Large-scale Conformational Changes Research Articles

Computational Recipe for Efficient Description of Large-Scale Conformational Changes in Biomolecular Systems.

Characterizing large-scale structural transitions in biomolecular systems poses major technical challenges to both experimental and computational approaches. On the computational side, efficient sampling of the configuration space along the transition pathway remains the most daunting challenge. Recognizing this issue, we introduce a knowledge-based computational approach toward describing large-scale conformational transitions using (i) nonequilibrium, driven simulations combined with work measurements and (ii) free energy calculations using empirically optimized biasing protocols. The first part is based on designing mechanistically relevant, system-specific reaction coordinates whose usefulness and applicability in inducing the transition of interest are examined using knowledge-based, qualitative assessments along with nonequilirbrium work measurements which provide an empirical framework for optimizing the biasing protocol. The second part employs the optimized biasing protocol resulting from the first part to initiate free energy calculations and characterize the transition quantitatively. Using a biasing protocol fine-tuned to a particular transition not only improves the accuracy of the resulting free energies but also speeds up the convergence. The efficiency of the sampling will be assessed by employing dimensionality reduction techniques to help detect possible flaws and provide potential improvements in the design of the biasing protocol. Structural transition of a membrane transporter will be used as an example to illustrate the workings of the proposed approach.

Correlated motions are a fundamental property of β-sheets.

Correlated motions in proteins can mediate fundamental biochemical processes such as signal transduction and allostery. The mechanisms that underlie these processes remain largely unknown due mainly to limitations in their direct detection. Here, based on a detailed analysis of protein structures deposited in the protein data bank, as well as on state-of-the art molecular simulations, we provide general evidence for the transfer of structural information by correlated backbone motions, mediated by hydrogen bonds, across β-sheets. We also show that the observed local and long-range correlated motions are mediated by the collective motions of β-sheets and investigate their role in large-scale conformational changes. Correlated motions represent a fundamental property of β-sheets that contributes to protein function.

Distance fluctuation of a single molecule in Lennard-Jones liquid based on generalized Langevin equation and mode coupling theory

Distance fluctuation of a single molecule, modeled as an idealized bead-spring chain, dissolved in a Lennard-Jones liquid is studied by using a multidimensional generalized Langevin equation, where the friction kernel ζ(t) is calculated from the kinetic mode coupling theory (MCT). Temporal behavior of the distance autocorrelation function shows three typical regimes of time dependence, starting with a constant, followed by a power law of t−α, and finally an exponential decay. Particular attentions are paid to the time span of the power law regime, which corresponds to anomalous subdiffusion behavior, and the MCT framework enables us to investigate thoroughly how this regime depends on microscopic details such as the bead-to-solvent mass ratio MR, chain spring frequency ω, and the chain length N. Interestingly, the exponent α is robust to be 1/2 against the change of these parameters, although the friction kernel ζ(t) shows nontrivial dependence on time. In addition, we find that the starting time of the power-law region t1 scales with Γ−1, with Γ = 4ω2/ζ0 where ζ0 is the zero-frequency friction which decreases rapidly with increasing bead mass. On the other hand, the ending time t2 is not sensitive to varying ω or ζ0, but it increases with N rapidly before it reaches a constant for N larger than some threshold value. Our work may provide a unified strategy starting from the microscopic level to understand the anomalous subdiffusive behavior regarding large scale conformational change of polymers or proteins.

Deep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome

The 26S proteasome is a 2.5 MDa molecular machine that executes the degradation of substrates of the ubiquitin-proteasome pathway. The molecular architecture of the 26S proteasome was recently established by cryo-EM approaches. For a detailed understanding of the sequence of events from the initial binding of polyubiquitylated substrates to the translocation into the proteolytic core complex, it is necessary to move beyond static structures and characterize the conformational landscape of the 26S proteasome. To this end we have subjected a large cryo-EM dataset acquired in the presence of ATP and ATP-γS to a deep classification procedure, which deconvolutes coexisting conformational states. Highly variable regions, such as the density assigned to the largest subunit, Rpn1, are now well resolved and rendered interpretable. Our analysis reveals the existence of three major conformations: in addition to the previously described ATP-hydrolyzing (ATPh) and ATP-γS conformations, an intermediate state has been found. Its AAA-ATPase module adopts essentially the same topology that is observed in the ATPh conformation, whereas the lid is more similar to the ATP-γS bound state. Based on the conformational ensemble of the 26S proteasome in solution, we propose a mechanistic model for substrate recognition, commitment, deubiquitylation, and translocation into the core particle.

A new mechanism in the binding between Homer3 EVH1 domain and inositol 1,4,5 trisphosphate receptor suppressor domain.

The suppressor domain of inositol 1,4,5 trisphosphate receptor (IP3R) has critical roles in regulating the calcium channel by interacting with many binding partners. The residue 49-53 (PPKKF) of the suppressor domain was suggested to be a canonical Homer EVH1 domain binding site and is also the first a part of calmodulin (CaM) binding site. As CaM-binding of the suppressor domain has been shown to involve large-scale conformational changes, we studied the binding characteristics of the Homer EVH1-suppressor domain with NMR spectroscopy and biochemical pull-down assays for mutants. Our data show that the suppressor domain employs the PPKKF motif in a similar but subtly different way compared to previously characterized interactions, and that the suppressor domain does not undergo large-scale conformational changes. Chemical shift assignments of the Homer3 EVH1 domain found that a new set of residues, located at the opposite side of the previously reported binding site, is also involved in binding, which was confirmed by mutant binding assays. Further analysis suggests that F40 in the new binding sites may have a critical role as a conformational lock-switch in Homer-target binding. The proposed mechanism is implicated in the signaling network involving calcium channels.

Ubiquitin code assembly and disassembly

Ubiquitin, a 76 amino-acid polypeptide, presents a compact three-dimensional structure, utilising a fold that recurs within larger polypeptides and in other protein modifiers, such as NEDD8 and SUMO. Ubiquitylation was initially recognised as a signal for proteasome-mediated degradation. We shall consider here how this view has evolved to appreciate that the dynamic appendage of different types of ubiquitin chains represents a versatile, three-dimensional code, fundamental to the control of many cellular processes.

THz time scale structural rearrangements and binding modes in lysozyme-ligand interactions

Predicting the conformational changes in proteins that are relevant for substrate binding is an ongoing challenge in the aim of elucidating the functional states of proteins. The motions that are induced by protein-ligand interactions are governed by the protein global modes. Our measurements indicate that the detected changes in the global backbone motion of the enzyme upon binding reflect a shift from the large-scale collective dominant mode in the unbound state towards a functional twisting deformation that assists in closing the binding cleft. Correlated motion in lysozyme has been implicated in enzyme function in previous studies, but detailed characterization of the internal fluctuations that enable the protein to explore the ensemble of conformations that ultimately foster large-scale conformational change is yet unknown. For this reason, we use THz spectroscopy to investigate the picosecond time scale binding modes and collective structural rearrangements that take place in hen egg white lysozyme (HEWL) when bound by the inhibitor (NAG)3. These protein thermal motions correspond to fluctuations that have a role in both selecting and sampling from the available protein intrinsic conformations that communicate function. Hence, investigation of these fast, collective modes may provide knowledge about the mechanism leading to the preferred binding process in HEWL-(NAG)3. Specifically, in this work we find that the picosecond time scale hydrogen-bonding rearrangements taking place in the protein hydration shell with binding modify the packing density within the hydrophobic core on a local level. These localized, intramolecular contact variations within the protein core appear to facilitate the large cooperative movements within the interfacial region separating the α- and β- domain that mediate binding. The THz time-scale fluctuations identified in the protein-ligand system may also reveal a molecular mechanism for substrate recognition.

PH-Induced Stability Switching of the Bacteriophage HK97 Maturation Pathway

Many viruses undergo large-scale conformational changes during their life cycles. Blocking the transition from one stage of the life cycle to the next is an attractive strategy for the development of antiviral compounds. In this work, we have constructed an icosahedrally symmetric, low-energy pathway for the maturation transition of bacteriophage HK97. By conducting constant-pH molecular dynamics simulations on this pathway, we identify which residues are contributing most significantly to shifting the stability between the states along the pathway under differing pH conditions. We further analyze these data to establish the connection between critical residues and important structural motifs which undergo reorganization during maturation. We go on to show how DNA packaging can induce spontaneous reorganization of the capsid during maturation.

Iterative Elastic 3D-to-2D Alignment Method Using Normal Modes for Studying Structural Dynamics of Large Macromolecular Complexes

This article presents a method to study large-scale conformational changes by combining electron microscopy (EM) single-particle image analysis and normal mode analysis (NMA). It is referred to as HEMNMA, which stands for hybrid electron microscopy normal mode analysis. NMA of a reference structure (atomic-resolution structure or EM volume) is used to predict possible motions that are then confronted with EM images within an automatic iterative elastic 3D-to-2D alignment procedure to identify actual motions in the imaged samples. HEMNMA can be used to extensively analyze the conformational changes and may be used in combination with classic discrete procedures. The identified conformations allow modeling of deformation pathways compatible with the experimental data. HEMNMA was tested with synthetic and experimental data sets of E.coli 70S ribosome, DNA polymerase Pol α and B subunit complex of the eukaryotic primosome, and tomato bushy stunt virus.

Sustained active site rigidity during synthesis by human DNA polymerase μ.

DNA polymerase μ (Pol μ) is the only template-dependent human DNA polymerase capable of repairing double-strand DNA breaks (DSBs) with unpaired 3' ends in nonhomologous end joining (NHEJ). To probe this function, we structurally characterized Pol μ's catalytic cycle for single-nucleotide incorporation. These structures indicate that, unlike other template-dependent DNA polymerases, Pol μ shows no large-scale conformational changes in protein subdomains, amino acid side chains or DNA upon dNTP binding or catalysis. Instead, the only major conformational change is seen earlier in the catalytic cycle, when the flexible loop 1 region repositions upon DNA binding. Pol μ variants with changes in loop 1 have altered catalytic properties and are partially defective in NHEJ. The results indicate that specific loop 1 residues contribute to Pol μ's unique ability to catalyze template-dependent NHEJ of DSBs with unpaired 3' ends.

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The EtfAf-NAD(+) complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH(-) is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH(-) by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD, which donates this electron further to Dh-FAD of BcdAf after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH(•), immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH(-) that converts crotonyl-CoA to butyryl-CoA.

Characterizing Conformational Ensemble and Free Energy Landscape of ABC Exporters using a Novel System-Specific Sampling Approach

During their transport cycle, ATP-binding cassette (ABC) exporters undergo large-scale conformational changes between inward-facing (IF) and outward-facing (OF) states. Using a novel approach based on designing system-specific reaction coordinates and employing nonequilibrium simulations, we have recently characterized the IF-OF conformational transition pathway of MsbA, a bacterial ABC exporter whose structure has been solved in multiple functional states including an OF conformation and two IF conformations (termed IF-closed and IF-open based on the extent to which the cytoplasmic side is open). Due to low resolution of MsbA crystal structures in IF conformation, only Cα atoms have been reported; however our unique system-specific sampling strategy results in a stable all-atom model of MsbA in the IF conformation in the presence of explicit membrane and water. In order to more accurately characterize the conformational ensemble of nucleotide-free apo MsbA, we also employ a free energy calculation technique based on a combination of umbrella sampling and replica exchange methods, using a reaction coordinate defined based on the orientation of transmembrane helices. The results indicate that the IF-closed conformation is associated with a relatively deep minimum and the IF-open-like conformations are also thermally accessible. However, the deepest free energy basin is associated with an IF conformation, more open than IF-closed and less open than IF-open, resembling the crystal structures of P-glycoprotein (a MsbA homolog) which are obtained at higher resolutions. The overall picture emerging from the reconstructed free energies is that MsbA is fairly flexible in its resting state in the absence of nucleotides and substrates. The approach proposed here provides a framework to study large-scale conformational changes of membrane transporters whose computational investigation at an atomic resolution may not be currently feasible using conventional methods.

A Meshfree Method For Mechanics and Conformational Change of Proteins and Their Assemblies

Mechanical properties of proteins play an important role in their biological function. For example, microtubules carry large loads to transport organelles inside the cell, and virus shells undergo changes in shape and mechanical properties during maturation which affect their infectivity. Various theoretical models including continuum elasticity have been applied to study these structural properties, and a significant success has been achieved. But, the previous frameworks lack a connection between the atomic and continuum descriptions. Here this is accomplished through the development of a meshfree framework based on reproducing kernel shape functions for the large deformation mechanics of protein structures. The framework is validated by comparing thermal fluctuations of small proteins against well established elastic network model. To demonstrate the usability of this framework, solutions to several other problems are presented. The response of virus shells to indentation under atomic force microscope tip is simulated and compared to the finite element results. Finally, the large scale conformational changes of viruses are analyzed by computing the deformations/strains associated with the conformational motions. Excellent agreement with the previously published results is observed while increasing the efficiency of numerical analysis. Furthermore, the results provide insights into the continuum behavior of proteins and the optimum amount of geometric details necessary for calculating their mechanical properties.

GNEIMO-Fixman: An Accurate Torsional Molecular Dynamics Simulation Method for Studying Biomolecular Dynamics

Torsion angle Molecular Dynamics simulations provide a powerful alternative to all-atom MD since they place constraints on the bond lengths and angles, and thereby efficiently simulate large scale conformational changes in torsional space. The previous uses of torsional MD have been severely limited by the lack of efficient algorithms to solve the mathematical complexities of torsional MD. Our development of an advanced Generalized Newton-Euler Inverse Mass Operator (GNEIMO) torsional MD method overcomes these limitations with several theoretical and algorithmic advancements. We will outline these advancements and the effectiveness of the GNEIMO method in critical applications such as protein homology model refinement and protein conformational dynamics.Due to the rigidity in the model the torsional MD simulations show a bias in the partition function, which results in inaccuracies in the calculated thermodynamic and kinetic properties. Fixman developed a compensating potential that rigorously annuls the bias in the partition function, but the calculation of the Fixman potential has remained mathematically intractable even for moderate size molecules. Recently, we have developed a low-cost algorithm, based on Spatial Operator Algebra for calculating Fixman potential and torque for any molecule. We will present results on the validation and the effect of the Fixman correction potential in recovering the correct population distribution function, transition rates, and free energy surface for large branched systems.We have designed a modular software platform for the GNEIMO-Fixman method that can be readily combined with any type of forcefield engine. We have combined GNEIMO algorithm with Rosetta forcefield and performed homology model refinement with torsional MD. Thus our GNEIMO-Fixman torsional MD method allows for the use of larger integration time steps than for all-atom MD simulations, and enables enhanced conformational sampling in the low frequency torsional degrees of freedom.

Conformational Transitions in ATP-Driven Calcium Pump SERCA

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is an integral membrane protein that uses ATP hydrolysis as a source of free energy to pump two calcium ions per ATP molecule from calcium poor cytoplasm of the muscle cell to the calcium rich lumen of the sarcoplasmic reticulum, thereby maintaining a ten thousand fold concentration gradient. Detailed structural studies of the pump under different conditions provided analogues of various intermediates in the reaction cycle and revealed important changes in the tertiary structure of the protein both in the cytoplasmic and in the transmembrane parts. Two major outstanding issues are the pathways of the ions to and from the transmembrane binding sites and a detailed understanding of the large scale conformational changes among various functionally relevant states. We have applied all-atom molecular dynamics (MD) and string method with swarms-of-trajectories to study transition pathways among various experimental structures. The size of the system (291,000) and the time-scale involved in the large scale motions prohibit the use of brute-force unbiased simulations to obtain statistically meaningful information. To circumvent this challenge, the string method with swarms-of-trajectories is used to discover the optimal minimum free energy path between the two end-states. The path, which is represented as a chain of states or images in the space of relevant collective variables, is optimized by iterating two steps: moving each image along the drift calculated from an ensemble (swarm) of short unbiased MD trajectories initiated from the image and a reparametrization procedure that keeps all the images equidistant. We hope to understand the molecular details of the complex motions involved in the conformational transitions by studying the pathways produced by the string method and correlating these observations to available structural and biochemical experiments.

Molecular Dynamics Simulations of Ribosomes: Integrating Theory and Experiment

We will report results from the first molecular simulations of eukaryotic ribosomes. Using an integrated approach, we combine data from X-ray crystallography, cryo-EM and SHAPE chemical probing. Over the past decade, we have developed a pipeline that begins with X-ray crystallographic structures and uses molecular simulation to produce all-atom models consistent with cryo-EM reconstructions. Theses models are then used as beginning and end points for simulations of large-scale conformational changes. Our strategy has been highly successful in the case of the accommodation conformational change during tRNA selection in bacteria. Here, we correctly predicted the universally conserved accommodation corridor, which has been verified in several independent experimental studies. We have also recently identified the hybrid corridor, responsible for tRNA hybrid state formation during translocation. Our latest addition to our pipeline is the incorporation of SHAPE probing data describing the mobility of the RNA backbone in solution. We have developed a novel algorithm to generate molecular dynamics simulations highly consistent with SHAPE probing data. We have applied these techniques to eukaryotic ribosomes to investigate their dynamics and conformational changes.

High-resolution modeling of protein structures based on flexible fitting of low-resolution structural data.

To circumvent the difficulty of directly solving high-resolution biomolecular structures, low-resolution structural data from Cryo-electron microscopy (EM) and small angle solution X-ray scattering (SAXS) are increasingly used to explore multiple conformational states of biomolecular assemblies. One promising venue to obtain high-resolution structural models from low-resolution data is via data-constrained flexible fitting. To this end, we have developed a new method based on a coarse-grained Cα-only protein representation, and a modified form of the elastic network model (ENM) that allows large-scale conformational changes while maintaining the integrity of local structures including pseudo-bonds and secondary structures. Our method minimizes a pseudo-energy which linearly combines various terms of the modified ENM energy with an EM/SAXS-fitting score and a collision energy that penalizes steric collisions. Unlike some previous flexible fitting efforts using the lowest few normal modes, our method effectively utilizes all normal modes so that both global and local structural changes can be fully modeled with accuracy. This method is also highly efficient in computing time. We have demonstrated our method using adenylate kinase as a test case which undergoes a large open-to-close conformational change. The EM-fitting method is available at a web server (http://enm.lobos.nih.gov), and the SAXS-fitting method is available as a pre-compiled executable upon request.

Thermodynamic Characterization of Protein Folding using Monte Carlo Methods

The study of protein folding has been a difficult challenge in molecular biology and simulation science.Recent publications showed the reproducible folding of small protein using molecular dynamics simulations. This large conformational changes can be only achieved by using specialized supercomputers [1]. In contrast to molecular dynamics simulations, Monte Carlo based simulations are not constrained solving Newton's equation of motion and therefore may be used conventional computer architectures to sample the protein folding process. Here we show fast reproducible all-atom folding transitions of the villin headpiece (Figure 1) simulated using SIMONA[2], a Monte Carlo based simulation package for nanoscale simulations including a variant of the Amber99SB∗-ILDN[3]. The results demonstrate the applicability of Monte Carlo simulation techniques to the investigation of large-scale conformational changes of proteins on conventional computer architectures. The computing time for observing folding/refolding events can be significantly reduced in comparison with molecular dynamics simulations. The thermodynamic characterization of simulated proteins can be compared with the experimental results.[1] Shaw, D. E. et al. Science 330, 341-346 (2010).[2] Wolf, M., Strunk, T. et al. J Comput Chem (2012).[3] Lindorff-Larsen et al. Science 334, 517-520 (2011).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Pulsed EPR Distance Measurements Resolve the Impact of Site-Specific Calmodulin Methionine Oxidation

We have examined the structural impact of oxidizing specific protein methionines (Met) in the C-ter lobe of calmodulin (CaM); these oxidation sites are known to abolish CaM regulation of the major calcium release channel, the ryanodine receptor complex (RyR). Protein oxidation by reactive oxygen species (ROS) is strongly associated with loss of strength in skeletal muscle and is proposed to play major roles in aging and degenerative muscle disease.We have linked oxidation-induced changes in RyR regulation to changes in CaM-RyR structure using (1) protein mutagenesis to mimic oxidation at specific sites and (2) spectroscopy to resolve oxidation-induced changes in protein structural dynamics. Pulsed EPR distance measurements across CaM's lobes (multiple pairs of labeling sites, one label on each lobe) were sensitive to large-scale conformational changes that accompany both calcium binding and RyR peptide binding. In the absence of calcium, CaM was highly disordered, populating a broad distribution of conformations. Calcium binding strongly stabilized the elongated conformation, while RyR peptide binding to calcium-loaded CaM strongly stabilized the compact conformation. CaM conformation, particularly the distribution over structural states, was sensitive to Met to Gln substitutions (M109Q and M124Q) designed to mimic CaM Met oxidation. Structural sensitivity to M-to-Q mutations was observed in both the presence and absence of calcium, and in complex with RyR peptide. We conclude that Met oxidation alters CaM's functional interaction with RyR through changes in the orientation and flexibility of CaM's lobes.This work is supported by a University of Wisconsin-La Crosse Faculty Research Grant to JC Klein, NIH grants to DD Thomas (R37AG026160 and P30AR057220), RJ Moen (F31AG037303), the Minnesota Biophysical Spectroscopy Center and the Minnesota Supercomputing Institute.

Reprogramming an ATP-Driven Biological Machine into a Light-Gated Protein Nanocage

Supramolecular protein assemblies are attractive materials for engineering nanoscale machines with controllable functions. Protein monomers have been engineered to self-assemble into symmetrical complexes, metal-templated structures and cage-like architectures. The mechanistic complexity of protein machines, however, has made it difficult to engineer assemblies with not only new architectures but also desired functions. Here we describe an approach to controlling functional states of a protein assembly with light. The approach uses covalently attached molecular spacers that reversibly switch interatomic distances upon illumination. We applied this strategy to convert an ATP-dependent homo-oligomeric group II chaperonin to a light-driven machine that undergoes large-scale conformational changes between open and closed states visualized by single particle cryo-electron microscopy. The resulting light-gated nanocontainer can capture and release non-native cargos. The design principle of alternately stabilizing conformational states by switching atomic distances illuminates the cooperativity of evolved protein assemblies and provides a strategy for engineering other light-controlled biologically inspired machines.View Large Image | View Hi-Res Image | Download PowerPoint Slide