Effect of Calcium on Calmodulin Bound to the IQ Motifs of Myosin V
2007; Elsevier BV; Volume: 282; Issue: 32 Linguagem: Inglês
10.1074/jbc.m701636200
ISSN1083-351X
AutoresKathleen M. Trybus, Marina I. Gushchin, HongJun Lui, Larnele Hazelwood, Elena B. Krementsova, Niels Volkmann, Dorit Hanein,
Tópico(s)Trace Elements in Health
ResumoThe long neck of unconventional myosin V is composed of six tandem "IQ motifs," which are fully occupied by calmodulin (CaM) in the absence of calcium. Calcium regulates the activity, the folded-to-extended conformational transition, and the processive run length of myosin V, and thus, it is important to understand how calcium affects CaM binding to the IQ motifs. Here we used electron cryomicroscopy together with computer-based docking of crystal structures into three-dimensional reconstructions of actin decorated with a motor domain-two IQ complex to provide an atomic model of myosin V in the presence of calcium. Calcium causes a major rearrangement of the bound CaMs, dissociation of CaM bound to IQ motif 2, and propagated changes in the motor domain. Tryptophan fluorescence spectroscopy showed that calcium-CaM binds to IQ motifs 1, 3, and 5 in a different conformation than apoCaM. Proteolytic cleavage was consistent with CaM preferentially dissociating from the second IQ motif. The enzymatic and mechanical functions of myosin V can, therefore, be modulated both by calcium-dependent conformational changes of bound CaM as well as by CaM dissociation. The long neck of unconventional myosin V is composed of six tandem "IQ motifs," which are fully occupied by calmodulin (CaM) in the absence of calcium. Calcium regulates the activity, the folded-to-extended conformational transition, and the processive run length of myosin V, and thus, it is important to understand how calcium affects CaM binding to the IQ motifs. Here we used electron cryomicroscopy together with computer-based docking of crystal structures into three-dimensional reconstructions of actin decorated with a motor domain-two IQ complex to provide an atomic model of myosin V in the presence of calcium. Calcium causes a major rearrangement of the bound CaMs, dissociation of CaM bound to IQ motif 2, and propagated changes in the motor domain. Tryptophan fluorescence spectroscopy showed that calcium-CaM binds to IQ motifs 1, 3, and 5 in a different conformation than apoCaM. Proteolytic cleavage was consistent with CaM preferentially dissociating from the second IQ motif. The enzymatic and mechanical functions of myosin V can, therefore, be modulated both by calcium-dependent conformational changes of bound CaM as well as by CaM dissociation. Myosin V is a double-headed, processive motor involved in transport of organelles, mRNA, and membrane trafficking (for review, see Ref. 1Reck-Peterson S.L. Provance Jr., D.W. Mooseker M.S. Mercer J.A. Biochim. Biophys. Acta. 2000; 1496: 36-51Crossref PubMed Scopus (243) Google Scholar). A striking feature of this motor is its elongated neck region that is composed of six IQ motifs (consensus sequence IQXXXRGXXXR, where X is any amino acid), each of which binds CaM 3The abbreviations used are: CaM, calmodulin; AVID, absolute values of individual differences; IHRSR, iterative helical real space refinement; MD, motor domain; MD-2IQ, complex containing the motor domain and the adjacent two IQ motifs of murine myosin V; GST, glutathione S-transferase. or a CaM-like light chain. The long neck region enables myosin V to take 36-nm steps on actin as it moves in a hand-over-hand fashion, with communication between the heads coordinated via a strain-dependent mechanism (for reviews, see Refs. 2Trybus K.M. Nat. Cell. Biol. 2005; 7: 854-856Crossref PubMed Scopus (6) Google Scholar and 3Sellers J.R. Veigel C. Curr. Opin. Cell Biol. 2006; 18: 68-73Crossref PubMed Scopus (120) Google Scholar). In addition to its mechanical role, the neck is also involved in regulating myosin V function. In the absence of calcium, all six of the IQ motifs of murine myosin V bind apoCaM (4Wang F. Chen L. Arcucci O. Harvey E.V. Bowers B. Xu Y. Hammer J.A. II I Sellers J.R. J. Biol. Chem. 2000; 275: 4329-4335Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Myosin V adopts a folded, inactive conformation under these conditions provided that cargo is not present (5Krementsov D.N. Krementsova E.B. Trybus K.M. J. Cell Biol. 2004; 164: 877-886Crossref PubMed Scopus (175) Google Scholar, 6Wang F. Thirumurugan K. Stafford W.F. Hammer J.A. II I Knight P.J. Sellers J.R. J. Biol. Chem. 2004; 279: 2333-2336Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 7Li X.D. Mabuchi K. Ikebe R. Ikebe M. Biochem. Biophys. Res. Commun. 2004; 315: 538-545Crossref PubMed Scopus (86) Google Scholar, 8Liu J. Taylor D.W. Krementsova E.B. Trybus K.M. Taylor K.A. Nature. 2006; 442: 208-211Crossref PubMed Scopus (172) Google Scholar, 9Thirumurugan K. Sakamoto T. Hammer J.A. II I Sellers J.R. Knight P.J. Nature. 2006; 442: 212-215Crossref PubMed Scopus (140) Google Scholar). Low calcium concentrations or cargo binding in the absence of calcium unfold and activate the molecule, whereas higher calcium concentrations inhibit motility and processive movement by dissociating CaM from one or more of the IQ motifs (5Krementsov D.N. Krementsova E.B. Trybus K.M. J. Cell Biol. 2004; 164: 877-886Crossref PubMed Scopus (175) Google Scholar, 10Nguyen H. Higuchi H. Nat. Struct. Mol. Biol. 2005; 12: 127-132Crossref PubMed Scopus (50) Google Scholar, 11Lu H. Krementsova E.B. Trybus K.M. J. Biol. Chem. 2006; 281: 31987-31994Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). It is important to know the structure of CaM bound to the IQ motifs in both the apo- and in the calcium-saturated states to fully understand how the neck functions. We have previously developed atomic models for a myosin V motor domain-two IQ complex bound to actin in the absence of calcium and in several nucleotide states that mimic the progression through the ATPase cycle (12Volkmann N. Liu H. Hazelwood L. Krementsova E.B. Lowey S. Trybus K.M. Hanein D. Mol. Cell. 2005; 19: 595-605Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The key results from that study were 1) ATP opens the long cleft dividing the motor domain, providing a mechanism by which the strong binding actomyosin interface is disrupted, 2) loop 2 at the actin interface is rearranged to act as a tether, when myosin is in the weak binding states, and 3) a pre-powerstroke transition state bound to actin was visualized for the first time through the use of nucleotide analogs and improved computational methods. The actin-bound MD-2IQ in the absence of calcium and nucleotide from our previous study (12Volkmann N. Liu H. Hazelwood L. Krementsova E.B. Lowey S. Trybus K.M. Hanein D. Mol. Cell. 2005; 19: 595-605Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) was used for comparison with the results presented here in the presence of calcium. A high resolution crystal structure of apoCaM bound to the first two IQ motifs of murine myosin V was also solved recently, which serves as a model for the entire neck of mammalian myosin V (13Houdusse A. Gaucher J.F. Krementsova E. Mui S. Trybus K.M. Cohen C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 19326-19331Crossref PubMed Scopus (112) Google Scholar). It was unclear how apoCaM could bind an IQ motif, because unbound apoCaM exists with both the N-lobe and the C-lobe in a closed, non-gripping conformation. Calcium binding opens both lobes, allowing CaM to adopt a gripping conformation, which is the typical way that CaM binds to and activates a target peptide (14Meador W.E. Means A.R. Quiocho F.A. Science. 1992; 257: 1251-1255Crossref PubMed Scopus (942) Google Scholar, 15Kuboniwa H. Tjandra N. Grzesiek S. Ren H. Klee C.B. Bax A. Nat. Struct. Biol. 1995; 2: 768-776Crossref PubMed Scopus (618) Google Scholar, 16Zhang M. Tanaka T. Ikura M. Nat. Struct. Biol. 1995; 2: 758-767Crossref PubMed Scopus (651) Google Scholar). The crystal structure revealed that apoCaM adopts a new conformation when bound to the IQ motifs. The C-terminal lobe of each CaM adopts a semi-open conformation that grips the first part of the IQ motif (IQXXXR; X indicates any amino acid), whereas the N-terminal lobe adopts a closed conformation that interacts more weakly with the second part of the motif (GXXXR). A surprising finding was the pivotal role played by the non-consensus residues in the IQ motif, which determine the precise structure of the bound CaM. Each IQ motif is, thus, expected to display some unique interactions with apoCaM, although the overall lobe conformation (semi-open C-lobe, closed N-lobe) remains the same. There has been no structural information to show how calcium affects the binding of CaM to the IQ motifs of myosin V. Here we use electron cryomicroscopy to show that calcium causes the lever arm of actin-bound myosin V to adopt a conformation distinct from that observed when apoCaM is bound to the IQ motifs of myosin V as well as causing propagated changes in the motor domain. Results from fluorescence spectroscopy also show that calcium-CaM bound to an IQ motif peptide adopts a different conformation from bound apoCaM. Microscopy and solution studies implicate IQ motif 2 as the site from which CaM dissociates in the presence of calcium. Calcium, thus, exerts its effect on myosin V function both by changing the conformation of bound CaM as well as by causing CaM dissociation. Protein Preparation—The murine myosin V construct consisting of the motor domain and two CaM binding IQ motifs (MD-2IQ) was truncated at amino acid 820 followed by a C-terminal FLAG epitope (DYKDDDDK) for purification by affinity chromatography. A similar construct containing one IQ motif (MD-1IQ) was truncated at amino acid 795. This construct was used for image processing to get an independent boundary between the two IQ motifs. Three mutant MD-2IQ constructs were also produced. In each of these, the wild-type DNA sequence at the second IQ motif after the motor domain was replaced by the sequence from the first, fourth, or sixth IQ motif of the wild-type molecule. Protein expression in Sf9 cells and purification is described in Krementsov et al. (5Krementsov D.N. Krementsova E.B. Trybus K.M. J. Cell Biol. 2004; 164: 877-886Crossref PubMed Scopus (175) Google Scholar). Chicken skeletal muscle actin was prepared and stored as described (17Volkmann N. Hanein D. Ouyang G. Trybus K.M. DeRosier D.J. Lowey S. Nat. Struct. Biol. 2000; 7: 1147-1155Crossref PubMed Scopus (135) Google Scholar). Adjacent pairs of IQ motifs derived by PCR from the heavy chain of murine myosin V were cloned into pGEX-2T (Amersham Biosciences), creating a fusion protein containing glutathione S-transferase (GST) at the N terminus followed by a thrombin cleavage site. The 1-2 IQ motif pair corresponds to amino acids 765-820 of the murine myosin V heavy chain, the 3-4 IQ pair to amino acids 813-867, and the 5-6 IQ pair to amino acids 861-908. CaM was cloned into a modified kanamycin-resistant vector (T7 RNA polymerase promoter, p15A origin of replication). The CaM-containing vector (kanamycin-resistant) and the IQ-containing pGEX-2T vector (ampicillin-resistant) were co-transformed into the Escherichia coli BL21 (DE3) strain and grown on LB plates containing both antibiotics. Cultures were grown overnight at 37 °C in enriched media (20 g/liter Tryptone, 10 g/liter yeast extract, 5 g/liter NaCl, 2 ml/liter glycerol, and 50 mm potassium phosphate, pH 7.2). The cells were disrupted by sonication. The clarified supernatant was incubated with glutathione-Sepharose 4B affinity matrix for 1 h at 4 °C. The resin-protein mixture was then poured into a column and washed with phosphate-buffered saline. The GST fusion protein was eluted with glutathione (0.154 g of reduced glutathione dissolved in 50 ml of 50 mm Tris-HCl, pH 8.0). The GST was removed by thrombin cleavage (10 units/mg of IQ-CaM complex) followed by a second glutathione-Sepharose 4B affinity column to remove the liberated GST. Electron Microscopy of Actomyosin V Complexes—F-actin was diluted to 30-50 μg/ml with 20 mm NaCl, 5 mm sodium phosphate, pH 7.0, 1 mm MgCl2, 0.1 mm EGTA, 3 mm NaN3 just before application to the glow-discharged 400-mesh copper grids coated with holey carbon film. After a 1-min incubation in a humid chamber, the grids were rinsed twice with 10 mm imidazole, pH 7.0, 10 mm NaCl, 1 mm EGTA, 1.5 mm CaCl2, 1 mm MgCl2, 1 mm dithiothreitol, 3 mm NaN3 (dilution buffer). The myosin sample was preincubated for 30 min on ice in dilution buffer. The myosin sample, diluted to ≈0.1 mg/ml in dilution buffer, was applied to the grid for 30 s and replaced by an additional drop of sample (30 s). Excess liquid was blotted off, and the grids were plunged into liquid ethane cooled by liquid N2. Low-dose images were recorded with a Tecnai G2 T12 electron microscope (FEI Electron Optics, The Netherlands) equipped with a LaB6 filament and a DH626 cryo-holder (Gatan, Pleasaton, CA) at a nominal magnification of 52,000× using 120 keV and a 1.5-μm defocus and with total electron dose of 10 e-/Å2. Micrographs were digitized with a SCAI scanner (Integraph, Phoenix, AZ) with a pixel size of 0.27 nm on the sample. Reconstruction Procedures—Helical reconstructions were obtained for all data sets with the Brandeis Helical Package (18Owen C.H. Morgan D.G. DeRosier D.J. J. Struct. Biol. 1996; 116: 167-175Crossref PubMed Scopus (86) Google Scholar) as described (17Volkmann N. Hanein D. Ouyang G. Trybus K.M. DeRosier D.J. Lowey S. Nat. Struct. Biol. 2000; 7: 1147-1155Crossref PubMed Scopus (135) Google Scholar). All reconstructions included 23 layer lines that were trimmed to 2.1-nm resolution. Because this resolution is within the first zero of the contrast transfer function, no phase correction was necessary. The abrupt edge that was introduced by this procedure was smoothed to zero by using a Gaussian falloff. Only layer lines that were found to be statistically significant in at least one of the individual filaments were used and included orders 2, -11, 4, -9, 6, -7, 8, -5, -3, -1, -12, 3, 14, 1, 5, -8, -4, -2, 7, -6, 13, and the equator. Individual filaments were reconstructed separately, aligned in real space (17Volkmann N. Hanein D. Ouyang G. Trybus K.M. DeRosier D.J. Lowey S. Nat. Struct. Biol. 2000; 7: 1147-1155Crossref PubMed Scopus (135) Google Scholar, 19Hanein D. Matsudaira P. DeRosier D.J. J. Cell Biol. 1997; 139: 387-396Crossref PubMed Scopus (88) Google Scholar), normalized, and averaged. The independent far and near side maps were kept separate and used for cross-validation. The iterative helical real space refinement (IHRSR) method (20Egelman E.H. Ultramicroscopy. 2000; 85: 225-234Crossref PubMed Scopus (387) Google Scholar) is a hybrid approach that uses real-space, single-particle processing and imposition of helical symmetry in an iterative manner. Our implementation uses EMAN (21Ludtke S.J. Baldwin P.R. Chiu W. J. Struct. Biol. 1999; 128: 82-97Crossref PubMed Scopus (2102) Google Scholar) for the single-particle reconstruction portion and routines adapted from the CoAn suite (22Volkmann N. Hanein D. J. Struct. Biol. 1999; 125: 176-184Crossref PubMed Scopus (174) Google Scholar, 23Volkmann N. Hanein D. Methods Enzymol. 2003; 374: 204-225Crossref PubMed Scopus (56) Google Scholar) to determine and impose the helical symmetry. A box size of 80 × 80 pixels with a 0.54 nm pixel size was used. This corresponds to about 15 asymmetric units of the helix, a little more than one actin crossover. An overlap of 60 pixels was chosen, allowing every asymmetric unit to contribute to four different views of the helix. To generate independent IHRSR reconstructions for cross-validation, the data were split into two random halves which where independently reconstructed. Docking and Modeling—At least two independently derived maps were used to cross-validate all docking, modeling, and segmentation results. An atomic model for filamentous actin, based on rigid-body refinement against fiber diffraction data (24Holmes K.C. Angert I. Kull F.J. Jahn W. Schroder R.R. Nature. 2003; 425: 423-427Crossref PubMed Scopus (311) Google Scholar) followed by refinement against constraints from electron microscopy and cross-linking studies (12Volkmann N. Liu H. Hazelwood L. Krementsova E.B. Lowey S. Trybus K.M. Hanein D. Mol. Cell. 2005; 19: 595-605Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), was docked into maps of undecorated actin. After real-space alignment (25Hanein D. DeRosier D. Ultramicroscopy. 1999; 76: 233-238Crossref PubMed Scopus (10) Google Scholar), the reconstruction of undecorated actin was subtracted from the respective myosin V-decorated maps. Single units of the actin-bound myosin V densities were isolated with the watershed transform segmentation program (26Volkmann N. J. Struct. Biol. 2002; 138: 123-129Crossref PubMed Scopus (144) Google Scholar). The resulting densities, due only to myosin V, were further subsegmented to delineate the densities for the motor domain (MD) and the CaMs. This was done to ensure that the refined positions of the CaM models are confined to the respective density unit and that they are not biased toward the higher density in the neighboring density unit. The boundary between the two CaMs was independently cross-validated by difference mapping of the MD-2IQ reconstruction with a reconstruction of an construct that lacked the second IQ motif (MD-1IQ, data not shown). Statistics-based modular docking (22Volkmann N. Hanein D. J. Struct. Biol. 1999; 125: 176-184Crossref PubMed Scopus (174) Google Scholar, 23Volkmann N. Hanein D. Methods Enzymol. 2003; 374: 204-225Crossref PubMed Scopus (56) Google Scholar) was used throughout to build models of the actin-bound myosin V. The structure was divided into the motor domain and the two CaM regions (IQ1 and IQ2). For the MD docking the available structures were divided into three groups. The post-power-stroke conformation was represented by PDB codes 1mma, 1mmn, 1mmd, 1mmg, 2mys, 1kk7, 1fmv, 1fmw, and 1w7j. The pre-power-stroke conformation was represented by PDB codes 1br1, 1br2, 1br4, 1dfl, 1mnd, and 1vom. The third group contains the myosin V crystal structures with a similar lever-arm position as the post-stroke conformation but a more tightly closed actin binding cleft in the absence of nucleotide; PDB codes 1oe9, 1w8j (four asymmetric units), and 1w7i. There was no significant difference (p < 0.001) between the first and the third group, implying that we cannot pick up the cleft state by docking alone, but the fit of the second group was significantly worse than for the other two groups. Modeling was continued using the docked 1oe9 coordinates. The two bound CaMs were modeled using the CaM-like essential light chain (LC1-sa) taken from the myosin V crystal structure. Docking was done into discrepancy maps that had the MD portion removed. The resulting MD/CaM boundary was very similar to that determined by segmentation. The initial placement of the first CaM was extrapolated from the docked crystal structure and then refined using the refinement module from CoAn. After docking, the contribution of the first CaM model was removed from the map by discrepancy mapping, and then the second CaM model was docked into the remainder without constraints. The discrepancy boundary was again very similar to the one determined independently by difference mapping and segmentation. Regularization with REFMAC5 (27Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13870) Google Scholar) was performed for all atomic models to relieve distortions in stereochemistry. Discrepancy, Difference, and Structural Flexibility Mapping—For "discrepancy mapping," density is first calculated from the docked atomic model using electronic scattering factors. Then image-formation and image-analysis artifacts present in the reconstruction are compensated for by matching the Fourier amplitude spectrum of the calculated density to that observed and by scaling the densities appropriately. Last, the modified density is subtracted voxel-wise from the observed reconstruction. The resulting discrepancy maps allow reliable localization of regions where the reconstruction has significantly more density than the model can explain. Through the use of multiple maps and crystal structures, an error estimate (S.D.) for each voxel in the discrepancy maps can be calculated. This feature allows assignment of statistical significance. Peaks were considered significant if their value was at least three times their S.D. The results were cross-validated between the helical reconstructions and the IHRSR maps. "Difference mapping" and error assessment were carried out using the real space averages and variances for helical reconstructions. Difference maps for the IHRSR maps were calculated without error assessment. Before subtracting, the helical reconstructions were aligned to each other using a hybrid real space reciprocal space alignment procedure that includes common features of the two maps in the alignment (19Hanein D. Matsudaira P. DeRosier D.J. J. Cell Biol. 1997; 139: 387-396Crossref PubMed Scopus (88) Google Scholar); alignment involving IHRSR maps was done using real-space correlation (22Volkmann N. Hanein D. J. Struct. Biol. 1999; 125: 176-184Crossref PubMed Scopus (174) Google Scholar, 23Volkmann N. Hanein D. Methods Enzymol. 2003; 374: 204-225Crossref PubMed Scopus (56) Google Scholar). For difference maps between helical reconstructions, we tested the significance of each difference between corresponding pairs of real space voxels using a classic t test at a confidence level of 99.9% (p < 0.001). Difference maps between different IHRSR maps and between IHRSR maps and helical maps were used for cross-validation. Only differences that were consistently present (and significant at 99.9% were applicable) were used for interpretation. Absolute values of individual differences (AVID) was used to map intensity variations of individual actomyosin units within filaments (28Rost L.E. Hanein D. DeRosier D.J. Ultramicroscopy. 1998; 72: 187-197Crossref PubMed Scopus (13) Google Scholar). These variations can either be caused by partial occupancy or by a mixture of conformations. Because the AVID procedure excludes the layer line data used for helical reconstruction, the resulting AVID map contains data that is independent of the data used for the three-dimensional reconstruction and difference mapping. Four AVID maps were generated of myosin V in the presence (two maps) and absence of calcium (two maps). Only peaks present in the two AVID maps of myosin V in the presence of calcium and not in the AVID maps of myosin V in the absence of calcium were considered for interpretation as effects of calcium addition. Fluorescence Spectroscopy—Fluorescence measurements were performed on an ISS Inc. spectrofluorometer (Model ISS K2; Champaign, IL) equipped with a 300-watt xenon arc lamp as an excitation source and a temperature controlled cell-housing (20 °C). Tryptophan fluorescence of the CaM-IQ complexes was excited at 290 nm, and the emission spectra were collected from 300 to 400 nm with a 320-nm cutoff filter in the emission path. The spectral bandwidths were 8 nm for excitation and 8 nm for emission. Emission spectra were recorded in ratio mode. Buffers contained final concentrations of either 1 mm EGTA or 1 mm EGTA and 1.5 mm CaCl2. Acrylamide Quenching of Trp Fluorescence—Acrylamide quenching was used to determine the degree of exposure of the single tryptophan residue present in each IQ-CaM complex. The decrease in fluorescence intensity at the wavelength maximum was measured as a function of increasing acrylamide concentration. The fluorescence intensity in the absence of quencher (F0) divided by the fluorescence intensity in the presence of quencher (F) was used to quantify the change in fluorescence resulting from acrylamide quenching (F0/F). F0/F was plotted as a function of acrylamide concentration, [Q], and fit to the Stern-Volmer relationship taking into account both static (V) and dynamic quenching (KSV) constants: F0/F = (1 + KSV[Q])(exp V[Q]) (29Eftink M.R. Ghiron C.A. Biochemistry. 1976; 15: 672-680Crossref PubMed Scopus (989) Google Scholar). Buffers contained final concentrations of either 1 mm EGTA or 1 mm EGTA and 1.5 mm CaCl2. Thrombin Cleavage—CaM-IQ complexes (0.4 mg/ml in 10 mm HEPES, pH 7.2, 10 mm NaCl, 3 mm CaCl2) were cleaved with human α-thrombin at 10 units/mg of complex (Hematologic Technologies Inc.) for 5 h at 37 °C. The proteolytic products were analyzed on a 15% SDS-PAGE gel. The wild-type and mutant MD-2IQ complexes (0.5 mg/ml in 10 mm HEPES, pH 7.8, 150 mm NaCl, 1 mm dithiothreitol, 37 °C with either 0.4 mm CaCl2 or 1 mm EGTA) were similarly cleaved with 10 units of thrombin/mg protein and analyzed on 6.5% SDS-polyacrylamide gels. Atomic Model of Myosin V Bound to Actin in the Presence of Calcium—Electron cryomicroscopy and helical reconstruction techniques were used to generate three-dimensional maps for a myosin V motor domain-2IQ (MD-2IQ) construct bound to actin in the presence of calcium and in the absence of nucleotide (Fig. 1). The comparable experiments done without calcium have been previously described and are used for comparison with the results described here (12Volkmann N. Liu H. Hazelwood L. Krementsova E.B. Lowey S. Trybus K.M. Hanein D. Mol. Cell. 2005; 19: 595-605Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). MD-2IQ contains the motor domain and the two IQ motifs adjacent to it together with bound CaM. The CaM molecules are sequentially numbered with respect to their position relative to the MD, with the first CaM adjacent to the MD. The data sets were first processed using standard helical reconstruction techniques. In contrast to the well defined density of the second CaM in the calcium-free actomyosin three-dimensional maps (12Volkmann N. Liu H. Hazelwood L. Krementsova E.B. Lowey S. Trybus K.M. Hanein D. Mol. Cell. 2005; 19: 595-605Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), the second CaM was difficult to resolve in the presence of calcium. Use of the iterative real space refinement method (20Egelman E.H. Ultramicroscopy. 2000; 85: 225-234Crossref PubMed Scopus (387) Google Scholar), a technique that includes classification procedures capable of sorting mixtures of actin-bound myosin conformations (12Volkmann N. Liu H. Hazelwood L. Krementsova E.B. Lowey S. Trybus K.M. Hanein D. Mol. Cell. 2005; 19: 595-605Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), was needed to resolve the second CaM. Even after sorting, the density for the second CaM is considerably less well defined than the one for the first CaM, but the definition is now good enough to model the CaM into the density (Fig. 1). An atomic model was generated using a quantitative fitting procedure, based on existing crystal structures (see "Materials and Methods"). The same approach was previously used to generate a model for actin-bound MD-2IQ in the absence of calcium (12Volkmann N. Liu H. Hazelwood L. Krementsova E.B. Lowey S. Trybus K.M. Hanein D. Mol. Cell. 2005; 19: 595-605Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). There are no significant differences in the docked orientation and position of the actin-bound motor domain when compared with the calcium-free state, but the CaMs do move substantially (see below). Calcium-dependent Changes in the Motor Domain—To analyze changes upon the addition of calcium, we calculated four types of maps in the motor-domain region (see also "Materials and Methods"); (i) discrepancy maps, obtained by subtracting the density calculated for the atomic model from the density of the three-dimensional reconstruction (magenta in Fig. 2; this approach identifies regions of higher density in the reconstruction that are not accounted for by the atomic models), (ii) difference maps subtracting the density of the maps in the absence of calcium from the density in its presence (yellow in Fig. 2; these maps indicate strengthening of density features when calcium is added), (iii) difference maps subtracting the density of the maps in the presence of calcium from the density in the absence of calcium (green in Fig. 2; these maps indicate weakening of density upon addition of calcium), (iv) AVID maps, indicating either partial occupancy or structural flexibility (blue peaks in Fig. 2; this type of map was calculated in the entire map, including the IQ region). Discrepancy mapping has been previously used to show whether the long cleft separating the upper and lower 50-kDa domains of the motor domain was closed or open in different nucleotide states in the actomyosin complex (12Volkmann N. Liu H. Hazelwood L. Krementsova E.B. Lowey S. Trybus K.M. Hanein D. Mol. Cell. 2005; 19: 595-605Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 17Volkmann N. Hanein D. Ouyang G. Trybus K.M. DeRosier D.J. Lowey S. Nat. Struct. Biol. 2000; 7: 1147-1155Crossref PubMed Scopus (135) Google Scholar, 30Volkmann N. Ouyang G. Trybus K.M. DeRosier D.J. Lowey S. Hanein D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3227-3232Crossref PubMed Scopus (37) Google Scholar). In the absence of nucleotide, the long cleft of actin-bound MD-2IQ in the presence of calcium is closed to the same extent as was seen in the absence of calcium. Loop 2, the ends of which are colored blue (Fig. 2E) shows the only discrepancy peak (Fig. 2, A-C and E, magenta density). This discrepancy peak is interpreted as showing that this region is stabilized upon binding to actin. Stabilization of loop 2 upon actin binding was previously reported for myosin V in the presence or absence of various nucleotides (12Volkmann N. Liu H. Hazelwood L. Krementsova E.B. Lowey S. Trybus K.M. Hanein D. Mol. Cell. 2005; 19: 595-605Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) as well as for smooth muscle and skeletal muscle myosin II (17Volkmann N. Hanein D. Ouyang G. T
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