RNA kink-turns as molecular elbows: hydration, cation binding, and large-scale dynamics

The presence of Kink-turns (Kt) at key functional sites in the ribosome (e.g., A-site finger and L7/L12 stalk) suggests that some Kink-turns can confer flexibility on RNA protuberances that regulate the traversal of tRNAs during translocation. Explicit solvent molecular dynamics demonstrates that Kink-turns can act as flexible molecular elbows. Kink-turns are associated with a unique network of long-residency static and dynamical hydration sites that is intimately involved in modulating their conformational dynamics. An implicit solvent conformational search confirms the flexibility of Kink-turns around their X-ray geometries and identifies a second low-energy region with open structures that could correspond to Kink-turn geometries seen in solution experiments. An extended simulation of Kt-42 with the factor binding site (helices 43 and 44) shows that the local Kt-42 elbow-like motion fully propagates beyond the Kink-turn, and that there is no other comparably flexible site in this rRNA region. Kink-turns could mediate large-scale adjustments of distant RNA segments.     

Proline-induced distortions of transmembrane helices

Proline residues in the transmembrane (TM) alpha-helices of integral membrane proteins have long been suspected to play a key role for helix packing and signal transduction by inducing regions of helix distortion and/or dynamic flexibility (hinges). In this study we try to characterise the effect of proline on the geometric properties of TM alpha-helices. We have examined 199 transmembrane alpha-helices from polytopic membrane proteins of known structure. After examining the location of proline residues within the amino acid sequences of TM helices, we estimated the helix axes either side of a hinge and hence identified a hinge residue. This enabled us to calculate helix kink and swivel angles. The results of this analysis show that proline residues occur with a significant concentration in the centre of sequences of TM alpha-helices. In this location, they may induce formation of molecular hinges, located on average about four residues N-terminal to the proline residue. A superposition of proline-containing TM helices structures shows that the distortion induced is anisotropic and favours certain relative orientations (defined by helix kink and swivel angles) of the two helix segments.     

Prokink: a protocol for numerical evaluation of helix distortions by proline

Proline residues are known to perturb the structure of helices by introducing a kink between the segments preceding and following the proline residue. The distortion of the helical structure results from the avoided steric clash between the ring of the proline at position (i) and the backbone carbonyl at position (i - 4), as well as the elimination of helix backbone H-bonds for the carbonyls at positions (i - 3) and (i - 4). Both the departure from the ideal helical pattern and the reduction in H-bond stabilization contribute to the observed flexibility of a proline-containing alpha-helix. The special local flexibility of the proline kink can confer an important role on the proline-containing helix in the conformational changes related to the function of the protein. As a useful tool in determining and evaluating the role of proline-induced flexibility and distortions in protein function, we present here a protocol to quantify the geometry of the distortion introduced in helices by prolines both as a time-averaged value and for individual 'snapshots' along a molecular dynamics simulation.     

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.     

Ordered opening of LDL receptor binding domain of human apolipoprotein E3 revealed by hydrogen/deuterium exchange mass spectrometry and fluorescence spectroscopy

Apolipoprotein E3 (apoE3) is an exchangeable apolipoprotein that plays a critical role in cholesterol homeostasis. The N-terminal (NT) domain of apoE3 (residues 1–191) is folded into a helix bundle comprised of 4 amphipathic α-helices: H1, H2, H3 and H4, flanked by flexible helices N1 and N2, and Hinge Helix 1 (Hinge H1), at the N-and C-terminal sides of the helix bundle, respectively. The NT domain plays a critical role in binding to the low density lipoprotein receptor (LDLR), which eventually leads to lowering of plasma cholesterol levels. In order to be recognized by the LDLR, the helix bundle has to open and undergo a conformational change. The objective of the study was to understand the mechanism of opening of the helix bundle. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) revealed that apoE3 NT domain adopts several disordered and unfolded regions, with H2 exhibiting relatively little protection against exchange-in compared to H1, H3, and H4. Site-directed fluorescence labeling indicated that H2 not only has the highest degree of solvent exposure but also the most flexibility in the helix bundle. It also indicated that the lipoprotein behavior of H1 was significnatly different from that of H2, H3 and H4. These results suggest that the opening of the helix bundle is likely initiated at the flexible end of H2 and the loop linking H2/H3, and involves movement of H2/H3 away from H1/H4. Together, these observations offer mechanistic insight suggesting a regulated helix bundle opening of apoE3 NT domain can be triggered by lipid binding.     

Influence of Nonconserved Regions of L1 Protuberance of <Emphasis Type="Italic">Thermus thermophilus</Emphasis> Ribosome on the Affinity of L1 Protein to 23s rRNA

The L1 protuberance of the ribosome includes two domain ribosomal protein L1 and three helices of 23S rRNA (H76, H77, and H78) with interconnecting loops A and B. Helix 78 consists of two parts, i.e., H78a and H78b. A comparison of the available structural data of L1-RNA complexes with the obtained kinetic data made it possible to determine the influence of the nonconserved regions of Thermus thermophilus L1-protuberance on the mutual affinity of the L1 protein and 23S rRNA. It has been shown that the N-terminal helix of the protein and 78b helix of 23S rRNA are essential for the formation of an additional intermolecular contact, which is separated in the protein from the main site of L1-rRNA interaction by a flexible connection. This results in a rise in the TthL1-rRNA affinity. At the same time, the elongation of the 76 helix has no effect on rRNA-protein binding.     

Folding of the cocaine aptamer studied by EPR and fluorescence spectroscopies using the bifunctional spectroscopic probe Ç

The cocaine aptamer is a DNA molecule that binds cocaine at the junction of three helices. The bifunctional spectroscopic probe Ç was incorporated independently into three different positions of the aptamer and changes in structure and dynamics upon addition of the cocaine ligand were studied. Nucleoside Ç contains a rigid nitroxide spin label and can be studied directly by electron paramagnetic resonance (EPR) spectroscopy and fluorescence spectroscopy after reduction of the nitroxide to yield the fluoroside Ç^f. Both the EPR and the fluorescence data for aptamer 2 indicate that helix III is formed before cocaine binding. Upon addition of cocaine, increased fluorescence of a fully base-paired Ç^f, placed at the three-way junction in helix III, was observed and is consistent with a helical tilt from a coaxial stack of helices II and III. EPR and fluorescence data clearly show that helix I is formed upon addition of cocaine, concomitant with the formation of the Y-shaped three-way helical junction. The EPR data indicate that nucleotides in helix I are more mobile than nucleotides in regular duplex regions and may reflect increased dynamics due to the short length of helix I.     

Hinge-like motions in RNA kink-turns: the role of the second a-minor motif and nominally unpaired bases

Kink-turn (K-turn) motifs are asymmetric internal loops found at conserved positions in diverse RNAs, with sharp bends in phosphodiester backbones producing V-shaped structures. Explicit-solvent molecular dynamics simulations were carried out for three K-turns from 23S rRNA, i.e., Kt-38 located at the base of the A-site finger, Kt-42 located at the base of the L7/L12 stalk, and Kt-58 located in domain III, and for the K-turn of human U4 snRNA. The simulations reveal hinge-like K-turn motions on the nanosecond timescale. The first conserved A-minor interaction between the K-turn stems is entirely stable in all simulations. The angle between the helical arms of Kt-38 and Kt-42 is regulated by local variations of the second A-minor (type I) interaction between the stems. Its variability ranges from closed geometries to open ones stabilized by insertion of long-residency waters between adenine and cytosine. The simulated A-minor geometries fully agree with x-ray data. Kt-58 and Kt-U4 exhibit similar elbow-like motions caused by conformational change of the adenosine from the nominally unpaired region. Despite the observed substantial dynamics of K-turns, key tertiary interactions are stable and no sign of unfolding is seen. We suggest that some K-turns are flexible elements mediating large-scale ribosomal motions during the protein synthesis cycle.     

Major centers of motion in the large ribosomal RNAs

Major centers of motion in the rRNAs of Thermus thermophilus are identified by alignment of crystal structures of EF-G bound and EF-G unbound ribosomal subunits. Small rigid helices upstream of these ‘pivots’ are aligned, thereby decoupling their motion from global rearrangements. Of the 21 pivots found, six are observed in the large subunit rRNA and 15 in the small subunit rRNA. Although the magnitudes of motion differ, with only minor exceptions equivalent pivots are seen in comparisons of Escherichia coli structures and one Saccharomyces cerevisiae structure pair. The pivoting positions are typically associated with structurally weak motifs such as non-canonical, primarily U-G pairs, bulge loops and three-way junctions. Each pivot is typically in direct physical contact with at least one other in the set and often several others. Moving helixes include rRNA segments in contact with the tRNA, intersubunit bridges and helices 28, 32 and 34 of the small subunit. These helices are envisioned to form a network. EF-G rearrangement would then provide directional control of this network propagating motion from the tRNA to the intersubunit bridges to the head swivel or along the same path backward.     

Solution Structure and Phospholipid Interactions of the Isolated Voltage-Sensor Domain from KvAP

Voltage-sensor domains (VSDs) are specialized transmembrane segments that confer voltage sensitivity to many proteins such as ion channels and enzymes. The activities of these domains are highly dependent on both the chemical properties and the physical properties of the surrounding membrane environment. To learn about VSD-lipid interactions, we used nuclear magnetic resonance spectroscopy to determine the structure and phospholipid interface of the VSD from the voltage-dependent K⁺ channel KvAP (prokaryotic Kv from Aeropyrum pernix). The solution structure of the KvAP VSD solubilized within phospholipid micelles is similar to a previously determined crystal structure solubilized by a nonionic detergent and complexed with an antibody fragment. The differences observed include a previously unidentified short amphipathic α-helix that precedes the first transmembrane helix and a subtle rigid-body repositioning of the S3-S4 voltage-sensor paddle. Using ¹⁵N relaxation experiments, we show that much of the VSD, including the pronounced kink in S3 and the S3-S4 paddle, is relatively rigid on the picosecond-to-nanosecond timescale. In contrast, the kink in S3 is mobile on the microsecond-to-millisecond timescale and may act as a hinge in the movement of the paddle during channel gating. We characterized the VSD-phospholipid micelle interactions using nuclear Overhauser effect spectroscopy and showed that the micelle uniformly coats the KvAP VSD and approximates the chemical environment of a phospholipid bilayer. Using paramagnetically labeled phospholipids, we show that bilayer-forming lipids interact with the S3 and S4 helices more strongly than with S1 and S2.     

Structure of a rare non-standard sequence k-turn bound by L7Ae protein

Kt-23 from Thelohania solenopsae is a rare RNA kink turn (k-turn) where an adenine replaces the normal guanine at the 2n position. L7Ae is a member of a strongly conserved family of proteins that bind a range of k-turn structures in the ribosome, box C/D and H/ACA small nucleolar RNAs and U4 small nuclear RNA. We have solved the crystal structure of T. solenopsae Kt-23 RNA bound to Archeoglobus fulgidus L7Ae protein at a resolution of 2.95 Å. The protein binds in the major groove displayed on the outer face of the k-turn, in a manner similar to complexes with standard k-turn structures. The k-turn adopts a standard N3 class conformation, with a single hydrogen bond from A2b N6 to A2n N3. This contrasts with the structure of the same sequence located in the SAM-I riboswitch, where it adopts an N1 structure, showing the inherent plasticity of k-turn structure. This potentially can affect any tertiary interactions in which the RNA participates.     

Slow formation of stable complexes during coincubation of minimal rRNA and ribosomal protein S4

Ribosomal protein S4 binds and stabilizes a five-helix junction or five-way junction (5WJ) in the 5′ domain of 16S ribosomal RNA (rRNA) and is one of two proteins responsible for nucleating 30S ribosome assembly. Upon binding, both protein S4 and 5WJ reorganize their structures. We show that labile S4 complexes rearrange into stable complexes within a few minutes at 42 °C, with longer coincubation leading to an increased population of stable complexes. In contrast, prefolding the rRNA has a smaller effect on stable S4 binding. Experiments with minimal rRNA fragments show that this structural change depends only on 16S residues within the S4 binding site. SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical probing experiments showed that S4 strongly stabilizes 5WJ and the helix (H) 18 pseudoknot, which become tightly folded within the first minute of S4 binding. However, a kink in H16 that makes specific contacts with the S4 N-terminal extension, as well as a right-angle motif between H3, H4, and H18, requires a minute or more to become fully structured. Surprisingly, S4 structurally reorganizes the 530-loop and increases the flexibility of H3, which is proposed to undergo a conformational switch during 30S assembly. These elements of the S4 binding site may require other 30S proteins to reach a stable conformation.     

Dynamics of the base of ribosomal A-site finger revealed by molecular dynamics simulations and Cryo-EM

Helix 38 (H38) of the large ribosomal subunit, with a length of 110 Å, reaches the small subunit through intersubunit bridge B1a. Previous cryo-EM studies revealed that the tip of H38 moves by more than 10 Å from the non-ratcheted to the ratcheted state of the ribosome while mutational studies implicated a key role of flexible H38 in attenuation of translocation and in dynamical signaling between ribosomal functional centers. We investigate a region including the elbow-shaped kink-turn (Kt-38) in the Haloarcula marismortui archaeal ribosome, and equivalently positioned elbows in three eubacterial species, located at the H38 base. We performed explicit solvent molecular dynamics simulations on the H38 elbows in all four species. They are formed by at first sight unrelated sequences resulting in diverse base interactions but built with the same overall topology, as shown by X-ray crystallography. The elbows display similar fluctuations and intrinsic flexibilities in simulations indicating that the eubacterial H38 elbows are structural and dynamical analogs of archaeal Kt-38. We suggest that this structural element plays a pivotal role in the large motions of H38 and may act as fulcrum for the abovementioned tip motion. The directional flexibility inferred from simulations correlates well with the cryo-EM results.     

Conformational Transition Pathway in the Inhibitor Binding Process of Human Monoacylglycerol Lipase

Human monoacylglycerol lipase (MGL) catalyzes the hydrolysis of 2-arachidonoylglycerol to arachidonic and glycerol, which plays a pivotal role in the normal biological processes of brain. Co-crystal structure of the MGL in complex with its inhibitor, compound 1, shows that the helix α4 undergoes large-scale conformational changes in response to the compound 1 binding compared to the apo MGL. However, the detailed conformational transition pathway of the helix α4 in the inhibitor binding process of MGL has remained unclear. Here, conventional molecular dynamics (MD) and nudged elastic band (NEB) simulations were performed to explore the conformational transition pathway of the helix α4. Conventional MD simulations unveiled that the compound 1 induced the closed conformation of the active site of MGL, reduced the conformational flexibility of the helix α4, and elicited the large-scale conformational rearrangement of the helix α4, leading to the complete folding of the helix α4. Moreover, NEB simulations revealed that the conformational transition pathway of helix α4 underwent an almost 180° counter-clockwise rotation of the helix α4. Our computational results advance the structural and mechanistic understanding of the inhibitory mechanism.     

Properties of aqueous solutions of lentinan in the absence and presence of zwitterionic surfactants

Morphological and conformational transitions of lentinan (LT), a β-glucan extracted from Shiitake mushrooms (Lentinula edodes), were investigated at different concentrations of aqueous NaOH, using Small Angle X-ray Scattering (SAXS) technique. At low NaOH_(aq) concentration LT chains are self-associated and adopt the triple helix form where as at higher NaOH concentrations the polymer chains undergo a transition to random coil chains. Also, the presence of fractal dimensions was observed through analysis of the exponential decay of the scattering intensity as a function of the scattering angle. In addition, the lateral radius of gyration was determined for LT in different concentrations of NaOH solution, indicating a rigid triple helix present as a small rod-like structure. Interactions of LT with two zwitterionic surfactants were investigated by surface tension, fluorescence, and static light scattering measurements. Experimental data showed that the formation of LT–(surfactant) complexes occurred through a cooperative process.     

Sequence-specific 1H and 15N resonance assignments and secondary structure of GDP-bound human c-Ha-Ras protein in solution

Summary All the backbone ¹H and ¹⁵N magnetic resonances (except for Pro residues) of the GDP-bound form of a truncated human c-Ha-ras proto-oncogene product (171 amino acid residues, the Ras protein) were assigned by ¹⁵N-edited two-dimensional NMR experiments on selectively ¹⁵N-labeled Ras proteins in combination with three-dimensional NMR experiments on the uniformly ¹⁵N-labeled protein. The sequence-specific assignments were made on the basis of the nuclear Overhauser effect (NOE) connectivities of amide protons with preceding amide and/or Cprotons. In addition to sequential NOEs, vicinal spin coupling constants for amide protons and C protons and deuterium exchange rates of amide protons were used to characterize the secondary structure of the GDP-bound Ras protein; six strands and five helices were identified and the topology of these elements was determined. The secondary structure of the Ras protein in solution was mainly consistent with that in crystal as determined by X-ray analyses. The deuterium exchange rates of amide protons were examined to elucidate the dynamic properties of the secondary structure elements of the Ras protein in solution. In solution, the -sheet structure in the Ras protein is rigid, while the second helix (A66-R73) is much more flexible, and the first and fifth helices (S17-124 and V152-L171) are more rigid than other helices. Secondary structure elements at or near the ends of the effector-region loop were found to be much more flexible in solution than in the crystalline state.     

Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin plantaricin EF

The three-dimensional structures of the two peptides plantaricin E (plnE; 33 residues) and plantaricin F (plnF; 34 residues) constituting the two-peptide bacteriocin plantaricin EF (plnEF) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles. PlnE has an N-terminal alpha-helix (residues 10-21), and a C-terminal alpha-helix-like structure (residues 25-31). PlnF has a long central alpha-helix (residues 7-32) with a kink of 38+/-7 degrees at Pro20. There is some flexibility in the helix in the kink region. Both helices in plnE are amphiphilic, while the helix in plnF is polar in its N-terminal half and amphiphilic in its C-terminal half. The alpha-helical content obtained by NMR spectroscopy is in agreement with CD studies. PlnE has two GxxxG motifs which are putative helix-helix interaction motifs, one at residues 5 to 9 and one at residues 20 to 24, while plnF has one such motif at residues 30 to 34. The peptides are flexible in these GxxxG regions. It is suggested that the two peptides lie parallel in a staggered fashion relative to each other and interact through helix-helix interactions involving the GxxxG motifs.     

Kinked structures of isolated nicotinic receptor M2 helices: A molecular dynamics study

The pore-lining M2 helix of the nicotinic acetylcholine receptor exhibits a pronounced kink when the corresponding ion channel is in a closed conformation [N. Unwin (1993) Journal of Molecular Biology, Vol. 229, pp. 1101–1124]. We have performed molecular dynamics simulations of isolated 22-residue M2 helices in order to identify a possible molecular origin of this kink. In order to sample a wide range of conformational space, a simulated annealing protocol was used to generate five initial M2 helix structures, each of which was subsequently used as the basis of 300 ps MD simulations. Two helix sequences (M2α and M2δ) were studied in this manner, resulting in a total often 300 ps trajectories. Kinked helices present in the trajectories were identified and energy minimized to yield a total of five different stable kinked structures. For comparison, a similar molecular dynamics simulation of a Leu₂₃ helix yielded no stable kinked structures. In four of the five kinked helices, the kink was stabilized by H bonds between the helix backbone and polar side-chain atoms. Comparison with data from the literature on site-directed mutagenesis of M2 residues suggests that such polar side-chain to main-chain H bonds may also contribute to kinking of M2 helices in the intact channel protein. © 1994 John Wiley & Sons, Inc.     

Ion-induced folding of a kink turn that departs from the conventional sequence

Kink turns (k-turns) are important structural motifs that create a sharp axial bend in RNA. Most conform to a consensus in which a three-nucleotide bulge is followed by consecutive G•A and A•G base pairs, and when these G•A pairs are modified in vitro this generally leads to a failure to adopt the k-turn conformation. Kt-23 in the 30S ribosomal subunit of Thermus thermophilus is a rare exception in which the bulge-distal A•G pair is replaced by a non-Watson–Crick A•U pair. In the context of the ribosome, Kt-23 adopts a completely conventional k-turn geometry. We show here that this sequence is induced to fold into a k-turn structure in an isolated RNA duplex by Mg²⁺ or Na⁺ ions. Therefore, the Kt-23 is intrinsically stable despite lacking the key A•G pair; its formation requires neither tertiary interactions nor protein binding. Moreover, the Kt-23 k-turn is stabilized by the same critical hydrogen-bonding interactions within the core of the structure that are found in more conventional sequences such as the near-consensus Kt-7. T. thermophilus Kt-23 has two further non-Watson–Crick base pairs within the non-canonical helix, three and four nucleotides from the bulge, and we find that the nature of these pairs influences the ability of the RNA to adopt k-turn conformation, although the base pair adjacent to the A•U pair is more important than the other.