Large-scale Conformational Motions Research Articles

Quantitative characterization of the path of glucose diffusion facilitated by human glucose transporter 1

Glucose transporter GLUT1 is ubiquitously expressed in the human body from the red cells to the blood-brain barrier to the skeletal muscles. It is physiologically relevant to understand how GLUT1 facilitates diffusion of glucose across the cell membrane. It is also pathologically relevant because GLUT1 deficiency causes neurological disorders and anemia and because GLUT1 overexpression fuels the abnormal growth of cancer cells. This article presents a quantitative investigation of GLUT1 based on all-atom molecular-dynamics (MD) simulations of the transporter embedded in lipid bilayers of asymmetric inner-and-outer-leaflet lipid compositions, subject to asymmetric intra-and-extra-cellular environments. This is in contrast with the current literature of MD studies that have not considered both of the aforementioned asymmetries of the cell membrane. The equilibrium (unbiased) dynamics of GLUT1 shows that it can facilitate glucose diffusion across the cell membrane without undergoing large-scale conformational motions. The Gibbs free-energy profile, which is still lacking in the current literature of GLUT1, quantitatively characterizes the diffusion path of glucose from the periplasm, through an extracellular gate of GLUT1, on to the binding site, and off to the cytoplasm. This transport mechanism is validated by the experimental data that GLUT1 has low water-permeability, uptake-efflux symmetry, and 10 kcal/mol Arrhenius activation barrier around 37 °C.

Protein-Bath Coupling of an Internal Reaction Coordinate at Intermediate Time Scales.

Thermally activated barrier-crossing processes are central to protein reaction kinetics. A determining factor for such kinetics is the extent to which the protein's motions are coupled to the surrounding bath. It is understood that slow large-scale conformational motions are strongly coupled to the environment, while fast librational motions are uncoupled. However, less is known about protein-bath coupling of reaction coordinates located on the interior of a protein and with dynamics on intermediate time scales. In this work, we use single molecule 2D fluorescence lifetime correlation spectroscopy to study the microsecond chemical reaction occurring in the chromophore pocket of eGFP. The equilibrium reaction involves a dihedral rotation of a glutamic acid residue and a rearrangement of the local hydrogen-bonding network surrounding the endogenous chromophore, with no accompanying large-scale conformational changes. We observe that the internal chemical reaction is coupled to the solvent viscosity, though the scaling deviates from Kramers' behavior. We attribute this deviation to the internal friction of the protein, which weakens the protein-solvent coupling at high viscosity and intermediate time scales.

Molecular modeling study of the induced-fit effect on kinase inhibition: the case of fibroblast growth factor receptor 3 (FGFR3).

Tyrosine kinases are a wide family of targets with strong pharmacological relevance. These proteins undergo large-scale conformational motions able to inactivate them. By the end of one of these structural processes, a new cavity is opened allowing the access to a specific type of inhibitors, called type II. The kinase domain of fibroblast growth factor receptor 3 (FGFR3) falls into this family of kinases. We describe here, for the first time, its inactivation process through target molecular dynamics. The transient cavity, at the crossroad between the DFGout and Cα helix out inactivation is herein explored. Molecular docking calculations of known ligands demonstrated that type II inhibitors are able to interact with this metastable transient conformation of FGFR3 kinase. Besides, supplemental computations were conducted and clearly show that type II inhibitors drive the kinase inactivation process through specific stabilization with the DFG triad. This induced-fit effect of type II ligands toward FGFR3 might be extrapolated to other kinase systems and provides meaningful structural information for future drug developments.

Energetics and structural characterization of the large-scale functional motion of adenylate kinase.

Adenylate Kinase (AK) is a signal transducing protein that regulates cellular energy homeostasis balancing between different conformations. An alteration of its activity can lead to severe pathologies such as heart failure, cancer and neurodegenerative diseases. A comprehensive elucidation of the large-scale conformational motions that rule the functional mechanism of this enzyme is of great value to guide rationally the development of new medications. Here using a metadynamics-based computational protocol we elucidate the thermodynamics and structural properties underlying the AK functional transitions. The free energy estimation of the conformational motions of the enzyme allows characterizing the sequence of events that regulate its action. We reveal the atomistic details of the most relevant enzyme states, identifying residues such as Arg119 and Lys13, which play a key role during the conformational transitions and represent druggable spots to design enzyme inhibitors. Our study offers tools that open new areas of investigation on large-scale motion in proteins.

Method of prediction and optimization of conformational motion of proteins based on mass transportation principle

The paper highlights approaches to fast prediction of protein conformational mobility. A new mathematical model based on the transportation principle is proposed. We describe an algorithm and soft-ware developed for a construction of the possible trajectories of the large-scale conformational motions of proteins (i.e. movements that occur within relatively large time intervals of the order of milliseconds). The modeling showed that the proposed method provides adequate, in terms of current knowledge of the biology and physics of proteins, results and allows simulation of large-scale conformational transitions for less time.

Mechanisms for Efficient TRNA Translocation through the Ribosome

After peptide bond formation the transfer RNAs (tRNAs) bound to the ribosome translocate by more than 7 nm to adjacent binding sites, accompanied by large-scale conformational motions of the ribosome. Combining cryo-EM reconstructions of translocation intermediates (Fischer, Nature 2010) with high resolution crystal structures, we obtained 13 near-atomic resolution structures. The quality of these structures was validated using recent crystal structures and subsequently all-atom molecular dynamics simulations of the fully solvated 70S ribosome were carried out for each of the 13 intermediate states, totaling 1.5 µs. The obtained dynamics within the intermediate states allowed us to estimate transition rates between states for motions of the L1-stalk, tRNAs and intersubunit rotations. These rates revealed rapid motions of the L1-stalk and the small subunit on sub-microsecond timescales, whereas the tRNA motions were seen to be rate-limiting for most transitions. By calculating the free energy of interaction between L1-stalk and tRNA, we obtained molecular forces revealing that the L1-stalk is actively pulling the tRNA from P to E site, thereby overcoming barriers for the tRNA motion. Further, ribosomal proteins L5 and L16 guide the tRNAs by ‘sliding’ and ‘stepping’ mechanisms involving key protein-tRNA contacts, explaining how tRNA binding affinity is kept sufficiently constant to allow rapid translocation despite large-scale displacements.

Correlated motion of protein subdomains and large-scale conformational flexibility of RecA protein filament

Based on X-ray crystallographic data available at Protein Data Bank, we have built molecular dynamics (MD) models of homologous recombinases RecA from E. coli and D. radiodurans. Functional form of RecA enzyme, which is known to be a long helical filament, was approximated by a trimer, simulated in periodic water box. The MD trajectories were analyzed in terms of large-scale conformational motions that could be detectable by neutron and X-ray scattering techniques. The analysis revealed that large-scale RecA monomer dynamics can be described in terms of relative motions of 7 subdomains. Motion of C-terminal domain was the major contributor to the overall dynamics of protein. Principal component analysis (PCA) of the MD trajectories in the atom coordinate space showed that rotation of C-domain is correlated with the conformational changes in the central domain and N-terminal domain, that forms the monomer-monomer interface. Thus, even though C-terminal domain is relatively far from the interface, its orientation is correlated with large-scale filament conformation. PCA of the trajectories in the main chain dihedral angle coordinate space implicates a co-existence of a several different large-scale conformations of the modeled trimer. In order to clarify the relationship of independent domain orientation with large-scale filament conformation, we have performed analysis of independent domain motion and its implications on the filament geometry.

Tracing conformational changes in proteins

BackgroundMany proteins undergo extensive conformational changes as part of their functionality. Tracing these changes is important for understanding the way these proteins function. Traditional biophysics-based conformational search methods require a large number of calculations and are hard to apply to large-scale conformational motions.ResultsIn this work we investigate the application of a robotics-inspired method, using backbone and limited side chain representation and a coarse grained energy function to trace large-scale conformational motions. We tested the algorithm on four well known medium to large proteins and we show that even with relatively little information we are able to trace low-energy conformational pathways efficiently. The conformational pathways produced by our methods can be further filtered and refined to produce more useful information on the way proteins function under physiological conditions.ConclusionsThe proposed method effectively captures large-scale conformational changes and produces pathways that are consistent with experimental data and other computational studies. The method represents an important first step towards a larger scale modeling of more complex biological systems.

Protein Conformational Transitions: The Closure Mechanism of a Kinase Explored by Atomistic Simulations

Kinase large-scale conformational rearrangement is an issue of enormous biological and pharmacological relevance. Atomistic simulations able to capture the dynamics and the energetics of kinase large-scale motions are still in their infancy. Here, we present a computational study in which the atomistic dynamics of the "open-to-closed" movement of the cyclin-dependent kinase 5 (CDK5) have been simulated. Simulations were carried out using a new sampling method that is able to find the lowest free-energy channel between an initial state and a final state. This large-scale movement has a two-step mechanism: first, the alphaC-helix rotates by approximately 45 degrees , allowing the interaction between Glu51 and Arg149; then the CDK5 activation loop refolds to assume the closed conformation. We have also estimated the free-energy profile associated with the global motion and identified a CDK5 intermediate, which could be exploited for drug-design purposes. Our new sampling method turned out to be well-suited for investigating at an atomistic level the energetics and dynamics of kinase large-scale conformational motions.

Dynamics of well-folded and natively disordered proteins in solution: a time-of-flight neutron scattering study

Casein proteins belong to the class of natively disordered proteins. The existence of disordered biologically active proteins questions the assumption that a well-folded structure is required for function. A hypothesis generally put forward is that the unstructured nature of these proteins results from the functional need of a higher flexibility. This interplay between structure and dynamics was investigated in a series of time-of-flight neutron scattering experiments, performed on casein proteins, as well as on three well-folded proteins with distinct secondary structures, namely, myoglobin (alpha), lysozyme (alpha/beta) and concanavalin A (beta). To illustrate the subtraction of the solvent contribution from the scattering spectra, we used the dynamic susceptibility spectra emphasizing the high frequency part of the spectrum, where the solvent dominates. The quality of the procedure is checked by comparing the corrected spectra to those of the dry and hydrated protein with negligible solvent contamination. Results of spectra analysis reveal differences in motional amplitudes of well-folded proteins, where beta-sheet structures appear to be more rigid than a cluster of alpha-helices. The disordered caseins display the largest conformational displacements. Moreover their global diffusion rates deviate from the expected dependence, suggesting further large-scale conformational motions.