Molecular dynamics simulation and coarse-grained analysis of the Arp2/3 complex

A molecular dynamics investigation and coarse-grained analysis of inactivated actin-related protein (Arp) 2/3 complex is presented. It was found that the nucleotide binding site within Arp3 remained in a closed position with bound ATP or ADP, but opened when simulation with no nucleotide was performed. In contrast, simulation of the isolated Arp3 subunit with bound ATP, showed a fast opening of the nucleotide binding cleft. A homology model for the missing subdomains 1 and 2 of Arp2 was constructed, and it was also found that the Arp2 binding cleft remained closed with bound nucleotide. Within the nucleotide binding cleft a distinct opening and closing period of 10 ns was observed in many of the simulations of Arp2/3 as well as isolated Arp3. Substitution studies were employed, and several alanine substitutions were found to induce a partial opening of the ATP binding cleft in Arp3 and Arp2, whereas only a single substitution was found to induce opening of the ADP binding cleft. It was also found that the nucleotide type did not cause a substantial change on interfacial contacts between Arp3 and the ArpC2, ArpC3 and ArpC4 subunits. Nucleotide-free Arp3 had generally less stable contacts, but the overall contact architecture was constant. Finally, nucleotide-dependent coarse-grained models for Arp3 are developed that serve to further highlight the structural differences induced in Arp3 by nucleotide hydrolysis.     

Insights into the influence of nucleotides on actin family proteins from seven structures of Arp2/3 complex

ATP is required for nucleation of actin filament branches by Arp2/3 complex, but the influence of ATP binding and hydrolysis are poorly understood. We determined crystal structures of bovine Arp2/3 complex cocrystallized with various bound adenine nucleotides and cations. Nucleotide binding favors closure of the nucleotide-binding cleft of Arp3, but no large-scale conformational changes in the complex. Thus, ATP binding does not directly activate Arp2/3 complex but is part of a network of interactions that contribute to nucleation. We compared nucleotide-induced conformational changes of residues lining the cleft in Arp3 and actin structures to construct a movie depicting the proposed ATPase cycle for the actin family. Chemical crosslinking stabilized subdomain 1 of Arp2, revealing new electron density for 69 residues in this subdomain. Steric clashes with Arp3 appear to be responsible for intrinsic disorder of subdomains 1 and 2 of Arp2 in inactive Arp2/3 complex.     

Nucleotide effects on the structure and dynamics of actin

Adenosine 5'-triphosphate or ATP is the primary energy source within the cell, releasing its energy via hydrolysis into adenosine 5'-diphosphate or ADP. Actin is an important ATPase involved in many aspects of cellular function, and the binding and hydrolysis of ATP regulates its polymerization into actin filaments as well as its interaction with a host of actin-associated proteins. Here we study the dynamics of monomeric actin in ATP, ADP-Pi, and ADP states via molecular dynamics simulations. As observed in some crystal structures we see that the DNase-I loop is an alpha-helix in the ADP state but forms an unstructured coil domain in the ADP-Pi and ATP states. We also find that this secondary structure change is reversible, and by mimicking nucleotide exchange we can observe the transition between the helical and coil states. Apart from the DNase-I loop, we also see several key structural differences in the nucleotide binding cleft as well as in the hydrophobic cleft between subdomains 1 and 3 where WH2-containing proteins have been shown to interact. These differences provide a structural basis for understanding the observed differences between the various nucleotide states of actin and provide some insight into how ATP regulates the interaction of actin with itself and other proteins.     

Stability and dynamics of G-actin: back-door water diffusion and behavior of a subdomain 3/4 loop.

Molecular dynamics simulations have been performed on solvated G-actin bound to ADP and ATP, starting with the crystal structure of the actin-DNase 1 complex, including a Ca2+ or Mg2+ ion at the high-affinity divalent cation-binding site. Water molecules have been found to enter the nucleotide-binding site (phosphate vicinity) along two pathways, from the side where the nucleotide base is exposed to water, as well as from the opposite side. The water channels suggest a "back-door" mechanism for ATP hydrolysis in which the phosphate is released to a side opposite that of nucleotide binding and unbinding. The simulations also reveal a propensity of G-actin to alter its crystallographic structure toward the filamentous structure. Domain movement closes the nucleotide cleft, the movement being more pronounced for bound Mg2+. The conformational change is interpreted as a response of the system to missing water molecules in the crystal structure. The structures arising in the simulations, classified according to nucleotide cleft separation and radius of gyration of the protein, fall into two distinct clusters: a cluster of states that are similar to the G-actin crystal structure, and a cluster of states with small cleft separation and with the subdomain 3/4 loop 264-273 detached from the protein. The latter states resemble the putative filamentous structure of actin, in which the loop connects the two strands of the actin filament.     

Crystal structure of monomeric actin in the ATP state. Structural basis of nucleotide-dependent actin dynamics

A nucleotide-dependent conformational change regulates actin filament dynamics. Yet, the structural basis of this mechanism remains controversial. The x-ray crystal structure of tetramethylrhodamine-5-maleimide-actin with bound AMPPNP, a non-hydrolyzable ATP analog, was determined to 1.85-A resolution. A comparison of this structure to that of tetramethylrhodamine-5-maleimide-actin with bound ADP, determined previously under similar conditions, reveals how the release of the nucleotide gamma-phosphate sets in motion a sequence of events leading to a conformational change in subdomain 2. The side chain of Ser-14 in the catalytic site rotates upon Pi release, triggering the rearrangement of the loop containing the methylated His-73, referred to as the sensor loop. This in turn causes a transition in the DNase I-binding loop in subdomain 2 from a disordered loop in ATP-actin to an ordered alpha-helix in ADP-actin. Despite this conformational change, the nucleotide cleft remains closed in ADP-actin, similar to ATP-actin. An analysis of the existing structures of members of the actin superfamily suggests that the cleft is open in the nucleotide-free state.     

A Nucleotide State-sensing Region on Actin

The nucleotide state of actin (ATP, ADP-P_i, or ADP) is known to impact its interactions with other actin molecules upon polymerization as well as with multiple actin binding proteins both in the monomeric and filamentous states of actin. Recently, molecular dynamics simulations predicted that a sequence located at the interface of subdomains 1 and 3 (W-loop; residues 165–172) changes from an unstructured loop to a β-turn conformation upon ATP hydrolysis (Zheng, X., Diraviyam, K., and Sept, D. (2007) Biophys. J. 93, 1277–1283). This region participates directly in the binding to other subunits in F-actin as well as to cofilin, profilin, and WH2 domain proteins and, therefore, could contribute to the nucleotide sensitivity of these interactions. The present study demonstrates a reciprocal communication between the W-loop region and the nucleotide binding cleft on actin. Point mutagenesis of residues 167, 169, and 170 and their site-specific labeling significantly affect the nucleotide release from the cleft region, whereas the ATP/ADP switch alters the fluorescence of probes located in the W-loop. In the ADP-P_i state, the W-loop adopts a conformation similar to that in the ATP state but different from the ADP state. Binding of latrunculin A to the nucleotide cleft favors the ATP-like conformation of the W-loop, whereas ADP-ribosylation of Arg-177 forces the W-loop into a conformation distinct from those in the ADP and ATP-states. Overall, our experimental data suggest that the W-loop of actin is a nucleotide sensor, which may contribute to the nucleotide state-dependent changes in F-actin and nucleotide state-modulated interactions of both G- and F-actin with actin-binding proteins.     

Structure and Dynamics of the Actin Filament

We used all-atom molecular dynamics simulations to investigate the structure and properties of the actin filament, starting with either the recent Oda model or the older Holmes model. Simulations of monomeric and polymerized actin show that polymerization changes the nucleotide-binding cleft, bringing together the Q137 side chain and bound ATP in a way that may enhance the ATP hydrolysis rate in the filament. Simulations with different bound nucleotides and conformations of the DNase I binding loop show that the persistence length of the filament depends only on loop conformation. Computational modeling reveals how bound phalloidin stiffens actin filaments and inhibits the release of γ-phosphate from ADP-P_i actin.     

Nucleotide dependent motion and mechanism of action of p97/VCP

The AAA (ATPases associated with a variety of cellular activities) family of proteins bind, hydrolyze, and release ATP to effect conformational changes, assembly, or disassembly upon their binding partners and substrate molecules. One of the members of this family, the hexameric p97/valosin-containing protein p97/VCP, is essential for the dislocation of misfolded membrane proteins from the endoplasmic reticulum. Here, we observe large motions and dynamic changes of p97/VCP as it proceeds through the ATP hydrolysis cycle. The analysis is based on crystal structures of four representative ATP hydrolysis states: APO, AMP-PNP, hydrolysis transition state ADP x AlF3, and ADP bound. Two of the structures presented herein, ADP and AMP-PNP bound, are new structures, and the ADP x AlF3 structure was re-refined to higher resolution. The largest motions occur at two stages during the hydrolysis cycle: after, but not upon, nucleotide binding and then following nucleotide release. The motions occur primarily in the D2 domain, the D1 alpha-helical domain, and the N-terminal domain, relative to the relatively stationary and invariant D1alpha/beta domain. In addition to the motions, we observed a transition from a rigid state to a flexible state upon loss of the gamma-phosphate group, and a further increase in flexibility within the D2 domains upon nucleotide release. The domains within each protomer of the hexameric p97/VCP deviate from strict 6-fold symmetry, with the more flexible ADP state exhibiting greater asymmetry compared to the relatively rigid ADP x AlF3 state, suggesting a mechanism of action in which hydrolysis and conformational changes move about the hexamer in a processive fashion.     

Rapid exchange of actin-bound nucleotide in perfused rat heart

In the rat heart the actin-bound nucleotide contained both ATP and ADP. The ratio of bound ATP to bound ADP depended on the functional state of the heart; it was higher in hearts stopped reversibly in diastole (low Ca(2+), high Mg(2+), or high K(+)), than in stimulated (inotropic agents or pacing) hearts. Immunoblotting and gel electrophoresis showed the existence of G-actin (30% of total actin) in the cytoplasm of the heart. Pure actin was isolated from rat hearts: in G-actin the bound nucleotide readily exchanged with ATP or ADP, and in F-actin the bound nucleotide did not exchange with ATP or ADP. The free and bound nucleotides were separated in the intact heart by extraction with 75% methanol at -15 degrees C. In rat hearts perfused with (32)P-labeled orthophosphate the actin-bound nucleotide rapidly exchanged with the cytoplasmic ATP. The full exchange of the bound ATP was immediate, whereas the full exchange of the bound ADP was slower. The full exchange of the bound ATP was independent of the heartbeat frequency, whereas the full exchange of the bound ADP was frequency dependent. The data suggest that the transformation of actin monomer-ATP to actin polymer-ADP is a part of the normal contraction-relaxation cycle of the rat heart.     

Mapping the Structural and Dynamical Features of Kinesin Motor Domains

Kinesin motor proteins drive intracellular transport by coupling ATP hydrolysis to conformational changes that mediate directed movement along microtubules. Characterizing these distinct conformations and their interconversion mechanism is essential to determining an atomic-level model of kinesin action. Here we report a comprehensive principal component analysis of 114 experimental structures along with the results of conventional and accelerated molecular dynamics simulations that together map the structural dynamics of the kinesin motor domain. All experimental structures were found to reside in one of three distinct conformational clusters (ATP-like, ADP-like and Eg5 inhibitor-bound). These groups differ in the orientation of key functional elements, most notably the microtubule binding α4–α5, loop8 subdomain and α2b-β4-β6-β7 motor domain tip. Group membership was found not to correlate with the nature of the bound nucleotide in a given structure. However, groupings were coincident with distinct neck-linker orientations. Accelerated molecular dynamics simulations of ATP, ADP and nucleotide free Eg5 indicate that all three nucleotide states could sample the major crystallographically observed conformations. Differences in the dynamic coupling of distal sites were also evident. In multiple ATP bound simulations, the neck-linker, loop8 and the α4–α5 subdomain display correlated motions that are absent in ADP bound simulations. Further dissection of these couplings provides evidence for a network of dynamic communication between the active site, microtubule-binding interface and neck-linker via loop7 and loop13. Additional simulations indicate that the mutations G325A and G326A in loop13 reduce the flexibility of these regions and disrupt their couplings. Our combined results indicate that the reported ATP and ADP-like conformations of kinesin are intrinsically accessible regardless of nucleotide state and support a model where neck-linker docking leads to a tighter coupling of the microtubule and nucleotide binding regions. Furthermore, simulations highlight sites critical for large-scale conformational changes and the allosteric coupling between distal functional sites.     

Crystal structures of Escherichia coli RecA in complex with MgADP and MnAMP-PNP

RecA catalyzes the DNA pairing and strand-exchange steps of homologous recombination, an important mechanism for repair of double-stranded DNA breaks. The binding of RecA to DNA is modulated by adenosine nucleotides. ATP increases the affinity of RecA for DNA, while ADP decreases the affinity. Previously, the crystal structures of E. coli RecA and its complex with ADP have been determined to resolutions of 2.3 and 3.0 A, respectively, but the model for the RecA-ADP complex did not include magnesium ion or side chains. Here, we have determined the crystal structures of RecA in complex with MgADP and MnAMP-PNP, a nonhydrolyzable analogue of ATP, at resolutions of 1.9 and 2.1 A, respectively. Both crystals grow in the same conditions and have RecA in a right-handed helical form with a pitch of approximately 82 A. The crystal structures show the detailed interactions of RecA with the nucleotide cofactors, including the metal ion and the gamma phosphate of AMP-PNP. There are very few conformational differences between the structures of RecA bound to ADP and AMP-PNP, which differ from uncomplexed RecA only in a slight opening of the P-loop residues 66-73 upon nucleotide binding. To interpret the functional significance of the structure of the MnAMP-PNP complex, a coprotease assay was used to compare the ability of different nucleotides to promote the active, extended conformation of RecA. Whereas ATPgammaS and ADP-AlF(4) facilitate a robust coprotease activity, ADP and AMP-PNP do not activate RecA at all. We conclude that the crystal structure of the RecA-MnAMP-PNP complex represents a preisomerization state of the RecA protein that exists after ATP has bound but before the conformational transition to the active state.     

Effects of Nucleotide and End-Dependent Actin Conformations on Polymerization

The regulation of actin is key for controlled cellular function. Filaments are regulated by actin-binding proteins, but the nucleotide state of actin is also an important factor. From extended molecular dynamics simulations, we find that both nucleotide states of the actin monomer have significantly less twist than their crystal structures and that the ATP monomer is flatter than the ADP form. We also find that the filament’s pointed end is flatter than the remainder of the filament and has a conformation distinct from G-actin, meaning that incoming monomers would need to undergo isomerization that would weaken the affinity and slow polymerization. Conversely, the barbed end of the filament takes on a conformation nearly identical to the ATP monomer, enhancing ATP G-actin’s ability to polymerize as compared with ADP G-actin. The thermodynamic penalty imposed by differences in isomerization for the ATP and ADP growth at the barbed end exactly matches experimental results.     

The structural basis of myosin V processive movement as revealed by electron cryomicroscopy

The processive motor myosin V has a relatively high affinity for actin in the presence of ATP and, thus, offers the unique opportunity to visualize some of the weaker, hitherto inaccessible, actin bound states of the ATPase cycle. Here, electron cryomicroscopy together with computer-based docking of crystal structures into three-dimensional (3D) reconstructions provide the atomic models of myosin V in both weak and strong actin bound states. One structure shows that ATP binding opens the long cleft dividing the actin binding region of the motor domain, thus destroying the strong binding actomyosin interface while rearranging loop 2 as a tether. Nucleotide analogs showed a second new state in which the lever arm points upward, in a prepower-stroke configuration (lever arm up) bound to actin before phosphate release. Our findings reveal how the structural elements of myosin V work together to allow myosin V to step along actin for multiple ATPase cycles without dissociating.     

Structural basis for profilin-mediated actin nucleotide exchange

Actin is a ubiquitous eukaryotic protein that is responsible for cellular scaffolding, motility, and division. The ability of actin to form a helical filament is the driving force behind these cellular activities. Formation of a filament depends on the successful exchange of actin's ADP for ATP. Mammalian profilin is a small actin binding protein that catalyzes the exchange of nucleotide and facilitates the addition of an actin monomer to a growing filament. Here, crystal structures of profilin-actin have been determined to show an actively exchanging ATP. Structural analysis shows how the binding of profilin to the barbed end of actin causes a rotation of the small domain relative to the large domain. This conformational change is propagated to the ATP site and causes a shift in nucleotide loops, which in turn causes a repositioning of Ca(2+) to its canonical position as the cleft closes around ATP. Reversal of the solvent exposure of Trp356 is also involved in cleft closure. In addition, secondary calcium binding sites were identified.     

Three myosin V structures delineate essential features of chemo-mechanical transduction

The molecular motor, myosin, undergoes conformational changes in order to convert chemical energy into force production. Based on kinetic and structural considerations, we assert that three crystal forms of the myosin V motor delineate the conformational changes that myosin motors undergo upon detachment from actin. First, a motor domain structure demonstrates that nucleotide-free myosin V adopts a specific state (rigor-like) that is not influenced by crystal packing. A second structure reveals an actomyosin state that favors rapid release of ADP, and differs from the rigor-like state by a P-loop rearrangement. Comparison of these structures with a third structure, a 2.0 angstroms resolution structure of the motor bound to an ATP analog, illuminates the structural features that provide communication between the actin interface and nucleotide-binding site. Paramount among these is a region we name the transducer, which is composed of the seven-stranded beta-sheet and associated loops and linkers. Reminiscent of the beta-sheet distortion of the F1-ATPase, sequential distortion of this transducer region likely controls sequential release of products from the nucleotide pocket during force generation.     

A structural pathway for activation of the kinesin motor ATPase

Molecular motors move along actin or microtubules by rapidly hydrolyzing ATP and undergoing changes in filament-binding affinity with steps of the nucleotide hydrolysis cycle. It is generally accepted that motor binding to its filament greatly increases the rate of ATP hydrolysis, but the structural changes in the motor associated with ATPase activation are not known. To identify the conformational changes underlying motor movement on its filament, we solved the crystal structures of three kinesin mutants that decouple nucleotide and microtubule binding by the motor, and block microtubule-activated, but not basal, ATPase activity. Conformational changes in the structures include a disordered loop and helices in the switch I region and a visible switch II loop, which is disordered in wild-type structures. Switch I moved closer to the bound nucleotide in two mutant structures, perturbing water-mediated interactions with the Mg2+. This could weaken Mg2+ binding and accelerate ADP release to activate the motor ATPASE: The structural changes we observe define a signaling pathway within the motor for ATPase activation that is likely to be essential for motor movement on microtubules.     

The real-time monitoring of the thermal unfolding of tetramethylrhodamine-labeled actin

Modification of actin at Cys (374) with tetramethylrhodamine maleimide (TMR-actin) has been used for visualization of actin filaments and to produce high-resolution crystal structures of actin. We show that TMR-actin exhibits a 21% decrease in absorbance at 557 nm upon thermal unfolding, likely due to the movement of TMR to a more hydrophobic environment upon rapid unfolding and protein aggregation. We took advantage of this property to test models of actin protein unfolding. A transition temperature ( T m) of 60.2 +/- 0.2 degrees C for Ca (2+).ATP.TMR-actin was determined using A 557 and agreed with our own determinations employing different techniques and previous work with unlabeled actin. Our data show that the dependence of TMR-actin thermal stability on the bound nucleotide and cations follows a trend of Ca (2+).ATP > Mg (2+).ATP > Ca (2+).ADP > Mg (2+).ADP. The activation energies and frequency factors for the thermal unfolding of TMR-actin determined with two methods were in good agreement with those previously determined for unlabeled actin. We observed a biphasic trend in the T m of TMR-actin with increasing nucleotide concentrations, supporting a two-pathway model for actin protein unfolding where one pathway dominates at different concentrations of nucleotide. Additionally, TMR-actin bound by DNase I or gelsolin segment-1 exhibited elevated transition temperatures.     

Activation of Arp2/3 complex: addition of the first subunit of the new filament by a WASP protein triggers rapid ATP hydrolysis on Arp2

In response to activation by WASP-family proteins, the Arp2/3 complex nucleates new actin filaments from the sides of preexisting filaments. The Arp2/3-activating (VCA) region of WASP-family proteins binds both the Arp2/3 complex and an actin monomer and the Arp2 and Arp3 subunits of the Arp2/3 complex bind ATP. We show that Arp2 hydrolyzes ATP rapidly—with no detectable lag—upon nucleation of a new actin filament. Filamentous actin and VCA together do not stimulate ATP hydrolysis on the Arp2/3 complex, nor do monomeric and filamentous actin in the absence of VCA. Actin monomers bound to the marine macrolide Latrunculin B do not polymerize, but in the presence of phalloidin-stabilized actin filaments and VCA, they stimulate rapid ATP hydrolysis on Arp2. These data suggest that ATP hydrolysis on the Arp2/3 complex is stimulated by interaction with a single actin monomer and that the interaction is coordinated by VCA. We show that capping of filament pointed ends by the Arp2/3 complex (which occurs even in the absence of VCA) also stimulates rapid ATP hydrolysis on Arp2, identifying the actin monomer that stimulates ATP hydrolysis as the first monomer at the pointed end of the daughter filament. We conclude that WASP-family VCA domains activate the Arp2/3 complex by driving its interaction with a single conventional actin monomer to form an Arp2–Arp3–actin nucleus. This actin monomer becomes the first monomer of the new daughter filament.     

Nucleotide binding to the homodimeric MJ0796 protein: a computational study of a prokaryotic ABC transporter NBD dimer

Transport by ABC proteins requires a cycle of ATP-driven conformational changes of the nucleotide binding domains (NBDs). We compare three molecular dynamics simulations of dimeric MJ0796: with ATP was present at both NBDs; with ATP at one NBD but ADP at the other; and without any bound ATP. In the simulation with ATP present at both NBDs, the dimeric protein interacts with the nucleotides in a symmetrical manner. However, if ADP is present at one binding site then both NBD-NBD and protein-ATP interactions are enhanced at the opposite site.     

Conformational Itinerary of Pseudomonas aeruginosa 1,6-Anhydro-N-acetylmuramic Acid Kinase during Its Catalytic Cycle

Anhydro-sugar kinases are unique from other sugar kinases in that they must cleave the 1,6-anhydro ring of their sugar substrate to phosphorylate it using ATP. Here we show that the peptidoglycan recycling enzyme 1,6-anhydro-N-acetylmuramic acid kinase (AnmK) from Pseudomonas aeruginosa undergoes large conformational changes during its catalytic cycle, with its two domains rotating apart by up to 32° around two hinge regions to expose an active site cleft into which the substrates 1,6-anhydroMurNAc and ATP can bind. X-ray structures of the open state bound to a nonhydrolyzable ATP analog (AMPPCP) and 1,6-anhydroMurNAc provide detailed insight into a ternary complex that forms preceding an operative Michaelis complex. Structural analysis of the hinge regions demonstrates a role for nucleotide binding and possible cross-talk between the bound ligands to modulate the opening and closing of AnmK. Although AnmK was found to exhibit similar binding affinities for ATP, ADP, and AMPPCP according to fluorescence spectroscopy, small angle x-ray scattering analyses revealed that AnmK adopts an open conformation in solution in the absence of ligand and that it remains in this open state after binding AMPPCP, as we had observed for our crystal structure of this complex. In contrast, the enzyme favored a closed conformation when bound to ADP in solution, consistent with a previous crystal structure of this complex. Together, our findings show that the open conformation of AnmK facilitates binding of both the sugar and nucleotide substrates and that large structural rearrangements must occur upon closure of the enzyme to correctly align the substrates and residues of the enzyme for catalysis.