Modeling the Structure, Dynamics, and Transformations of Proteins with the UNRES Force Field.

The physics-based united-residue (UNRES) model of proteins ( www.unres.pl ) has been designed to carry out large-scale simulations of protein folding. The force field has been derived and parameterized based on the principles of statistical-mechanics, which makes it independent of structural databases and applicable to treat nonstandard situations such as, proteins that contain D-amino-acid residues. Powered by Langevin dynamics and its replica-exchange extensions, UNRES has found a variety of applications, including ab initio and database-assisted protein-structure prediction, simulating protein-folding pathways, exploring protein free-energy landscapes, and solving biological problems. This chapter provides a summary of UNRES and a guide for potential users regarding the application of the UNRES package in a variety of research tasks.

Substitutions at position 263 within the von Willebrand factor type A domain determine the functionality of complement C2 protein.

The complement system is one of the first defense lines protecting from invading pathogens. However, it may turn offensive to the body's own cells and tissues when deregulated by the presence of rare genetic variants that impair physiological regulation and/or provoke abnormal activity of key enzymatic components. Factor B and complement C2 are examples of paralogs engaged in the alternative and classical/lectin complement pathway, respectively. Pathogenic mutations in the von Willebrand factor A domain (vWA) of FB have been known for years. Despite substantial homology between two proteins and the demonstration that certain substitutions in FB translated to C2 result in analogous phenotype, there was a limited number of reports on pathogenic C2 variants in patients. Recently, we studied a cohort of patients suffering from rare kidney diseases and confirmed the existence of two gain-of-function and three loss-of-function mutations within the C2 gene sequences coding for the vWA domain (amino acids 254-452) or nearly located unstructured region (243-253) of C2 protein. Herein, we report the functional consequences of amino acid substitution of glutamine at position 263. The p.Q263G variant resulted in the gain-of-function phenotype, similarly to a homologous mutation p.D279G in FB. Conversely, the p.Q263P variant found in a patient with C3 glomerulopathy resulted in the loss of C2 function. Our results confirm that the N-terminal part of the vWA domain is a hot spot crucial for the complement C2 function.

Gain-of-Function Mutations R249C and S250C in Complement C2 Protein Increase C3 Deposition in the Presence of C-Reactive Protein.

The impairment of the alternative complement pathway contributes to rare kidney diseases such as atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). We recently described an aHUS patient carrying an exceptional gain-of-function (GoF) mutation (S250C) in the classical complement pathway component C2 leading to the formation of hyperactive classical convertases. We now report the identification of the same mutation and another C2 GoF mutation R249C in two other patients with a glomerulopathy of uncertain etiology. Both mutations stabilize the classical C3 convertases by a similar mechanism. The presence of R249C and S250C variants in serum increases complement-dependent cytotoxicity (CDC) in antibody-sensitized human cells and elevates deposition of C3 on ELISA plates coated with C-reactive protein (CRP), as well as on the surface of glomerular endothelial cells. Our data justify the inclusion of classical pathway genes in the genetic analysis of patients suspected of complement-driven renal disorders. Also, we point out CRP as a potential antibody-independent trigger capable of driving excessive complement activation in carriers of the GoF mutations in complement C2.

Compatibility of Distinct Label-Free Proteomic Workflows in Absolute Quantification of Proteins Linked to the Oocyte Quality in Human Follicular Fluid.

We present two separate label-free quantitative workflows based on different high-resolution mass spectrometers and LC setups, which are termed after the utilized instrument: Quad-Orbitrap (nano-LC) and Triple Quad-TOF (micro-LC) and their directed adaptation toward the analysis of human follicular fluid proteome. We identified about 1000 proteins in each distinct workflow using various sample preparation methods. With assistance of the Total Protein Approach, we were able to obtain absolute protein concentrations for each workflow. In a pilot study of twenty samples linked to diverse oocyte quality status from four donors, 455 and 215 proteins were quantified by the Quad-Orbitrap and Triple Quad-TOF workflows, respectively. The concentration values obtained from both workflows correlated to a significant degree. We found reasonable agreement of both workflows in protein fold changes between tested groups, resulting in unified lists of 20 and 22 proteins linked to oocyte maturity and blastocyst development, respectively. The Quad-Orbitrap workflow was best suited for an in-depth analysis without the need of extensive fractionation, especially of low abundant proteome, whereas the Triple Quad-TOF workflow allowed a more robust approach with a greater potential to increase in effectiveness with the growing number of analyzed samples after the initial effort of building a comprehensive spectral library.

AfsK-Mediated Site-Specific Phosphorylation Regulates DnaA Initiator Protein Activity in Streptomyces coelicolor.

In all organisms, chromosome replication is regulated mainly at the initiation step. Most of the knowledge about the mechanisms that regulate replication initiation in bacteria has come from studies on rod-shaped bacteria, such as Escherichia coli and Bacillus subtilisStreptomyces is a bacterial genus that is characterized by distinctive features and a complex life cycle that shares some properties with the developmental cycle of filamentous fungi. The unusual lifestyle of streptomycetes suggests that these bacteria use various mechanisms to control key cellular processes. Here, we provide the first insights into the phosphorylation of the bacterial replication initiator protein, DnaA, from Streptomyces coelicolor We suggest that phosphorylation of DnaA triggers a conformational change that increases its ATPase activity and decreases its affinity for the replication origin, thereby blocking the formation of a functional orisome. We suggest that the phosphorylation of DnaA is catalyzed by Ser/Thr kinase AfsK, which was shown to regulate the polar growth of S. coelicolor Together, our results reveal that phosphorylation of the DnaA initiator protein functions as a negative regulatory mechanism to control the initiation of chromosome replication in a manner that presumably depends on the cellular localization of the protein.IMPORTANCE This work provides insights into the phosphorylation of the DnaA initiator protein in Streptomyces coelicolor and suggests a novel bacterial regulatory mechanism for initiation of chromosome replication. Although phosphorylation of DnaA has been reported earlier, its biological role was unknown. This work shows that upon phosphorylation, the cooperative binding of the replication origin by DnaA may be disturbed. We found that AfsK kinase is responsible for phosphorylation of DnaA. Upon upregulation of AfsK, chromosome replication occurred further from the hyphal tip. Orthologs of AfsK are exclusively found in mycelial actinomycetes that are related to Streptomyces and exhibit a complex life cycle. We propose that the AfsK-mediated regulatory pathway serves as a nonessential, energy-saving mechanism in S. coelicolor.

A general method for the derivation of the functional forms of the effective energy terms in coarse-grained energy functions of polymers. III. Determination of scale-consistent backbone-local and correlation potentials in the UNRES force field and force-field calibration and validation.

The general theory of the construction of scale-consistent energy terms in the coarse-grained force fields presented in Paper I of this series has been applied to the revision of the UNRES force field for physics-based simulations of proteins. The potentials of mean force corresponding to backbone-local and backbone-correlation energy terms were calculated from the ab initio energy surfaces of terminally blocked glycine, alanine, and proline, and the respective analytical expressions, derived by using the scale-consistent formalism, were fitted to them. The parameters of all these potentials depend on single-residue types, thus reducing their number and preventing over-fitting. The UNRES force field with the revised backbone-local and backbone-correlation terms was calibrated with a set of four small proteins with basic folds: tryptophan cage variant (TRP1; α), Full Sequence Design (FSD; α + ß), villin headpiece (villin; α), and a truncated FBP-28 WW-domain variant (2MWD; ß) (the NEWCT-4P force field) and, subsequently, with an enhanced set of 9 proteins composed of TRP1, FSD, villin, 1BDC (α), 2I18 (α), 1QHK (α + ß), 2N9L (α + ß), 1E0L (ß), and 2LX7 (ß) (the NEWCT-9P force field). The NEWCT-9P force field performed better than NEWCT-4P in a blind-prediction-like test with a set of 26 proteins not used in calibration and outperformed, in a test with 76 proteins, the most advanced OPT-WTFSA-2 version of UNRES with former backbone-local and backbone-correlation terms that contained more energy terms and more optimizable parameters. The NEWCT-9P force field reproduced the bimodal distribution of backbone-virtual-bond angles in the simulated structures, as observed in experimental protein structures.

Human follicular fluid proteomic and peptidomic composition quantitative studies by SWATH-MS methodology. Applicability of high pH RP-HPLC fractionation.

Analysis of proteomic composition of human follicular fluid (hFF) has been previously proposed as a potential tool of oocyte quality evaluation. In order to develop an efficient method to investigate the hFF proteome and peptidome components, we applied and tested a few prefractionation schemes of hFF material consisting of ultrafiltration, optional immunodepletion, and high pH RP-HPLC separation by building spectral libraries and comparing their quantification capabilities of unfractionated samples. Low Molecular-Weight Fraction peptides (LMWF, <10 kDa) and High Molecular-Weight Fraction proteins (HMWF, >10 kDa) resulting from ultrafiltration were analyzed separately. We identified 302 proteins in HMWF and 161 proteins in LMWF in all qualitative experiments. All LMWF peptidomic libraries turned out to be of poor quantification quality, however they enabled measurement of higher numbers of peptides with increasing input of experiment data, in contrast to HMWF proteomic libraries. We were able to quantify a total of 108 HMWF proteins and 250 LMWF peptides (from 84 proteins) in all experiments. Employment of high RP-HPLC fractionation allowed for identification of a much broader set of proteins, however did not significantly improve the quantification capabilities of the applied method. Data are available via ProteomeXchange with identifier PXD008073. SIGNIFICANCE: In the search of biomarkers for assessment of oocyte quality in assisted reproductive technology, many studies are devoted to analysis of follicular fluid composition. Candidates for such biomarkers can be located in both the proteome and the recently investigated peptidome of hFF. Reliable qualitative and especially quantitative analysis of complex mixtures such as hFF, requires development of a fast and preferably inexpensive analytical procedure. The powerful SWATH-MS technique is well suited for quantitative label-free analysis of complex protein and peptide mixtures. However, for efficient usage it needs well designed and constructed MS-spectral libraries as well as a proper protocol for sample preparation. We investigated the influence of the size and quality of MS-spectral libraries (different spectral libraries are constructed using various sample prefractionation protocols) on SWATH experiments on hFF proteome and peptidome. In the case of peptidome investigation, increasing the size of spectral libraries led to quantification of more peptides in a single experiment. For the proteome, increasing the size of spectral libraries improved quantification only to a limited extend, and further extension of spectral libraries even worsened results. Nevertheless, using the best selected prefractionation schemes and spectral libraries we were able to quantify as many as 79 proteins of hFF proteome and 106 peptides (from 53 proteins) of hFF peptidome in single experiments. The spectral libraries and prefractionation protocols we developed allow for a large scale fast scan of hundreds of clinical hFF samples in the search for biomarkers for evaluation of oocyte quality.

Maximum Likelihood Calibration of the UNRES Force Field for Simulation of Protein Structure and Dynamics.

By using the maximum likelihood method for force-field calibration recently developed in our laboratory, which is aimed at achieving the agreement between the simulated conformational ensembles of selected training proteins and the corresponding ensembles determined experimentally at various temperatures, the physics-based coarse-grained UNRES force field for simulations of protein structure and dynamics was optimized with seven small training proteins exhibiting a variety of secondary and tertiary structures. Four runs of optimization, in which the number of optimized force-field parameters was gradually increased, were carried out, and the resulting force fields were subsequently tested with a set of 22 α-, 12 ß-, and 12 α + ß-proteins not used in optimization. The variant in which energy-term weights, local, and correlation potentials, side-chain radii, and anisotropies were optimized turned out to be the most transferable and outperformed all previous versions of UNRES on the test set.

Qualitative and Quantitative Analysis of Proteome and Peptidome of Human Follicular Fluid Using Multiple Samples from Single Donor with LC-MS and SWATH Methodology.

Human follicular fluid (hFF) is a natural environment of oocyte maturation, and some components of hFF could be used to judge oocyte capability for fertilization and further development. In our pilot small-scale study three samples from four donors (12 samples in total) were analyzed to determine which hFF proteins/peptides could be used to differentiate individual oocytes and which are patient-specific. Ultrafiltration was used to fractionate hFF to high-molecular-weight (HMW) proteome (>10 kDa) and low-molecular-weight (LMW) peptidome (<10 kDa) fractions. HMW and LMW compositions were analyzed using LC-MS in SWATH data acquisition and processing methodology. In total we were able to identify 158 proteins, from which 59 were never reported before as hFF components. 55 (45 not reported before) proteins were found by analyzing LMW fraction, 67 (14 not reported before) were found by analyzing HMW fraction, and 36 were identified in both fractions of hFF. We were able to perform quantitative analysis for 72 proteins from HMW fraction of hFF. We found that concentrations of 11 proteins varied substantially among hFF samples from single donors, and those proteins are promising targets to identify biomarkers useful in oocyte quality assessment.

Unique and Universal Features of Epsilonproteobacterial Origins of Chromosome Replication and DnaA-DnaA Box Interactions.

In bacteria, chromosome replication is initiated by the interaction of the initiator protein DnaA with a defined region of a chromosome at which DNA replication starts (oriC). While DnaA proteins share significant homology regardless of phylogeny, oriC regions exhibit more variable structures. The general architecture of oriCs is universal, i.e., they are composed of a cluster of DnaA binding sites, a DNA-unwinding element, and sequences that bind regulatory proteins. However, detailed structures of oriCs are shared by related species while being significantly different in unrelated bacteria. In this work, we characterized Epsilonproteobacterial oriC regions. Helicobacter pylori was the only species of the class for which oriC was characterized. A few unique features were found such as bipartite oriC structure, not encountered in any other Gram-negative species, and topology-sensitive DnaA-DNA interactions, which have not been found in any other bacterium. These unusual H. pylori oriC features raised questions of whether oriC structure and DnaA-DNA interactions are unique to this bacterium or whether they are common to related species. By in silico and in vitro analyses we identified putative oriCs in three Epsilonproteobacterial species: pathogenic Arcobacter butzleri, symbiotic Wolinella succinogenes, and free-living Sulfurimonas denitrificans. We propose that oriCs typically co-localize with ruvC-dnaA-dnaN in Epsilonproteobacteria, with the exception of Helicobacteriaceae species. The clusters of DnaA boxes localize upstream (oriC1) and downstream (oriC2) of dnaA, and they likely constitute bipartite origins. In all cases, DNA unwinding was shown to occur in oriC2. Unlike the DnaA box pattern, which is not conserved in Epsilonproteobacterial oriCs, the consensus DnaA box sequences and the mode of DnaA-DnaA box interactions are common to the class. We propose that the typical Epsilonproteobacterial DnaA box consists of the core nucleotide sequence 5'-TTCAC-3' (4-8 nt), which, together with the significant changes in the DNA-binding motif of corresponding DnaAs, determines the unique molecular mechanism of DnaA-DNA interaction. Our results will facilitate identification of oriCs and subsequent identification of factors which regulate chromosome replication in other Epsilonproteobacteria. Since replication is controlled at the initiation step, it will help to better characterize life cycles of these species, many of which are considered as emerging pathogens.

A Maximum-Likelihood Approach to Force-Field Calibration.

A new approach to the calibration of the force fields is proposed, in which the force-field parameters are obtained by maximum-likelihood fitting of the calculated conformational ensembles to the experimental ensembles of training system(s). The maximum-likelihood function is composed of logarithms of the Boltzmann probabilities of the experimental conformations, calculated with the current energy function. Because the theoretical distribution is given in the form of the simulated conformations only, the contributions from all of the simulated conformations, with Gaussian weights in the distances from a given experimental conformation, are added to give the contribution to the target function from this conformation. In contrast to earlier methods for force-field calibration, the approach does not suffer from the arbitrariness of dividing the decoy set into native-like and non-native structures; however, if such a division is made instead of using Gaussian weights, application of the maximum-likelihood method results in the well-known energy-gap maximization. The computational procedure consists of cycles of decoy generation and maximum-likelihood-function optimization, which are iterated until convergence is reached. The method was tested with Gaussian distributions and then applied to the physics-based coarse-grained UNRES force field for proteins. The NMR structures of the tryptophan cage, a small α-helical protein, determined at three temperatures (T = 280, 305, and 313 K) by Halabis et al. ( J. Phys. Chem. B 2012 , 116 , 6898 - 6907 ), were used. Multiplexed replica-exchange molecular dynamics was used to generate the decoys. The iterative procedure exhibited steady convergence. Three variants of optimization were tried: optimization of the energy-term weights alone and use of the experimental ensemble of the folded protein only at T = 280 K (run 1); optimization of the energy-term weights and use of experimental ensembles at all three temperatures (run 2); and optimization of the energy-term weights and the coefficients of the torsional and multibody energy terms and use of experimental ensembles at all three temperatures (run 3). The force fields were subsequently tested with a set of 14 α-helical and two α + ß proteins. Optimization run 1 resulted in better agreement with the experimental ensemble at T = 280 K compared with optimization run 2 and in comparable performance on the test set but poorer agreement of the calculated folding temperature with the experimental folding temperature. Optimization run 3 resulted in the best fit of the calculated ensembles to the experimental ones for the tryptophan cage but in much poorer performance on the training set, suggesting that use of a small α-helical protein for extensive force-field calibration resulted in overfitting of the data for this protein at the expense of transferability. The optimized force field resulting from run 2 was found to fold 13 of the 14 tested α-helical proteins and one small α + ß protein with the correct topologies; the average structures of 10 of them were predicted with accuracies of about 5 Å C(α) root-mean-square deviation or better. Test simulations with an additional set of 12 α-helical proteins demonstrated that this force field performed better on α-helical proteins than the previous parametrizations of UNRES. The proposed approach is applicable to any problem of maximum-likelihood parameter estimation when the contributions to the maximum-likelihood function cannot be evaluated at the experimental points and the dimension of the configurational space is too high to construct histograms of the experimental distributions.

Theoretical Studies of Interactions between O-Phosphorylated and Standard Amino-Acid Side-Chain Models in Water.

Phosphorylation is a common post-translational modification of the amino-acid side chains (serine, tyrosine, and threonine) that contain hydroxyl groups. The transfer of the negatively charged phosphate group from an ATP molecule to such amino-acid side chains leads to changes in the local conformations of proteins and the pattern of interactions with other amino-acid side-chains. A convenient characteristic of the side chain-side chain interactions in the context of an aqueous environment is the potential of mean force (PMF) in water. A series of umbrella-sampling molecular dynamic (MD) simulations with the AMBER force field were carried out for pairs of O-phosphorylated serine (pSer), threonine (pThr), and tyrosine, (pTyr) with natural amino acids in a TIP3P water model as a solvent at 298 K. The weighted-histogram analysis method was used to calculate the four-dimensional potentials of mean force. The results demonstrate that the positions and depths of the contact minima and the positions and heights of the desolvation maxima, including their dependence on the relative orientation depend on the character of the interacting pairs. More distinct minima are observed for oppositely charged pairs such as, e.g., O-phosphorylated side-chains and positively charged ones, such as the side-chains of lysine and arginine.

Chelating ability and biological activity of hesperetin Schiff base.

Hydrazone hesperetin Schiff base (HHSB) - N-[(±)-[5,7-dihydroxy-2-(3-hydroxy-4-methoxy-phenyl)chroman-4-ylidene]amino]benzamide has been synthesized and its crystal structure was determined. This compound was used for the formation of Cu(II) complexes in solid state and in solution which were characterized using different spectroscopic methods. The analyses of potentiometric titration curves revealed that monomeric and dimeric complexes of Cu(II) are formed above pH7. The ESI-MS (electrospray ionization-mass spectrometry) spectra confirmed their formation. The EPR and UV-visible spectra evidenced the involvement of oxygen and nitrogen atoms in Cu(II) coordination. Hydrazone hesperetin Schiff base can show keto-enol tautomerism and coordinate Cu(II) in the keto (O(-), N, Oket) and in the enolate form (O(-), N, O(-)enol). The semi-empirical molecular orbital method PM6 and DFT (density functional theory) calculations have revealed that the more stable form of the dimeric complex is that one in which the ligand is present in the enol form. The CuHHSB complex has shown high efficiency in the cleavage of plasmid DNA in aqueous solution, indicating its potential as chemical nuclease. Studies on DNA interactions, antimicrobial and cytotoxic activities have been undertaken to gain more information on the biological significance of HHSB and copper(II)-HHSB chelate species.

A unified coarse-grained model of biological macromolecules based on mean-field multipole-multipole interactions.

A unified coarse-grained model of three major classes of biological molecules--proteins, nucleic acids, and polysaccharides--has been developed. It is based on the observations that the repeated units of biopolymers (peptide groups, nucleic acid bases, sugar rings) are highly polar and their charge distributions can be represented crudely as point multipoles. The model is an extension of the united residue (UNRES) coarse-grained model of proteins developed previously in our laboratory. The respective force fields are defined as the potentials of mean force of biomacromolecules immersed in water, where all degrees of freedom not considered in the model have been averaged out. Reducing the representation to one center per polar interaction site leads to the representation of average site-site interactions as mean-field dipole-dipole interactions. Further expansion of the potentials of mean force of biopolymer chains into Kubo's cluster-cumulant series leads to the appearance of mean-field dipole-dipole interactions, averaged in the context of local interactions within a biopolymer unit. These mean-field interactions account for the formation of regular structures encountered in biomacromolecules, e.g., α-helices and ß-sheets in proteins, double helices in nucleic acids, and helicoidally packed structures in polysaccharides, which enables us to use a greatly reduced number of interacting sites without sacrificing the ability to reproduce the correct architecture. This reduction results in an extension of the simulation timescale by more than four orders of magnitude compared to the all-atom representation. Examples of the performance of the model are presented.

Lessons from application of the UNRES force field to predictions of structures of CASP10 targets.

The performance of the physics-based protocol, whose main component is the United Residue (UNRES) physics-based coarse-grained force field, developed in our laboratory for the prediction of protein structure from amino acid sequence, is illustrated. Candidate models are selected, based on probabilities of the conformational families determined by multiplexed replica-exchange simulations, from the 10th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP10). For target T0663, classified as a new fold, which consists of two domains homologous to those of known proteins, UNRES predicted the correct symmetry of packing, in which the domains are rotated with respect to each other by 180° in the experimental structure. By contrast, models obtained by knowledge-based methods, in which each domain is modeled very accurately but not rotated, resulted in incorrect packing. Two UNRES models of this target were featured by the assessors. Correct domain packing was also predicted by UNRES for the homologous target T0644, which has a similar structure to that of T0663, except that the two domains are not rotated. Predictions for two other targets, T0668 and T0684_D2, are among the best ones by global distance test score. These results suggest that our physics-based method has substantial predictive power. In particular, it has the ability to predict domain-domain orientations, which is a significant advance in the state of the art.

Mean-field interactions between nucleic-acid-base dipoles can drive the formation of a double helix.

A proposed coarse-grained model of nucleic acids demonstrates that average interactions between base dipoles, together with chain connectivity and excluded-volume interactions, are sufficient to form double-helical structures of DNA and RNA molecules. Additionally, local interactions determine helix handedness and direction of strand packing. This result, and earlier research on reduced protein models, suggests that mean-field multipole-multipole interactions are the principal factors responsible for the formation of regular structure of biomolecules.

The unusual stabilization of the Ni2+ and Cu2+ complexes with NSFRY.

The binding mode provided by an unprotected peptide with non-coordinating side-chains is simple and well understood. However, when particular residues are inserted into the peptide sequence, they can have a significant impact on the stability of the formed complexes. The presence of non-bonding side chains of amino acids close to the metal binding centre in the peptide/protein can provide special interactions which result in increasing the stabilization of the formed species. Moreover, these interactions can play a crucial role in generating particular protein structures and in influencing biological activity. In the present paper it is shown how peptides with no specific predisposition for metal binding, like ANF peptides, can form metal complexes with a very high thermodynamic stability. For better understanding this peculiar behavior, a combined pH-metric and spectroscopic method was used to determine the stability and the solution structure of Cu(2+) and Ni(2+) complexes with NSFRY-NH(2) (ANF peptide) and a series of analogue peptides. All obtained data support the hypothesis that the complex-formation process is very similar for both metal ions and all the ligands, involving some intramolecular interactions among the different side chains. The two-dimensional NMR analysis of nickel complexes showed the occurrence of many inter-residue correlations and suggested the presence of a direct interaction between the d electrons of the metal ion and the π-ring system of the aromatic side-chains of the ligand.

Simulation of the opening and closing of Hsp70 chaperones by coarse-grained molecular dynamics.

Heat-shock proteins 70 (Hsp70s) are key molecular chaperones which assist in the folding and refolding/disaggregation of proteins. Hsp70s, which consist of a nucleotide-binding domain (NBD, consisting of NBD-I and NBD-II subdomains) and a substrate-binding domain [SBD, further split into the ß-sheet (SBD-ß) and α-helical (SBD-α) subdomains], occur in two major conformations having (a) a closed SBD, in which the SBD and NBD domains do not interact, (b) an open SBD, in which SBD-α interacts with NBD-I and SBD-ß interacts with the top parts of NBD-I and NBD-II. In the SBD-closed conformation, SBD is bound to a substrate protein, with release occurring after transition to the open conformation. While the transition from the closed to the open conformation is triggered efficiently by binding of adenosine triphosphate (ATP) to the NBD, it also occurs, although less frequently, in the absence of ATP. The reverse transition occurs after ATP hydrolysis. Here, we report canonical and multiplexed replica exchange simulations of the conformational dynamics of Hsp70s using a coarse-grained molecular dynamics approach with the UNRES force field. The simulations were run in the following three modes: (i) with the two halves of the NBD unrestrained relative to each other, (ii) with the two halves of the NBD restrained in an "open" geometry as in the SBD-closed form of DnaK (2KHO), and (iii) the two halves of NBD restrained in a "closed" geometry as in known experimental structures of ATP-bound NBD forms of Hsp70. Open conformations, in which the SBD interacted strongly with the NBD, formed spontaneously during all simulations; the number of transitions was largest in simulations carried out with the "closed" NBD domain, and smallest in those carried out with the "open" NBD domain; this observation is in agreement with the experimentally-observed influence of ATP-binding on the transition of Hsp70's from the SBD-closed to the SBD-open form. Two kinds of open conformations were observed: one in which SBD-α interacts with NBD-I and SBD-ß interacts with the top parts of NBD-I and NBD-II (as observed in the structures of nucleotide exchange factors), and another one in which this interaction pattern is swapped. A third type of motion, in which SBD-α binds to NBD without dissociating from SBD-ß was also observed. It was found that the first stage of interdomain communication (approach of SBD-ß, to NBD) is coupled with the rotation of the long axes of NBD-I and NBD-II towards each other. To the best of our knowledge, this is the first successful simulation of the full transition of an Hsp70 from the SBD-closed to the SBD-open conformation.