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• W4256249882 abstract "Actin filament growth and disassembly, as well as affinity for actin-binding proteins, is mediated by the nucleotide-bound state of the component actin monomers. The structural differences between ATP-actin and ADP-actin, however, remain controversial. We expressed a cytoplasmic actin in Sf9 cells, which was rendered non-polymerizable by virtue of two point mutations in subdomain 4 (A204E/P243K). This homogeneous monomer, called AP-actin, was crystallized in the absence of toxins, binding proteins, or chemical modification, with ATP or ADP at the active site. The two surface mutations do not perturb the structure. Significant differences between the two states are confined to the active site region and sensor loop. The active site cleft remains closed in both states. Minor structural shifts propagate from the active site toward subdomain 2, but dissipate before reaching the DNase binding loop (D-loop), which remains disordered in both the ADP and ATP states. This result contrasts with previous structures of actin made monomeric by modification with tetramethylrhodamine, which show formation of an α-helix at the distal end of the D-loop in the ADP-bound but not the ATP-bound form (Otterbein, L. R., Graceffa, P., and Dominguez, R. (2001) Science 293, 708-711). Our reanalysis of the TMR-modified actin structures suggests that the nucleotide-dependent formation of the D-loop helix may result from signal propagation through crystal packing interactions. Whereas the observed nucleotide-dependent changes in the structure present significantly different surfaces on the exterior of the actin monomer, current models of the actin filament lack any actin-actin interactions that involve the region of these key structural changes. Actin filament growth and disassembly, as well as affinity for actin-binding proteins, is mediated by the nucleotide-bound state of the component actin monomers. The structural differences between ATP-actin and ADP-actin, however, remain controversial. We expressed a cytoplasmic actin in Sf9 cells, which was rendered non-polymerizable by virtue of two point mutations in subdomain 4 (A204E/P243K). This homogeneous monomer, called AP-actin, was crystallized in the absence of toxins, binding proteins, or chemical modification, with ATP or ADP at the active site. The two surface mutations do not perturb the structure. Significant differences between the two states are confined to the active site region and sensor loop. The active site cleft remains closed in both states. Minor structural shifts propagate from the active site toward subdomain 2, but dissipate before reaching the DNase binding loop (D-loop), which remains disordered in both the ADP and ATP states. This result contrasts with previous structures of actin made monomeric by modification with tetramethylrhodamine, which show formation of an α-helix at the distal end of the D-loop in the ADP-bound but not the ATP-bound form (Otterbein, L. R., Graceffa, P., and Dominguez, R. (2001) Science 293, 708-711). Our reanalysis of the TMR-modified actin structures suggests that the nucleotide-dependent formation of the D-loop helix may result from signal propagation through crystal packing interactions. Whereas the observed nucleotide-dependent changes in the structure present significantly different surfaces on the exterior of the actin monomer, current models of the actin filament lack any actin-actin interactions that involve the region of these key structural changes. ATP hydrolysis drives actin filament dynamics. The effect known as treadmilling arises from the preferential addition of ATP-actin monomers to the plus or barbed end of actin and the preferential dissociation of ADP-actin from the minus or pointed end. Hydrolysis of ATP occurs after the monomer is incorporated into the filament. In addition, actin-binding proteins often have significantly different affinities for the ADP-bound versus ATP-bound forms of actin. Both of these lines of evidence strongly suggest that there must be significant structural changes in actin induced by ATP hydrolysis. Indirect solution techniques such as proteolytic digestion rates and fluorescence studies (reviewed in Ref. 1Dominguez R. Graceffa P. Biophys. J. 2003; 85: 2073-2074Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar) are in agreement with conformational differences between the ADP and ATP states, but direct structural evidence has been lacking until recently. Based on crystal structures of a tetramethylrhodamine-labeled monomeric actin (TMR-actin) 2The abbreviations used are: TMR-actin, tetramethylrhodamine-Cys374-labeled monomeric actin; AP-actin, actin-A204E/P243K; ε-ATP, etheno-ATP; DTT, dithiothreitol; RMSD, root mean square deviation; AMPPNP, adenosine 5′-(β,γ-imino)triphosphate. 2The abbreviations used are: TMR-actin, tetramethylrhodamine-Cys374-labeled monomeric actin; AP-actin, actin-A204E/P243K; ε-ATP, etheno-ATP; DTT, dithiothreitol; RMSD, root mean square deviation; AMPPNP, adenosine 5′-(β,γ-imino)triphosphate. with ADP (2Otterbein L.R. Graceffa P. Dominguez R. Science. 2001; 293: 708-711Crossref PubMed Scopus (411) Google Scholar) or AMP-PNP (3Graceffa P. Dominguez R. J. Biol. Chem. 2003; 278: 34172-34180Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) at the active site, Dominguez and co-workers proposed the provocative idea that ATP hydrolysis initiates a series of changes originating at the active site, that ultimately cause a loop-to-helix transition in the DNase binding loop in subdomain 2 of actin (“D-loop,” residues in subdomain 2 of actin which comprise part of the DNase I binding site). They suggested that this was the long sought after change in structure between ADP and ATP actin. The cleft between subdomains 2 and 4 of actin remained closed in both nucleotide states. Despite this observation, an opposing point of view (4Sablin E.P. Dawson J.F. VanLoock M.S. Spudich J.A. Egelman E.H. Fletterick R.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10945-10947Crossref PubMed Scopus (58) Google Scholar) held that the more important conformational change is an opening of the cleft upon ATP hydrolysis, a change that would be compatible with that observed for other nucleotide hydrolyzing proteins. Docking of actin crystal structures into negatively stained images of F-actin was also consistent with the idea that ADP-actin had a more open cleft than the triphosphate state (5Belmont L.D. Orlova A. Drubin D.G. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 29-34Crossref PubMed Scopus (139) Google Scholar). If the latter view is correct, one would have to suppose that the modification of Cys374 by TMR stabilized the closed conformation and inactivated the nucleotide sensing mechanism by virtue of the binding of rhodamine between subdomains 1 and 3, a potential hinge region (6Page R. Lindberg U. Schutt C.E. J. Mol. Biol. 1998; 280: 463-474Crossref PubMed Scopus (84) Google Scholar) of the molecule. It has been suggested (4Sablin E.P. Dawson J.F. VanLoock M.S. Spudich J.A. Egelman E.H. Fletterick R.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10945-10947Crossref PubMed Scopus (58) Google Scholar) that the short helix observed in the crystal structure of the ADP state of TMR-actin could be formed as a result of contacts with neighboring molecules in the crystal. We provide further evidence to support this idea, and suggest a pathway through which nucleotide-dependent changes may propagate through fortuitous crystal packing interactions from one monomer to the D-loop region of another in the TMR-actin crystals. Here we crystallized and expressed a cytoplasmic actin that was rendered incapable of polymerization by virtue of two surface mutations in subdomain 4 (A204E/P243K). This strategy negates concerns raised about the TMR modification of actin. De novo crystallization of AP-actin with either ATP or ADP at the active site reveals obligatory nucleotide-dependent conformational changes that are localized to the active site and the so-called “sensor loop” (residues 71-73 in actin, Ref. 3Graceffa P. Dominguez R. J. Biol. Chem. 2003; 278: 34172-34180Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), and propagate only short distances from there. A helix is not observed in the D-loop in either the ADP- or ATP-bound states, and the cleft remains closed in both states. Our data show that a change in the D-loop conformation and/or a change in cleft disposition is not an obligatory nucleotide-dependent change in the actin monomer. Expression and Purification of AP-actin—A modified plasmid pAcUW2B containing the coding sequence for the Drosophila 5C actin gene was a generous gift from Volkman et al. (7Volkman L. Storm K. Aivazachvili V. Oppenheimer D. Virology. 1996; 225: 369-376Crossref PubMed Scopus (19) Google Scholar). Site-directed mutagenesis was used to create a mutant construct of the 5C actin in which Ala204 was replaced by Glu and Pro243 was replaced by Lys (AP-actin). The numbering system used here corresponds to that of skeletal muscle actin. 5C actin is a cytoplasmic actin, and thus it has one less acidic residue at the N terminus compared with striated muscle actins, which have four. Drosophila 5C actin is 98.7% identical with human γ-cytoplasmic actin. Infection of Sf9 cells with recombinant baculovirus encoding the AP-actin construct, lysis of the cells, and dialysis of the cell lysate have been described (8Joel P.B. Fagnant P.M. Trybus K.M. Biochemistry. 2004; 43: 11554-11559Crossref PubMed Scopus (44) Google Scholar). The dialysate was fractionated on a Q-Sepharose column (1.5 × 30 cm for a 4 billion cell culture preparation) using a 500-ml gradient from 0.1 to 0.4 m KCl (10 mm imidazole, pH 7.5, 0.1 mm CaCl2, 0.5 mm DTT, 0.5 mm Na2ATP, 1 μg/ml leupeptin). Pooled fractions from the Q Sepharose were concentrated with an Amicon Ultacentrifugal filter device (Millipore Corp.) and applied to a Sephacryl S300 column as described (8Joel P.B. Fagnant P.M. Trybus K.M. Biochemistry. 2004; 43: 11554-11559Crossref PubMed Scopus (44) Google Scholar). Some preparations were run on a second Q-Sepharose column following the Sephacryl S300 column. The final preparation of AP-actin was dialyzed into G buffer (5 mm Tris-HCl, pH 8.2 at 4 °C, 0.2 mm CaCl2, 0.1 mm sodium azide, 0.5 mm DTT, 1 μg/ml leupeptin, and 0.2 mm Na2ATP or Na2ADP and stored in liquid nitrogen. AP-actin concentration was determined from the absorbance at 290 nm by use of an extinction coefficient of 0.63 ml mg-1 cm-1. Chicken skeletal muscle actin was prepared as described in Ref. 9Pardee J.D. Spudich J.A. Methods Enzymol. 1982; 85: 164-181Crossref PubMed Scopus (980) Google Scholar. Conversion of ATP-actin to ADP-actin—To prepare AP-actin with ADP in the active site, AP-actin dialyzed into G buffer with Na2ADP was diluted to 9 or 12 mg/ml with G Buffer. ADP was increased to 0.5 mm. 4 mm MgCl2 and 0.5 mm EGTA were added, and the solution was incubated 10 min on ice. 2 mm glucose and 40 units/ml hexokinase (Sigma) were added, and the solution was incubated on ice for at least 40 h (10Pollard T.D. J. Cell Biol. 1986; 103: 2747-2754Crossref PubMed Scopus (596) Google Scholar). The solution was then centrifuged 20 min at 300,000 × g, the concentration was adjusted to 7 or 10 mg/ml, and 5 mm DTT was added. To prepare AP-actin with ATP in the active site, AP-actin dialyzed into G buffer with Na2ATP was treated the same as the AP-actin-ADP except that ATP rather than ADP was added, and no hexokinase was used. Nucleotide Hydrolysis—Free external nucleotides were removed from CaATP-AP-actin in G buffer by addition of 10% by volume of a 50% slurry of Dowex AG-1 × 8. The suspension was mixed 1 min on ice and centrifuged 20 s at 10,000 × g. The Dowex treatment was repeated two more times. The final supernatant was centrifuged 20 min at 300,000 × g. The CaATP-AP-actin was adjusted to 4 mg/ml. A portion of this protein was converted to MgATP-AP-actin by addition of 0.2 mm MgCl2 and 0.5 mm EGTA. To determine the rate of nucleotide hydrolysis, samples were incubated on ice, and 50-μl samples were taken at various time intervals. To liberate nucleotide from the active site of actin and precipitate the protein, 50 μlof 10% perchloric acid was added to the sample. After 5 min on ice, 20 μl of 4 m potassium acetate in 10 m KOH was added to neutralize the solution, and the sample was centrifuged for 10 min at 12,000 × g followed by 10 min at 300,000 × g. The supernatant was diluted to 200 μl with water and loaded on a 1-ml Mono Q HR5/5 column (Amersham Biosciences) equilibrated with 5 mm triethylammonium bicarbonate pH 8.5 (Sigma-Aldrich) using an AKTA-FPLC system (GE Healthcare). The nucleotides were eluted with a 10-ml gradient of 5 mm to 0.5 m triethylammonium bicarbonate pH 8.5, followed by an additional 4 ml of 0.5 m triethylammonium bicarbonate pH 8.5. ATP and ADP standards were 25 μm. Nucleotide Exchange—The rate of nucleotide exchange in AP-actin and in tissue-purified skeletal G-actin was determined by measuring the decrease in fluorescence on release of etheno-ATP (ε-ATP) (Molecular Probes) from actin. Unbound ATP was removed from 5 μm actin by addition of 10% by volume of 50% Dowex AG-1 × 8 followed by centrifugation to remove the resin. 100 μm ε-ATP was then added to the actin solution, and the solution was left on ice for 2 h to allow ε-ATP to exchange into the active site. The actin solution was treated with Dowex AG-1 × 8 to remove unbound nucleotide. The fluorescence of bound ε-ATP (2 μm actin, 5 mm Hepes, pH 7.5, 50 μm CaCl2, 1 mm DTT, 20 °C) was measured in a fluorometer (model K2, ISS, Inc; 340 nm excitation, 415 nm emission). 100 μm Na2ATP was added, and the decrease in fluorescence as ATP was exchanged for ε-ATP was followed as a function of time. Data were fitted to a single exponential. Subtilisin Digestion—Skeletal G-actin and AP-actin (10 μm) were cleaved by subtilisin at an enzyme/protein mass ratio of 1:1500 at 25 °C, as described in Ref. 11Kudryashov D.S. Reisler E. Biophys. J. 2003; 85: 2466-2475Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar. ATP-actin and ADP-actin were prepared as described in a previous section. Crystallization—Crystals of AP-actin (10-11 mg/ml in G buffer) were first obtained by vapor diffusion at 4 °C. The protein was mixed with an equal volume of reservoir buffer composed of 25% 2-methyl-2,4-pentanediol, 100 mm NaAc, pH 4.8, 100 mm NaCl, 40 mm CaCl2. Optimal crystals were obtained by several rounds of microseeding. Preparation of ADP-actin or ATP-actin for microseeding is described above. The composition of the reservoir buffer for the microseeding experiments was 20% 2-methyl-2,4-pentanediol, 100 mm NaAc, pH 5.4, 100 mm NaCl, 20 mm CaCl2 for ADP-actin crystals, and the same solution but at pH 5.2 for the ATP-actin crystals. Crystals were flash frozen in liquid nitrogen directly from the crystallization buffer. Structure Determination and Refinement—Diffraction data were collected on a Rigaku RUH3R generator and MAR 345 detector at a temperature of 100K maintained by a CryoIndustries cryocooler. Crystals of ADP- and ATP-bound forms of AP-actin belong to space group C2, with cell parameters a = 199.7 Å, b = 54.07 Å, c = 39.59 Å, and β = 93.16°. Data collection statistics are provided in Table 1. The ATP-bound diffraction data and refinement statistics are of higher quality despite a higher overall B because these data were collected using Xenocs multilayer optics, while data for the ADP-bound crystal were collected using double mirror optics.TABLE 1Data collection and model refinement statisticsAP-Actin·ADPAP-Actin·ATPData Collection:Resolution20-1.8 Å20-1.8 ÅRmergeaRmerge = Σ(|Ii – 〈I 〉|)/Σ(I), where Σ is over all reflections measured more than once, and 〈I 〉 is the mean intensity of all measured observations equivalent to reflection Ii8.0% (39.0%)bValues in parentheses are for the highest resolution shell (1.86-1.80Å)6.6% (27.6%)No. unique reflections3678937194Coverage98.4% (86.8%)99.7% (97.3%)Mean redundancy3.53.9〈I 〉/ 〈σ 〉15.1 (2.1)20.7 (2.5)Riso on I (/on F)cRiso on I = Σ(|IATP – IADP|/Σ 0.5(IATP + IADP), where Σ is over all reflections for which intensities from both the actin-ADP and actin-ATP crystals are measured. Riso on F is analogous to Riso on I, substituting observed amplitudes for intensities23.9%/17.0%Refinement:No. protein atoms28442843No. nucleotide atoms2731No. calcium ions22No. solvent atoms494434RcrystdRcryst = Σ(|Fobs – Fcalc|)/Σ(Fobs), where Σ is over all reflections used in refinement, Fobs are the observed diffraction amplitudes, and Fcalc are their corresponding calculated amplitudes from inverse Fourier transformation of the model17.5%18.2%RfreeeRfree is defined identically to Rcryst, but involves 9% of the measured reflections not used in refinement and set aside for cross-validation purposes (15). The same set of reflections were set aside from both datasets21.3%21.1%Estimated coordinate errorfEstimated coordinate error is via a Luzzati plot (37) based on cross-validated reflections only0.23 Å0.23 ÅDeviations from ideal stereochemistry:RMSD bonds0.0047 Å0.0047 ÅRMSD angles1.18°1.17°RMSD B-factorsgRMSD in B-factor between covalently-bonded main chain atoms and side chain atoms1.2 Å2/2.0 Å21.2 Å2/2.1 Å2Average B-factorshAverage B-factors for protein/solvent/nucleotide and Ca2+ atoms15.6 Å2/29.7 Å2/11.5 Å220.4 Å2/34.5 Å2/14.9 Å2Ramachandran plot analysis:iRamachandran plot analysis is from PROCHECK (38)Core region93.7%94.3%Allowed region6.3%5.7%Generously allowed region00Disallowed region00a Rmerge = Σ(|Ii – 〈I 〉|)/Σ(I), where Σ is over all reflections measured more than once, and 〈I 〉 is the mean intensity of all measured observations equivalent to reflection Iib Values in parentheses are for the highest resolution shell (1.86-1.80Å)c Riso on I = Σ(|IATP – IADP|/Σ 0.5(IATP + IADP), where Σ is over all reflections for which intensities from both the actin-ADP and actin-ATP crystals are measured. Riso on F is analogous to Riso on I, substituting observed amplitudes for intensitiesd Rcryst = Σ(|Fobs – Fcalc|)/Σ(Fobs), where Σ is over all reflections used in refinement, Fobs are the observed diffraction amplitudes, and Fcalc are their corresponding calculated amplitudes from inverse Fourier transformation of the modele Rfree is defined identically to Rcryst, but involves 9% of the measured reflections not used in refinement and set aside for cross-validation purposes (15Brunger A.T. Methods Enzymol. 1997; 277: 366-396Crossref PubMed Scopus (274) Google Scholar). The same set of reflections were set aside from both datasetsf Estimated coordinate error is via a Luzzati plot (37Luzzati V. Acta Crystallogr. 1952; 5: 802-810Crossref Google Scholar) based on cross-validated reflections onlyg RMSD in B-factor between covalently-bonded main chain atoms and side chain atomsh Average B-factors for protein/solvent/nucleotide and Ca2+ atomsi Ramachandran plot analysis is from PROCHECK (38Laskowski R. MacArthur M. Moss D. Thornton J. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) Open table in a new tab The structure of ADP-bound AP-actin was solved by molecular replacement via EPMR (12Kissinger C.R. Gehlhaar D.K. Fogel D.B. Acta Crystallogr. D. Biol. Crystallogr. 1999; 55: 484-491Crossref PubMed Scopus (690) Google Scholar) using only the protein portion of TMR-Actin-ADP (PDB: 1J6Z) as the search model. All atomic B-factors were reset to 10 Å2 and the model was rebuilt into simulated-annealing Fo-Fc omit maps (T0 = 9000 K) (13Hodel A. Kim S. Brunger A. Acta Crystallographica Section A. 1992; 48: 851-858Crossref Scopus (393) Google Scholar) after rigid-body refinement. Cycles of rebuilding into SA-omit maps using the molecular graphics program vuSette zc (M.A.R.) and refinement with CNS (14Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D. Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar) yielded a final model with statistics given in Table 1. A model of the ADP state above, at an early stage in refinement, with all water molecules and ligands removed and all B-factors reset, served as the initial model for building the ATP-bound state of AP-actin. The model was rebuilt into simulated-annealing omit maps (T0 = 9000 K) and refined as above, independently of the ATP-bound structure. The same set of reflections (about 10% of the total) were set aside from both the ATP- and ADP-actin data sets for cross-validation (i.e. for the “free R-factor” calculation (15Brunger A.T. Methods Enzymol. 1997; 277: 366-396Crossref PubMed Scopus (274) Google Scholar)), prior to any refinement. Because the ATP- and ADP-bound AP-actin crystals are isomorphous (i.e. belong to the same space group, have the same unit cell parameters, and show a close agreement in diffraction amplitudes), we are able to make use of isomorphous differential crystallography to accurately visualize the differences between the two structures. These methods allow a direct comparison of the structures at the level of differences in their electron density, free of model bias (16Rould M.A. Carter C.W. Methods Enzymol. 2003; 374: 145-163Crossref PubMed Scopus (16) Google Scholar). Given that the root mean square deviation (RMSD) after superposition of the two α-carbon backbones is only 0.23 Å and the estimated model coordinate error is also 0.23 Å (Table 1), differential crystallography is essential for the detection of any subtle yet significant propagated structural changes arising from the change in nucleotide state. To generate an isomorphous difference electron density map, the scaled differences between the observed diffraction amplitudes from the ATP- and ADP-bound actin crystals are used as coefficients in a Fourier transform, along with phases calculated from either of the refined models. Positive density (shown as green contours, as in Fig. 4) in the resulting isomorphous difference Fourier map indicates regions where the density in the ATP state is greater than in the ADP state, and vice versa for negative density (red contours). Peaks of positive difference density adjacent to peaks of negative difference density indicate that an atom or group has shifted, with a detection limit of less than a tenth of an angstrom. Further details of the procedure are in Ref. 17Rould M.A. Methods Mol. Biol. 2006; 364 (in press)Google Scholar. The isomorphous character of the AP-actin-ATP and -ADP crystals allows for an additional means of validating the small coordinate shifts seen between the independently refined models of the two structures. After refinement is complete, the two models and their corresponding diffraction data are swapped and re-refined; that is, a new “swapped” AP-actin-ATP model is refined starting from the final refined AP-actin-ADP coordinates, and vice versa. Crystallographic refinement of atomic positions and individual B-factors via CNS (14Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D. Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar) is applied to convergence for the swapped models of both states, using the corresponding diffraction amplitudes (i.e. the new swapped ATP-state model is the original ADP-state model refined against the ATP-state diffraction data). Validation of the differences between the ATP- and ADP-bound crystal structures of actin is determined by the degree to which the atomic shift vectors between refined swapped ATP- and ADP-bound models recapitulate the atomic shift vectors between the original (final refined unswapped) models. Quantitatively this is assessed as the vector correlation between ATP-to-ADP atomic shift vectors for the original and swapped models, summed over all α-carbons in Equation 1,Σ(O·S)/(Σ(O·O)×Σ(S·S))0.5(Eq. 1) where the vector O is the coordinate difference of an atom between the original refined ADP-bound and ATP-bound crystal structures, and S is the same coordinate difference after swapping models and re-refining. Intuitively, the dot product O·S represents the extent to which the two vectors point in the same direction, weighted by the product of the magnitude of the two vectors. This swapped-refined-shift-vector-correlation statistic is analogous to the common scalar correlation coefficient and thus has the range of -1 (perfect anti-correlation) to +1 (perfect correlation.) For the ATP- and ADP-bound AP-actin models reported herein, the swapped-refined-shift-vector-correlation is 0.80 (excluding residues 70-73 of the sensor loop, whose shifts are outside the radius of convergence of crystallographic refinement without manual re-building), suggesting that the modeled coordinate differences between the states represent well the true differences, however subtle, present in the crystals. Further details of the procedure are in Ref. 17Rould M.A. Methods Mol. Biol. 2006; 364 (in press)Google Scholar. Biochemical Properties of AP-actin—Two surface mutations in subdomain 4 of actin (A204E/P243K) are sufficient to render actin completely non-polymerizable (8Joel P.B. Fagnant P.M. Trybus K.M. Biochemistry. 2004; 43: 11554-11559Crossref PubMed Scopus (44) Google Scholar). As previously described (8Joel P.B. Fagnant P.M. Trybus K.M. Biochemistry. 2004; 43: 11554-11559Crossref PubMed Scopus (44) Google Scholar), these mutations were chosen based on their presence in the sequence of a non-polymerizable actin-related protein, Arp3 (18Kelleher J.F. Atkinson S.J. Pollard T.D. J. Cell Biol. 1995; 131: 385-397Crossref PubMed Scopus (159) Google Scholar). The mutant actin, named AP-actin for the residues present in the wild-type structure, was expressed in high yield (≥ 60 mg/liter culture) in the baculovirus/insect cell expression system. The actin isoform used here is 98.7% identical with human γ-cytoplasmic actin. Our approach to crystallizing this protein is unique in that it is the first monomeric actin crystallized without chemical cross-linking, toxins, or actin-binding proteins to prevent polymerization. Because AP-actin shows no tendency to polymerize, ATP hydrolysis is very slow. Rapid hydrolysis of actin-bound ATP only occurs after a monomer has added onto the filament (reviewed in (19Carlier M.F. Adv. Biophys. 1990; 26: 51-73Crossref PubMed Scopus (66) Google Scholar)). Analysis of the nucleotide content of AP-actin with Ca2+ as the divalent cation in the active site showed little hydrolysis of CaATP over the course of 72 h. With Mg2+ as the divalent cation, ATP hydrolysis occurred at a rate of 0.025 h-1 (Fig. 1A). This result established that AP-actin is catalytically active, despite being polymerization-incompetent. One proposed explanation for the reduced tendency of CaATP to be hydrolyzed is that the bond distance and angle between the γ-phosphate of ATP and a key water molecule that is poised to act as a nucleophile is more optimally aligned when magnesium is coordinated than when calcium is the divalent cation (20Vorobiev S. Strokopytov B. Drubin D.G. Frieden C. Ono S. Condeelis J. Rubenstein P.A. Almo S.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5760-5765Crossref PubMed Scopus (127) Google Scholar). In contrast, it was necessary to use the non-hydrolyzable analog AMPPNP to crystallize TMR-actin in the nucleotide triphosphate state, presumably because TMR modification does not render the molecule as non-polymerizable as the two surface mutations in AP-actin (21Kudryashov D.S. Phillips M. Reisler E. Biophys. J. 2004; 87: 1136-1145Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The rate of ATP release from the active site of AP-actin is very similar to that of tissue-purified skeletal G-actin, implying that the two point mutations have little effect on this fundamental property of actin (Fig. 1B). Subtilisin digestion between Met47 and Gly48 in subdomain 2 was also used to show that the point mutations had no long range effect on other domains of actin. The time course of the digestion pattern of AP-actin and tissue-purified skeletal actin with ATP at the active site was essentially indistinguishable (Fig. 1C, circles). Moreover, the rate of digestion of both AP-actin and tissue-purified skeletal actin was considerably slower in the ADP than in the ATP state (Fig. 1C, triangles). The slower digestion in ADP has been considered to be evidence that the D-loop undergoes a nucleotide-dependent change in conformation (22Strzelecka-Golaszewska H. Moraczewska J. Khaitlina S.Y. Mossakowska M. Eur. J. Biochem. 1993; 211: 731-742Crossref PubMed Scopus (126) Google Scholar). We also tested whether cofilin would bind more tightly to the ADP than the ATP form of AP-actin, as has been established for skeletal muscle actin (23Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (829) Google Scholar). Gel filtration chromatography in G buffer with 0.2 m NaCl was used to show that ADP-AP-actin forms a stoichiometric complex with cofilin. Under the same conditions, no complex formation was observed with ATP-AP-actin (data not shown). These biochemical assays established that AP-actin retains many important properties of skeletal muscle G-actin, except for its ability to remain monomeric at high concentrations, which was the intended goal of the mutations. Structure of AP-actin with ATP or ADP at the Active Site— AP-actin was first crystallized with CaATP at the active site, the nucleotide that remained bound throughout the purification procedure. Calcium was also a component of the solution used for c" @default.
• W4256249882 created "2022-05-12" @default.
• W4256249882 creator A5035579449 @default.
• W4256249882 creator A5051567182 @default.
• W4256249882 creator A5066067174 @default.
• W4256249882 creator A5084134263 @default.
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• W4256249882 date "2006-10-01" @default.
• W4256249882 modified "2023-10-18" @default.
• W4256249882 title "Crystal Structures of Expressed Non-polymerizable Monomeric Actin in the ADP and ATP States" @default.
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