A classical and ab initio study of the interaction of the myosin triphosphate binding domain with ATP.

We used classical molecular mechanics (MM) simulations and quantum mechanical (QM) structural relaxations to examine the active site of myosin when bound to ATP. Two conformations of myosin have been determined by x-ray crystallography. In one, there is no direct interaction between switch 2 and the nucleotide (open state). In the other (closed state), the universally conserved switch 2 glycine forms a hydrogen bond with a gamma-phosphate oxygen. MM simulations indicate that the two states are thermodynamically stable and allow us to investigate the extent to which the P-loop, switch 1, and switch 2 are involved in hydrolysis. We find that the open structure has a higher affinity for ATP than the closed structure, and that ATP is distorted toward a transition state by interactions with the protein. We also examine how the structure of the binding site changes with either MgATP or CaATP as the nucleotide in myosin in the open conformer. Our analyses suggest that higher CaATPase rates occur because the leaving phosphate (P(i)) group is more weakly bound and dissociation occurs faster. Finally, we validate the use of a particular formulation of a QM methodology (Car-Parrinello) to further refine the structures of the active site.     

EPR spectra and molecular dynamics agree that the nucleotide pocket of myosin V is closed and that it opens on binding actin

We have used EPR spectroscopy and computational modeling of nucleotide-analog spin probes to investigate conformational changes at the nucleotide site of myosin V. We find that, in the absence of actin, the mobility of a spin-labeled diphosphate analog [spin-labeled ADP (SLADP)] bound at the active site is strongly hindered, suggesting a closed nucleotide pocket. The mobility of the analog increases when the MV·SLADP complex (MV = myosin V) binds to actin, implying an opening of the active site in the A·MV·SLADP complex (A = actin). The probe mobilities are similar to those seen with myosin II, despite the fact that myosin V has dramatically altered kinetics. Molecular dynamics (MD) simulation was used to understand the EPR spectra in terms of the X-ray database. The X-ray structure of MV·ADP·BeFx shows a closed nucleotide site and has been proposed to be the detached state. The MV·ADP structure shows an open nucleotide site and has been proposed to be the A·MV·ADP state at the end of the working powerstroke. MD simulation of SLADP docked in the closed conformation gave a probe mobility comparable to that seen in the EPR spectrum of the MV·SLADP complex. The simulation of the open conformation gave a probe mobility that was 35-40° greater than that observed experimentally for the A·MV·SLADP state. Thus, EPR, X-ray diffraction, and computational analysis support the closed conformation as a myosin V state that is detached from actin. The MD results indicate that the MV·ADP crystal structure, which may correspond to the strained actin-bound post-powerstroke conformation resulting from head-head interaction in the dimeric processive motor, is superopened.     

Crystal structure analysis,covalent docking,and molecular dynamics calculations reveal a conformational switch in PhaZ7 PHB depolymerase

An open and a closed conformation of a surface loop in PhaZ7 extracellular poly(3‐hydroxybutyrate) depolymerase were identified in two high‐resolution crystal structures of a PhaZ7 Y105E mutant. Molecular dynamics (MD) simulations revealed high root mean square fluctuations (RMSF) of the 281–295 loop, in particular at residue Asp289 (RMSF 7.62 Å). Covalent docking between a 3‐hydroxybutyric acid trimer and the catalytic residue Ser136 showed that the binding energy of the substrate is significantly more favorable in the open loop conformation compared to that in the closed loop conformation. MD simulations with the substrate covalently bound depicted 1 Å RMSF higher values for the residues 281–295 in comparison to the apo (substrate‐free) form. In addition, the presence of the substrate in the active site enhanced the ability of the loop to adopt a closed form. Taken together, the analysis suggests that the flexible loop 281–295 of PhaZ7 depolymerase can act as a lid domain to control substrate access to the active site of the enzyme. Proteins 2017; 85:1351–1361. © 2017 Wiley Periodicals, Inc.     

Stabilization of Helical Order in the Thick Filaments by Blebbistatin: Further Evidence of Coexisting Multiple Conformations of Myosin

The degree of helical order of the thick filament of mammalian skeletal muscle is highly dependent on temperature and the nature of the ligand. Previously, we showed that there was a close correlation between the conformation of the myosin heads on the surface of the thick filaments and the extent of their helical order. Helical order required the heads to be in the closed conformation. In addition, we showed that, with the same ligand bound at the active site, three conformations of myosin coexisted in equilibrium. Hitherto, however, there was no detectable helical order as measured by x-ray diffraction under the temperatures studied for myosin with MgADP and the nucleotide-free myosin, raising the possibility that the concept of multiple conformations has limited validity. In this study, blebbistatin was used to stabilize the closed conformation of myosin. The degree of helical order is substantially improved with MgATP at low temperature or with MgADP or in the absence of nucleotide. The thermodynamic parameters of the disorder↔order transition and the characteristics of the ordered array were not significantly altered by binding blebbistatin. The simplest explanation is that the binding of blebbistatin increases the proportion of myosin in the closed conformation from being negligible to substantial. These results provide further evidence for the coexistence of multiple conformations of myosin under a wide range of conditions and for the closed conformation being directly coupled to helical order.     

Switch I closure simultaneously promotes strong binding to actin and ADP in smooth muscle myosin

The motor protein myosin uses energy derived from ATP hydrolysis to produce force and motion. Important conserved components (P-loop, switch I, and switch II) help propagate small conformational changes at the active site into large scale conformational changes in distal regions of the protein. Structural and biochemical studies have indicated that switch I may be directly responsible for the reciprocal opening and closing of the actin and nucleotide-binding pockets during the ATPase cycle, thereby aiding in the coordination of these important substrate-binding sites. Smooth muscle myosin has displayed the ability to simultaneously bind tightly to both actin and ADP, although it is unclear how both substrate-binding clefts could be closed if they are rigidly coupled to switch I. Here we use single tryptophan mutants of smooth muscle myosin to determine how conformational changes in switch I are correlated with structural changes in the nucleotide and actin-binding clefts in the presence of actin and ADP. Our results suggest that a closed switch I conformation in the strongly bound actomyosin-ADP complex is responsible for maintaining tight nucleotide binding despite an open nucleotide-binding pocket. This unique state is likely to be crucial for prolonged tension maintenance in smooth muscle.     

Switch II mutants reveal coupling between the nucleotide- and actin-binding regions in myosin V

Conserved active-site elements in myosins and other P-loop NTPases play critical roles in nucleotide binding and hydrolysis; however, the mechanisms of allosteric communication among these mechanoenzymes remain unresolved. In this work we introduced the E442A mutation, which abrogates a salt-bridge between switch I and switch II, and the G440A mutation, which abolishes a main-chain hydrogen bond associated with the interaction of switch II with the γ phosphate of ATP, into myosin V. We used fluorescence resonance energy transfer between mant-labeled nucleotides or IAEDANS-labeled actin and FlAsH-labeled myosin V to examine the conformation of the nucleotide- and actin-binding regions, respectively. We demonstrate that in the absence of actin, both the G440A and E442A mutants bind ATP with similar affinity and result in only minor alterations in the conformation of the nucleotide-binding pocket (NBP). In the presence of ADP and actin, both switch II mutants disrupt the formation of a closed NBP actomyosin.ADP state. The G440A mutant also prevents ATP-induced opening of the actin-binding cleft. Our results indicate that the switch II region is critical for stabilizing the closed NBP conformation in the presence of actin, and is essential for communication between the active site and actin-binding region.     

Cofactor assisted gating mechanism in the active site of NADH oxidase from Thermus thermophilus

NADH oxidase (NOX) from Thermus thermophilus is a member of a structurally homologous flavoprotein family of nitroreductases and flavin reductases. The importance of local conformational dynamics in the active site of NOX has been recently demonstrated. The enzyme activity was increased by 250% in the presence of 1 M urea with no apparent perturbation of the native structure of the protein. The present in silico results correlate with the in vitro data and suggest the possible explanation about the effect of urea on NOX activity at the molecular level. Both, X-ray structure and molecular dynamics (MD) simulations, show open conformation of the active site represented by approximately 0.9 nm distance between the indole ring of Trp47 and the isoalloxazine ring of FMN412. In this conformation, the substrate molecule can bind in the active site without sterical restraints. MD simulations also indicate more stable conformation of the active site called "closed" conformation. In this conformation, Trp47 and the isoalloxazine ring of FMN412 are so close to each other (approximately 0.5 nm) that the substrate molecule is unable to bind between them without perturbing this conformation. The open/close transition of the active site between Trp47 and the flavin ring is accompanied by release of the "tightly" bound water molecule from the active site--cofactor assisted gating mechanism. The presence of urea in aqueous solutions of NOX prohibits closing of the active site and even unlocks the closed active site because of the concomitant binding of a urea molecule in the active site cavity. The binding of urea in the active site is stabilized by formation of one/two persistent hydrogen bonds involving the carbonyl group of the urea molecule. Our report represents the first MD study of an enzyme from the novel flavoprotein family of nitroreductases and flavin reductases. The common occurrence of aromatic residues covering the active sites in homologous enzymes suggests the possibility of a general gating mechanism and the importance of local dynamics within this flavoprotein family.     

Dynamic reorganization of the motor domain of myosin subfragment 1 in different nucleotide states.

Atomic models of the myosin motor domain with different bound nucleotides have revealed the open and closed conformations of the switch 2 element [Geeves, M.A. & Holmes, K.C. (1999) Annu. Rev. Biochem.68, 687-728]. The two conformations are in dynamic equilibrium, which is controlled by the bound nucleotide. In the present work we attempted to characterize the flexibility of the motor domain in the open and closed conformations in rabbit skeletal myosin subfragment 1. Three residues (Ser181, Lys553 and Cys707) were labelled with fluorophores and the probes identified three fluorescence resonance energy transfer pairs. The effect of ADP, ADP.BeFx, ADP.AlF4- and ADP.Vi on the conformation of the motor domain was shown by applying temperature-dependent fluorescence resonance energy transfer methods. The 50 kDa lower domain was found to maintain substantial rigidity in both the open and closed conformations to provide the structural basis of the interaction of myosin with actin. The flexibility of the 50 kDa upper domain was high in the open conformation and further increased in the closed conformation. The converter region of subfragment 1 became more rigid during the open-to-closed transition, the conformational change of which can provide the mechanical basis of the energy transduction from the nucleotide-binding pocket to the light-chain-binding domain.     

Early stages of the recovery stroke in myosin II studied by molecular dynamics simulations

The recovery stroke is a key step in the functional cycle of muscle motor protein myosin, during which pre-recovery conformation of the protein is changed into the active post-recovery conformation, ready to exersice force. We study the microscopic details of this transition using molecular dynamics simulations of atomistic models in implicit and explicit solvent. In more than 2 μs of aggregate simulation time, we uncover evidence that the recovery stroke is a two-step process consisting of two stages separated by a time delay. In our simulations, we directly observe the first stage at which switch II loop closes in the presence of adenosine triphosphate at the nucleotide binding site. The resulting configuration of the nucleotide binding site is identical to that detected experimentally. Distribution of inter-residue distances measured in the force generating region of myosin is in good agreement with the experimental data. The second stage of the recovery stroke structural transition, rotation of the converter domain, was not observed in our simulations. Apparently it occurs on a longer time scale. We suggest that the two parts of the recovery stroke need to be studied using separate computational models.     

Effects of protonation state of Asp181 and position of active site water molecules on the conformation of PTP1B

In protein tyrosine phosphatase 1B (PTP1B), the flexible WPD loop adopts a closed conformation (WPD_closed) in the active state of PTP1B, bringing the catalytic Asp181 close to the active site pocket, while WPD loop is in an open conformation (WPD_open) in the inactive state. Previous studies showed that Asp181 may be protonated at physiological pH, and ordered water molecules exist in the active site. In the current study, molecular dynamics simulations are employed at different Asp181 protonation states and initial positions of active site water molecules, and compared with the existing crystallographic data of PTP1B. In WPD_closed conformation, the active site is found to maintain its conformation only in the protonated state of Asp181 in both free and liganded states, while Asp181 is likely to be deprotonated in WPD_open conformation. When the active site water molecule network that is a part of the free WPD_closed crystal structure is disrupted, intermediate WPD loop conformations, similar to that in the PTPRR crystal structure, are sampled in the MD simulations. In liganded PTP1B, one active site water molecule is found to be important for facilitating the orientation of Cys215 and the phosphate ion, thus may play a role in the reaction. In conclusion, conformational stability of WPD loop, and possibly catalytic activity of PTP1B, is significantly affected by the protonation state of Asp181 and position of active site water molecules, showing that these aspects should be taken into consideration both in MD simulations and inhibitor design. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Molecular Dynamics Simulation Studies of dTTP Binding and Catalysis Mediated by YhdE Dimerization

YhdE is a Maf-like (multicopy associated filamentation) protein that primarily acts as dTTPase to hydrolyze dTTP into dTMP and two phosphate molecules in cell metabolism pathway. Two crystal structures of YhdE have been previously determined, representing the open and closed active site conformations, respectively. Based on the structures, we have carried out molecular dynamics simulations and free energy calculations to investigate dTTP binding to and hydrolysis by YhdE. Our results suggest that YhdE closed state is structurally more compact than its open state at room temperature. YhdE open state is a favorable conformation for dTTP binding and closed state is a structurally favorable conformation for catalytic reaction. This observation is supported by the structure of YhdE homolog in complex with a nucleotide analog. Free energy calculations reveal that YhdE dimerization occurs preferentially in dTTP binding and is favorable for successive cooperative reaction. The key residues R11, R12 and K80, are found to contribute to the substrate stabilization. Further, YhdE dimerization and binding of dTTP induce the cooperative effect through a direct allosteric communication network in YhdE from the dTTP binding sites in the catalytic center to the intermolecular β-strand in YhdE dimer.     

Inhibitory effect of ATP analogs and actin on the modification of myosin subfragment 1 with 9-anthroylnitrile

The fluorescent probe, 9-anthroylnitrile (ANN), can selectively attach to Ser-180 at the ATP-binding site of subfragment 1 (S1) of skeletal muscle myosin [J. Biol. Chem. 278 (2003) 31891]. We have found that MgATP, MgATPgammaS, MgADP.AlF(4) or MgPP(i), but not MgADP, inhibit the incorporation of ANN into S1. The inhibitory effect of the nucleotide gamma-phosphate group (or its analog) on the modification of S1 with ANN can be explained by the contribution of Ser-180 to the binding of the nucleotide gamma-phosphate at the active site of S1. We have also observed that the incorporation of ANN into S1.MgADP complex is inhibited by actin. These experimental data strongly support the existence of nucleotide-promoted conformational changes revealed by crystal structures of S1 complexes with various nucleotide analogs. They also convincingly show an effect of actin on the environment of Ser-180 at the nucleotide binding site of S1.     

Monomerization alters the dynamics of the lid region in Campylobacter jejuni CstII: an MD simulation study

CstII, a bifunctional (α2,3/8) sialyltransferase from Campylobacter jejuni, is a homotetramer. It has been reported that mutation of the interface residues Phe121 (F121D) or Tyr125 (Y125Q) leads to monomerization and partial loss of enzyme activity, without any change in the secondary or tertiary structures. MD simulations of both tetramer and monomer, with and without bound donor substrate, were performed for the two mutants and WT to understand the reasons for partial loss of activity due to monomerization since the active site is located within each monomer. RMSF values were found to correlate with the crystallographic B-factor values indicating that the simulations are able to capture the flexibility of the molecule effectively. There were no gross changes in either the secondary or tertiary structure of the proteins during MD simulations. However, interface is destabilized by the mutations, and more importantly the flexibility of the lid region (Gly152-Lys190) is affected. The lid region accesses three major conformations named as open, intermediate, and closed conformations. In both Y121Q and F121D mutants, the closed conformation is accessed predominantly. In this conformation, the catalytic base His188 is also displaced. Normal mode analysis also revealed differences in the lid movement in tetramer and monomer. This provides a possible explanation for the partial loss of enzyme activity in both interface mutants. The lid region controls the traffic of substrates and products in and out of the active site, and the dynamics of this region is regulated by tetramerization. Thus, this study provides valuable insights into the role of loop dynamics in enzyme activity of CstII.     

Combining EPR with Fluorescence Spectroscopy to Monitor Conformational Changes at the Myosin Nucleotide Pocket

We used spin-labeled nucleotide analogs and fluorescence spectroscopy to monitor conformational changes at the nucleotide-binding site of wild-type Dictyostelium discoideum (WT) myosin and a construct containing a single tryptophan at position F239 near the switch 1 loop. Electron paramagnetic resonance (EPR) spectroscopy and tryptophan fluorescence have been used previously to investigate changes at the myosin nucleotide site. A limitation of fluorescence spectroscopy is that it must be done on mutated myosins containing only a single tryptophan. A limitation of EPR spectroscopy is that one infers protein conformational changes from alterations in the mobility of an attached probe. These limitations have led to controversies regarding conclusions reached by the two approaches. For the first time, the data presented here allow direct correlations to be made between the results from the two spectroscopic approaches on the same proteins and extend our previous EPR studies to a nonmuscle myosin. EPR probe mobility indicates that the conformation of the nucleotide pocket of the WT⋅SLADP (spin-labeled ADP) complex is similar to that of skeletal myosin. The pocket is closed in the absence of actin for both diphosphate and triphosphate nucleotide states. In the actin⋅myosin⋅diphosphate state, the pocket is in equilibrium between closed and open conformations, with the open conformation slightly more favorable than that seen for fast skeletal actomyosin. The EPR spectra for the mutant show similar conformations to skeletal myosin, with one exception: in the absence of actin, the nucleotide pocket of the mutant displays an open component that was approximately 4-5 kJ/mol more favorable than in skeletal or WT myosin. These observations resolve the controversies between the two techniques. The data from both techniques confirm that binding of myosin to actin alters the conformation of the myosin nucleotide pocket with similar but not identical energetics in both muscle and nonmuscle myosins.     

A structural model for actin-induced nucleotide release in myosin

Myosins are molecular motor proteins that harness the chemical energy stored in ATP to produce directed force along actin filaments. Complex communication pathways link the catalytic nucleotide-binding region, the structures responsible for force amplification and the actin-binding domain of myosin. We have crystallized the nucleotide-free motor domain of myosin II in a new conformation in which switch I and switch II, conserved loop structures involved in nucleotide binding, have moved away from the nucleotide-binding pocket. These movements are linked to rearrangements of the actin-binding region, which illuminate a previously unobserved communication pathway between the nucleotide-binding pocket and the actin-binding region, explain the reciprocal relationship between actin and nucleotide affinity and suggest a new mechanism for product release in myosin family motors.     

X-ray Crystallographic and MD Simulation Studies on the Mechanism of Interfacial Activation of a Family I.3 Lipase with Two Lids

The interfacial activation mechanism of family I.3 lipase from Pseudomonas sp. MIS38 (PML), which has two α-helical lids (lid1 and lid2), was investigated using a combination of X-ray crystallography and molecular dynamics (MD) simulation. The crystal structure of PML in an open conformation was determined at 2.1 Å resolution in the presence of Ca²⁺ and Triton X-100. Comparison of this structure with that in the closed conformation indicates that both lids greatly change their positions and lid1 is anchored by the calcium ion (Ca1) in the open conformation. This structure was not seriously changed even when the protein was dialyzed extensively against the Ca²⁺-free buffer containing Triton X-100 before crystallization, indicating that the open conformation is fairly stable unless a micellar substance is removed. The crystal structure of the PML derivative, in which the active site serine residue (Ser207) is diethylphosphorylated by soaking the crystal of PML in the open conformation in a solution containing diethyl p-nitrophenyl phosphate, was also determined. This structure greatly resembles that in the open conformation, indicating that PML structure in the open conformation represents that in the active form. MD simulation of PML in the open conformation in the absence of micelles showed that lid2 closes first, while lid1 maintains its open conformation. Likewise, MD simulation of PML in the closed conformation in the absence of Ca²⁺ and in the presence of octane or trilaurin micelles showed that lid1 opens, while lid2 remains closed. These results suggest that Ca1 functions as a hook for stabilization of a fully opened conformation of lid1 and for initiation of subsequent opening of lid2.     

Closed conformation of the active site loop of rabbit muscle triosephosphate isomerase in the absence of substrate: evidence of conformational heterogeneity

The active site loop of triosephosphate isomerase (TIM) exhibits a hinged-lid motion, alternating between the two well defined "open" and "closed" conformations. Until now the closed conformation had only been observed in protein complexes with substrate analogues. Here, we present the first rabbit muscle apo TIM structure, refined to 1.5A resolution, in which the active site loop is either in the open or in the closed conformation in different subunits of the enzyme. In the closed conformation described here, the lid loop residues participate in stabilizing hydrogen bonds characteristic of holo TIM structures, whereas chemical interactions observed in the open loop conformation are similar to those found in the apo structures of TIM. In the closed conformation, a number of water molecules are observed at the projected ligand atom positions that are hydrogen bonded to the active site residues. Additives used during crystallization (DMSO and Tris molecules and magnesium atoms) were modeled in the electron density maps. However, no specific binding of these molecules is observed at, or close to, the active site and the lid loop. To further investigate this unusual closed conformation of the apo enzyme, two more rabbit muscle TIM structures, one in the same and another in a different crystal form, were determined. These structures present the open lid conformation only, indicating that the closed conformation cannot be explained by crystal contact effects. To rationalize why the active site loop is closed in the absence of ligand in one of the subunits, extensive comparison with previously solved TIM structures was carried out, supported by the bulk of available experimental information about enzyme kinetics and reaction mechanism of TIM. The observation of both open and closed lid conformations in TIM crystals might be related to a persistent conformational heterogeneity of this protein in solution.     

Resolution of conformational states of spin-labeled myosin during steady-state ATP hydrolysis

We have measured the conventional electron paramagnetic resonance (EPR) spectrum of spin-labeled myosin filaments as a function of the nucleotide occupancy of the active site of the enzyme. The probe used was 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1-oxyl (IASL), which reacts specifically with sulfhydryl 1 of the myosin head. In the absence of nucleotide, the probe remains strongly immobilized (rigidly attached to the myosin head) so that no nanosecond rotational motions are detectable. When MgADP is added to IASL-labeled myosin filaments (T = 20 degrees C), the probe mobility increases slightly. During steady-state MgADP hydrolysis (T = 20 degrees C), the probe undergoes large-amplitude nanosecond rotational motion. These results are consistent with previous studies of myosin monomers, heavy meromyosin, and myosin subfragment 1. Isoclinic points observed in overlays of sequential EPR spectra recorded during ATP hydrolysis strongly suggest that the probes fall into two motional classes, separated by approximately an order of magnitude in effective rotational correlation time. Both of the observed states are distinct from the conformation of myosin in the absence of nucleotides, and the spectrum of the less mobile population is indistinguishable from that observed in the presence of MgADP. The addition of ADP and vanadate to IASL-myosin gives rise to two motional classes virtually identical with those observed in the presence of ATP, but the relative concentrations of the spin populations are significantly different. We have quantitated the percentage of myosin in each motional state during ATP hydrolysis. The result agrees well with the predicted percentages in the two predominant chemical states in the myosin ATPase cycle. Spectra obtained in the presence of nucleotide analogues permit us to assign the conformational states to specific chemical states. We propose that the two motional classes represent two distinct local conformations of myosin that are in exchange with one another during the ATP hydrolysis reaction cycle.