Dominant folding pathways of a WW domain

To demonstrate the utility of the coarse-grained united-residue (UNRES) force field to compare experimental and computed kinetic data for folding proteins, we have performed long-time millisecondtimescale canonical Langevin molecular dynamics simulations of the triple β-strand from the Formin binding protein 28 WW domain and six nonnatural variants, using UNRES. The results have been compared with available experimental data in both a qualitative and a quantitative manner. Complexities of the folding pathways, which cannot be determined experimentally, were revealed. The folding mechanisms obtained from the simulated folding kinetics are in agreement with experimental results, with a few discrepancies for which we have accounted. The origins of single-and double-exponential kinetics and their correlations with two-and three-state folding scenarios are shown to be related to the relative barrier heights between the various states. The rate constants obtained from time profiles of the fractions of the native, intermediate, and unfolded structures, and the kinetic equations fitted to them, correlate with the experimental values; however, they are about three orders of magnitude larger than the experimental ones for most of the systems. These differences are in agreement with the timescale extension derived by scaling down the friction of water and averaging out the fast degrees of freedom when passing from all-atom to a coarse-grained representation. Our results indicate that the UNRES force field can provide accurate predictions of folding kinetics of these WW domains, often used as models for the study of the mechanisms of proein folding.FBP28 WW domain | nonnatural variants | folding rates | free-energy landscapes | millisecond-timescale canonical MD simulations R ecent advances in computer simulation techniques have facilitated the direct study of the folding process of small fastfolding proteins, using all-atom force fields (1). However, it is important to validate the simulation methodologies, and the only way to accomplish this is a quantitative comparison with experimental data with proper statistics. The validation of all-atom simulation methodologies is still a major problem because of the differences between the experimental timescale (from multiple microseconds to seconds) and the theoretical one (from hundreds of nanoseconds to microseconds). To overcome this problem, many approximate coarse-grained methods have been developed during the past decade (2-5). One of them makes use of a physics-based united-residue (UNRES) force field developed in our group over the past years (6-14) (SI Appendix, Fig. S1 and SI Materials and Methods).The folding and unfolding rates are among the most accessible quantitative observables for two-and multistate folding proteins; therefore, a study of protein folding kinetics can bridge microscopic motions and the world of experimental measurements. In analyzing protein folding kinetics, the differential rate equations and their integrated forms become more complex as the num...

Section: Significancementioning

confidence: 99%

Folding kinetics of WW domains with the united residue force field for bridging microscopic motions and experimental measurements

Zhou

Maisuradze

Suñol

et al. 2014

“…The DRP method is a path integral-based approach that yields the reaction pathways with the highest probability of being realized within Langevin dynamics. This scheme has been extensively tested against the results of MD protein folding simulations using both reduced (16,17) and realistic atomistic force fields (15). It was then successfully applied to much more complex processes, such as the folding of a natively knotted protein (14).…”

mentioning

confidence: 99%

Serpin latency transition at atomic resolution

Cazzolli

Wang

Beccara

et al. 2014

Self Cite

Protease inhibition by serpins requires a large conformational transition from an active, metastable state to an inactive, stable state. Similar reactions can also occur in the absence of proteases, and these latency transitions take hours, making their time scales many orders of magnitude larger than are currently accessible using conventional molecular dynamics simulations. Using a variational path sampling algorithm, we simulated the entire serpin active-to-latent transition in all-atom detail with a physically realistic force field using a standard computing cluster. These simulations provide a unifying picture explaining existing experimental data for the latency transition of the serpin plasminogen activator inhibitor-1 (PAI-1). They predict a long-lived intermediate that resembles a previously proposed, partially loop-inserted, prelatent state; correctly predict the effects of PAI-1 mutations on the kinetics; and provide a potential means to identify ligands able to accelerate the latency transition. Interestingly, although all of the simulated PAI-1 variants readily access the prelatent intermediate, this conformation is not populated in the active-to-latent transition of another serpin, α 1 -antitrypsin, which does not readily go latent. Thus, these simulations also help elucidate why some inhibitory serpin families are more conformationally labile than others. T he serpin plasminogen activator inhibitor 1 (PAI-1) negatively regulates blood clot clearance (fibrinolysis) by mechanically inhibiting important serine proteases, including tissue type plasminogen activator and urokinase type plasminogen activator (1). Suicide inhibition, initiated by proteolytic cleavage of the PAI-1 reactive center loop (RCL), requires insertion of the cleaved RCL into the central β-sheet (sheet A). This process expands sheet A, inhibits the covalently attached protease by mechanical disruption of the active site (2), and results in a thermodynamically stable serpin conformation. Alternatively, PAI-1 can spontaneously deactivate by inserting its intact, uncleaved RCL into sheet A, resulting in the more stable but inactive latent conformation (1) (Fig. 1).The latency transition provides a facile way to regulate PAI-1 activity. Physiologically, this regulation is achieved by binding to the cell adhesion factor vitronectin, leading to an ∼50% increase in the active state t 1/2 (3). Because high levels of active PAI-1 are associated both with poor prognoses for some cancers, presumably due to interactions with vitronectin, and with cardiovascular diseases (1), PAI-1 inhibitors that accelerate the latency transition are under development (1, 4, 5). However, drug design efforts are hampered by the lack of detailed molecular mechanisms for PAI-1 conformational changes. Numerous studies have identified mutations that either accelerate or retard the conformational transition, as well as antibodies that can accelerate latency. Despite these efforts, the molecular details of the latency transition and the residues involved in the key i...

“…1N), has been shown to fold with biphasic kinetics exhibiting intermediates during folding (3,5,6,(11)(12)(13)(14)(15)(16). We address this problem here with the design of new FBP28 WW domain mutants and by examining their structural properties and folding kinetics.Because of the small size, fast folding kinetics, and biological importance, the formation of intermolecular β-sheets is thought to be a crucial event in the initiation and propagation of amyloid diseases, such as Alzheimer's disease, and spongiform encephalopathy, FBP28, and other WW domain proteins (e.g., Pin1 and FiP35) have been the subjects of extensive experimental (4,11,(17)(18)(19)(20)(21)(22)(23) and theoretical (3,5,6,(12)(13)(14)(15)(16)(24)(25)(26)(27) studies. However, a folding mechanism of the FBP28 was debatable for a long time because of its complexity.…”

mentioning

confidence: 99%

Preventing fibril formation of a protein by selective mutation

Maisuradze

Medina

Kachlishvili

et al. 2015

The origins of formation of an intermediate state involved in amyloid formation and ways to prevent it are illustrated with the example of the Formin binding protein 28 (FBP28) WW domain, which folds with biphasic kinetics. Molecular dynamics of protein folding trajectories are used to examine local and global motions and the time dependence of formation of contacts between C α s and C β s of selected pairs of residues. Focus is placed on the WT FBP28 WW domain and its six mutants (L26D, L26E, L26W, E27Y, T29D, and T29Y), which have structures that are determined by high-resolution NMR spectroscopy. The origins of formation of an intermediate state are elucidated, viz. as formation of hairpin 1 by a hydrophobic collapse mechanism causing significant delay of formation of both hairpins, especially hairpin 2, which facilitates the emergence of an intermediate state. It seems that three-state folding is a major folding scenario for all six mutants and WT. Additionally, two-state and downhill folding scenarios were identified in ∼15% of the folding trajectories for L26D and L26W, in which both hairpins are formed by the Matheson-Scheraga mechanism much faster than in three-state folding. These results indicate that formation of hairpins connecting two antiparallel β-strands determines overall folding. The correlations between the local and global motions identified for all folding trajectories lead to the identification of the residues making the main contributions in the formation of the intermediate state. The presented findings may provide an understanding of protein folding intermediates in general and lead to a procedure for their prevention.A n intermediate state in protein folding is involved in amyloid fibril formation, which is responsible for a number of neurodegenerative diseases (1-7). Therefore, prevention of the aggregation of folding intermediates is one of the most important problems to surmount. Hence, it is necessary to determine the mechanism by which an intermediate state is formed. For example, one of the members of the WW domain family (8, 9), the triple β-stranded WW domain from the Formin binding protein 28 (FBP28; Protein Data Bank ID code 1E0L) (10) (Fig. 1N), has been shown to fold with biphasic kinetics exhibiting intermediates during folding (3,5,6,(11)(12)(13)(14)(15)(16). We address this problem here with the design of new FBP28 WW domain mutants and by examining their structural properties and folding kinetics.Because of the small size, fast folding kinetics, and biological importance, the formation of intermolecular β-sheets is thought to be a crucial event in the initiation and propagation of amyloid diseases, such as Alzheimer's disease, and spongiform encephalopathy, FBP28, and other WW domain proteins (e.g., Pin1 and FiP35) have been the subjects of extensive experimental (4,11,(17)(18)(19)(20)(21)(22)(23) and theoretical (3,5,6,(12)(13)(14)(15)(16)(24)(25)(26)(27) studies. However, a folding mechanism of the FBP28 was debatable for a long time because of its complexity. There ar...