C Subunits Binding to the Protein Kinase A RIα Dimer Induce a Large Conformational Change
2004; Elsevier BV; Volume: 279; Issue: 18 Linguagem: Inglês
10.1074/jbc.m313405200
ISSN1083-351X
AutoresWilliam T. Heller, Dominico Vigil, Simon H. J. Brown, Donald Blumenthal, Susan S. Taylor, Jill Trewhella,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoWe present structural data on the RIα isoform of the cAMP-dependent protein kinase A that reveal, for the first time, a large scale conformational change within the RIα homodimer upon catalytic subunit binding. This result infers that the inhibition of catalytic subunit activity is not the result of a simple docking process but rather is a multi-step process involving local conformational changes both in the cAMP-binding domains as well as in the linker region of the regulatory subunit that impact the global structure of the regulatory homodimer. The results were obtained using small-angle neutron scattering with contrast variation and deuterium labeling. From these experiments we derived information on the shapes and dispositions of the catalytic subunits and regulatory homodimer within a holoenzyme reconstituted with a deuterated regulatory subunit. The scattering data also show that, despite extensive sequence homology between the isoforms, the overall structure of the type Iα holoenzyme is significantly more compact than the type IIα isoform. We present a model of the type Iα holoenzyme, built using available high-resolution structures of the component subunits and domains, which best fits the neutron-scattering data. In this model, the type Iα holoenzyme forms a flattened V shape with the RIα dimerization domain at the point of the V and the cAMP-binding domains of the RIα subunits with their bound catalytic subunits at the ends. We present structural data on the RIα isoform of the cAMP-dependent protein kinase A that reveal, for the first time, a large scale conformational change within the RIα homodimer upon catalytic subunit binding. This result infers that the inhibition of catalytic subunit activity is not the result of a simple docking process but rather is a multi-step process involving local conformational changes both in the cAMP-binding domains as well as in the linker region of the regulatory subunit that impact the global structure of the regulatory homodimer. The results were obtained using small-angle neutron scattering with contrast variation and deuterium labeling. From these experiments we derived information on the shapes and dispositions of the catalytic subunits and regulatory homodimer within a holoenzyme reconstituted with a deuterated regulatory subunit. The scattering data also show that, despite extensive sequence homology between the isoforms, the overall structure of the type Iα holoenzyme is significantly more compact than the type IIα isoform. We present a model of the type Iα holoenzyme, built using available high-resolution structures of the component subunits and domains, which best fits the neutron-scattering data. In this model, the type Iα holoenzyme forms a flattened V shape with the RIα dimerization domain at the point of the V and the cAMP-binding domains of the RIα subunits with their bound catalytic subunits at the ends. Many cellular signaling pathways in eukaryotes involve protein kinases, which phosphorylate Ser, Thr, and Tyr residues in a variety of target proteins. The cAMP-dependent protein kinase A (PKA 1The abbreviations used are: PKA, protein kinase A; R, regulator subunit; C, catalytic subunit; RC, R and C heterodimer; AKAP, A-kinase anchoring protein; D/D, dimerization/docking; SANS, small-angle neutron scattering; and SAXS, small-angle x-ray scattering. or protein kinase A) is one of the best-studied members of the Ser/Thr protein kinase family. PKA is known to be involved in the regulation of a large number of cellular processes including metabolism, contractile activity, growth, apoptosis, and ion flux (1Shabb J.B. Chem. Rev. 2001; 101: 2381-2411Google Scholar). Mutations in PKA can lead to diseases such as Carney complex (2Kirschner L.S. Carney J.A. Pack S.D. Taymans S.E. Giatzakis C. Cho Y.S. Cho-Chung Y.S. Stratakis C.A. Nat. Genet. 2000; 26: 89-92Google Scholar) and lupus (3Laxminarayana D. Kammer G.M. Int. Immunol. 2000; 12: 1521-1529Google Scholar). The catalytic (C) subunits of PKA are responsible for catalyzing the phospho-transfer reaction, whereas the regulatory (R) subunits serve both to confer cAMP dependence and to localize the holoenzyme to discrete subcellular locations via interactions with A-kinase anchoring proteins (AKAPs) (4Skälhegg B.S. Tasken K. Front. Biosci. 2000; 5: D678-D693Google Scholar). At low cAMP concentrations, PKA is maintained as an inactive tetrameric holoenzyme complex (R2C2) consisting of a homodimeric R2 subunit and two C subunits. When intracellular concentrations of cAMP increase in response to specific cellular stimuli, four cAMP molecules bind to each R2 subunit. This event causes a release of inhibition of C by R, allowing the C subunits to phosphorylate their target proteins. There are four major isoforms of PKA, types Iα, Iβ, IIα, and IIβ, which differ with respect to their R subunits (RIα, RIβ, RIIα, and RIIβ, respectively). The isoforms have different biological functions, as determined by genetic studies using mice. For instance, mice lacking the RIα gene die in utero (5Amieux P.S. McKnight G.S. Ann. N. Y. Acad. Sci. 2002; 968: 75-92Google Scholar), whereas mice lacking the RIIβ gene are viable, lean, and resistant to diet-induced obesity (6Cummings D.E. Brandon E.P. Planas J.V. Motamed K. Idzerda R.L. McKnight G.S. Nature. 1996; 382: 622-626Google Scholar). Despite their differing biological functions, all of the R isoforms share the same domain organization. At the N terminus of each R subunit is a dimerization/docking (D/D) domain that serves both to dimerize and anchor the R subunits to AKAPs. The C terminus of the R subunit consists of tandem cAMP-binding domains. Between the D/D domain and the cAMP-binding domains is a linker region that contains a pseudo-substrate inhibitor region that binds to the active site of the C subunits. Mutagenesis (7Gibson R.M. Ji-Buechler Y. Taylor S.S. J. Biol. Chem. 1997; 272: 16343-16350Google Scholar) and hydrogen/deuterium exchange experiments (8Anand G.S. Hughes C.A. Jones J.M. Taylor S.S. Komives E.A. J. Mol. Biol. 2002; 323: 377-386Google Scholar) have identified the helical subdomain in the more N-terminal cAMP-binding domain (domain A) of RIα as an additional region of contact between the R and C subunits. There is high sequence homology between the isoforms in the D/D domain and the cAMP-binding domains, but the linker regions are quite different in length and sequence (9Canaves J.M. Taylor S.S. J. Mol. Evol. 2002; 54: 17-29Google Scholar). High-resolution structures of the C subunit (10Knighton D.R. Zheng J.H. Ten Eyck L.F. Ashford V.A. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Google Scholar), cAMP-binding domains of RIα (11Su Y. Dostmann W.R. Herberg F.W. Durick K. Xuong N.-H. Ten Eyck L. Taylor S.S. Varughese K.I. Science. 1995; 269: 807-813Google Scholar) and RIIβ (12Diller T.C. Madhusudan Xuong N.-H. Taylor S.S. Structure. 2001; 9: 73-82Google Scholar), and the D/D domains of RIIα (13Newlon M.G. Roy M. Morikis D. Hausken Z.E. Coghlan V. Scott J.D. Jennings P.A. Nat. Struct. Biol. 1999; 6: 222-227Google Scholar) and RIα (14Banky P. Roy M. Newlon M.G. Morikis D. Haste N.M. Taylor S.S. Jennings P.A. J. Mol. Biol. 2003; 330: 1117-1129Google Scholar) are available and provide important molecular insights into the activation process. The C subunit is a globular, bilobal protein with the active site in a cleft between the lobes. The cAMP-binding domains of the R subunits each consist of a β-barrel region that binds cAMP, and a helical region, whereas the D/D domain consists of an antiparallel four-helix bundle. There are no high-resolution structures of the linker regions, which are thought to be relatively flexible in solution (15Li F. Gangal M. Jones J.M. Deich J. Lovett K.E. Taylor S.S. Johnson D.A. Biochemistry. 2000; 39: 15626-15632Google Scholar). Similarly, there are no high-resolution structures of the full-length R homodimers or holoenzyme complexes, which is likely due to the inherent flexibility of the linker regions. Ultracentrifugation and analytical gel filtration indicate that there are significant structural differences between the isoforms. Specifically, the type IIα holoenzyme is more elongated than the type Iα. The reported Stokes radii range from 56.8-60 Å for the type IIα holoenzyme, and 47.4-53.8 Å for the type Iα holoenzyme (16Erlichman J. Rubin C.S. Rosen O.M. J. Biol. Chem. 1973; 248: 7607-7609Google Scholar, 17Zoller M.J. Kerlavage A.R. Taylor S.S. J. Biol. Chem. 1979; 254: 2408-2412Google Scholar, 18Herberg F.W. Dostmann W.R.G. Zorn M. Davis S.J. Taylor S.S. Biochemistry. 1994; 33: 7485-7494Google Scholar). This difference in Stokes radii is larger than expected for the relatively small difference in isoform molecular weight. Neutron scattering with contrast variation and deuterium labeling is a useful technique for determining the relative shapes and positions of subunits within a molecular complex. The technique was used previously to investigate the subunit arrangement of the PKA type IIα holoenzyme (19Zhao J.K. Hoye E. Boylan S Walsh D.A. Trewhella J. J. Biol. Chem. 1998; 273: 30448-30459Google Scholar). The neutron data and associated modeling revealed an extended dumbbell shape for the holoenzyme, with the C subunits located in the lobes of the dumbbell and separated by ∼120 Å. To further investigate the structural differences between the PKA isoforms and the conformational changes involved in the activation process, we completed small-angle neutron scattering experiments with contrast variation on the type Iα holoenzyme with deuterated R subunits. To aid in the interpretation of the scattering data, we built models of the RIα holoenzyme using the NMR structure of the RIα D/D domain (14Banky P. Roy M. Newlon M.G. Morikis D. Haste N.M. Taylor S.S. Jennings P.A. J. Mol. Biol. 2003; 330: 1117-1129Google Scholar) and two previously published models of the RC heterodimer (20Tung C.S. Walsh D.A. Trewhella J. J. Biol. Chem. 2002; 277: 12423-12431Google Scholar, 21Anand G.S. Law D. Mandell J.G. Snead A.N. Tsigelny I. Taylor S.S. Ten Eyck L.F. Komives E.A. Proc. Natl. Acad. Sci. 2003; 100: 13264-13269Google Scholar) to determine the arrangements of subunits that best fit the contrast series data. The models and the structural parameters derived from the scattering data (Rg, dmax, and P(r)) indicate that the RIα holoenzyme is significantly more compact than the type IIα holoenzyme studied previously by small-angle neutron scattering with contrast variation (19Zhao J.K. Hoye E. Boylan S Walsh D.A. Trewhella J. J. Biol. Chem. 1998; 273: 30448-30459Google Scholar) as well as a recombinant RIIα holoenzyme prepared similarly to the RIα holoenzyme studied here. The two C subunits in the RIα complex are well separated and do not interact with each other, just as in the type IIα isoform. However, the R subunit homodimer in the type Iα complex forms a flattened V-shape in contrast to the more extended dumbbell shape seen for the RIIα homodimer. Interestingly, the RIα homodimer is more extended when bound to the C-subunits than when it is free in solution (22Vigil D. Blumenthal D.K. Heller W.T. Brown S. Canaves J.M. Taylor S.S. Trewhella J. J. Mol. Biol. 2004; 337: 1183-1194Google Scholar). Our results clearly demonstrate that the interaction of the C subunits with the R subunits plays a critical role in defining the domain organization within the holoenzyme. Cloning, Protein Expression, and Purification—To create an isopropyl-1-thio-β-d-galactopyranoside-inducible construct that makes more efficient use of the expensive deuterated media required to produce deuterated R, the wild-type bovine RIα gene was subcloned out of a wild-type RIα pUC118 vector into an ampicillin-resistant pRSET vector (Invitrogen) using standard methods. After optimizing media deuteration levels to maximize protein yield, large scale expression was performed in 2 liters of 97% deuterated Martek-9 media and unlabeled Martek-9 media at a 7:3 ratio. Recombinant bovine R (RIα and RIIα) was purified from Escherichia coli Bl21-DE3 cells using cAMP-agarose resin (23Diller T.C. Xuong N.-H. Taylor S.S. Protein Expr. Purif. 2000; 20: 357-364Google Scholar) and eluted with 25 mm cGMP instead of cAMP. Recombinant, non-myristoylated bovine catalytic subunit was purified from E. coli Bl21-DE3 cells as described previously (24Slice L.W. Taylor S.S. J. Biol. Chem. 1989; 264: 20940-20946Google Scholar). Holoenzymes were then reconstituted as described elsewhere (8Anand G.S. Hughes C.A. Jones J.M. Taylor S.S. Komives E.A. J. Mol. Biol. 2002; 323: 377-386Google Scholar). Small-Angle X-ray Scattering (SAXS) Measurements—The SAXS data were collected using the line source instrument at Los Alamos National Laboratory (25Heidorn D.B. Trewhella J. Biochemistry. 1998; 27: 909-915Google Scholar). Intensity data were reduced to I(q) versus q using standard procedures to correct for detector sensitivity and background signal, and a smearing procedure was used to correct for the slit geometry of the instrument (25Heidorn D.B. Trewhella J. Biochemistry. 1998; 27: 909-915Google Scholar). The SAXS data from the holoenzyme were compared with a lysozyme standard (26Krigbaum W.R. Kugler F.R. Biochemistry. 1970; 9: 1216-1223Google Scholar) to verify that the samples were free from nonspecific aggregation and the influence of inter-particle interference. Small-Angle Neutron Scattering (SANS) Measurements—Samples of the type Iα holoenzyme with deuterated R subunit in H2O/D2O mixtures containing 0, 20, 40, 80, and 90% D2O were prepared by mixing stock solutions of the holoenzyme dissolved in H2O and D2O to provide five neutron contrast values. The samples and corresponding solvent blanks with 0, 20, and 40% D2O were measured in 1-mm path length cells, whereas 20-mm path length cells were used for the samples with 80 and 90% D2O buffer. The precise D2O content of each sample was determined by comparing the neutron absorbance, μ, to that of pure H2O and D2O. The neutron absorbance is obtained from the measured sample transmission using the relationship μ = ln(T)/d, where μ is the neutron absorbance, T is the transmittance, and d is the sample path length. Neutron absorbance varies linearly with hydrogen content of the buffer; thus, using the relation presented below in Equation 1, P=1-((μsam-μD2O)/(μH2O-μD2O))(Eq. 1) the solvent deuteration fraction of the sample, P, can be determined. The D2O content of each sample determined in this manner was used throughout the analysis of the data. The SANS data were collected at the Center for Neutron Research at the National Institute of Standards and Technology (Gaithersburg, MD) using the NG-3 30m SANS instrument (27Hammouda B. Barker J.G. Krueger S. Small Angle Neutron Scattering Manuals. National Institute of Standards and Technology, Gaithersburg, MD1996Google Scholar). A neutron wavelength of 5.5 Å was used with a wavelength spread Δλ/λ of 0.26 to provide the maximum flux. Sample and background intensities were collected at sample-to-detector distances of 1.5 and 6 m to obtain data over the required q-range. Data reduction to one-dimensional scattered intensity profiles, I(q) versus q, followed standard procedures to correct for detector sensitivity and sample background (27Hammouda B. Barker J.G. Krueger S. Small Angle Neutron Scattering Manuals. National Institute of Standards and Technology, Gaithersburg, MD1996Google Scholar). The reduced intensities from the two detector distances were merged using routines included with the data reduction software provided by the National Institute of Standards and Technology. Small-Angle Scattering Data Analysis—The small-angle scattering intensity profile of monodisperse, identical particles in solution is given by Equation 2, I(q)=|〈∫V(ρ(r→)-ρs)e-iq→⋅r→d3r〉|2(Eq. 2) where ρ(r→) is the scattering length density of the particle as a function of position r→ within the volume V, ρs is the average scattering length density of the solvent, and q→ is the momentum transfer, having the magnitude 4π(sinθ)/λ, where 2θ is the scattering angle and λ is the wavelength. The integration is averaged over all conformations and orientations of the particles in solution, because small-angle solution scattering measures the time- and ensemble-averaged information from all of the particles in the sampled volume. As shown below in Equation 3, P(r)=12π2∫0∞dq⋅(qr)⋅I(q)sin(qr)(Eq. 3) the inverse Fourier transform of I(q) versus q gives P(r) versus r, the probable distribution of vector lengths, r, between scattering centers within the scattering object. P(r) is readily interpreted in terms of the shape of the scattering object. The indirect Fourier transform algorithm originally described by Moore (28Moore P.B. J. Appl. Crystallogr. 1980; 13: 168-175Google Scholar) was used to determine P(r) from I(q). The boundary conditions P(r)/r = 0 at r = 0 and the maximum linear dimension, dmax, are applied to P(r). The contrast series intensity profiles I(q) can be written as a set of linear equations in the three basic scattering functions, i.e. the scattering functions of the labeled and unlabeled components and a cross-term (29Olah G.A. Rokop S.E. Wang C.-L.A. Blechner S.L. Trewhella J. Biochemistry. 1994; 33: 8233-8239Google Scholar, 30Ibel K. Stuhrmann H B. J. Mol. Biol. 1975; 93: 255-265Google Scholar). The P(r) calculated from the cross-term gives the distribution of vector lengths between the labeled and unlabeled components of the complex. A multiple linear regression routine (31Bevington P.R. Data Reduction and Error Analysis for the Physical Sciences. McGraw-Hill, New York1969: 164-176Google Scholar) implemented in the C programming language at Los Alamos National Laboratory was used to solve for the three basic scattering functions using the contrast series data. These basic scattering functions were used to derive information on the shapes and relative dispositions of deuterated and non-deuterated components in the system. Structural Models—Structural models of the isotopically labeled subunits and the overall complex were determined from the SANS data using an algorithm developed at Los Alamos National Laboratory and implemented in the program CONTRAST (32Heller W.T. Abusamhadneh E. Finley N. Rosevear P.R. Trewhella J. Biochemistry. 2002; 41: 15654-15663Google Scholar). This program can take a set of known high-resolution structures, and/or shapes, and find the relative position and orientation of the components that give the best fit to a set of intensity profiles measured in a neutron contrast series. The best fit model structures are generated using a Monte Carlo approach employed previously (19Zhao J.K. Hoye E. Boylan S Walsh D.A. Trewhella J. J. Biol. Chem. 1998; 273: 30448-30459Google Scholar, 25Heidorn D.B. Trewhella J. Biochemistry. 1998; 27: 909-915Google Scholar). To evaluate the quality of the fit of each model to the data, CONTRAST uses the fitting parameter F, as shown below in Equation 4, F=1Npts(∑Npts(I(q)-Im(q))2σ(q)-2)(Eq. 4) where Npts is the number of points in the data set, I(q) and Im(q) are the experimental and model intensities, respectively, and σ(q) is the experimental uncertainty of I(q). The D/D domain used was the NMR structure of the RIα isoform (Protein Data Bank entry 1R2A, Ref. 14Banky P. Roy M. Newlon M.G. Morikis D. Haste N.M. Taylor S.S. Jennings P.A. J. Mol. Biol. 2003; 330: 1117-1129Google Scholar). The modeling was done using two different high-resolution models of the Δ(1-91)RIIα/C heterodimer based on the crystal structures of the RIα cAMP binding domain and C subunit (Protein Data Bank entry 1PVK, Ref. 20Tung C.S. Walsh D.A. Trewhella J. J. Biol. Chem. 2002; 277: 12423-12431Google Scholar, and Protein Data Bank entry 1KMU, Ref. 21Anand G.S. Law D. Mandell J.G. Snead A.N. Tsigelny I. Taylor S.S. Ten Eyck L.F. Komives E.A. Proc. Natl. Acad. Sci. 2003; 100: 13264-13269Google Scholar). To simulate the volume occupied by the linker region, cylinders of 8, 10, 12, and 14 Å radii were generated with a volume calculated from the molecular weight of the linker sequence missing from the structures. The cylinders were filled with random points to the same density of points as used by the other structures. The structure was chained together in the sequence RC heterodimer:linker cylinder:D/D domain:linker cylinder:RC heterodimer. All of these structural components were then allowed to rotate freely with respect to each other about their connection points. The angles were chosen randomly with no constraints applied. A 2-fold axis of symmetry about the natural axis of symmetry of dimerization/docking domain was enforced for all models. Two independent runs of CONTRAST were performed per cylinder length for each of the two RC heterodimer models, each testing in excess of 250,000 possible models. SAXS Data from the Type Iα and IIα Holoenzymes—The small-angle x-ray scattering intensity profiles and associated distance distribution functions, P(r), for the type Iα PKA holoenzyme (in H2O and in D2O) and the type IIα PKA (in H2O) holoenzyme, which was similarly prepared for this study, are shown in Fig. 1. The P(r) functions of the type Iα holoenzyme in H2O and D2O are identical within the error of the measurements, indicating that the overall conformation of the holoenzyme is unaltered by D2O. All of the samples were free of nonspecific aggregation and the effects of inter-particle interference, as judged by the lack of an upturn or downturn, respectively, in the low q region of the scattering intensity plot. Comparison of the forward scattering (I(0)) intensity with that from a lysozyme standard also supported this conclusion. The P(r) function for the type Iα holoenzyme peaks at ∼40 Å, has a strong shoulder at 112 Å, goes to zero at 150 Å, and is indicative of a bilobal object. The P(r) curve for the type IIα holoenzyme peaks just below 40 Å with shoulders at 112 and 140 Å and extends out to ∼210 Å, indicative of a significantly more extended, asymmetric shape. The structural parameters Rg and dmax, derived from the P(r) plots, are summarized in Table I. Two sets of parameters are given for the type IIα holoenzyme; one is derived from samples prepared for this study, and the other one is from an earlier study (19Zhao J.K. Hoye E. Boylan S Walsh D.A. Trewhella J. J. Biol. Chem. 1998; 273: 30448-30459Google Scholar). Although both data sets show a more extended structure than what we see for the type Iα isoform, the earlier study found an even more extended structure than what we see here for the type IIα samples reconstituted using the same methods employed for the type Iα isoform. The samples used in the earlier study of the type IIα holoenzyme were reconstituted using native, myristoylated C subunits, and a urea unfolding step was used to remove cAMP from R in order to reconstitute holoenzyme, whereas this study used a recombinant, unmyristoylated C subunit and no urea unfolding step. These differences in sample preparation are the likely source of differences in the type II holoenzyme structures. However, both studies show the type IIα holoenzyme to be significantly more extended than the type Iα holoenzyme. Thus, our observation of the large isoform differences between the two holoenzymes is not an artifact of the sample preparation conditions.Table IScattering parametersRgaRg and dmax were determined from the R(r) as discussed under "Materials and Methods." P(r) was determined using the method of Moore (28). The C subunit pair and R homodimer in holoenzyme values were calculated from the basic scattering functions derived from SANS.dmaxbData taken from x-ray scattering study of the free RIα homodimer (22).ÅÅType Iα holoenzyme in H2O51.2 ± 2.5150 ± 5Type Iα holoenzyme in D2O50.0 ± 2.3150 ± 6C subunit pair in holoenzyme54.3 ± 2.4140 ± 4RIα homodimer in holoenzyme44.3 ± 0.3140 ± 10Free RIα homodimerbData taken from x-ray scattering study of the free RIα homodimer (22).40.8 ± 1.5117 ± 5Type IIα holoenzyme with unmyristoylated CcR and non-myristoylated C subunits are bacterially expressed, and buffer conditions are very similar to those used for the type Iα holoenzyme61.6 ± 2.1225 ± 10Type IIα holoenzyme with myristoylateddData from Zhao et al. (19), which used native myristoylated C subunits and urea-treated R subunits.73.5 ± 6.3220 ± 10a Rg and dmax were determined from the R(r) as discussed under "Materials and Methods." P(r) was determined using the method of Moore (28Moore P.B. J. Appl. Crystallogr. 1980; 13: 168-175Google Scholar). The C subunit pair and R homodimer in holoenzyme values were calculated from the basic scattering functions derived from SANS.b Data taken from x-ray scattering study of the free RIα homodimer (22Vigil D. Blumenthal D.K. Heller W.T. Brown S. Canaves J.M. Taylor S.S. Trewhella J. J. Mol. Biol. 2004; 337: 1183-1194Google Scholar).c R and non-myristoylated C subunits are bacterially expressed, and buffer conditions are very similar to those used for the type Iα holoenzymed Data from Zhao et al. (19Zhao J.K. Hoye E. Boylan S Walsh D.A. Trewhella J. J. Biol. Chem. 1998; 273: 30448-30459Google Scholar), which used native myristoylated C subunits and urea-treated R subunits. Open table in a new tab SANS Data, with Contrast Variation, from the Type Iα Holoenzyme—The intensity profiles collected for the neutron contrast variation series on the type Iα holoenzyme reconstituted with deuterated R are plotted in Fig. 2. The quality of the data is good as judged by the counting statistics. The 40 and 80% D2O data sets are close to the match points of the unlabeled C and deuterium-labeled R components, respectively. As a result, these two intensity profiles closely match the basic scattering functions for the R and C components within the holoenzyme complex, respectively. The basic scattering functions extracted from the full contrast series data and corresponding to the scattering functions for the deuterated and non-deuterated components in the complex, plus a cross-term, are plotted in Fig. 3A. The P(r) functions derived from the basic scattering functions corresponding to the scattering profiles of the deuterated and non-deuterated components in the holoenzyme are shown in Fig. 3B, and the corresponding structural parameters are presented in Table I along with those derived from SAXS data for the free RIα homodimer (22Vigil D. Blumenthal D.K. Heller W.T. Brown S. Canaves J.M. Taylor S.S. Trewhella J. J. Mol. Biol. 2004; 337: 1183-1194Google Scholar). As observed previously for the type IIα holoenzyme (19Zhao J.K. Hoye E. Boylan S Walsh D.A. Trewhella J. J. Biol. Chem. 1998; 273: 30448-30459Google Scholar), the P(r) curve for the C subunits in the complex shows two well separated peaks, indicating that they are not in contact with each other. The peak at ∼25 Å resembles the P(r) expected for the isolated C subunit (19Zhao J.K. Hoye E. Boylan S Walsh D.A. Trewhella J. J. Biol. Chem. 1998; 273: 30448-30459Google Scholar) and corresponds to the intermolecular vector lengths within each individual C subunit. The peak at ∼112 Å corresponds to the inter-atomic distances between the two C subunits in the holoenzyme complex. The areas under the two peaks are the same, as is expected for two identical subunits separated in space. The P(r) of the R dimer in the complex has a peak at ∼37 Å with a broad shoulder ∼85-90 Å, indicating a bilobal structure. There is a striking difference between this shape and RIα dimer free in solution, which is significantly more compact as evidenced by its P(r), which has a peak at ∼33 Å, a high shoulder at ∼80 Å, and goes to zero at 117 Å. Thus, we see that the C subunit binding to the RIα homodimer results in increases in Rg by∼10 Å (25%) and dmax by 33 Å (28%) (Table I). Taken together, the scattering parameters and the P(r) functions indicate that the RIα homodimer undergoes a large extension upon binding C subunits.Fig. 3Scattering functions for the RIα PKA subunits. Basic scattering functions derived from the contrast variation series data are plotted along with the SAXS data for the RIα homodimer (A) for comparison and the corresponding P(r) functions (B). The C subunit pair (▪), the R homodimer (○), and the cross-term (▵) from the neutron-derived basic scattering functions are shown along with the SAXS data for the free RIα homodimer (▾), which has been offset for clarity. The free RIα homodimer SAXS data are taken from another study (22Vigil D. Blumenthal D.K. Heller W.T. Brown S. Canaves J.M. Taylor S.S. Trewhella J. J. Mol. Biol. 2004; 337: 1183-1194Google Scholar). Simplified versions of the models shown in Fig. 4 are shown on the right to help visualize which P(r) function represents which subunit within the holoenzyme. For the C subunit basic scattering function, the C subunits within the model to the right are highlighted in black, and the R dimer is in light gray. For the R homodimer basic scattering function, the color scheme is reversed.View Large Image Figure ViewerDownload (PPT) Structural Models of the Type Iα Holoenzyme—Using high-resolution structures of the RIα D/D domain (14Banky P. Roy M. Newlon M.G. Morikis D. Haste N.M. Taylor S.S. Jennings P.A. J. Mol. Biol. 2003; 330: 1117-1129Google Scholar) and the RC heterodimer model (20Tung C.S. Walsh D.A. Trewhella J. J. Biol. Chem. 2002; 277: 12423-12431Google Scholar, 21Anand G.S. Law D. Mandell J.G. Snead A.N. Tsigelny I. Taylor S.S. Ten Eyck L.F. Komives E.A. Proc. Natl. Acad. Sci. 2003; 100: 13264-13269Google Scholar) connected to the D/D domain by cylinders to represent the linker regions, we developed models for the holoenzyme and tested possible configurations of the components, as described
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