Organization of the Cores of the Mammalian Pyruvate Dehydrogenase Complex Formed by E2 and E2 Plus the E3-binding Protein and Their Capacities to Bind the E1 and E3 Components
2004; Elsevier BV; Volume: 279; Issue: 8 Linguagem: Inglês
10.1074/jbc.m308172200
ISSN1083-351X
AutoresYasuaki Hiromasa, Tetsuro Fujisawa, Yoichi Aso, Thomas E. Roche,
Tópico(s)Diet and metabolism studies
ResumoThe subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex can form a 60-mer via association of the C-terminal I domain of E2 at the vertices of a dodecahedron. Exterior to this inner core structure, E2 has a pyruvate dehydrogenase component (E1)-binding domain followed by two lipoyl domains, all connected by mobile linker regions. The assembled core structure of mammalian pyruvate dehydrogenase complex also includes the dihydrolipoyl dehydrogenase (E3)-binding protein (E3BP) that binds the I domain of E2 by its C-terminal I′ domain. E3BP similarly has linker regions connecting an E3-binding domain and a lipoyl domain. The composition of E2·E3BP was thought to be 60 E2 plus ∼12 E3BP. We have prepared homogenous human components. E2 and E2·E3BP have s20,w values of 36 S and 31.8 S, respectively. Equilibrium sedimentation and small angle x-ray scattering studies indicate that E2·E3BP has lower total mass than E2, and small angle x-ray scattering showed that E3 binds to E2·E3BP outside the central dodecahedron. In the presence of saturating levels of E1, E2 bound ∼60 E1 and maximally sedimented 64.4 ± 1.5 S faster than E2, whereas E1-saturated E2·E3BP maximally sedimented 49.5 ± 1.4 S faster than E2·E3BP. Based on the impact on sedimentation rates by bound E1, we estimate fewer E1 (∼12) were bound by E2·E3BP than by E2. The findings of a smaller E2·E3BP mass and a lower capacity to bind E1 support the smaller E3BP substituting for E2 subunits rather than adding to the 60-mer. We describe a substitution model in which 12 I′ domains of E3BP replace 12 I domains of E2 by forming 6 dimer edges that are symmetrically located in the dodecahedron structure. Twelve E3 dimers were bound per E248·E3BP12 mass, which is consistent with this model. The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex can form a 60-mer via association of the C-terminal I domain of E2 at the vertices of a dodecahedron. Exterior to this inner core structure, E2 has a pyruvate dehydrogenase component (E1)-binding domain followed by two lipoyl domains, all connected by mobile linker regions. The assembled core structure of mammalian pyruvate dehydrogenase complex also includes the dihydrolipoyl dehydrogenase (E3)-binding protein (E3BP) that binds the I domain of E2 by its C-terminal I′ domain. E3BP similarly has linker regions connecting an E3-binding domain and a lipoyl domain. The composition of E2·E3BP was thought to be 60 E2 plus ∼12 E3BP. We have prepared homogenous human components. E2 and E2·E3BP have s20,w values of 36 S and 31.8 S, respectively. Equilibrium sedimentation and small angle x-ray scattering studies indicate that E2·E3BP has lower total mass than E2, and small angle x-ray scattering showed that E3 binds to E2·E3BP outside the central dodecahedron. In the presence of saturating levels of E1, E2 bound ∼60 E1 and maximally sedimented 64.4 ± 1.5 S faster than E2, whereas E1-saturated E2·E3BP maximally sedimented 49.5 ± 1.4 S faster than E2·E3BP. Based on the impact on sedimentation rates by bound E1, we estimate fewer E1 (∼12) were bound by E2·E3BP than by E2. The findings of a smaller E2·E3BP mass and a lower capacity to bind E1 support the smaller E3BP substituting for E2 subunits rather than adding to the 60-mer. We describe a substitution model in which 12 I′ domains of E3BP replace 12 I domains of E2 by forming 6 dimer edges that are symmetrically located in the dodecahedron structure. Twelve E3 dimers were bound per E248·E3BP12 mass, which is consistent with this model. The mitochondrial pyruvate dehydrogenase complex (PDC) 1The abbreviations used are: PDC, pyruvate dehydrogenase complex; E1, pyruvate dehydrogenase component; E2, dihydrolipoyl acetyltransferase component; L1 domain, NH2-lipoyl domain of E2; L2 domain, interior lipoyl domain of E2; B, binding domain in E2 for E1; H1, H2, and H3 are the linker (hinge) regions of E2 (Fig. 1A); scE2, E2 with PreScission protease site introduced in H3 domain of E2; t-E2, truncated form of E2 with I domain and small part of H3 linker region; E3, dihydrolipoyl dehydrogenase; E3BP, E3-binding protein; L3, lipoyl domain of E3BP; B′, binding domain of E3BP that binds E3; I′ domain, inner domain of E3BP; H1′ and H2′ linker (hinge) regions in E3BP (Fig. 1A); AUC, analytical ultracentrifugation; SAXS, small angle x-ray scattering; R0, Rs, Rg, and Re are, respectively, the unhydrated spherical Stokes radius, Stokes radius, radius of gyration, and particle excluded volume radius. 1The abbreviations used are: PDC, pyruvate dehydrogenase complex; E1, pyruvate dehydrogenase component; E2, dihydrolipoyl acetyltransferase component; L1 domain, NH2-lipoyl domain of E2; L2 domain, interior lipoyl domain of E2; B, binding domain in E2 for E1; H1, H2, and H3 are the linker (hinge) regions of E2 (Fig. 1A); scE2, E2 with PreScission protease site introduced in H3 domain of E2; t-E2, truncated form of E2 with I domain and small part of H3 linker region; E3, dihydrolipoyl dehydrogenase; E3BP, E3-binding protein; L3, lipoyl domain of E3BP; B′, binding domain of E3BP that binds E3; I′ domain, inner domain of E3BP; H1′ and H2′ linker (hinge) regions in E3BP (Fig. 1A); AUC, analytical ultracentrifugation; SAXS, small angle x-ray scattering; R0, Rs, Rg, and Re are, respectively, the unhydrated spherical Stokes radius, Stokes radius, radius of gyration, and particle excluded volume radius. catalyzes the irreversible conversion of pyruvate to acetyl-CoA along with the reduction of NAD+. PDCs from all known sources contain the pyruvate dehydrogenase (E1), the dihydrolipoyl acetyltransferase (E2), and the dihydrolipoyl dehydrogenase (E3) components. Mammalian PDC has a highly organized structure in which the E2 component plays a central role in the organization, integrated chemical reactions, and regulation of the complex (1Reed L.J. Hackert M.L. J. Biol. Chem. 1990; 265: 8971-8974Abstract Full Text PDF PubMed Google Scholar, 2Patel M.S. Roche T.E. FASEB J. 1990; 4: 3224-3233Crossref PubMed Scopus (485) Google Scholar, 3Roche T.E. Cox D.J. Agius L. Sherratt H.S.A. Channeling in Intermediary Metabolism. Portland Press Ltd., London1996: 115-132Google Scholar, 4Roche T.E. Baker J. Yan X. Hiromasa Y. Gong X. Peng T. Dong J. Turkan A. Kasten S.A. Prog. Nucleic Acids Res. Mol. Biol. 2001; 70: 33-75Crossref PubMed Google Scholar, 5Roche T.E. Hiromasa Y. Turkan A. Gong X. Peng T. Yan X. Kasten S.A. Bao H. Dong J. Eur. J. Biochem. 2003; 270: 1050-1056Crossref PubMed Scopus (61) Google Scholar). Besides those universal components, the mammalian and subsequently some other eukaryotic PDCs were shown to contain another component, called E3-binding protein (E3BP); this protein was originally designated protein X (6De Marcuuci O. Lindsay G.J. Eur. J. Biochem. 1985; 149: 641-648Crossref PubMed Scopus (102) Google Scholar, 7Jilka J.M. Rahmatullah M. Kazemi M. Roche T.E. J. Biol. Chem. 1986; 261: 1858-1867Abstract Full Text PDF PubMed Google Scholar). Mammalian E3BP was first characterized as a component with a reactive lipoyl group on a single lipoyl domain (7Jilka J.M. Rahmatullah M. Kazemi M. Roche T.E. J. Biol. Chem. 1986; 261: 1858-1867Abstract Full Text PDF PubMed Google Scholar, 8Rahmatullah M. Roche T.E. J. Biol. Chem. 1987; 262: 10265-10271Abstract Full Text PDF PubMed Google Scholar, 9Hodgson J.A. De Marcucci O.G. Lindsay J.G. Eur. J. 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Chang C.L. Radke G.A. Roche T.E. J. Biol. Chem. 1989; 264: 2221-2227Abstract Full Text PDF PubMed Google Scholar, 14Powers-Greenwood S.L. Rahmatullah M. Radke G.A. Roche T.E. J. Biol. Chem. 1989; 264: 3655-3657Abstract Full Text PDF PubMed Google Scholar, 15Gopalakrishnan S. Rahmatullah M. Radke G.A. Powers-Greenwood S.L. Roche T.E. Biochem. Biophys. Res. Commun. 1989; 160: 715-721Crossref PubMed Scopus (39) Google Scholar, 16Sanderson S.J. Miller C.M. Lindsay G.J. Eur. J. Biochem. 1996; 236: 68-77Crossref PubMed Scopus (49) Google Scholar, 17Sanderson S.J. Khan S.S. McCartney R.G. Miller C.M. Lindsay G.J. Biochem. J. 1996; 319: 109-116Crossref PubMed Scopus (31) Google Scholar, 18Harris R.A. Bowker-Kinley M.M. Wu P. Jeng J. Popov K.M. J. Biol. Chem. 1997; 272: 19746-19751Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). This work provides new insights into the integration of E3BP into the central framework of the mammalian complex. Although first characterized in the bovine complex, the most thoroughly characterized E3BP is that associated with the yeast PDC (19Behal R.H. Browning K.S. Hall T.B. Reed L.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8732-8736Crossref PubMed Scopus (52) Google Scholar, 20Lawson J.E. Niu X.-D. Reed L.J. Biochemistry. 1991; 30: 11249-11254Crossref PubMed Scopus (33) Google Scholar, 21Stoops J.K. Baker T.S. Schroeter J.P. Kolodziej S.J. Niu X. Reed L.J. J. Biol. Chem. 1992; 267: 24769-24775Abstract Full Text PDF PubMed Google Scholar, 22Maeng C.-Y. Yazdi M.A. Niu X.-D. Lee H.Y. Reed L.J. Biochemistry. 1994; 33: 13801-13807Crossref PubMed Scopus (42) Google Scholar, 23Stoops J.K. Cheng R.H. Yazdi M.A. Maeng C.-Y. Schroeter J.P. Klueppelberg U. Kolodziej Baker T.S. Reed L.J. J. Biol. Chem. 1997; 272: 5757-5764Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Both the yeast (19Behal R.H. Browning K.S. Hall T.B. Reed L.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8732-8736Crossref PubMed Scopus (52) Google Scholar, 20Lawson J.E. Niu X.-D. Reed L.J. Biochemistry. 1991; 30: 11249-11254Crossref PubMed Scopus (33) Google Scholar, 22Maeng C.-Y. Yazdi M.A. Niu X.-D. Lee H.Y. Reed L.J. Biochemistry. 1994; 33: 13801-13807Crossref PubMed Scopus (42) Google Scholar) and mammalian E3BP (2Patel M.S. Roche T.E. FASEB J. 1990; 4: 3224-3233Crossref PubMed Scopus (485) Google Scholar, 7Jilka J.M. Rahmatullah M. Kazemi M. Roche T.E. J. Biol. Chem. 1986; 261: 1858-1867Abstract Full Text PDF PubMed Google Scholar, 10Rahmatullah M. Gopalakrishnan S. Radke G.A. Roche T.E. J. Biol. Chem. 1989; 264: 1245-1251Abstract Full Text PDF PubMed Google Scholar, 13Rahmatullah M. Gopalakrishnan S. Andrews P.C. Chang C.L. Radke G.A. Roche T.E. J. Biol. Chem. 1989; 264: 2221-2227Abstract Full Text PDF PubMed Google Scholar, 18Harris R.A. Bowker-Kinley M.M. Wu P. Jeng J. Popov K.M. J. Biol. Chem. 1997; 272: 19746-19751Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) consist of three globular domains connected by two linker (or hinge) regions (Fig. 1). At the N terminus is a lipoyl domain (designated L3) which is connected by linker region to the E3-binding domain (B′) which, in turn, is connected to a C-terminal domain (I′). The E2 component of the mammalian complex has a similar structure with two lipoyl domains (L1 and L2), an E1-binding domain (B), and a C-terminal inner (I) domain (Fig. 1) (1Reed L.J. Hackert M.L. J. Biol. Chem. 1990; 265: 8971-8974Abstract Full Text PDF PubMed Google Scholar, 2Patel M.S. Roche T.E. FASEB J. 1990; 4: 3224-3233Crossref PubMed Scopus (485) Google Scholar, 3Roche T.E. Cox D.J. Agius L. Sherratt H.S.A. Channeling in Intermediary Metabolism. Portland Press Ltd., London1996: 115-132Google Scholar, 4Roche T.E. Baker J. Yan X. Hiromasa Y. Gong X. Peng T. Dong J. Turkan A. Kasten S.A. Prog. Nucleic Acids Res. Mol. Biol. 2001; 70: 33-75Crossref PubMed Google Scholar, 12Rahmatullah M. Radke G.A. Andrews P.C. Roche T.E. J. Biol. Chem. 1990; 265: 14512-14517Abstract Full Text PDF PubMed Google Scholar, 13Rahmatullah M. Gopalakrishnan S. Andrews P.C. Chang C.L. Radke G.A. Roche T.E. J. Biol. Chem. 1989; 264: 2221-2227Abstract Full Text PDF PubMed Google Scholar, 24Bleile C.M. Hackert M.L. Pettit F.H. Reed L.J. J. Biol. Chem. 1981; 256: 514-519Abstract Full Text PDF PubMed Google Scholar, 25Thekkumkara T.J. Ho L. Wexler I.D. Pons G. Lui T.-C. Patel M.S. FEBS Lett. 1988; 240: 45-48Crossref PubMed Scopus (73) Google Scholar, 26Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1997; 272: 6361-6369Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). With E2 alone, the I domain assembles via 20 trimers (27Behal R.H. DeBuysere M.S. Demeler B. Hansen J.C. Olson M.S. J. Biol. Chem. 1994; 269: 31372-31377Abstract Full Text PDF PubMed Google Scholar) connecting as the corners of a pentagonal dodecahedron (28Oliver R.M. Reed L.J. Harris R. Electron Microscopy of Proteins. 2. Academic Press, London1982: 1-48Google Scholar, 29Wagenknecht T. Grassucci R. Radke G.A. Roche T.E. J. Biol. Chem. 1991; 266: 24650-24656Abstract Full Text PDF PubMed Google Scholar, 30Izard T. Aevarsson A. Allen M.D. Westphal A.H. Perham R.N. de Kok A. Hol W.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1240-1245Crossref PubMed Scopus (136) Google Scholar, 31Zhou Z.H. Wangcai L. Cheng R.H. Lawson J.E. McCarthy D.B. Reed L.J. Stoops J.K. J. Biol. Chem. 2001; 276: 21704-21713Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The C-terminal inner (I′) domain of E3BP associates with this inner core dodecahedron formed by the I domain of E2s (10Rahmatullah M. Gopalakrishnan S. Radke G.A. Roche T.E. J. Biol. Chem. 1989; 264: 1245-1251Abstract Full Text PDF PubMed Google Scholar, 20Lawson J.E. Niu X.-D. Reed L.J. Biochemistry. 1991; 30: 11249-11254Crossref PubMed Scopus (33) Google Scholar, 22Maeng C.-Y. Yazdi M.A. Niu X.-D. Lee H.Y. Reed L.J. Biochemistry. 1994; 33: 13801-13807Crossref PubMed Scopus (42) Google Scholar). In the yeast PDC, convincing evidence has been presented that the E3BP component is bound by its I domain binding inside the dodecahedron formed completely by the I domain of E2 (21Stoops J.K. Baker T.S. Schroeter J.P. Kolodziej S.J. Niu X. Reed L.J. J. Biol. Chem. 1992; 267: 24769-24775Abstract Full Text PDF PubMed Google Scholar, 22Maeng C.-Y. Yazdi M.A. Niu X.-D. Lee H.Y. Reed L.J. Biochemistry. 1994; 33: 13801-13807Crossref PubMed Scopus (42) Google Scholar, 23Stoops J.K. Cheng R.H. Yazdi M.A. Maeng C.-Y. Schroeter J.P. Klueppelberg U. Kolodziej Baker T.S. Reed L.J. J. Biol. Chem. 1997; 272: 5757-5764Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The free yeast E3BP could be added to the E2 60-mer and was bound in this manner. A 60/12 addition model has been proposed for mammalian E2·E3BP (16Sanderson S.J. Miller C.M. Lindsay G.J. Eur. J. Biochem. 1996; 236: 68-77Crossref PubMed Scopus (49) Google Scholar). In part, this model was favored based on the results with the yeast system, and the fact that the smallest symmetry element in the dodecahedron has 12 pentagonal faces. In contrast to the yeast system, resolved bovine E3BP, which retained the capacity to bind E3 and had a functional lipoyl domain, failed to bind back to assembled E2 under non-chaotropic conditions or after a rapid transition to less chaotropic conditions (8Rahmatullah M. Roche T.E. J. Biol. Chem. 1987; 262: 10265-10271Abstract Full Text PDF PubMed Google Scholar, 14Powers-Greenwood S.L. Rahmatullah M. Radke G.A. Roche T.E. J. Biol. Chem. 1989; 264: 3655-3657Abstract Full Text PDF PubMed Google Scholar, 27Behal R.H. DeBuysere M.S. Demeler B. Hansen J.C. Olson M.S. J. Biol. Chem. 1994; 269: 31372-31377Abstract Full Text PDF PubMed Google Scholar, 32Li L. Radke G.A. Ono K. Roche T.E. Arch. Biochem. Biophys. 1992; 296: 497-504Crossref PubMed Scopus (9) Google Scholar, 33McCartney R.G. Sanderson S.J. Lindsay J.G. Biochemistry. 1997; 36: 6819-6826Crossref PubMed Scopus (26) Google Scholar) (see “Discussion”). The sequence alignments of the human and yeast E2 and E3BP components (see Supplemental Material Fig. 1S) show that mammalian E3BP is closely related to mammalian E2 (18Harris R.A. Bowker-Kinley M.M. Wu P. Jeng J. Popov K.M. J. Biol. Chem. 1997; 272: 19746-19751Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The alignment shows that both lipoyl domains in human E2 are equally related to the L3 domain of human E3BP, that there is little basis for aligning linker regions, and that the linker regions are very different in yeast components. Of particular importance for assembly of the E2·E3BP complex, the I and I′ domains of mammalian E2 and E3BP, respectively, are closely related in sequence and size. In marked contrast, the inner domain of yeast E3BP has no clear relationship to the inner domain of human or yeast E2 or to human E3BP based on its amino acid sequence and size (Supplemental Material, Fig. 1S). Although it is clear that the I domain of human E2 and I′ domain of human E3BP are homologs, it is interesting to note that there is a large increase in the number of charged residues in the I′ domain of E3BP and a decrease in histidine residues (Fig. 1), including one required for catalysis of the transacetylation reaction (18Harris R.A. Bowker-Kinley M.M. Wu P. Jeng J. Popov K.M. J. Biol. Chem. 1997; 272: 19746-19751Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Differences in the Stokes radii, apparent density, and non-ideality in equilibrium sedimentation between human E2 and E2·E3BP, uncovered in this work, may be related to these differences in the electrostatic properties of E2 and E3BP (Fig. 1) and amino acid composition of the linker regions (Supplemental Material Fig. 1S) (see “Discussion”). Given these structural relationships, the previously proposed 60/12 addition model (16Sanderson S.J. Miller C.M. Lindsay G.J. Eur. J. Biochem. 1996; 236: 68-77Crossref PubMed Scopus (49) Google Scholar, 18Harris R.A. Bowker-Kinley M.M. Wu P. Jeng J. Popov K.M. J. Biol. Chem. 1997; 272: 19746-19751Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 19Behal R.H. Browning K.S. Hall T.B. Reed L.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8732-8736Crossref PubMed Scopus (52) Google Scholar) should not be considered an expected outcome for the organization of mammalian E2·E3BP. The E2-binding I′ domain of mammalian E3BP might take advantage of structural properties that derive from its close structural relationship to the I domain of E2. Here we provide evidence from a combination of studies that association of E3BP in the mammalian complex involves a substitution process rather than addition of E3BP to the E2 60-mer. Materials and Methods—The construction of vectors for preparation of His tag free E1 (tag removed by PreScission protease) and the vectors for expressing E2, E2 plus E3BP, sc-E2 that contains a PreScission site in the third linker region and a series of modified constructs will be described elsewhere. 2Y. Hiromasa, H. Bao, X. Yan, X. Gong, A. Yakhnin, J. Dong, S. A. Kasten, L. Hu, T. Peng, J. C. Baker, M. Sadler, and T. E. Roche, manuscript in preparation. A critical feature for removal of a truncated form of E2 (26Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1997; 272: 6361-6369Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) involved introducing silent mutations in the coding region for the L1 domain that removed a Shine-Dalgarno sequence, thereby preventing the codon for Met-59 serving as an internal start site. t-E2 is prepared from sc-E2 that is prepared as described for E2 below. Human E3 was prepared as described previously (34Kim H. Te-Cung L. Patel M.S. J. Biol. Chem. 1991; 266: 9367-9373Abstract Full Text PDF PubMed Google Scholar, 35Te-Cung L. Korotchkina L.G. Hyatt S.L. Vettakkourmakankav N.N. Patel M.S. J. Biol. Chem. 1995; 270: 15545-15550Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). E1 from which the His tag is removed by PreScission protease is prepared as will be described elsewhere. 2Y. Hiromasa, H. Bao, X. Yan, X. Gong, A. Yakhnin, J. Dong, S. A. Kasten, L. Hu, T. Peng, J. C. Baker, M. Sadler, and T. E. Roche, manuscript in preparation. We have developed extensively revised conditions for the preparation of human E2 and E2·E3BP (see Supplemental Material, Experimental Procedures). Slab Gel Electrophoresis and Densitometric Analysis—SDS-PAGE was performed with the standard Laemmli system (36Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205523) Google Scholar) with 10% acrylamide in the separating gel. Electrophoresis was performed at 14 mA and 4 °C. Gels were stained with Coomassie Brilliant Blue R-250. Band intensities were quantified with a Hewlett-Packard scanner, and the area density was analyzed by ImageQuant version 5.2 (Amersham Biosciences). Analytical Ultracentrifugation (AUC) Studies of E2 and E2·E3BP and Their Complexes—Sedimentation velocity experiments were conducted at 20 °C by using an Optima XL-I ultracentrifuge using the 4-hole An-60 Ti rotor with loading of 370-400 μl of sample and 400-450 μl of matching buffer into 12-mm charcoal-filled double sector cells. Following an initial scan at 3,000 rpm to check the total absorbance and detect very large aggregates, sedimentation velocity runs were conducted at the desired speed with scans continuously collected at a 2-7-min interval with absorption optics (usually at 280 nm but also at 450 nm) and/or interference optics that are analyzed by time derivative procedures (37Stafford W.F. Anal. Biochem. 1992; 203: 295-301Crossref PubMed Scopus (515) Google Scholar, 38Stafford W.F. Methods Enzymol. 2000; 323: 303-325Google Scholar, 39Philo J.S. Anal. Biochem. 2000; 279: 151-163Crossref PubMed Scopus (237) Google Scholar, 40Stafford W.F. Schuster T.M. Laue T.M. Modern Analytical Ultracentrifugation: Acquisition and Interpretation of Data for Biological and Synthetic Polymer Systems. Birkhauser Boston, Inc., Cambridge, MA1994: 119-137Crossref Google Scholar, 41Correia J.J. Methods Enzymol. 2000; 321: 81-100Crossref PubMed Google Scholar). Sedimentation profiles, including a consecutive set of at least four scans, were analyzed to obtain the apparent distribution of sedimentation coefficient g(s*) by using DCDT+ software version 1.16 provided by J. S. Philo (www.jphilo.mailway.com) (39Philo J.S. Anal. Biochem. 2000; 279: 151-163Crossref PubMed Scopus (237) Google Scholar). The sedimentation coefficient was calculated by using g(s*) fitting function in DCDT+ software (39Philo J.S. Anal. Biochem. 2000; 279: 151-163Crossref PubMed Scopus (237) Google Scholar, 40Stafford W.F. Schuster T.M. Laue T.M. Modern Analytical Ultracentrifugation: Acquisition and Interpretation of Data for Biological and Synthetic Polymer Systems. Birkhauser Boston, Inc., Cambridge, MA1994: 119-137Crossref Google Scholar). Buffer density and viscosity were calculated by Sednterp version 1.08 (www.jphilo.mailway.com). Partial specific volumes for E1, E3, E2, t-E2, E248·E3BP12, and E260·E3BP12, calculated based on their amino acid compositions using Sednterp, are 0.7339, 0.7414, 0.7462, 0.7485, 0.7459, and 0.7459 ml/g at 20 °C, respectively. In the calculation of the translational frictional coefficient ratios, f/f0, and Stokes radii, Rs, of protein components and their complexes, masses based on the amino acid sequence of constructs (below) and the above partial specific volumes of protein components were used to calculate R0 (i.e. Stokes radii for the unhydrated sphere of the same mass and density). Two different procedures (42Hiromasa Y. Roche T.E. J. Biol. Chem. 2003; 278: 33681-33693Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) were used to analyze sedimentation velocity results in order to estimate the fraction of E1 or E3 component bound to the E2 or E2·E3BP. When a significant portion of a component was both bound and free, the level of free component was estimated by determining the change in concentration of trailing component in the presence versus the absence of E2 oligomer using absorption optics. The base line was determined by over-speed methods at the end of the run. The bound component was also estimated from the increase in the rapidly sedimenting complex due to bound component (fringe ratio method). The g(s*) analysis in DCDT+ software gave the g(s*) distribution and extrapolated to the initial fringe. The initial fringe of complex fraction (i.e. E1-E260) was calculated above the 20 S region of the g(s*) distribution. The initial fringe of the complex fraction was then compared with that of E2 or E2·E3BP alone determined in the same manner. Refractive increments (fringe) are nearly constant for proteins (43Babul J. Stellwagen E. Anal. Biochem. 1969; 28: 216-221Crossref PubMed Scopus (305) Google Scholar, 44Schachman H.K. Rowe A.J. Horton J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge, UK1992: 3-15Google Scholar, and 59Laue T.M. Beckman Application Information A-1821A. 1996; : 1-17Google Scholar). The fringe ratio between E2 or E2·E3BP complex to E1 or E3 and E2 or E2·E3BP alone provides a weight concentration ratio. The number of bound E1 per E2 60-mer is as follows: ((fringe of E1-E2/ fringe of E2)-1) × M of E2 60-mer/M of E1 tetramer, where M is the calculated molecular mass. In the case of E1 or E3 binding to E2·E3BP, a model-dependent molecular mass for E2·E3BP is substituted for the M of E2 in this equation. Sedimentation equilibrium studies on E2 and E2·E3BP were conducted using three charcoal-filled Epon centerpieces with 6-sector cells. Normally, 50-μl samples were overlaid over 25 μl of Fluorinert® FC-43 silicon oil (3M Industrial Chemical Products Division), and 100 μl of matched solvent was introduced in the reference position. Each protein was evaluated at three concentrations, and equilibrium was attained at six rotor speeds. Following equilibration at the lowest speed during the 30-h period, at least an 8-h equilibration time was used for the transition to a higher speed. The stability of the concentration gradient established was confirmed over at least a 4-h period before making final measurements. The equilibrated protein gradients and their solvent controls were scanned 10 times at 0.001-cm intervals; the difference between the averaged sample and solvent boundaries yielded the equilibrium boundary of the solute protein. Sedimentation equilibrium data were evaluated with Beckman software (version 4) that was provided with the Optima XL-I ultracentrifuge. Additional analyses were required because there was a decrease in mass with increasing concentration of sample. This indicates a non-ideality (molecular crowding) that is characteristic of a large solvent-filled or asymmetric molecule with substantial electrostatic repulsion (see “Discussion”). For the different speeds and concentrations, the apparent molecular weight, Mapp, was plotted against the concentration (A280 nm); these Mapp values were based on 20 points (0.02 cm) and were recalculated at each 0.001-cm interval. Mapp estimates within a 0.01 A280 nm interval were averaged for all the above. From plots of these average Mapp and of 1/Mapp versus concentration in mg/ml, the molecular weight, M, was estimated by extrapolation to zero concentration (45Chatelier R.C. Minton A.P. Biopolymers. 1987; 26: 507-524Crossref PubMed Scopus (57) Google Scholar, 46Cole N. Ralston G.B. Biochim. Biophys. Acta. 1992; 1121: 23-30Crossref PubMed Scopus (21) Google Scholar, 47Minton A.P. Biophys. Chem. 1995; 26: 65-70Crossref Scopus (49) Google Scholar, 48Adams E.T. Fujita H. Williams J.W. Ultracentrifuge Analysis in Theory and Experiment. Academic Press, New York1962: 119-129Google Scholar). For conversion of A280 nm values to mg/ml, 1.34 mg/ml has a A280 nm of 1 for 1-cm light path (or 0.75 A280 nm per mg/ml). With 1.2-cm light path in the centrifuge, this was 1.154 mg/ml E2 per/1 A at 280 nm. The concentration dependence of Mapp, for a single solute is described by Equation 1, Mapp=M/(1+c(dlnγ/dc)) where c is the concentration of protein solute (w/v, g/liter), and γ is its activity coefficient (45Chatelier R.C. Minton A.P. Biopolymers. 1987; 26: 507-524Crossref PubMed Scopus (57) Google Scholar). The estimated d lnγ/dc, the slope of the 1/Mapp versus c plot, was evaluated by using a simple model in which the excluded volume of the solute was estimated using the first virial coefficient (B2) for an equivalent hard sphere (45Chatelier R.C. Minton A.P. Biopolymers. 1987; 26: 507-524Crossref PubMed Scopus (57) Google Scholar), so that M(d lnγ/dc) = B2 = 8 Ve, where Ve, like B2, has units of liter/g and is an approximation of the effective excluded volume occupied by 1 g of solute. We applied a linear fit to the change in 1/Mapp with concentration, so that we did not fit our results with higher order virial coefficients that have a higher order dependence on concentration. We used Ve to calculate the excluded volume radius, Re. Calculated Subunit, Component, and E2 Oligomer Masses—Masses of components and complexes were calculated from the amino acid sequence of our constructs using the PeptideMass program at the ExPASy site (us.expasy.org/) with cofactors (lipoyl groups on E2 and E3BP and FAD on E3) added for masses used in evaluating AUC data. The
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