glu at the adjacent g′ position. Our results demonstrate that this potentially attractive interhelical Coulombic interaction has little or no influence on helix orientation. Instead, we show that burying a single Arg residue at an interior position is sufficient to specify a dimeric state at a significantly lower thermodynamic cost than burial of two interacting Asn residues.

A general method for selection and screening of coiled coils on the basis of relative helix orientation.

We have developed a method for selecting coiled coils that associate with a given relative helix orientation from a randomized pool of proteins. To select for antiparallel dimers, we have designed a model basic region-leucine zipper (bZip) heterodimer capable of binding DNA only when the coiled coil associates with an antiparallel relative helix alignment. The dimerization domain for this bZip heterodimer is the model antiparallel coiled coil Acid-a1-Base-a1 (Oakley, M. G.; Kim, P. S. Biochemistry 1998, 37, 12603), and both monomers contain the GCN4 basic region. Although the basic regions in naturally occurring bZip proteins are located N-terminal to the leucine zipper, we have attached the GCN4 basic region to the C-terminus of Acid-a1 to allow both basic regions to contact DNA in an antiparallel heterodimer. The resulting heterodimer, BR-Base-a1-Acid-a1-BR, can bind to a direct repeat of the GCN4 half-site in vivo, leading to spectinomycin resistance in the transcription interference assay of Elledge et al. (Elledge, S. J.; Sugiono, P.; Guarente, L.; Davis, R. W. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 3689). A buried interhelical polar interaction between two Asn residues in the Acid-a1-Base-a1 heterodimer is known to specify an antiparallel helix orientation. The position of one of these buried Asn residues was randomized, and bZip heterodimers containing antiparallel coiled coils were selected using the transcription interference assay. All of the selected colonies contained Asn at the randomized position, suggesting that the selection is specific for antiparallel coiled coils.

Evaluation of the energetic contribution of interhelical coulombic interactions for coiled coil helix orientation specificity

Coiled coils are formed by two or more α-helices that align in a parallel or an antiparallel relative orientation. The factors that determine a preference for a given relative helix orientation are incompletely understood. The helix orientation preference for the designed coiled coil, Acid-a1-Base-a1, was measured previously. This model system therefore provides a means for the experimental determination of the energetic contribution of a variety of interactions to helix orientation specificity. The antiparallel preference for Acid-a1-Base-a1 is imparted by a single buried polar interaction. Interhelical Coulombic interactions between residues at the e and g positions have been proposed to influence helix orientation preference. In the Acid-a1-Base-a1 heterodimer, potentially attractive Coulombic interactions are expected in both orientations. To determine the energetic consequences of Coulombic interactions for helix orientation preference, we have positioned a single charged residue in each peptide such that exclusively favorable interhelical Coulombic interactions can occur only in the parallel orientation. In contrast, two potentially repulsive interactions are expected in the antiparallel orientation. Because the buried polar interaction can occur only in the antiparallel orientation, interhelical Coulombic interactions favor the parallel orientation and the potential to form a buried polar interaction favors the antiparallel orientation. We find no clear preference for an antiparallel orientation in the resulting heterodimer, Acid-K e-Base-E g, suggesting that interhelical Coulombic interactions and a buried polar interaction are of approximately equal importance for helix orientation specificity. Stability measurements indicate that maintenance of all favorable electrostatic interactions and/or avoidance of two potentially repulsive interactions contributes approximately 2.1 kcal/mol to helix orientation preference.

Design and Characterization of a Heterodimeric Coiled Coil that Forms Exclusively with an Antiparallel Relative Helix Orientation

Design and Characterization of a Heterodimeric Coiled Coil that Forms Exclusively with an Antiparallel Relative Helix Orientation

A buried polar interaction can direct the relative orientation of helices in a coiled coil.

Coiled coils consist of bundles of two or more alpha-helices that are aligned in a parallel or an antiparallel relative orientation. The designed peptides, Acid-p1 and Base-p1, associate in solution to form a parallel, heterodimeric two-stranded coiled coil [O'Shea, E. K., Lumb, K. J., and Kim, P. S. (1993) Curr. Biol. 3, 658]. The buried interface of this complex is formed by hydrophobic Leu residues, with the exception of an Asn residue from each strand that is positioned to engage in a buried polar interaction. Substitution of these buried Asn residues by Leu residues results in a loss of structural uniqueness, as evidenced by a lack of a particular helix orientation in the Acid-Base coiled-coil complex [Lumb, K. J., and Kim, P. S. (1995) Biochemistry 34, 8642]. Here, we alter the positions of the Asn residues in the Acid and Base peptides such that a buried polar interaction is only expected to occur when the helices are in an antiparallel orientation. The resulting peptides, Acid-a1 and Base-a1, associate to form a helical heterodimer, as shown by circular dichroism (CD) and equilibrium sedimentation centrifugation. The helix orientation preference has been measured using covalently linked, disulfide-containing heterodimers in which the constituent peptides are constrained to interact in either a parallel or an antiparallel orientation. Although both the parallel and antiparallel heterodimers form stable, helical structures, the antiparallel heterodimer is the predominant species at equilibrium when the heterodimers are allowed to undergo thiol-disulfide exchange. In addition, the antiparallel heterodimer is more stable to chemical denaturation than the parallel counterpart by approximately 2.3 kcal/mol. These results demonstrate that a single buried polar interaction in the interface between the helices of a coiled coil is sufficient to determine the relative orientation of its constituent helices.

Aggregation and conformational transition in aqueous solution of a bombolitin III analogue containing a photoreactive side-chain group.

The peptide toxin bombolitin III [B(III)], originally isolated from bumblebee venom, has been shown to undergo a concentration-dependent conformational change from a random structure to an alpha-helix induced by aggregation. The aggregation process and the consequent folding results from a delicate balance of electrostatic and hydrophobic interactions. The conformational change is strongly dependent on pH and salt concentration. In order to gain insight on the structure of the aggregates, and in particular, on the aggregation number and relative orientation of helices in the molecular complexes, the following analogue of bombolitin III was designed and synthesized: Ile-Lys-Bpa-Met-Asp-Ile-Leu-Ala-Lys-Leu-Gly-Lys-Val-Leu-Ala-His-Val-NH2 Bpa3-B(III) where Bpa is benzoylphenylalanine. Bpa3-B(III) aggregates were investigated by CD and nmr techniques. The observed nuclear Overhauser effect pattern accounts for an antiparallel orientation of two distinct helices. The Bpa side chain allows for the photoinduced cross reaction with any aliphatic proton in spatial proximity. After irradiation, the reaction mixture was analyzed by high performance liquid chromatography and electrospray mass spectrometry. The results confirmed the presence of dimeric and trimeric complexes of bombolitin III formed upon interhelix cross-linking.

Transcription factor-induced, phased bending of the E-selectin promoter.

E-selectin is an endothelial adhesion molecule that is critically involved in neutrophil adhesion and recruitment. All DNA elements required for interleukin-1 inducibility have been located in the proximal promoter: an NF-ELAM1/ATF site, two NF-kappa B sites (I and II), the NF-ELAM2 element and a TATA box. We show here that interleukin-1 induced promoter activity is exquisitely sensitive to the spatial arrangements of these elements. Phasing of the ATF and NF-kappa B II elements indicates that their relative helix orientation is more important than distance per se. This sensitivity is partly due to a requirement for correctly oriented, transcription factor-induced DNA-bending. (i) Band shift analyses with permuted ATF- and NF-kappa B elements show that their associated factors all bend DNA. (ii) One can functionally replace the NF-ELAM1/ATF element by a subset of a panel of DNA fragments that contain defined bends in various planes. We conclude that the main role of the factors binding at the NF-ELAM1/ATF element is to alter the conformation of the E-selectin promoter, presumably looping distant enhancer elements into each other's proximity.