Harmonic Analysis of the Fluorescence Response of Bimane Adducts of Colicin E1 at Helices 6, 7, and 10

The pre-channel state of helices 6, 7, and 10 (Val⁴⁴⁷–Gly⁴⁷⁵ and Ile⁵⁰⁸–Ile⁵²²) of colicin E1 was investigated by a site-directed fluorescence labeling technique. A total of 44 cysteine variants were purified and covalently labeled with monobromobimane fluorescent probe. A variety of fluorescence properties of the bimane fluorophore were measured for both the soluble and membrane-bound states of the channel peptide, including the fluorescence emission maximum, fluorescence anisotropy, and membrane bilayer penetration depth. Using site-directed fluorescence labeling combined with our novel helical periodicity analysis method, the data revealed that helices 6, 7, and 10 are separate amphipathic α-helices with a calculated periodicity of T = 3.34 ± 0.08 for helix 6, T = 3.56 ± 0.03 for helix 7, and T = 2.99 ± 0.12 for helix 10 in the soluble state. In the membrane-bound state, the helical periodicity was determined to be T = 3.00 ± 0.15 for helix 6, T = 3.68 ± 0.03 for helix 7, and T = 3.47 ± 0.04 for helix 10. Dual fluorescence quencher analysis showed that both helices 6 and 7 adopt a tilted topology that correlates well with the analysis based on the fluorescence anisotropy profile. These data provide further support for the umbrella model of the colicin E1 channel domain.     

The k-junction motif in RNA structure

The k-junction is a structural motif in RNA comprising a three-way helical junction based upon kink turn (k-turn) architecture. A computer program written to examine relative helical orientation identified the three-way junction of the Arabidopsis TPP riboswitch as an elaborated k-turn. The Escherichia coli TPP riboswitch contains a related k-junction, and analysis of >11 000 sequences shows that the structure is common to these riboswitches. The k-junction exhibits all the key features of an N1-class k-turn, including the standard cross-strand hydrogen bonds. The third helix of the junction is coaxially aligned with the C (canonical) helix, while the k-turn loop forms the turn into the NC (non-canonical) helix. Analysis of ligand binding by ITC and global folding by gel electrophoresis demonstrates the importance of the k-turn nucleotides. Clearly the basic elements of k-turn structure are structurally well suited to generate a three-way helical junction, retaining all the key features and interactions of the k-turn.     

Measurement of the Change in Twist at a Helical Junction in RNA Using the?Orientation Dependence of FRET

Indocarbocyanine fluorophores attached via the 5′ terminus of double-stranded nucleic acids have a strong propensity to stack onto the terminal basepair. We previously demonstrated that the efficiency of fluorescence resonance energy transfer between cyanine 3 and 5 terminally attached to duplex species exhibits a pronounced modulation with helix length. This results from a systematic variation in the orientation parameter κ² as the relative rotation of the fluorophore transition moments changes due to the helical geometry. Analysis of such profiles provides a rich source of orientational information. In this work, we applied this methodology to the structure of a three-way helical junction that plays an important role in the hepatitis C virus internal ribosome entry site. By comparing matched pairs of duplex and junction species, we were able to measure the change in rotation at the junction. The data reveal a 29.5° overwinding and a small axial extension. This shows the power of this approach for measuring orientational information in biologically important RNA junctions.     

Structure of the three-way helical junction of the hepatitis C virus IRES element

The hepatitis C virus internal ribosome entry site (IRES) element contains a three-way junction that is important in the overall RNA conformation, and for its role in the internal initiation of translation. The junction also illustrates some important conformational principles in the folding of three-way helical junctions. It is formally a 3HS₄ junction, with the possibility of two alternative stacking conformers. However, in principle, the junction can also undergo two steps of branch migration that would form 2HS₁HS₃ and 2HS₂HS₂ junctions. Comparative gel electrophoresis and ensemble fluorescence resonance energy transfer (FRET) studies show that the junction is induced to fold by the presence of Mg²⁺ ions in low micromolar concentrations, and suggest that the structure adopted is based on coaxial stacking of the two helices that do not terminate in a hairpin loop (i.e., helix IIId). Single-molecule FRET studies confirm this conclusion, and indicate that there is no minor conformer present based on an alternative choice of helical stacking partners. Moreover, analysis of single-molecule FRET data at an 8-msec resolution failed to reveal evidence for structural transitions. It seems probable that this junction adopts a single conformation as a unique and stable fold.     

Pronounced instability of tandem IU base pairs in RNA

Optical melting was used to determine the stabilities of three series of RNA oligomers containing tandem XU base pairs, GGCXUGCC (5′XU3′), GGCUXGCC (5′UX3′) and GGCXXGGC/CCGUUCCG (5′XX3′), where X is either A, G or I (inosine). The helices containing tandem AU base pairs were the most stable in the first two series (5′XU3′ and 5′UX3′), with an average melting temperature ~11°C higher than the helices with tandem 5′GU3′ base pairs and 25°C higher than the helices with tandem 5′IU3′ base pairs. For the third series (5′XX3′), the helix containing tandem GG is the most stable, with an average melting temperature ~2°C higher than the helix with tandem AA base pairs and ~24°C higher than the helix with tandem II base pairs. The thermodynamic stability of the oligomers with tandem IU base pairs was also investigated as a function of magnesium ion concentration. As with normal A–U or G–U tandem duplexes, the data could best be interpreted as non-specific binding of magnesium ions to the inosine-containing RNA oligonucleotides.     

Computational de novo design of a four-helix bundle protein—DND_4HB

The de novo design of proteins is a rigorous test of our understanding of the key determinants of protein structure. The helix bundle is an interesting de novo design model system due to the diverse topologies that can be generated from a few simple α-helices. Previously, noncomputational studies demonstrated that connecting amphipathic helices together with short loops can sometimes generate helix bundle proteins, regardless of the bundle''s exact sequence. However, using such methods, the precise positions of helices and side chains cannot be predetermined. Since protein function depends on exact positioning of residues, we examined if sequence design tools in the program Rosetta could be used to design a four-helix bundle with a predetermined structure. Helix position was specified using a folding procedure that constrained the design model to a defined topology, and iterative rounds of rotamer-based sequence design and backbone refinement were used to identify a low energy sequence for characterization. The designed protein, DND_4HB, unfolds cooperatively (T_m >90°C) and a NMR solution structure shows that it adopts the target helical bundle topology. Helices 2, 3, and 4 agree very closely with the design model (backbone RMSD = 1.11 Å) and >90% of the core side chain χ1 and χ2 angles are correctly predicted. Helix 1 lies in the target groove against the other helices, but is displaced 3 Å along the bundle axis. This result highlights the potential of computational design to create bundles with atomic-level precision, but also points at remaining challenges for achieving specific positioning between amphipathic helices.     

Conformation dependent binding of netropsin and distamycin to DNA and DNA model polymers

The binding of the antibiotics netropsin and distamycin A to DNA has been studied by thermal melting, CD and sedimentation analysis. Netropsin binds strongly at antibiotic/nucleotide ratios up to at least 0.05. CD spectra obtained using DNA model polymers reveal that netropsin binds tightly to poly (dA) · poly (dT), poly (dA-dT) · poly(dA-dT) and poly (dI-dC) · poly (dI-dC) but poorly, if at all, to poly (dG) · poly (dC). Binding curves obtained with calf thymus DNA reveal one netropsin-binding site per 6.0 nucleotides (K_a=2.9 · 10⁵ M⁻¹); corresponding values for distamycin A are one site per 6.1 nucleotides with K_a= 11.6 · 10⁵ M⁻¹. Binding sites apparently involve predominantly A·T-rich sequences whose specific conformation determines their high affinity for the two antibiotics. It is suggested that the binding is stabilized primarily by hydrogen bonding and electrostatic interactions probably in the narrow groove of the DNA helix, but without intercalation. Any local structural deformation of the helix does not involve unwinding greater than approximately 3° per bound antibiotic molecule.     

Measurement of the Change in Twist at a Helical Junction in RNA Using the Orientation Dependence of FRET

Indocarbocyanine fluorophores attached via the 5′ terminus of double-stranded nucleic acids have a strong propensity to stack onto the terminal basepair. We previously demonstrated that the efficiency of fluorescence resonance energy transfer between cyanine 3 and 5 terminally attached to duplex species exhibits a pronounced modulation with helix length. This results from a systematic variation in the orientation parameter κ² as the relative rotation of the fluorophore transition moments changes due to the helical geometry. Analysis of such profiles provides a rich source of orientational information. In this work, we applied this methodology to the structure of a three-way helical junction that plays an important role in the hepatitis C virus internal ribosome entry site. By comparing matched pairs of duplex and junction species, we were able to measure the change in rotation at the junction. The data reveal a 29.5° overwinding and a small axial extension. This shows the power of this approach for measuring orientational information in biologically important RNA junctions.     

The global structure of the VS ribozyme

The VS ribozyme comprises five helical segments (II-VI) in a formal H shape, organized by two three-way junctions. It interacts with its stem-loop substrate (I) by tertiary interactions. We have determined the global shape of the 3-4-5 junction (relating helices III-V) by electrophoresis and FRET. Estimation of the dihedral angle between helices II and V electrophoretically has allowed us to build a model for the global structure of the complete ribozyme. We propose that the substrate is docked into a cleft between helices II and VI, with its loop making a tertiary interaction with that of helix V. This is consistent with the dependence of activity on the length of helix III. The scissile phosphate is well placed to interact with the probable active site of the ribozyme, the loop containing A730.     

Crystal structure of a poly(rA) staggered zipper at acidic pH: evidence that adenine N1 protonation mediates parallel double helix formation

We have solved at 1.07 Å resolution the X-ray crystal structure of a polyriboadenylic acid (poly(rA)) parallel and continuous double helix. Fifty-nine years ago, double helices of poly(rA) were first proposed to form at acidic pH. Here, we show that 7-mer oligo(rA), i.e. rA₇, hybridizes and overlaps in all registers at pH 3.5 to form stacked double helices that span the crystal. Under these conditions, rA₇ forms well-ordered crystals, whereas rA₆ forms fragile crystalline-like structures, and rA₅, rA₈ and rA₁₁ fail to crystallize. Our findings support studies from ∼50 years ago: one showed using spectroscopic methods that duplex formation at pH 4.5 largely starts with rA₇ and begins to plateau with rA₈; another proposed a so-called ‘staggered zipper’ model in which oligo(rA) strands overlap in multiple registers to extend the helical duplex. While never shown, protonation of adenines at position N1 has been hypothesized to be critical for helix formation. Bond angles in our structure suggest that N1 is protonated on the adenines of every other rAMP−rAMP helix base pair. Our data offer new insights into poly(rA) duplex formation that may be useful in developing a pH sensor.     

High-Resolution Crystal Structures of Protein Helices Reconciled with Three-Centered Hydrogen Bonds and Multipole Electrostatics

Theoretical and experimental evidence for non-linear hydrogen bonds in protein helices is ubiquitous. In particular, amide three-centered hydrogen bonds are common features of helices in high-resolution crystal structures of proteins. These high-resolution structures (1.0 to 1.5 Å nominal crystallographic resolution) position backbone atoms without significant bias from modeling constraints and identify Φ = -62°, ψ = -43 as the consensus backbone torsional angles of protein helices. These torsional angles preserve the atomic positions of α-β carbons of the classic Pauling α-helix while allowing the amide carbonyls to form bifurcated hydrogen bonds as first suggested by Némethy et al. in 1967. Molecular dynamics simulations of a capped 12-residue oligoalanine in water with AMOEBA (Atomic Multipole Optimized Energetics for Biomolecular Applications), a second-generation force field that includes multipole electrostatics and polarizability, reproduces the experimentally observed high-resolution helical conformation and correctly reorients the amide-bond carbonyls into bifurcated hydrogen bonds. This simple modification of backbone torsional angles reconciles experimental and theoretical views to provide a unified view of amide three-centered hydrogen bonds as crucial components of protein helices. The reason why they have been overlooked by structural biologists depends on the small crankshaft-like changes in orientation of the amide bond that allows maintenance of the overall helical parameters (helix pitch (p) and residues per turn (n)). The Pauling 3.6₁₃ α-helix fits the high-resolution experimental data with the minor exception of the amide-carbonyl electron density, but the previously associated backbone torsional angles (Φ, Ψ) needed slight modification to be reconciled with three-atom centered H-bonds and multipole electrostatics. Thus, a new standard helix, the 3.6_13/10-, Némethy- or N-helix, is proposed. Due to the use of constraints from monopole force fields and assumed secondary structures used in low-resolution refinement of electron density of proteins, such structures in the PDB often show linear hydrogen bonding.     

Electrostatic free energy landscapes for nucleic acid helix assembly

Metal ions are crucial for nucleic acid folding. From the free energy landscapes, we investigate the detailed mechanism for ion-induced collapse for a paradigm system: loop-tethered short DNA helices. We find that Na⁺ and Mg²⁺ play distinctive roles in helix–helix assembly. High [Na⁺] (>0.3 M) causes a reduced helix–helix electrostatic repulsion and a subsequent disordered packing of helices. In contrast, Mg²⁺ of concentration >1 mM is predicted to induce helix–helix attraction and results in a more compact and ordered helix–helix packing. Mg²⁺ is much more efficient in causing nucleic acid compaction. In addition, the free energy landscape shows that the tethering loops between the helices also play a significant role. A flexible loop, such as a neutral loop or a polynucleotide loop in high salt concentration, enhances the close approach of the helices in order to gain the loop entropy. On the other hand, a rigid loop, such as a polynucleotide loop in low salt concentration, tends to de-compact the helices. Therefore, a polynucleotide loop significantly enhances the sharpness of the ion-induced compaction transition. Moreover, we find that a larger number of helices in the system or a smaller radius of the divalent ions can cause a more abrupt compaction transition and a more compact state at high ion concentration, and the ion size effect becomes more pronounced as the number of helices is increased.     

The electrostatic characteristics of G.U wobble base pairs

G·U wobble base pairs are the most common and highly conserved non-Watson–Crick base pairs in RNA. Previous surface maps imply uniformly negative electrostatic potential at the major groove of G·U wobble base pairs embedded in RNA helices, suitable for entrapment of cationic ligands. In this work, we have used a Poisson–Boltzmann approach to gain a more detailed and accurate characterization of the electrostatic profile. We found that the major groove edge of an isolated G·U wobble displays distinctly enhanced negativity compared with standard GC or AU base pairs; however, in the context of different helical motifs, the electrostatic pattern varies. G·U wobbles with distinct widening have similar major groove electrostatic potentials to their canonical counterparts, whereas those with minimal widening exhibit significantly enhanced electronegativity, ranging from 0.8 to 2.5kT/e, depending upon structural features. We propose that the negativity at the major groove of G·U wobble base pairs is determined by the combined effect of the base atoms and the sugar-phosphate backbone, which is impacted by stacking pattern and groove width as a result of base sequence. These findings are significant in that they provide predictive power with respect to which G·U sites in RNA are most likely to bind cationic ligands.     

An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates

RNA aptamers are being developed as inhibitors of macromolecular and cellular function, diagnostic tools, and potential therapeutics. Our understanding of the physical nature of this emerging class of nucleic acid–protein complexes is limited; few atomic resolution structures have been reported for aptamers bound to their protein target. Guided by chemical mapping, we systematically minimized an RNA aptamer (Lys1) selected against hen egg white lysozyme. The resultant 59-nucleotide compact aptamer (Lys1.2minE) retains nanomolar binding affinity and the ability to inhibit lysozyme''s catalytic activity. Our 2.0-Å crystal structure of the aptamer–protein complex reveals a helical stem stabilizing two loops to form a protein binding platform that binds lysozyme distal to the catalytic cleft. This structure along with complementary solution analyses illuminate a novel protein–nucleic acid interface; (1) only 410 Ų of solvent accessible surface are buried by aptamer binding; (2) an unusually small fraction (∼18%) of the RNA-protein interaction is electrostatic, consistent with the limited protein phosphate backbone contacts observed in the structure; (3) a single Na⁺ stabilizes the loops that constitute the protein-binding platform, and consistent with this observation, Lys1.2minE–lysozyme complex formation takes up rather than displaces cations at low ionic strength; (4) Lys1.2minE inhibits catalysis of large cell wall substrates but not catalysis of small model substrates; and (5) the helical stem of Lys1.2minE can be shortened to four base pairs (Lys1.2minF) without compromising binding affinity, yielding a 45-nucleotide aptamer whose structure may be an adaptable protein binding platform.     

Conformation and Orientation of Gramicidin a in Oriented Phospholipid Bilayers Measured by Solid State Carbon-13 NMR

Three analogues of the helical ionophore gramicidin A have been synthesized with ¹³C-labeled carbonyls (¹³C=O) incorporated at either Gly², Ala³, or Val⁷. A fourth compound incorporated ¹³C at both the carbonyl and α-carbon of Gly² within the same molecule. These labels were studied using solid-state, proton-enhanced, ¹³C nuclear magnetic resonance (NMR) in hydrated dispersions of dimyristoylphosphatidylcholine (DMPC)-gramicidin A. The dispersions were aligned on glass coverslips whose orientation to the magnetic field could be varied through 180°. The orientation dependence of the NMR spectrum was used to obtain an accurate measurement of the ¹³C chemical shift anisotropy (CSA), and in the case of the fourth compound, the ¹³C—¹³C dipolar coupling constant. From the measured CSA and estimates of the orientation of the ¹³C shielding tensor, we are able to determine the direction of the ¹³C=O bonds and to compare these with the predictions of the various reported models for the configuration of gramicidin A in phospholipid bilayers. Our results are consistent with the left-handed ππ^6.3_LD single-stranded helix (Urry, D. W., J. T. Walker, and T. L. Trapane. 1982. J. Membr. Biol. 69:225-231). The right-handed ππ^6.3_LD single-stranded helix observed for gramicidin A in sodium dodecyl sulfate micelles (Arseniev, A. S., I. L. Barsukov, V. F. Bystrov, A. L. Loize, and Yu A. Ovchinnikov. 1985. FEBS (Fed. Eur. Biochem. Soc.) Lett. 186:168-174) yields a poorer fit to the data. However, the width of the carbonyl resonances suggests a distribution of molecular geometries possibly resulting from a spread in the helix pitch and handedness. Double-stranded helices and β sheet structures are excluded. In dispersions in which the lipid is in the L_α phase, the gramicidin A undergoes rapid reorientation about an axis which is centered on the normal to the plane of the coverslips. When the supporting lipid is in the L_β′ phase the helices are rigid on the timescale of ¹³C-NMR. The configuration of gramicidin A is unaltered by L_α-L_β′ phase transition of the bilayer lipid.     

Conformation and interactions of uteroglobin fragments 4–14 and 49–65 in aqueous solution containing surfactant micelles

The conformation of two fragments of rabbit uteroglobin is described. The peptides are PRFAHVIENLL and PQTTRENIMKLTEKIVK, corresponding to helices I and IV in the crystal structure. CD shows that both peptides interact with sodium dodecyl sulfate (SDS) micelles and change their conformation to an α-helix. The helical content estimated from the CD band at 222 nm is about 40% in each peptide. Surface tension measurements show that both peptides lower the critical micellar concentration (cmc) of SDS, with a more dramatic effect in the case of helix I. This peptide by itself acts as a surfactant, and is able to interact with SDS even below the observed cmc, forming β aggregates. Proton magnetic resonance (¹H-nmr) suggests that flexible helices are present. The longest helical stretches compatible with ¹H-nmr data extend from Phe⁶ to Leu¹⁴ for helix I and from Arg⁵³ to Ile⁶³ for helix IV. © 1993 John Wiley & Sons, Inc.     

Epiregulin Recognition Mechanisms by Anti-epiregulin Antibody 9E5: STRUCTURAL,FUNCTIONAL, AND MOLECULAR DYNAMICS SIMULATION ANALYSES*

Epiregulin (EPR) is a ligand of the epidermal growth factor (EGF) family that upon binding to its epidermal growth factor receptor (EGFR) stimulates proliferative signaling, especially in colon cancer cells. Here, we describe the three-dimensional structure of the EPR antibody (the 9E5(Fab) fragment) in the presence and absence of EPR. Among the six complementarity-determining regions (CDRs), CDR1–3 in the light chain and CDR2 in the heavy chain predominantly recognize EPR. In particular, CDR3 in the heavy chain dramatically moves with cis-trans isomerization of Pro¹⁰³. A molecular dynamics simulation and mutational analyses revealed that Arg⁴⁰ in EPR is a key residue for the specific binding of 9E5 IgG. From isothermal titration calorimetry analysis, the dissociation constant was determined to be 6.5 nm. Surface plasmon resonance analysis revealed that the dissociation rate of 9E5 IgG is extremely slow. The superimposed structure of 9E5(Fab)·EPR on the known complex structure of EGF·EGFR showed that the 9E5(Fab) paratope overlaps with Domains I and III on the EGFR, which reveals that the 9E5(Fab)·EPR complex could not bind to the EGFR. The 9E5 antibody will also be useful in medicine as a neutralizing antibody specific for colon cancer.     

Differential backbone dynamics of companion helices in the extended helical coiled‐coil domain of a bacterial chemoreceptor

Cytoplasmic domains of transmembrane bacterial chemoreceptors are largely extended four‐helix coiled coils. Previous observations suggested the domain was structurally dynamic. We probed directly backbone dynamics of this domain of the transmembrane chemoreceptor Tar from Escherichia coli using site‐directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. Spin labels were positioned on solvent‐exposed helical faces because EPR spectra for such positions reflect primarily polypeptide backbone movements. We acquired spectra for spin‐labeled, intact receptor homodimers solubilized in detergent or inserted into native E. coli lipid bilayers in Nanodiscs, characterizing 16 positions distributed throughout the cytoplasmic domain and on both helices of its helical hairpins, one amino terminal to the membrane‐distal tight turn (N‐helix), and the other carboxyl terminal (C‐helix). Detergent solubilization increased backbone dynamics for much of the domain, suggesting that loss of receptor activities upon solubilization reflects wide‐spread destabilization. For receptors in either condition, we observed an unanticipated difference between the N‐ and C‐helices. For bilayer‐inserted receptors, EPR spectra from sites in the membrane‐distal protein‐interaction region and throughout the C‐helix were typical of well‐structured helices. In contrast, for approximately two‐thirds of the N‐helix, from its origin as the AS‐2 helix of the membrane‐proximal HAMP domain to the beginning of the membrane‐distal protein‐interaction region, spectra had a significantly mobile component, estimated by spectral deconvolution to average approximately 15%. Differential helical dynamics suggests a four‐helix bundle organization with a pair of core scaffold helices and two more dynamic partner helices. This newly observed feature of chemoreceptor structure could be involved in receptor function.     

Complete integrin headpiece opening in eight steps

Carefully soaking crystals with Arg-Gly-Asp (RGD) peptides, we captured eight distinct RGD-bound conformations of the α_IIbβ₃ integrin headpiece. Starting from the closed βI domain conformation, we saw six intermediate βI conformations and finally the fully open βI with the hybrid domain swung out in the crystal lattice. The β1-α1 backbone that hydrogen bonds to the Asp side chain of RGD was the first element to move followed by adjacent to metal ion-dependent adhesion site Ca²⁺, α1 helix, α1’ helix, β6-α7 loop, α7 helix, and hybrid domain. We define in atomic detail how conformational change was transmitted over long distances in integrins, 40 Å from the ligand binding site to the opposite end of the βI domain and 80 Å to the far end of the hybrid domain. During these movements, RGD slid in its binding groove toward α_IIb, and its Arg side chain became ordered. RGD concentration requirements in soaking suggested a >200-fold higher affinity after opening. The thermodynamic cycle shows how higher affinity pays the energetic cost of opening.