The Structure of a Mutant Insulin Uncouples Receptor Binding from Protein Allostery
2008; Elsevier BV; Volume: 283; Issue: 30 Linguagem: Inglês
10.1074/jbc.m800235200
ISSN1083-351X
AutoresZhu‐li Wan, Kun Huang, Shi-Quan Hu, Jonathan Whittaker, Michael A. Weiss,
Tópico(s)Protein Structure and Dynamics
ResumoThe zinc insulin hexamer undergoes allosteric reorganization among three conformational states, designated T6, T3R3f, and R6. Although the free monomer in solution (the active species) resembles the classical T-state, an R-like conformational change is proposed to occur upon receptor binding. Here, we distinguish between the conformational requirements of receptor binding and the crystallographic TR transition by design of an active variant refractory to such reorganization. Our strategy exploits the contrasting environments of HisB5 in wild-type structures: on the T6 surface but within an intersubunit crevice in R-containing hexamers. The TR transition is associated with a marked reduction in HisB5 pKa, in turn predicting that a positive charge at this site would destabilize the R-specific crevice. Remarkably, substitution of HisB5 (conserved among eutherian mammals) by Arg (occasionally observed among other vertebrates) blocks the TR transition, as probed in solution by optical spectroscopy. Similarly, crystallization of ArgB5-insulin in the presence of phenol (ordinarily a potent inducer of the TR transition) yields T6 hexamers rather than R6 as obtained in control studies of wild-type insulin. The variant structure, determined at a resolution of 1.3Å, closely resembles the wild-type T6 hexamer. Whereas ArgB5 is exposed on the protein surface, its side chain participates in a solvent-stabilized network of contacts similar to those involving HisB5 in wild-type T-states. The substantial receptor-binding activity of ArgB5-insulin (40% relative to wild type) demonstrates that the function of an insulin monomer can be uncoupled from its allosteric reorganization within zinc-stabilized hexamers. The zinc insulin hexamer undergoes allosteric reorganization among three conformational states, designated T6, T3R3f, and R6. Although the free monomer in solution (the active species) resembles the classical T-state, an R-like conformational change is proposed to occur upon receptor binding. Here, we distinguish between the conformational requirements of receptor binding and the crystallographic TR transition by design of an active variant refractory to such reorganization. Our strategy exploits the contrasting environments of HisB5 in wild-type structures: on the T6 surface but within an intersubunit crevice in R-containing hexamers. The TR transition is associated with a marked reduction in HisB5 pKa, in turn predicting that a positive charge at this site would destabilize the R-specific crevice. Remarkably, substitution of HisB5 (conserved among eutherian mammals) by Arg (occasionally observed among other vertebrates) blocks the TR transition, as probed in solution by optical spectroscopy. Similarly, crystallization of ArgB5-insulin in the presence of phenol (ordinarily a potent inducer of the TR transition) yields T6 hexamers rather than R6 as obtained in control studies of wild-type insulin. The variant structure, determined at a resolution of 1.3Å, closely resembles the wild-type T6 hexamer. Whereas ArgB5 is exposed on the protein surface, its side chain participates in a solvent-stabilized network of contacts similar to those involving HisB5 in wild-type T-states. The substantial receptor-binding activity of ArgB5-insulin (40% relative to wild type) demonstrates that the function of an insulin monomer can be uncoupled from its allosteric reorganization within zinc-stabilized hexamers. Insulin is a small globular protein containing two chains, A (21 residues) and B (30 residues). In pancreatic β-cells, the hormone is stored as Zn2+-stabilized hexamers, which form microcrystalline arrays within specialized secretory granules (1Dodson G. Steiner D. Curr. Opin. Struct. Biol. 1998; 8: 189-194Crossref PubMed Scopus (444) Google Scholar). The hexamers dissociate upon secretion into the portal circulation, enabling the hormone to function as a zinc-free monomer. Structure-function relationships have been inferred from patterns of sequence conservation (2Baker E.N. Blundell T.L. Cutfield J.F. Cutfield S.M. Dodson E.J. Dodson G.G. Hodgkin D.M. Hubbard R.E. Isaacs N.W. Reynolds C.D. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1988; 319: 369-456Crossref PubMed Scopus (693) Google Scholar) and extensively probed by mutagenesis (2Baker E.N. Blundell T.L. Cutfield J.F. Cutfield S.M. Dodson E.J. Dodson G.G. Hodgkin D.M. Hubbard R.E. Isaacs N.W. Reynolds C.D. Philos. Trans. R. Soc. 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Biol. 2002; 316: 435-441Crossref PubMed Scopus (42) Google Scholar, 10Huang K. Xu B. Hu S.Q. Chu Y.C. Hua Q.X. Qu Y. Li B. Wang S. Wang R.Y. Nakagawa S.H. Theede A.M. Whittaker J. De Meyts P. Katsoyannis P.G. Weiss M.A. J. Mol. Biol. 2004; 341: 529-550Crossref PubMed Scopus (74) Google Scholar). A variety of evidence suggests that insulin undergoes a change in conformation on binding to the insulin receptor (IR) 2The abbreviations used are: IR, insulin receptor; DKP-insulin, insulin analog containing three substitutions in B chain (AspB10, LysB28, and ProB29); pH*, pD value uncorrected for isotope effect; Rf, frayed R-state in which residues B1 and B2 are not well ordered. (11De Meyts P. Whittaker J. Nat. Rev. Drug Discov. 2002; 1: 769-783Crossref PubMed Scopus (486) Google Scholar). A model for induced fit is provided by the TR transition, a long range allosteric reorganization of zinc insulin hexamers (12Brader M.L. Dunn M.F. Trends Biochem. Sci. 1991; 16: 341-345Abstract Full Text PDF PubMed Scopus (75) Google Scholar). The structural basis of the TR transition has been extensively investigated by x-ray crystallography (13Bentley G. Dodson E. Dodson G. Hodgkin D. Mercola D. Nature. 1976; 261: 166-168Crossref PubMed Scopus (238) Google Scholar, 14Derewenda U. Derewenda Z. Dodson E.J. Dodson G.G. Reynolds C.D. Smith G.D. Sparks C. Swenson D. Nature. 1989; 338: 594-596Crossref PubMed Scopus (313) Google Scholar, 15Ciszak E. Beals J.M. Frank B.H. Baker J.C. Carter N.D. Smith G.D. Structure. 1995; 3: 615-622Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). In this paper, we investigate the relationship between such allosteric reorganization and biological activity. Experimental design exploits the contrasting structures of classical hexamers to introduce an electrostatic block to the TR transition. The TR transition encompasses three families of zinc insulin hexamers, designated T6, T3R3f, and R6 (Fig. 1). Interconversion among these families is regulated by ionic strength (13Bentley G. Dodson E. Dodson G. Hodgkin D. Mercola D. Nature. 1976; 261: 166-168Crossref PubMed Scopus (238) Google Scholar, 16Blundell T.L. Cutfield J.F. Cutfield S.M. Dodson E.J. Dodson G.G. Hodgkin D.C. Mercola D.A. Vijayan M. Nature. 1971; 231: 506-511Crossref PubMed Scopus (272) Google Scholar) and the binding of small cyclic alcohols (14Derewenda U. Derewenda Z. Dodson E.J. Dodson G.G. Reynolds C.D. Smith G.D. Sparks C. Swenson D. Nature. 1989; 338: 594-596Crossref PubMed Scopus (313) Google Scholar). NMR studies have established that the structure of an insulin monomer in solution resembles the crystallographic T-state (Fig. 2, left) (17Hua Q.X. Shoelson S.E. Kochoyan M. Weiss M.A. Nature. 1991; 354: 238-241Crossref PubMed Scopus (235) Google Scholar, 18Hua Q.X. Hu S.Q. Frank B.H. Jia W. Chu Y.C. Wang S.H. Burke G.T. Katsoyannis P.G. Weiss M.A. J. Mol. Biol. 1996; 264: 390-403Crossref PubMed Scopus (112) Google Scholar, 19Olsen H.B. Ludvigsen S. Kaarsholm N.C. Biochemistry. 1996; 35: 8836-8845Crossref PubMed Scopus (123) Google Scholar). The TR transition is characterized by a change in the secondary structure of the B-chain (Fig. 2, right). 3The altered orientation between the A-chain and N-terminal segment of the B-chain also entails a change in the handedness of the A7-B7 disulfide bridge, which is in turn coupled to a change in the conformation of GlyB8 (from the right to left side of the Ramachandran plane). The A7-B7 sulfur atoms are exposed in the T-state but buried in a nonpolar crevice in the R-state. Although extensive contacts stabilize the hexamer, within an individual protomer the TR transition is associated with a loss of interchain contacts. Such R-state features have been observed only in zinc hexamers; monomeric R-like conformers have not been detected in solution by NMR (18Hua Q.X. Hu S.Q. Frank B.H. Jia W. Chu Y.C. Wang S.H. Burke G.T. Katsoyannis P.G. Weiss M.A. J. Mol. Biol. 1996; 264: 390-403Crossref PubMed Scopus (112) Google Scholar, 19Olsen H.B. Ludvigsen S. Kaarsholm N.C. Biochemistry. 1996; 35: 8836-8845Crossref PubMed Scopus (123) Google Scholar, 20Wan Z. Xu B. Chu Y.C. Katsoyannis P.G. Weiss M.A. Biochemistry. 2003; 42: 12770-12783Crossref PubMed Scopus (18) Google Scholar). Dodson and colleagues (14Derewenda U. Derewenda Z. Dodson E.J. Dodson G.G. Reynolds C.D. Smith G.D. Sparks C. Swenson D. Nature. 1989; 338: 594-596Crossref PubMed Scopus (313) Google Scholar) have nonetheless proposed that an insulin monomer may adopt an R-like conformation upon receptor binding. Such induced fit could extend the potential receptor-binding surface of insulin by exposing side chains otherwise inaccessible in the T-state. Indirect evidence favoring this hypothesis has been obtained by nonstandard mutagenesis (21Nakagawa S.H. Zhao M. Hua Q.X. Hu S.Q. Wan Z.L. Jia W. Weiss M.A. Biochemistry. 2005; 44: 4984-4999Crossref PubMed Scopus (55) Google Scholar, 22Hua Q.X. Liu M. Hu S.Q. Jia W. Arvan P. Weiss M.A. J. Biol. Chem. 2006; 281: 24889-24899Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). d-Amino acid substitutions at GlyB8 (black circles in Fig. 2) markedly impair receptor binding in association with a shift in the conformational equilibrium among T6, T3R3f, and R6 hexamers favoring the T-state. The low biological activity of such analogs (reduced by 102 to 103-fold) (21Nakagawa S.H. Zhao M. Hua Q.X. Hu S.Q. Wan Z.L. Jia W. Weiss M.A. Biochemistry. 2005; 44: 4984-4999Crossref PubMed Scopus (55) Google Scholar) has been ascribed to thermodynamic stabilization of a native-like but inactive T-state conformation (22Hua Q.X. Liu M. Hu S.Q. Jia W. Arvan P. Weiss M.A. J. Biol. Chem. 2006; 281: 24889-24899Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar).FIGURE 2Cylinder models of T- and R-state protomers. Shown is the crystal structure of dimer comprising T-state (left) and Rf-state (right) as extracted from T3R3f hexamer. The A-chains are shown in black, and B-chains are shown in green. In each panel, the positions of residues B5 and B8 (Ca atoms) are shown as filled red and black circles, respectively. Dimerization is mediated by an anti-parallel β-sheet (central ribbons) and nonpolar interactions between central B-chain α-helices. In the T state conformation, the A-chain contains an N-terminal α-helix (residues A1-A8) followed by a noncanonical turn, second helix (A12-A18), and C-terminal segment (A19-A21); the B-chain contains an N-terminal segment (residues B1-B6), type II′ β-turn (B7-B10), central α-helix (B9-B19), type I β-turn (B20-B23), and C-terminal β-strand (B24-B28), extended by less well ordered terminal residues B29 and B30. In the R-state, the N-terminal portion of the B-chain participates in a single long α-helix. The resulting B1-B19 α-helix (or B3-B19 in the frayed Rf state) projects from the globular core of the protomer to make extensive hexamer contacts, including formation of a specific phenol-binding pocket at a trimer interface (14Derewenda U. Derewenda Z. Dodson E.J. Dodson G.G. Reynolds C.D. Smith G.D. Sparks C. Swenson D. Nature. 1989; 338: 594-596Crossref PubMed Scopus (313) Google Scholar). The side chain of HisB5 packs within an R-state-specific subunit interface in T3R3f and R6 hexamers; the side chain neither contacts a zinc ion nor contacts a bound phenolic ligand.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The present study focuses on the role of residue B5, conserved as His among eutherian mammals. Previous studies have shown that HisB5 contributes to the foldability of human proinsulin but is not required for biological activity (22Hua Q.X. Liu M. Hu S.Q. Jia W. Arvan P. Weiss M.A. J. Biol. Chem. 2006; 281: 24889-24899Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Experimental design exploits the contrasting environments of HisB5 in wild-type structures: on the T6 surface but within an intersubunit crevice in R-containing hexamers. In the T-state, HisB5 lies within the N-terminal extended strand (Fig. 2, left, filled red circle). In the T6 hexamer (Fig. 3A, red side chains), HisB5 is surrounded in part by water molecules (blue spheres); the imidazole ring packs against an interchain crevice near IleA10 and the solvent-exposed A7-B7 disulfide bridge (shown in stereo view in Fig. 3C). 4In 1H NMR spectra of engineered insulin monomers (such as DKP-insulin) (18Hua Q.X. Hu S.Q. Frank B.H. Jia W. Chu Y.C. Wang S.H. Burke G.T. Katsoyannis P.G. Weiss M.A. J. Mol. Biol. 1996; 264: 390-403Crossref PubMed Scopus (112) Google Scholar), prominent B5-related nuclear Overhauser effects are observed within this crevice, indicating that such packing does not require dimerization or hexamer assembly. In addition, NOESY spectra obtained at pD 1.9 and pD 7.6 exhibit similar patterns of B5-related interresidue contacts, demonstrating that its T-state-specific packing along the surface of the A-chain can accommodate either a neutral or protonated imidazole ring. Participation of HisB5 in the R-specific α-helix (Fig. 2, right, red circle) causes its side chain to move from the T6 surface to pack within a trimer interface; the B5 side chain does not participate in zinc coordination or the immediate binding of phenol. The structural environment of HisB5 within an R-specific trimer interface is shown in Fig. 4 (see also supplemental Figs. S1 and S2).FIGURE 4Environment of HisB5 in R-related subunit interface in R6 zinc insulin hexamer. A and B, structure of an R6 hexamer (Protein Data Bank code 1ZNJ). A, space-filling representations showing (left to right) front, back, and side views. The six B5 side chains are shown in green, and zinc ions are shown in yellow. The hexamer contains six independent molecules. The corresponding A and B chains of molecules 1, 3, and 5 are shown in black and gray, respectively; the A and B chains of molecules 2, 4, and 6 are shown in light and dark blue, respectively. B, stereo pair, stick model of the intersubunit crevice. The position of HisB5 (green) is shown in relation to the B-chains of molecule 1 (M1-B; gray), molecule 2 (M2-B; blue), and molecule 4 (M4-B; purple) and to the A-chain of molecule 4 (M4-A; black). The analogous R3f trimer interface in a T3R3f hexamer is shown in supplemental Fig. S1. Specific residue labeling and corresponding space-filling models are provided in supplemental Fig. S2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our study has two parts. We first describe pH-dependent 1H NMR studies of the wild-type R6 hexamer. R-specific burial of HisB5 is shown to be associated with a marked reduction in its side chain pKa. These results in turn predict that a positive charge at B5 would destabilize the associated R-specific trimer interface. To test this prediction, a human insulin analog was prepared in which HisB5 was substituted by Arg, a variant observed in some hystricomorph mammals, fish, and reptiles (23Conlon J.M. Peptides. 2001; 22: 1183-1193Crossref PubMed Scopus (108) Google Scholar). 5Hystricomorph mammals exhibit divergent insulin sequences, including at B5. Such insulins exhibit low affinity for the human IR and may not form zinc hexamers due to substitution of HisB10 (68Bajaj M. Blundell T.L. Horuk R. Pitts J.E. Wood S.P. Gowan L.K. Schwabe C. Wollmer A. Gliemann J. Gammeltoft S. Biochem. J. 1986; 238: 345-351Crossref PubMed Scopus (26) Google Scholar). Fibrillation of a divergent and presumed monomeric insulin in the degu rat is associated with islet amyloidosis and age-associated diabetes mellitus (69Hellman U. Wernstedt C. Westermark P. O'Brien T.D. Rathbun W.B. Johnson K.H. Biochem. Biophys. Res. Commun. 1990; 169: 571-577Crossref PubMed Scopus (43) Google Scholar, 70Nishi M. Steiner D.F. Mol. Endocrinol. 1990; 4: 1192-1198Crossref PubMed Scopus (67) Google Scholar). Although the variant hormone retains substantial activity, ArgB5-insulin is unable to undergo the TR transition in solution. Similarly, crystals of ArgB5-insulin, grown under conditions leading to formation of wild-type R6 hexamers, contain only T6 hexamers. The variant structure, determined by molecular replacement at a resolution of 1.3 Å, closely resembles the wild-type T6 hexamer. The side chains of HisB5 and ArgB5, despite their differences in size and shape, pack within corresponding interchain crevices and participate in part in analogous networks of van der Waals interactions and hydrogen bonds. Despite such similarities, ArgB5-insulin is less stable than the wild-type protein, as probed by chemical denaturation studies, presumably due either to loss of weakly polar interactions associated with the imidazole ring (24Burley S.K. Petsko G.A. Adv. Protein Chem. 1988; 39: 125-189Crossref PubMed Scopus (803) Google Scholar) or to less efficient packing of the linear ArgB5 side chain within the crevice. Together, the properties of ArgB5-insulin uncouple the mechanism of receptor binding from the choreography of conformational changes in the classical TR transition. Impairment of hexamer reorganization by introduction of a charged side chain, reminiscent of classical mutations in hemoglobin that impair its cooperativity (25Bonaventura J. Bonaventura C. Amiconi G. Tentori L. Brunori M. Antonini E. J. Biol. Chem. 1975; 250: 6278-6281Abstract Full Text PDF PubMed Google Scholar, 26Bonaventura C. Bonaventura J. Amiconi G. Tentori L. Brunori M. Antonini E. J. Biol. Chem. 1975; 250: 6273-6277Abstract Full Text PDF PubMed Google Scholar, 27Poyart C. Bursaux E. Arnone A. Bonaventura J. Bonaventura C. J. Biol. Chem. 1980; 255: 9465-9473Abstract Full Text PDF PubMed Google Scholar, 28Bonaventura J. Bonaventura C. Sullivan B. Godette C. J. Biol. Chem. 1975; 250: 9250-9255Abstract Full Text PDF PubMed Google Scholar), demonstrates the utility of “electrostatic engineering” in studies of protein allostery. Synthesis of Insulin Analogs—Human insulin and AspB28-insulin were obtained from Lilly and Novo-Nordisk (Bagsværd, Denmark), respectively. Synthesis of ArgB5-insulin was performed as described (9Xu B. Hua Q.X. Nakagawa S.H. Jia W. Chu Y.C. Katsoyannis P.G. Weiss M.A. J. Mol. Biol. 2002; 316: 435-441Crossref PubMed Scopus (42) Google Scholar, 10Huang K. Xu B. Hu S.Q. Chu Y.C. Hua Q.X. Qu Y. Li B. Wang S. Wang R.Y. Nakagawa S.H. Theede A.M. Whittaker J. De Meyts P. Katsoyannis P.G. Weiss M.A. J. Mol. Biol. 2004; 341: 529-550Crossref PubMed Scopus (74) Google Scholar); see also the supplemental materials. Receptor Binding Assays—The human IR was expressed and purified as described (29Whittaker J. Whittaker L. J. Biol. Chem. 2005; 280: 20932-20936Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Competitive IR binding assays were performed by a microtiter plate antibody capture assay. Microtiter strip plates (Nunc Maxisorb) were incubated overnight at 4 °C with anti-FLAG IgG (100 ml/well of a 40 μg/ml solution in phosphate-buffered saline). Washing, blocking, receptor binding, and competitive binding assays with labeled and unlabeled peptides were performed as described (29Whittaker J. Whittaker L. J. Biol. Chem. 2005; 280: 20932-20936Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Binding data were analyzed by a two-site sequential model with homologous or heterologous labeled and unlabeled ligands to obtain dissociation constants. The percentage of tracer bound in the absence of competing ligand was <15% to avoid ligand depletion artifacts. Visible Absorption Spectroscopy—To probe the TR transition of Co2+-substituted insulin hexamers, the d-d optical absorption bands of Co2+ (a characteristic feature of a tetrahedral complex) (30Roy M. Brader M.L. Lee R.W. Kaarsholm N.C. Hansen J.F. Dunn M.F. J. Biol. Chem. 1989; 264: 19081-19085Abstract Full Text PDF PubMed Google Scholar, 31Wollmer A. Gilge G. Brandenburg D. Gattner H.G. Biol. Chem. Hoppe-Seyler. 1994; 375: 219-222PubMed Google Scholar) were monitored as described (32Nakagawa S.H. Tager H.S. J. Biol. Chem. 1991; 266: 11502-11509Abstract Full Text PDF PubMed Google Scholar). Solutions contained 0.2 mm insulin or insulin analog in a buffer consisting of 0.07 mm CoCl2 and 50 mm phenol in 50 mm Tris-HCl (pH 8). Absorption spectra were obtained in the presence of 0.8 m NaSCN; the thiocyanate anion contributes a fourth ligand (in addition to three symmetry-related HisB10 side chains) to the coordination of each axial metal ion and so enhances the d-d band intensity (30Roy M. Brader M.L. Lee R.W. Kaarsholm N.C. Hansen J.F. Dunn M.F. J. Biol. Chem. 1989; 264: 19081-19085Abstract Full Text PDF PubMed Google Scholar, 31Wollmer A. Gilge G. Brandenburg D. Gattner H.G. Biol. Chem. Hoppe-Seyler. 1994; 375: 219-222PubMed Google Scholar). 1H NMR pH Titrations—Spectra were obtained at 700 MHz with a high sensitivity cryogenic probe (Bruker Biospin, Inc., Billerica, MA). Free histidine (Sigma-Aldrich, St. Louis, MO), human insulin (calculated pI 5.4), or AspB28-insulin (calculated pI 4.9) were first dissolved in 500 μl of 99.0% D2O containing 10 mm deuterated Tris-HCl (pH* 8.5, direct meter reading; Isotec, Miamisburg, OH) at respective sample concentrations of 10 mm, 1 mm, and 1 mm. After ∼12 h of hydrogen-deuterium amide proton exchange at room temperature, samples were lyophilized and redissolved in 500 μl of 99.9% D2O containing 50 mm deuterated phenol (Sigma-Aldrich); 0.33 mm ZnCl2 was then added to the protein solutions to form phenol-stabilized R6 hexamers. The apparent pD of the samples (designated pH*; uncorrected for isotope effects) was in each case measured with a microelectrode (Ingold 6030; Toledo-Mettler, Columbus, OH) at 40 °C and adjusted to pH* 8.3 with DCl or NaOD. 1H NMR spectra were acquired at 40 °C; chemical shift values are shown relative to 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS). Successive pH* values were obtained by adding an aliquot of dilute DCl; serial spectra were obtained at each pH* tested between pH* 8.5 and 2.0. Titration curves were fitted to a modified Henderson-Hasselbalch equation using Kaleida-Graph, Synergy Software (Reading, PA) by nonlinear least squares analysis as follows, δobs=δAH++δA10(pH−pKa)1+10(pH−pKa) (Eq. 1) in which δobs is the chemical shift observed at each pH* value, and δAH+ and δA are the chemical shifts of the protonated and deprotonated histidines, respectively. The relationship, pKaH=0.929×pKaH∗+0.42 (Eq. 2) was applied to convert the apparent pKa estimates obtained in D2O to corresponding values in H2O as described (33Krezel A. Bal W. J. Inorg. Biochem. 2004; 98: 161-166Crossref PubMed Scopus (394) Google Scholar). Circular Dichroism—Far-UV CD spectra were obtained as described (34Hua Q.X. Chu Y.C. Jia W. Phillips N.B. Wang R.Y. Katsoyannis P.G. Weiss M.A. J. Biol. Chem. 2002; 277: 43443-43453Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Spectra, acquired with an Aviv spectropolarimeter (Aviv Biomedical Inc., Lakewood, NJ), were normalized by mean residue ellipticity. Samples were dissolved in 10 mm potassium phosphate (pH 7.4) and 50 mm KCl at a protein concentration of ∼25 μm. ZnCl2 was added to provide 2.2 zinc ions/insulin hexamer. For equilibrium denaturation studies, samples were diluted in the same buffer to 5 μm; guanidine HCl was employed as denaturant (34Hua Q.X. Chu Y.C. Jia W. Phillips N.B. Wang R.Y. Katsoyannis P.G. Weiss M.A. J. Biol. Chem. 2002; 277: 43443-43453Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Data were obtained at 4 °C and fitted by nonlinear least squares to a two-state model (35Sosnick T.R. Fang X. Shelton V.M. Methods Enzymol. 2000; 317: 393-409Crossref PubMed Google Scholar). X-ray Crystallography—Crystals were grown by hanging drop vapor diffusion under intended R6 conditions (see supplemental materials). Data were collected from single crystals mounted in a rayon loop and flash-frozen to 100 K. Reflections from 30.6 to 1.3 Å were measured with a CCD detector system using synchrotron radiation at the Advanced Light Source (Lawrence Berkeley National Laboratory, Berkeley, CA). Data were processed with the program DTREK. Crystals belong to space group R3 with unit cell parameters a = b = 81.39 Å, c = 34.00 Å, α = β = 90, and γ = 120°. These dimensions are typical of T6 crystals under cryoconditions. The structure was determined by molecular replacement using the CNS suite of programs (36Brunger 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. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). A model was obtained using the native T2 dimer (Protein Data Bank identifier 4INS) following removal of all water molecules and zinc ions. A translation-function search was performed using coordinates from the best solution for the rotation function following analysis of data between 15.0 and 4.0 Å resolution. Rigid body refinement using CNS, employing overall anisotropic temperature factors and bulk solvent correction, yielded respective values of 0.31 and 0.30 for R and Rfree for data between 19.2 and 3.0 Å resolution. Between refinement cycles, 2Fo - Fc and Fo - Fc maps were calculated using data to 3.0 Å resolution; zinc ions were then built into the structure using the program O (37Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). The geometry was monitored with PROCHECK (38Laskowski R.A. Macarthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar); zinc ions and water molecules were built into the difference map as the refinement proceeded. Calculation of omit maps (of particular importance in relation to the N-terminal segment of the B-chain) and further refinements were carried out using CNS (36Brunger 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. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar), which implements maximum likelihood torsion angle dynamics and conjugate-gradient refinement. X-ray diffraction and refinement statistics are provided in supplemental Table S1. 1H NMR Studies of the R6 Hexamer—We first describe pH-dependent 1H NMR studies of the wild-type R6 zinc insulin hexamer as a probe of the electrostatic environment of HisB5. Due to isoelectric precipitation of the wild-type hexamer near pH 6, these studies were extended through the use of a variant R6 hexamer with lower isoelectric point (AspB28-insulin) (39Brange J. Andersen L. Laursen E.D. Meyn G. Rasmussen E. J. Pharmacol. Sci. 1997; 86: 517-525Abstract Full Text PDF PubMed Scopus (449) Google Scholar, 40Heinemann L. Heise T. Jorgensen L.N. Starke A.A. Diabet. Med. 1993; 10: 535-539Crossref PubMed Scopus (97) Google Scholar). Crystallographic studies have established that AspB28-insulin (a fast acting analog in clinical use for treatment of diabetes mellitus) forms R6 hexamers that (with the exception of a local distortion at the dimer interface) are essentially identical to those of wild-type insulin. Human insulin contains two histidine residues (positions B5 and B10). Whereas HisB10 mediates the binding of axial zinc ions in each family of insulin hexamer, the environment of HisB5 varies among T- and R-states. Although 1H NMR spectra of T6 and T3R3f hexamers are not tractable, the spectrum of the phenol-stabilized R6 hexamer exhibits high resolution features and has been well characterized (supplemental Fig. S3) (41Jacoby E. Hua Q.X. Stern A.S. Frank B.H. Weiss M.A. J. Mol. Biol. 1996; 258: 136-157Crossref PubMed Scopus (54) Google Scholar). To probe the pKa values of the histidine residues, 1H NMR spectra were obtained in D2Oat 40 °C as a function of pD; reference spectra of free histidine were also obtained. (A temperature of 40 °C was chosen to enhance resonance line widths relative to room temperature with retention of native pattern of chemical shifts (41Jacoby E. Hua Q.X.
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