The Crystal Structure of Nitrophorin 2
2000; Elsevier BV; Volume: 275; Issue: 39 Linguagem: Inglês
10.1074/jbc.m002857200
ISSN1083-351X
AutoresJohn F. Andersen, W.R. Montfort,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoNitrophorin 2 (NP2) (also known as prolixin-S) is a salivary protein that transports nitric oxide, binds histamine, and acts as an anticoagulant during blood feeding by the insect Rhodnius prolixus. The 2.0-Å crystal structure of NP2 reveals an eight-stranded antiparallel β-barrel containing a ferric heme coordinated through His57, similar to the structures of NP1 and NP4. All four Rhodnius nitrophorins transport NO and sequester histamine through heme binding, but only NP2 acts as an anticoagulant. Here, we demonstrate that recombinant NP2, but not recombinant NP1 or NP4, is a potent anticoagulant; recombinant NP3 also displays minor activity. Comparison of the nitrophorin structures suggests that a surface region near the C terminus and the loops between β strands B-C and E-F is responsible for the anticoagulant activity. NP2 also displays larger NO association rates and smaller dissociation rates than NP1 and NP4, which may result from a more open and more hydrophobic distal pocket, allowing more rapid solvent reorganization on ligand binding. The NP2 protein core differs from NP1 and NP4 in that buried Glu53, which allows for larger NO release rates when deprotonated, hydrogen bonds to invariant Tyr81. Surprisingly, this tyrosine lies on the protein surface in NP1 and NP4. Nitrophorin 2 (NP2) (also known as prolixin-S) is a salivary protein that transports nitric oxide, binds histamine, and acts as an anticoagulant during blood feeding by the insect Rhodnius prolixus. The 2.0-Å crystal structure of NP2 reveals an eight-stranded antiparallel β-barrel containing a ferric heme coordinated through His57, similar to the structures of NP1 and NP4. All four Rhodnius nitrophorins transport NO and sequester histamine through heme binding, but only NP2 acts as an anticoagulant. Here, we demonstrate that recombinant NP2, but not recombinant NP1 or NP4, is a potent anticoagulant; recombinant NP3 also displays minor activity. Comparison of the nitrophorin structures suggests that a surface region near the C terminus and the loops between β strands B-C and E-F is responsible for the anticoagulant activity. NP2 also displays larger NO association rates and smaller dissociation rates than NP1 and NP4, which may result from a more open and more hydrophobic distal pocket, allowing more rapid solvent reorganization on ligand binding. The NP2 protein core differs from NP1 and NP4 in that buried Glu53, which allows for larger NO release rates when deprotonated, hydrogen bonds to invariant Tyr81. Surprisingly, this tyrosine lies on the protein surface in NP1 and NP4. nitrophorin root mean square deviation Blood feeding in insects has evolved independently multiple times, resulting in a diverse array of molecules designed to circumvent host hemostatic defenses (1Montfort, W. R., Weichsel, A., and Andersen, J. F. (2000) Biochim. Biophys. Acta, in pressGoogle Scholar, 2Law J. Ribeiro J.M.C. Wells M. Annu. Rev. Biochem. 1992; 61: 87-111Crossref PubMed Scopus (151) Google Scholar, 3Champagne D.E. Nussenzvieg R.H. Ribeiro J.M.C. J. Biol. Chem. 1995; 270: 8691-8695Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Among the most remarkable of these substances are the nitrophorins (NPs),1 a group of heme proteins found in the saliva of the bug Rhodnius prolixus(4Ribeiro J.M.C. Hazzard J.M.H. Nussenzveig R.H. Champagne D.E. Walker F.A. Science. 1993; 260: 539-541Crossref PubMed Scopus (290) Google Scholar, 5Ribeiro J.M.C. Walker F.A. J. Exp. Med. 1994; 180: 2251-2257Crossref PubMed Scopus (134) Google Scholar, 6Champagne D.E. Parasitol. Today. 1994; 10: 430-433Abstract Full Text PDF PubMed Scopus (53) Google Scholar, 7Sun J. Yamaguchi M. Yuda M. Miura K. Takeya H. Hirai M. Matsuoka H. Ando K. Watanabe T. Suzuki K. Chinzei Y. Thromb. Hemostasis. 1996; 75: 573-577Crossref PubMed Scopus (41) Google Scholar). NPs are unique in being multifunctional, in that multiple anti-hemostatic activities are combined into a single protein. NPs possess a potent vasodilatory activity due to transport and release of nitric oxide (4Ribeiro J.M.C. Hazzard J.M.H. Nussenzveig R.H. Champagne D.E. Walker F.A. Science. 1993; 260: 539-541Crossref PubMed Scopus (290) Google Scholar). NO binding with NPs occurs in the salivary gland lumen, and release occurs at the site of feeding after being transported through the insect mouth parts. There, NO induces relaxation of the vascular endothelium (vasodilation) through activation of soluble guanylate cyclase. The NPs also bind histamine that is released from mast cells around the bite (5Ribeiro J.M.C. Walker F.A. J. Exp. Med. 1994; 180: 2251-2257Crossref PubMed Scopus (134) Google Scholar). The affinity of the proteins for histamine is extremely high, which may serve both to displace the NO and to provide a significant local antihistaminic activity. Finally, NPs possess potent anticoagulation activity via inhibition of the factor Xase complex of the intrinsic pathway (7Sun J. Yamaguchi M. Yuda M. Miura K. Takeya H. Hirai M. Matsuoka H. Ando K. Watanabe T. Suzuki K. Chinzei Y. Thromb. Hemostasis. 1996; 75: 573-577Crossref PubMed Scopus (41) Google Scholar, 8Ribeiro J.M.C. Schneider M. Guimaraes J.A. Biochem. J. 1995; 308: 243-249Crossref PubMed Scopus (134) Google Scholar, 9Zhang Y. Ribeiro J.M. Guimaraes J.A. Walsh P.N. Biochemistry. 1998; 37: 10681-10690Crossref PubMed Scopus (50) Google Scholar). There are four NPs in the R. prolixus saliva that are designated as NP1–NP4 and can be divided into two groups based on sequence relationships (3Champagne D.E. Nussenzvieg R.H. Ribeiro J.M.C. J. Biol. Chem. 1995; 270: 8691-8695Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 7Sun J. Yamaguchi M. Yuda M. Miura K. Takeya H. Hirai M. Matsuoka H. Ando K. Watanabe T. Suzuki K. Chinzei Y. Thromb. Hemostasis. 1996; 75: 573-577Crossref PubMed Scopus (41) Google Scholar, 10Sun J. Yuda M. Miura K. Chinzei Y. Insect Biochem. Mol. Biol. 1998; 28: 191-200Crossref PubMed Scopus (14) Google Scholar). NP1 and NP4 are 90% identical, and NP2 (also known as prolixin-S) and NP3 are 80% identical (Fig.1). The two groups are more distantly related, with NP1 and NP2 being 47% identical. The NPs are all Fe(III) heme proteins, but the sequence groups differ in both their NO binding and anticoagulation properties. Although all four proteins bind nitric oxide and histamine, only NP2 possesses strong anticoagulation activity (7Sun J. Yamaguchi M. Yuda M. Miura K. Takeya H. Hirai M. Matsuoka H. Ando K. Watanabe T. Suzuki K. Chinzei Y. Thromb. Hemostasis. 1996; 75: 573-577Crossref PubMed Scopus (41) Google Scholar, 8Ribeiro J.M.C. Schneider M. Guimaraes J.A. Biochem. J. 1995; 308: 243-249Crossref PubMed Scopus (134) Google Scholar). NP1 and NP4 bind NO with lower affinity (0.5–1 μm at pH 8.0) than do NP2 and NP3 (0.02 μmat pH 8.0) (11Andersen J.F. Ding X.D. Balfour C. Shokhireva T.K. Champagne D.E. Walker F.A. Montfort W.R. Biochemistry. 2000; 39: 10118-10131Crossref PubMed Scopus (124) Google Scholar). The differences in affinity are due to both larger association rate constants and smaller dissociation rate constants in NP2 and NP3. The differences in ligand release rates may serve to extend the duration of the NO signal in the host or increase the effective radius of the signal around the bite. NP4 binding to NO (but not to cyanide or ammonia) induces a large conformational change in the protein that results in burial of the NO ligand in the distal pocket and a substantial increase in NO binding affinity (12Weichsel A. Andersen J.F. Roberts S.A. Montfort W.R. Nat. Struct. Biol. 2000; 7: 551-554Crossref PubMed Scopus (117) Google Scholar). The mechanism for this is not yet clear, but it appears to involve a change in distal pocket polarity. Interestingly, activation of the soluble guanylate cyclase catalytic domain is also thought to occur through a NO-induced conformational change, in this case through the Fe(II) heme center in the regulatory domain of the protein (13Sharma V.S. Magde D. Methods. 1999; 19: 494-505Crossref PubMed Scopus (78) Google Scholar). The different NP forms provide naturally occurring variants that differ in the kinetics and thermodynamics of ligand binding and therefore provide clues to the structural basis for NO-induced conformational changes (11Andersen J.F. Ding X.D. Balfour C. Shokhireva T.K. Champagne D.E. Walker F.A. Montfort W.R. Biochemistry. 2000; 39: 10118-10131Crossref PubMed Scopus (124) Google Scholar). NP2 has the highest affinity for NO, so structural comparisons with NP4, which binds NO less strongly, are of particular interest. The NP-NO complexes are also resistant to the autoreductive reactions that are seen with NO complexes of Fe(III) globins (11Andersen J.F. Ding X.D. Balfour C. Shokhireva T.K. Champagne D.E. Walker F.A. Montfort W.R. Biochemistry. 2000; 39: 10118-10131Crossref PubMed Scopus (124) Google Scholar, 14Ding X.D. Weichsel A. Andersen J.F. Shokhireva T.K. Balfour C. Pierik A.J. Averill B.A. Montfort W.R. Walker F.A. J. Am. Chem. Soc. 1999; 121: 128-138Crossref Scopus (149) Google Scholar). The structural basis for stability of the Fe(III) form of the protein is not yet known. NP2 has special importance in being the only anticoagulant among the nitrophorins. The protein acts by inhibiting the intrinsic factor Xase complex, and its activity is independent of the heme moiety (8Ribeiro J.M.C. Schneider M. Guimaraes J.A. Biochem. J. 1995; 308: 243-249Crossref PubMed Scopus (134) Google Scholar). Zhang et al. (9Zhang Y. Ribeiro J.M. Guimaraes J.A. Walsh P.N. Biochemistry. 1998; 37: 10681-10690Crossref PubMed Scopus (50) Google Scholar) showed NP2 to be a hyperbolic mixed-type inhibitor that inhibits factor IXa-catalyzed cleavage of factor X in the presence of factor VIIIa or phospholipid or both. However, it has no effect against the basal proteolytic activity of factor IXa in the absence of both factor VIIIa and phospholipids, suggesting that complex formation is interfered with. NP2 also appears to bind more tightly with the enzyme-substrate complex than the substrate-free complex (9Zhang Y. Ribeiro J.M. Guimaraes J.A. Walsh P.N. Biochemistry. 1998; 37: 10681-10690Crossref PubMed Scopus (50) Google Scholar). These observations suggest that NP2 interacts with a specific conformation of factor IXa found in the factor Xase complex. Surface plasmon resonance analyses performed by Isawa et al. (15Isawa H. Yuda M. Yoneda K. Chinzei Y. J. Biol. Chem. 2000; 275: 6636-6641Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) showed specific binding of NP2 with factors IX and IXa and also showed that NP2 inhibits assembly of the factor Xase complex. Additionally, these studies revealed a previously uncharacterized inhibition of the factor VIIa-tissue factor complex by NP2, suggesting that NP2 also may inhibit activation of factor X via the extrinsic pathway. The NPs have been placed in the lipocalin protein family based on the structures of NP1 and NP4 (16Weichsel A. Andersen J.F. Champagne D.E. Walker F.A. Montfort W.R. Nat. Struct. Biol. 1998; 5: 304-309Crossref PubMed Scopus (160) Google Scholar, 17Andersen J.F. Weichsel A. Balfour C.A. Champagne D.E. Montfort W.R. Structure. 1998; 6: 1315-1327Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The NP structure is comprised of an eight-stranded anti-parallel β-barrel containing a large ligand-binding cavity that contains a single ferriheme molecule. The heme is bound to the protein through proximal coordination of a histidine side chain with the iron atom. A single NO molecule is coordinated with the heme iron in the distal pocket of the NO complex, and this ligand is replaced by water after release (12Weichsel A. Andersen J.F. Roberts S.A. Montfort W.R. Nat. Struct. Biol. 2000; 7: 551-554Crossref PubMed Scopus (117) Google Scholar). Histamine also occupies the distal pocket, to the exclusion of NO, and is bound by coordination of the imidazole ring and hydrogen bonding of the alkylamino group (16Weichsel A. Andersen J.F. Champagne D.E. Walker F.A. Montfort W.R. Nat. Struct. Biol. 1998; 5: 304-309Crossref PubMed Scopus (160) Google Scholar). In this study, we have determined the crystal structure of recombinant NP2 and compared the anticoagulation activity of this protein with recombinant samples of NP1–NP4. This reveals significant differences between NP2 and both NP1 and NP4, including the surprising use of invariant amino acid residues in completely new ways. NP1–NP4 were prepared as described previously (11Andersen J.F. Ding X.D. Balfour C. Shokhireva T.K. Champagne D.E. Walker F.A. Montfort W.R. Biochemistry. 2000; 39: 10118-10131Crossref PubMed Scopus (124) Google Scholar, 17Andersen J.F. Weichsel A. Balfour C.A. Champagne D.E. Montfort W.R. Structure. 1998; 6: 1315-1327Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 18Andersen J.F. Champagne D.E. Weichsel A. Ribeiro J.M.C. Balfour C.A. Dress V. Montfort W.R. Biochemistry. 1997; 36: 4423-4428Crossref PubMed Scopus (89) Google Scholar). Briefly, each of the four cDNAs was modified by polymerase chain reaction to remove the sequence encoding the signal peptide. In each case, an ATG codon was added immediately 5′ of the coding sequence of the mature protein in order to initiate translation. The modified cDNAs were cloned into the expression vector pET17b and expressed in the strain BL21(DE3). Inclusion bodies obtained from these cultures were denatured, refolded, and reconstituted with heme as described previously (11Andersen J.F. Ding X.D. Balfour C. Shokhireva T.K. Champagne D.E. Walker F.A. Montfort W.R. Biochemistry. 2000; 39: 10118-10131Crossref PubMed Scopus (124) Google Scholar, 17Andersen J.F. Weichsel A. Balfour C.A. Champagne D.E. Montfort W.R. Structure. 1998; 6: 1315-1327Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 18Andersen J.F. Champagne D.E. Weichsel A. Ribeiro J.M.C. Balfour C.A. Dress V. Montfort W.R. Biochemistry. 1997; 36: 4423-4428Crossref PubMed Scopus (89) Google Scholar). Recombinant NP2 was then purified by chromatography on Q-Sepharose (Amersham Pharmacia Biotech) and Sephacryl S-200 (Amersham Pharmacia Biotech). Each of the four NPs was assayed using the activated partial thromboplastin time assay. The activated partial thromboplastin time reagent was obtained from Sigma and contains rabbit brain cephalin in a buffered 0.1 mmsolution of ellagic acid. NPs at various concentrations in either 100 mm sodium phosphate, pH 7.5, or water were added to human normal coagulation control serum (Sigma). After incubation for 1 min at 37 °C, the activated partial thromboplastin time reagent was added to the serum-NP mixture and incubated at 30 °C for 3 min. Coagulation was initiated by adding 0.1 ml of 20 mmCaCl2. The formation of clots was determined visually. Crystals were obtained by the hanging drop vapor diffusion method using 2.8 mammonium phosphate, 0.1 m Tris-HCl, pH 7.7, as precipitant. Initially, small plate-like crystals were obtained using 2.8m ammonium phosphate. These were then introduced by macroseeding into drops equilibrated with 2.6 m ammonium phosphate, 0.1 m Tris-HCl, pH 7.7. The plates grew slowly, eventually reaching a size of 0.8 × 0.2 × 0.04 mm, and diffracted to 2.0 Å nominal resolution. Data were collected at room temperature using a FAST area detector and an Enraf Nonius rotating anode generator operated at 40 kV and 95 mA. Images were collected and reflections were integrated and indexed using MADNES (19Messerschmidt A. Pflugrath J.W. J. Appl. Crystallogr. 1987; 20: 306-315Crossref Scopus (457) Google Scholar). The data were reduced using PROCOR (20Kabsch W. J. Appl. Crystallogr. 1988; 21: 916-934Crossref Scopus (1680) Google Scholar) followed by SCALA (21Collaborative Computational Project Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). The crystals were found to be orthorhombic with unit cell dimensions of a = 40.3 Å, b = 128.0 Å, and c = 33.7 Å. The pattern of systematic absences placed the crystal in the space group P21212, with one NP2 molecule in the asymmetric unit (Table I).Table IData measurement and refinement statistics for NP2Data measurement Resolution range (Å)9.2–2.0 Total observations49,410 Unique observations12,186 Completeness (%)1-aValues in parentheses are for the highest resolution shell: 2.1–2.0 Å.96.9 (90.3) Multiplicity4.2 (3.2) Rsym(%)1-bRsym = (Σh ‖ Ih − 〈I〉 ‖)/(ΣhIh), where 〈I〉 is the mean intensity of all symmetry-related reflections Ih.10.6 (34.8) I/ς(I)4.5 (1.9)Structure refinement Rcryst1-cRcryst = (Σh ‖Fo −Fc ‖)/(ΣFo).0.19 Rfree1-dRfree as for Rcryst, using a random subset of the data (5%) not included in the refinement.0.24 Most favorable φ/ψ (%)91.4 allowed φ/ψ (%)100 RMSD bond lengths (Å)0.006 RMSD bond angles (°)1.27Average B-factor Main chain25.2 Side chains27.8 Heme22.3 Solvent36.41-a Values in parentheses are for the highest resolution shell: 2.1–2.0 Å.1-b Rsym = (Σh ‖ Ih − 〈I〉 ‖)/(ΣhIh), where 〈I〉 is the mean intensity of all symmetry-related reflections Ih.1-c Rcryst = (Σh ‖Fo −Fc ‖)/(ΣFo).1-d Rfree as for Rcryst, using a random subset of the data (5%) not included in the refinement. Open table in a new tab Molecular replacement was performed using XPLOR (22Fujinaga M. Read R.J. J. Appl. Crystallogr. 1987; 20: 517-521Crossref Scopus (178) Google Scholar, 23Brunger A.T. Acta Crystallogr. Sect. A. 1990; 46: 46-57Crossref Scopus (361) Google Scholar). Two search models were employed: a model of NP4 with protein side chains differing between NP4 and NP2 changed to alanine and containing heme, and a polyalanine model of NP1 with heme removed. Rotation solutions were subjected to Patterson correlation refinement, and the best solutions were entered into the translation search. Both models gave the same solution to the rotation function, but the correlation coefficients were low. Different translation solutions were obtained with the two models, and the solution obtained with the NP4 model was rejected on the basis of an unlikely crystal packing arrangement. A number of solutions having similar magnitudes of the translation function were obtained with the NP1 model, and the top solution (function value = 0.24) was subjected to rigid body refinement using data from 8.0–3.0 Å, resulting in an Rfactor of 0.54. A model of NP2 was constructed based on the NP1 structure in which side chains conserved between NP1 and NP2 were included, but nonconserved residues other than glycine were modeled as alanine. This model was refined by simulated annealing using XPLOR and CNS (24Pannu N.S. Read R.J. Acta Crystallogr. Sect. A. 1996; 52: 659-668Crossref Scopus (319) Google Scholar, 25Adams P.D. Pannu N.S. Read R.J. Brunger A.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5018-5023Crossref PubMed Scopus (383) Google Scholar), with data from 9.0 to 3.0 Å included, and the R factor dropped to 0.38. Additional cycles of simulated annealing and manual rebuilding were performed using 2 Fo – Fc ,Fo – Fc , and annealed Fo – Fc omit maps. Density for side chains that differed between NP1 and NP2 was observed, indicating that the molecular replacement solution was correct. The side chain of Ile120 in the distal heme pocket was not included in the model until the structure was near completion, in order to independently monitor the quality of electron density as the resolution was extended to 2.0 Å. In the latter stages, maximum likelihood refinement and refinement of individual temperature factors were included using CNS, resulting in a final Rcryst of 0.19 and Rfreeof 0.24 (Table I). Good electron density was seen in 2 Fo – Fc maps for all parts of the molecule (Fig. 2), except around residues 126 and 127, which showed some main chain disorder. Various data manipulations, calculations, and superpositioning of models were performed using programs obtained from the Uppsala Software Factory (26Kleywegt G.J. Jones T.A. Structure. 1995; 3: 535-540Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Structural figures were drawn with MOLSCRIPT (27Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), BOBSCRIPT (28Esnouf R.M. J Mol Graph Model. 1997; 15 (, 112–113): 132-134Crossref PubMed Scopus (1795) Google Scholar), and RASTER 3-D (29Merritt E.A. Murphy M.E.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2859) Google Scholar). Refined coordinates and structure factor amplitudes have been deposited in the Protein Data Bank with accession code 1EUO. The NP2 structure was determined by molecular replacement using a polyalanine model of NP1 as a starting point. The final model displayed good refinement statistics (TableI) and exhibited good electron density for all but two of the 180 residues in the final model (Fig. 2). Like NP1 and NP4, NP2 has a lipocalin fold consisting of an eight-stranded antiparallel β-barrel containing a central ligand-binding cavity (Fig. 3). At the C-terminal end of the barrel, two α-helices are present, and a disulfide bond tethers the C terminus to the β-barrel. The N-terminal portion of the protein is also disulfide bonded to the barrel, and contains a single turn of α-helix prior to the first strand of the β-sheet. For expression in Escherichia coli, methionine was added to the N terminus of the protein, and this residue was found to be well ordered in the crystal and stabilized through interaction with an adjacent NP2 molecule. An N-terminal methionine was not visible in either the NP1 or NP4 crystal structures (16Weichsel A. Andersen J.F. Champagne D.E. Walker F.A. Montfort W.R. Nat. Struct. Biol. 1998; 5: 304-309Crossref PubMed Scopus (160) Google Scholar, 17Andersen J.F. Weichsel A. Balfour C.A. Champagne D.E. Montfort W.R. Structure. 1998; 6: 1315-1327Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), although Edman degradation indicated that it was present in NP1 but not in NP4 (17Andersen J.F. Weichsel A. Balfour C.A. Champagne D.E. Montfort W.R. Structure. 1998; 6: 1315-1327Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 18Andersen J.F. Champagne D.E. Weichsel A. Ribeiro J.M.C. Balfour C.A. Dress V. Montfort W.R. Biochemistry. 1997; 36: 4423-4428Crossref PubMed Scopus (89) Google Scholar). A heme ligand is contained within the central cavity of NP2 and is bonded to the protein via coordination with the imidazole portion of His57 on the proximal side of the heme (Fig.4). A distal ligand is also evident and is considered to be an ammonia molecule, based on previous results with NP1 and NP4 (16Weichsel A. Andersen J.F. Champagne D.E. Walker F.A. Montfort W.R. Nat. Struct. Biol. 1998; 5: 304-309Crossref PubMed Scopus (160) Google Scholar, 17Andersen J.F. Weichsel A. Balfour C.A. Champagne D.E. Montfort W.R. Structure. 1998; 6: 1315-1327Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The Cα positions of the β-barrel are very similar in NP4 and NP2. When a core is defined containing residues from each of the eight strands of the barrel (Figs. 1 and 3), the RMSD for these positions is 0.6 Å. When all Cα atoms are compared, the RMSD increases to 1.5 Å, due mainly to changes in loop positions (Fig. 5).Figure 5RMSDs for Cα atoms of the NP2 model as compared with NP4. Superpositioning was performed using the core residues identified in Fig. 1. Loop positions are labeled using the scheme used in Fig. 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The loops surrounding the heme-binding pocket show a variety of conformations in the NP structures determined to date (16Weichsel A. Andersen J.F. Champagne D.E. Walker F.A. Montfort W.R. Nat. Struct. Biol. 1998; 5: 304-309Crossref PubMed Scopus (160) Google Scholar, 17Andersen J.F. Weichsel A. Balfour C.A. Champagne D.E. Montfort W.R. Structure. 1998; 6: 1315-1327Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Unlike the case with NP4, the large ω loop that lies between strands A and B of the β-barrel (Fig. 3, loop A-B) is well ordered in the NP2 crystals. This is probably due to stabilization of the region through contacts with an adjacent protein molecule. In previous studies with NP4, loops A-B and G-H were found to undergo a major conformational change on NO binding that buries the NO ligand within the distal pocket (12Weichsel A. Andersen J.F. Roberts S.A. Montfort W.R. Nat. Struct. Biol. 2000; 7: 551-554Crossref PubMed Scopus (117) Google Scholar). This conformational change appears to be related to stabilization of the NO complex and to the biphasic association kinetics observed in ligand binding experiments with NO. In NP2 the position of loop A-B is similar to the "open" NO-free conformation seen in NP1 and NP4. As with other NPs, loop G-H (residues 123–128) shows disorder in the NP2 structure that is consistent with NO-induced movement, as previously observed with NP4 (12Weichsel A. Andersen J.F. Roberts S.A. Montfort W.R. Nat. Struct. Biol. 2000; 7: 551-554Crossref PubMed Scopus (117) Google Scholar, 16Weichsel A. Andersen J.F. Champagne D.E. Walker F.A. Montfort W.R. Nat. Struct. Biol. 1998; 5: 304-309Crossref PubMed Scopus (160) Google Scholar, 17Andersen J.F. Weichsel A. Balfour C.A. Champagne D.E. Montfort W.R. Structure. 1998; 6: 1315-1327Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The heme moiety of NP4 has orientational disorder in that half of the hemes are "right-side-up," and half are "upside-down" (12Weichsel A. Andersen J.F. Roberts S.A. Montfort W.R. Nat. Struct. Biol. 2000; 7: 551-554Crossref PubMed Scopus (117) Google Scholar). With NP2, omit electron density maps calculated after simulated annealing of a model from which the vinyl methylene groups were removed show strong density for one heme orientation and no density for the other. This indicates that, unlike NP4, NP2 has a single heme orientation. The heme conformation in NP2, like that for NP1 and NP4, is severely distorted from planar. This distortion arises in large part from a rotation of the pyrrole rings about their Fe-N bonds, referred to as heme "ruffling," which may serve to stabilize the ferric iron center with respect to reduction (30Shelnutt J.A. Song X.-Z. Ma J.-G. Jia S.-l. Jentzen W. Medforth C.J. Chem. Soc. Reviews. 1998; 27: 31-41Crossref Scopus (778) Google Scholar). The NP2 pyrroles are rotated to about the same or to a slightly greater degree than those of NP4 (12Weichsel A. Andersen J.F. Roberts S.A. Montfort W.R. Nat. Struct. Biol. 2000; 7: 551-554Crossref PubMed Scopus (117) Google Scholar). The distal pocket ligand-binding environment of NP2 is quite similar to that found in NP1 and NP4 (16Weichsel A. Andersen J.F. Champagne D.E. Walker F.A. Montfort W.R. Nat. Struct. Biol. 1998; 5: 304-309Crossref PubMed Scopus (160) Google Scholar, 17Andersen J.F. Weichsel A. Balfour C.A. Champagne D.E. Montfort W.R. Structure. 1998; 6: 1315-1327Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) (Fig. 4). The side chains of Leu122, Leu129, and Leu132 occupy positions similar to those of the corresponding residues in the previously determined NP structures. These hydrophobic residues surround the NO ligand in the NP4-NO complex (12Weichsel A. Andersen J.F. Roberts S.A. Montfort W.R. Nat. Struct. Biol. 2000; 7: 551-554Crossref PubMed Scopus (117) Google Scholar) and sandwich the imidazole ring in the NP1-histamine complex (16Weichsel A. Andersen J.F. Champagne D.E. Walker F.A. Montfort W.R. Nat. Struct. Biol. 1998; 5: 304-309Crossref PubMed Scopus (160) Google Scholar). Also, the residues interacting with the alkylamino group of histamine are in similar positions, consistent with the near identity of kinetic and thermodynamic values for histamine binding to the three proteins. However, replacement of Thr121 of NP1 and NP4 with isoleucine (Ile120) adds a bulkier nonpolar group to the NP2 distal pocket that increases its hydrophobicity (Fig. 4). The conformational change associated with binding of NO appears to be mediated by hydrophobic interactions, because no hydrogen bonds are formed with the ligand. An increase in the hydrophobicity of the pocket may in part be responsible for the at least 10-fold greater affinity for NO in NP2 when compared with NP1 and NP4 (11Andersen J.F. Ding X.D. Balfour C. Shokhireva T.K. Champagne D.E. Walker F.A. Montfort W.R. Biochemistry. 2000; 39: 10118-10131Crossref PubMed Scopus (124) Google Scholar). Additionally, the larger size of the Ile120 side chain and the placement of Tyr38 in contact with the proximal side of the heme cause the heme to rotate slightly in the protein with respect to its position in NP4 (12Weichsel A. Andersen J.F. Roberts S.A. Montfort W.R. Nat. Struct. Biol. 2000; 7: 551-554Crossref PubMed Scopus (117) Google Scholar) (Fig. 4). It is not yet apparent whether differences in heme position or conformation among the NPs gives rise to differences in their reduction potentials (11Andersen J.F. Ding X.D. Balfour C. Shokhireva T.K. Champagne D.E. Walker F.A. Montfort W.R. Biochemistry. 2000; 39: 10118-10131Crossref PubMed Scopus (124) Google Scholar, 14Ding X.D. Weichsel A. Andersen J.F. Shokhireva T.K. Balfour C. Pierik A.J. Averill B.A. Montfort W.R. Walker F.A. J. Am. Chem. Soc. 1999; 121: 128-138Crossref Scopus (149) Google Scholar). The structure of the proximal pocket of NP2 is also very similar to those described for NP1 and NP4. The imidazole ring of the proximal ligand (His 57) is hydrogen bonded to the side chain of Asn68 through an intervening water molecule (Fig. 4). The structure of this network is virtually identical to that
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