Ribosomal RNA Kink-turn Motif—A Flexible Molecular Hinge

Abstract

Ribosomal RNA K-turn motifs are asymmetric internal loops characterized by a sharp bend in the phosphodiester backbone resulting in “V” shaped structures, recurrently observed in ribosomes and showing a high degree of sequence conservation. We have carried out extended explicit solvent molecular dynamics simulations of selected K-turns, in order to investigate their intrinsic structural and dynamical properties. The simulations reveal an unprecedented dynamical flexibility of the K-turns around their X-ray geometries. The K-turns sample, on the nanosecond timescale, different conformational substates. The overall behavior of the simulations suggests that the sampled geometries are essentially isoenergetic and separated by minimal energy barriers. The nanosecond dynamics of isolated K-turns can be qualitatively considered as motion of two rigid helix stems controlled by a very flexible internal loop which then leads to substantial hinge-like motions between the two stems. This internal dynamics of K-turns is strikingly different for example from the bacterial 5S rRNA Loop E motif or BWYV frameshifting pseudoknot which appear to be rigid in the same type of simulations. Bistability and flexibility of K-turns was also suggested by several recent biochemical studies. Although the results of MD simulations should be considered as a qualitative picture of the K-turn dynamics due to force field and sampling limitations, the main advantage of the MD technique is its ability to investigate the region close to K-turn riboso- mal-like geometries. This part of the conformational space is not well characterized by the solution experiments due to large-scale conformational changes seen in the experiments. We suggest that K-turns are well suited to act as flexible structural elements of ribosomal RNA. They can for example be involved in mediation of large-scale motions or they can allow a smooth assembling of the other parts of the ribosome.     

Ribosomal RNA kink-turn motif--a flexible molecular hinge

Ribosomal RNA K-turn motifs are asymmetric internal loops characterized by a sharp bend in the phosphodiester backbone resulting in "V" shaped structures, recurrently observed in ribosomes and showing a high degree of sequence conservation. We have carried out extended explicit solvent molecular dynamics simulations of selected K-turns, in order to investigate their intrinsic structural and dynamical properties. The simulations reveal an unprecedented dynamical flexibility of the K-turns around their X-ray geometries. The K-turns sample, on the nanosecond timescale, different conformational substates. The overall behavior of the simulations suggests that the sampled geometries are essentially isoenergetic and separated by minimal energy barriers. The nanosecond dynamics of isolated K-turns can be qualitatively considered as motion of two rigid helix stems controlled by a very flexible internal loop which then leads to substantial hinge-like motions between the two stems. This internal dynamics of K-turns is strikingly different for example from the bacterial 5S rRNA Loop E motif or BWYV frameshifting pseudoknot which appear to be rigid in the same type of simulations. Bistability and flexibility of K-turns was also suggested by several recent biochemical studies. Although the results of MD simulations should be considered as a qualitative picture of the K-turn dynamics due to force field and sampling limitations, the main advantage of the MD technique is its ability to investigate the region close to K-turn ribosomal-like geometries. This part of the conformational space is not well characterized by the solution experiments due to large-scale conformational changes seen in the experiments. We suggest that K-turns are well suited to act as flexible structural elements of ribosomal RNA. They can for example be involved in mediation of large-scale motions or they can allow a smooth assembling of the other parts of the ribosome.     

Role of flexibility and polarity as determinants of the hydration of internal cavities and pockets in proteins

Molecular dynamics simulations of Staphylococcal nuclease and of 10 variants with internal polar or ionizable groups were performed to investigate systematically the molecular determinants of hydration of internal cavities and pockets in proteins. In contrast to apolar cavities in rigid carbon structures, such as nanotubes or buckeyballs, internal cavities in proteins that are large enough to house a few water molecules will most likely be dehydrated unless they contain a source of polarity. The water content in the protein interior can be modulated by the flexibility of protein elements that interact with water, which can impart positional disorder to water molecules, or bias the pattern of internal hydration that is stabilized. This might explain differences in the patterns of hydration observed in crystal structures obtained at cryogenic and room temperature conditions. The ability of molecular dynamics simulations to determine the most likely sites of water binding in internal pockets and cavities depends on its efficiency in sampling the hydration of internal sites and alternative protein and water conformations. This can be enhanced significantly by performing multiple molecular dynamics simulations as well as simulations started from different initial hydration states.     

Probing the flexibility of the bacterial reaction center: the wild-type protein is more rigid than two site-specific mutants

Experimental and theoretical studies have stressed the importance of flexibility for protein function. However, more local studies of protein dynamics, using temperature factors from crystallographic data or elastic models of protein mechanics, suggest that active sites are among the most rigid parts of proteins. We have used quasielastic neutron scattering to study the native reaction center protein from the purple bacterium Rhodobacter sphaeroides, over a temperature range of 4-260 K, in parallel with two nonfunctional mutants both carrying the mutations L212Glu/L213Asp --> Ala/Ala (one mutant carrying, in addition, the M249Ala --> Tyr mutation). The so-called dynamical transition temperature, Td, remains the same for the three proteins around 230 K. Below Td the mean square displacement, u2, and the dynamical structure factor, S(Q,omega), as measured respectively by backscattering and time-of-flight techniques are identical. However, we report that above Td, where anharmonicity and diffusive motions take place, the native protein is more rigid than the two nonfunctional mutants. The higher flexibility of both mutant proteins is demonstrated by either their higher u2 values or the notable quasielastic broadening of S(Q,omega) that reveals the diffusive nature of the motions involved. Remarkably, we demonstrate here that in proteins, point genetic mutations may notably affect the overall protein dynamics, and this effect can be quantified by neutron scattering. Our results suggest a new direction of investigation for further understanding of the relationship between fast dynamics and activity in proteins. Brownian dynamics simulations we have carried out are consistent with the neutron experiments, suggesting that a rigid core within the native protein is specifically softened by distant point mutations. L212Glu, which is systematically conserved in all photosynthetic bacteria, seems to be one of the key residues that exerts a distant control over the rigidity of the core of the protein.     

A Direct Coupling between Global and Internal Motions in a Single Domain Protein? MD Investigation of Extreme Scenarios

Proteins are not rigid molecules, but exhibit internal motions on timescales ranging from femto- to milliseconds and beyond. In solution, proteins also experience global translational and rotational motions, sometimes on timescales comparable to those of the internal fluctuations. The possibility that internal and global motions may be directly coupled has intriguing implications, given that enzymes and cell signaling proteins typically associate with binding partners and cellular scaffolds. Such processes alter their global motion and may affect protein function. Here, we present molecular dynamics simulations of extreme case scenarios to examine whether a possible relationship exists. In our model protein, a ubiquitin-like RhoGTPase binding domain of plexin-B1, we removed either internal or global motions. Comparisons with unrestrained simulations show that internal and global motions are not appreciably coupled in this single-domain protein. This lack of coupling is consistent with the observation that the dynamics of water around the protein, which is thought to permit, if not stimulate, internal dynamics, is also largely independent of global motion. We discuss implications of these results for the structure and function of proteins.     

Translational hydration water dynamics drives the protein glass transition

Experimental and computer simulation studies have revealed the presence of a glass-like transition in the internal dynamics of hydrated proteins at approximately 200 K involving an increase of the amplitude of anharmonic dynamics. This increase in flexibility has been correlated with the onset of protein activity. Here, we determine the driving force behind the protein transition by performing molecular dynamics simulations of myoglobin surrounded by a shell of water. A dual heat bath method is used with which, in any given simulation, the protein and solvent are held at different temperatures, and sets of simulations are performed varying the temperature of the components. The results show that the protein transition is driven by a dynamical transition in the hydration water that induces increased fluctuations primarily in side chains in the external regions of the protein. The water transition involves activation of translational diffusion and occurs even in simulations where the protein atoms are held fixed.     

Solvent effects in the slow dynamics of proteins

The influence of solvent on the slow internal dynamics of proteins is studied by comparing molecular dynamics simulations of solvated and unsolvated lysozyme. The dynamical trajectories are projected onto the protein's normal modes in order to obtain a separate analysis for each of the associated time scales. The results show that solvent effects are important for the slowest motions (below approximately 1 ps(-1)) but negligible for faster motions. The damping effects seen in the latter show that the principal source of friction in protein dynamics is not the solvent, but the protein itself.     

Biophysical study of thermal denaturation of apo-calmodulin: dynamics of native and unfolded states

Apo-calmodulin, a small, mainly α, soluble protein is a calcium-dependent protein activator. This article presents a study of internal dynamics of native and thermal unfolded apo-calmodulin, using quasi-elastic neutron scattering. This technique can probe protein internal dynamics in the picosecond timescale and in the nanometer length-scale. It appears that a dynamical transition is associated with thermal denaturation of apo-calmodulin. This dynamical transition goes together with a decrease of the confinement of hydrogen atoms, a decrease of immobile protons proportion and an increase of dynamical heterogeneity. The comparison of native and unfolded states dynamics suggests that the dynamics of protein atoms is more influenced by their distance to the backbone than by their solvent exposure.     

Virtual rigid body dynamics

An important direction in biological simulations is the development of methods that permit the study of larger systems and/or longer simulation time scales than is currently feasible by molecular dynamics. One such method designed with this objective in mind is stochastic boundary molecular dynamics (SBMD). SBMD was developed for the characterization of spatially localized processes in proteins, and has been shown to successfully reproduce structural and dynamical properties of these macromolecules, as compared to a molecular dynamics control simulation, when concerted or global motions are not present. The virtual rigid body dynamics method presented in this work extends the range of applicability of the SBMD method, by providing a framework to include these important long time scale conformational transitions. In this paper we describe the two-step implementation of the virtual rigid body model: first, the reduction of the full atomic representation to a reduced particle (virtual bond) model, and second, the propagation of the dynamics of flexibly connected rigid bodies containing virtual atom sites.     

Molecular dynamics study of water penetration in staphylococcal nuclease

The ionization properties of Lys and Glu residues buried in the hydrophobic core of staphylococcal nuclease (SN) suggest that the interior of this protein behaves as a highly polarizable medium with an apparent dielectric constant near 10. This has been rationalized previously in terms of localized conformational relaxation concomitant with the ionization of the internal residue, and with contributions by internal water molecules. Paradoxically, the crystal structure of the SN V66E variant shows internal water molecules and the structure of the V66K variant does not. To assess the structural and dynamical character of interior water molecules in SN, a series of 10-ns-long molecular dynamics (MD) simulations was performed with wild-type SN, and with the V66E and V66K variants with Glu66 and Lys66 in the neutral form. Internal water molecules were identified based on their coordination state and characterized in terms of their residence times, average location, dipole moment fluctuations, hydrogen bonding interactions, and interaction energies. The locations of the water molecules that have residence times of several nanoseconds and display small mean-square displacements agree well with the locations of crystallographically observed water molecules. Additional, relatively disordered water molecules that are not observed crystallographically were found in internal hydrophobic locations. All of the interior water molecules that were analyzed in detail displayed a distribution of interaction energies with higher mean value and narrower width than a bulk water molecule. This underscores the importance of protein dynamics for hydration of the protein interior. Further analysis of the MD trajectories revealed that the fluctuations in the protein structure (especially the loop elements) can strongly influence protein hydration by changing the patterns or strengths of hydrogen bonding interactions between water molecules and the protein. To investigate the dynamical response of the protein to burial of charged groups in the protein interior, MD simulations were performed with Glu66 and Lys66 in the charged state. Overall, the MD simulations suggest that a conformational change rather than internal water molecules is the dominant determinant of the high apparent polarizability of the protein interior.     

Internal Dynamics of Dynactin CAP-Gly Is Regulated by Microtubules and Plus End Tracking Protein EB1

CAP-Gly domain of dynactin, a microtubule-associated activator of dynein motor, participates in multiple cellular processes, and its point mutations are associated with neurodegenerative diseases. Recently, we have demonstrated that conformational plasticity is an intrinsic property of CAP-Gly. To understand its origin, we addressed internal dynamics of CAP-Gly assembled on polymeric microtubules, bound to end-binding protein EB1 and free, by magic angle spinning NMR and molecular dynamics simulations. The analysis of residue-specific dynamics of CAP-Gly on time scales spanning nano- through milliseconds reveals its unusually high mobility, both free and assembled on polymeric microtubules. On the contrary, CAP-Gly bound to EB1 is significantly more rigid. Molecular dynamics simulations indicate that these motions are strongly temperature-dependent, and loop regions are surprisingly mobile. These findings establish the connection between conformational plasticity and internal dynamics in CAP-Gly, which is essential for the biological functions of CAP-Gly and its ability to bind to polymeric microtubules and multiple binding partners. In this work, we establish an approach, for the first time, to probe atomic resolution dynamic profiles of a microtubule-associated protein assembled on polymeric microtubules. More broadly, the methodology established here can be applied for atomic resolution analysis of dynamics in other microtubule-associated protein assemblies, including but not limited to dynactin, dynein, and kinesin motors assembled on microtubules.     

Molecular dynamics simulation of deoxy and carboxy murine neuroglobin in water

Globins are respiratory proteins that reversibly bind dioxygen and other small ligands at the iron of a heme prosthetic group. Hemoglobin and myoglobin are the most prominent members of this protein family. Unexpectedly a few years ago a new member was discovered and called neuroglobin (Ngb), being predominantly expressed in the brain. Ngb is a single polypeptide of 151 amino acids and despite the small sequence similarity with other globins, it displays the typical globin fold. Oxygen, nitric oxide, or carbon monoxide can displace the distal histidine which, in ferrous Ngb as well as in ferric Ngb, is bound to the iron, yielding a reversible adduct. Recent crystallographic data on carboxy Ngb show that binding of an exogenous ligand is associated to structural changes involving heme sliding and a topological reorganization of the internal cavities; in particular, the huge internal tunnel that connects the bulk with the active site, peculiar to Ngb, is heavily reorganized. We report the results of extended (90 ns) molecular dynamics simulations in water of ferrous deoxy and carboxy murine neuroglobin, which are both coordinated on the distal site, in the latter case by CO and in the former one by the distal His(64)(E7). The long timescale of the simulations allowed us to characterize the equilibrated protein dynamics and to compare protein structure and dynamical behavior coupled to the binding of an exogenous ligand. We have characterized the heme sliding motion, the topological reorganization of the internal cavities, the dynamics of the distal histidine, and particularly the conformational change of the CD loop, whose flexibility depends ligand binding.     

600 ps Molecular dynamics reveals stable substructures and flexible hinge points in cAMP dependent protein kinase

Molecular dynamics simulations of the catalytic subunit of cAMP dependent protein kinase (cAPK) have been performed in an aqueous environment. The relations among the protein hydrogen‐bonding network, secondary structural elements, and the internal motions of rigid domains were examined. The values of fluctuations of protein dihedral angles during dynamics show quite distinct maxima in the regions of loops and minima in the regions of α‐helices and β‐strands. Analyses of conformation snapshots throughout the run show stable subdomains and indicate that these rigid domains are constrained during the dynamics by a stable network of hydrogen bonds. The most stable subdomain during the dynamics was in the small lobe including part of the carboxy‐terminal tail. The most significant flexible region was the highly conserved glycine‐rich loop between β strands 1 and 2 in the small lobe. Many of the main chain dihedral angle changes measured in a comparison of the crystallographic structures of “open” and “closed” conformations of cAPK correspond to the highly flexible residues found during dynamics. © 1999 John Wiley & Sons, Inc. Biopoly 50: 513–524, 1999     

The role of protein–solvent hydrogen bond dynamics in the structural relaxation of a protein in glycerol versus water

We used MD simulations to investigate the dependence of the dynamics of a soluble protein, RNase A, on temperature and solvent environment. Consistent with neutron scattering data, the simulations predict that the protein undergoes a dynamical transition in both glycerol and aqueous solutions that is absent in the dry protein. The temperature of the transition is higher, while the rate of increase with temperature of the amplitudes of motion on the 100 ps timescale is lower, in glycerol versus water. Analysis of the dynamics of hydrogen bonds revealed that the protein dynamical transition is connected to the relaxation of the protein-solvent hydrogen bond network, which, in turn, is associated with solvent translational diffusion. Thus, it appears that the role of solvent dynamics in affecting the protein dynamical transition is qualitatively similar in water and glycerol.     

MD — Simulation of Diffusion of Methane in Zeolites of Type LTA

Using molecular dynamical computer simulations (MD) the dynamics of kinetic processes in zeolites will be discussed on a molecular level. Small changes in lattice parameters can cause dramatical changes in the diffusion coefficient. The presence of cations Na⁺, Ca²⁺ also strongly influences the diffusion. Changes of the self-diffusivities will be discussed that appear if a vibrating lattice instead of a rigid one is used. Nonequilibrium simulations show the correlation between transport-diffusion and self-diffusion in zeolites.     

Molecular dynamics simulation of the neuroglobin crystal: comparison with the simulation in solution

Neuroglobin (Ngb) is a monomeric protein that, despite the small sequence similarity with other globins, displays the typical globin fold. In the absence of exogenous ligands, the ferric and the ferrous forms of Ngb are both hexacoordinated to the distal and proximal histidines. In the ferrous form, oxygen, nitric oxide or carbon monoxide can displace the distal histidine, yielding a reversible adduct. Crystallographic data show that the binding of an exogenous ligand is associated to structural changes involving heme sliding and a topological reorganization of the internal cavities. Molecular dynamics (MD) simulations in solution show that the heme oscillates between two positions, much as the ones observed in the crystal structure, although the occupancy is different. The simulations also suggest that ligand binding in solution can affect the flexibility and conformation of residues connecting the C and D helices, referred to as the CD corner, which is coupled to the configuration adopted by the distal histidine. In this study, we report the results of 30 ns MD simulations of CO-bound Ngb in the crystal. Our goal was to compare the protein dynamical behavior in the crystal with the results supplied by the previous MD simulation of CO-bound Ngb in solution and the x-ray experimental data. The results show that the different environments (crystal or solution) affect the dynamics of the heme group and of the CD corner.     

Dynamic and Structural Changes in the Minimally Restructuring EcoRI Bound to a Minimally Mutated DNA Chain

Abstract

The dynamics of a protein plays an important role in protein functionality. Here, we examine the differences in the dynamics of a minimally restructuring protein, EcoRI, when it is bound to its cognate DNA and to a noncognate sequence which differs by just a single basepair. Molecular dynamics simulations of the complexes and essential dynamics analyses reveal that the overall dynamics of the protein subunits change from a coordinated motion in the cognate complex to a scrambled motion in the noncognate complex. This dynamical difference extends to the protein-DNA interface where EcoRI tries to constrict the DNA in the cognate complex. In the noncognate complex, absence of the constricting motion of interfacial residues, overall change in backbone dynamics and structural relaxation of the arms enfolding the DNA leave the DNA less-kinked relative to the situation in the cognate complex, thus indicating that the protein is poised for linear diffusion along the DNA rather than for catalytic action. In a larger context, the results imply that the DNA sequences dictate protein dynamics and that when a protein chances upon the recognition sequence some of the key domains of the protein undergo dynamical changes that prepare the protein for eventual catalytic action.     

Interplay between structural rigidity and electrostatic interactions in the ligand binding domain of GluR2

Using molecular dynamics (MD) simulations, computational protein modifications, and a novel theoretical methodology that determines structural rigidity/flexibility (the FIRST algorithm), we investigate how molecular structure and dynamics of the glutamate receptor ligand binding domain (GluR2 S1S2) facilitate its conformational transition. S1S2 is a two-lobe protein, which undergoes a cleft closure conformational transition upon binding an agonist in the cleft between the two lobes; hence it is expected that the mechanism of this conformational transition can be characterized as a hinge-type. However, in the rigidity analysis one lobe of the protein is identified as a single rigid cluster while the other one is structurally flexible, inconsistent with a presumed mechanical hinge mechanism. Instead, we characterize the cleft-closing transition as a load and lock mechanism. We find that when two cross-cleft hydrogen bonds are disrupted the protein undergoes a rapid cleft opening transition. At the same time, the dynamical behavior of the cleft in the presence of the glutamate ligand is only weakly affected by the S652 peptide bond in its flipped conformation observed in the crystal structure. The residue E705 plays significant role in stabilization of the closed conformation via electrostatic interactions. The presence of the E705-K730 salt bridge seems to correlate strongly withthe cleft opening transition in the MD simulations.