S-Nitrosylation-induced Conformational Change in Blackfin Tuna Myoglobin

S-Nitrosylation is a post-translational protein modification that can alter the function of a variety of proteins. Despite the growing wealth of information that this modification may have important functional consequences, little is known about the structure of the moiety or its effect on protein tertiary structure. Here we report high-resolution x-ray crystal structures of S-nitrosylated and unmodified blackfin tuna myoglobin, which demonstrate that in vitro S-nitrosylation of this protein at the surface-exposed Cys-10 directly causes a reversible conformational change by “wedging” apart a helix and loop. Furthermore, we have demonstrated in solution and in a single crystal that reduction of the S-nitrosylated myoglobin with dithionite results in NO cleavage from the sulfur of Cys-10 and rebinding to the reduced heme iron, showing the reversibility of both the modification and the conformational changes. Finally, we report the 0.95-Å structure of ferrous nitrosyl myoglobin, which provides an accurate structural view of the NO coordination geometry in the context of a globin heme pocket. S-Nitrosylation is a post-translational protein modification that can alter the function of a variety of proteins. Despite the growing wealth of information that this modification may have important functional consequences, little is known about the structure of the moiety or its effect on protein tertiary structure. Here we report high-resolution x-ray crystal structures of S-nitrosylated and unmodified blackfin tuna myoglobin, which demonstrate that in vitro S-nitrosylation of this protein at the surface-exposed Cys-10 directly causes a reversible conformational change by “wedging” apart a helix and loop. Furthermore, we have demonstrated in solution and in a single crystal that reduction of the S-nitrosylated myoglobin with dithionite results in NO cleavage from the sulfur of Cys-10 and rebinding to the reduced heme iron, showing the reversibility of both the modification and the conformational changes. Finally, we report the 0.95-Å structure of ferrous nitrosyl myoglobin, which provides an accurate structural view of the NO coordination geometry in the context of a globin heme pocket. Protein S-nitrosylation, or the formation of an S–NO bond involving the sulfur of a cysteine residue, is an important post-translational modification. Numerous proteins whose functions are altered by S-nitrosylation have been identified, including enzymes, transcription factors, receptors, channels, and structural proteins (reviewed in Ref. 1Hess D.T. Matsumoto A. Kim S.O. Marshall H.E. Stamler J.S. Nat. Rev. Mol. Cell Biol. 2005; 6: 150-166Crossref PubMed Scopus (1731) Google Scholar). Parallels have been drawn between S-nitrosylation and O-phosphorylation, a ubiquitous biological signal (2Lane P. Hao G. Gross S.S. Science's STKE. 2001; : RE1PubMed Google Scholar, 3Mannick J.B. Schonhoff C.M. Arch. Biochem. Biophys. 2002; 408: 1-6Crossref PubMed Scopus (96) Google Scholar). Regulation of the function of many proteins by O-phosphorylation is a consequence of structural changes that take place in the protein following phosphorylation. The molecular consequences of protein S-nitrosylation are less well characterized. Enzymes such as caspases that require a cysteine thiol in the active site can be modulated in their activity by S-nitrosylation, which alters the reactive properties of the cysteine (4Mannick J.B. Schonhoff C. Papeta N. Ghafourifar P. Szibor M. Fang K. Gaston B. J. Cell Biol. 2001; 154: 1111-1116Crossref PubMed Scopus (328) Google Scholar). S-Nitrosylation can also influence protein-protein interactions, as recently demonstrated with yeast two-hybrid screening (5Matsumoto A. Comatas K.E. Liu L. Stamler J.S. Science. 2003; 301: 657-661Crossref PubMed Scopus (131) Google Scholar). Finally, S-nitrosylation may have the ability to modulate the function of a protein via allosteric changes in its structure. Although this mechanism has been hypothesized for several systems, there has thus far been little structural evidence published (6Kim S.O. Merchant K. Nudelman R. Beyer Jr., W.F. Keng T. DeAngelo J. Hausladen A. Stamler J.S. Cell. 2002; 109: 383-396Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 7Williams J.G. Pappu K. Campbell S.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6376-6381Crossref PubMed Scopus (84) Google Scholar). Another critical role for NO in cell physiology involves its interaction with heme proteins. The first well defined mechanism for a NO-dependent physiological effect was the relaxation of vascular smooth muscle following the binding of NO to the heme iron of guanylate cyclase, which activates this enzyme to convert guanosine triphosphate to the second messenger cGMP (8Denninger J.W. Marletta M.A. Biochim. Biophys. Acta. 1999; 1411: 334-350Crossref PubMed Scopus (878) Google Scholar). The reaction of the NO group (NO, NO+, NO-) with heme and thiol is complex (9Williams D.L.H. Nitrosation Reactions and the Chemistry of Nitric Oxide. Elsevier Science, Oxford2004Google Scholar). However, under various in vitro and in vivo conditions, the NO group is capable of binding at one or the other or exchanging between the two. It has been shown that the bioavailability of NO when bound to Cysβ93 of hemoglobin is dependent upon blood O2 concentration as well as the allosteric and redox state and that it acts as an important regulator of vessel tone and blood flow (10Gow A.J. Luchsinger B.P. Pawloski J.R. Singel D.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9027-9032Crossref PubMed Scopus (378) Google Scholar, 11Stamler J.S. Jia L. Eu J.P. McMahon T.J. Demchenko I.T. Bonaventura J. Gernert K. Piantadosi C.A. Science. 1997; 276: 2034-2037Crossref PubMed Scopus (956) Google Scholar, 12Jia L. Bonaventura C. Bonaventura J. Stamler J.S. Nature. 1996; 380: 221-226Crossref PubMed Scopus (1471) Google Scholar, 13Singel D.J. Stamler J.S. Annu. Rev. Physiol. 2005; 67: 99-145Crossref PubMed Scopus (397) Google Scholar). Sperm whale myoglobin (Mb), 2The abbreviation used is: Mb, myoglobin. the first myoglobin whose x-ray structure was determined, lacks a cysteinyl residue. Human myoglobin has a single reactive cysteine at position 110 and is known to form an S-nitrosylated species in vitro and in vascular smooth muscle cell culture when exposed to physiological concentrations of a nitric oxide donor (14Rayner B.S. Wu B.J. Raftery M. Stocker R. Witting P.K. J. Biol. Chem. 2005; 280: 9985-9993Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 15Witting P.K. Douglas D.J. Mauk A.G. J. Biol. Chem. 2001; 276: 3991-3998Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Myoglobins with reactive cysteines are seen scattered throughout the vertebrate species, but there seems to be little to suggest a functional pattern. In fishes, the group that includes tunas frequently possesses a single cysteine within the N-terminal helix. It has been suggested that this reactive -SH plays a role in S-nitrosylation in vivo (16Marcinek D.J. Bonaventura J. Wittenberg J.B. Block B.A. Am. J. Physiol. 2001; 280: R1123-R1133Google Scholar), although there are no hard data yet to support this hypothesis. With the recent advancement in methodologies for the detection and identification of S-nitrosylated proteins in cell culture and tissue samples, such as the S-nitrosylation site identification (17Hao G. Derakhshan B. Shi L. Campagne F. Gross S.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1012-1017Crossref PubMed Scopus (299) Google Scholar, 18Greco T.M. Hodara R. Parastatidis I. Heijnen H.F. Dennehy M.K. Liebler D.C. Ischiropoulos H. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7420-7425Crossref PubMed Scopus (237) Google Scholar) extension of the original biotin switch method (19Jaffrey S.R. Snyder S.H. Science's STKE. 2001; : PL1PubMed Google Scholar), an extensive data base of S-nitrosylation sites within proteins is being accumulated. In light of these advancements, a more detailed knowledge of the structural consequences of this modification will be critical for understanding the molecular mechanisms by which S-nitrosylation alters protein function. In this work, we have demonstrated with atomic resolution the effects of S-nitrosylation on the x-ray crystal structure of blackfin tuna myoglobin. In addition, we have identified conformational changes in this protein that are the direct result of the formation of the S-nitrosothiol group. We also demonstrate that, upon reduction with dithionite, the NO group is transferred from the surface-exposed Cys-10 to the reduced heme iron, showing the reversibility of the S–NO modification and the conformational changes. Finally, we have provided the first atomic resolution structure of nitric oxide bound to the heme iron of a globin protein, which allows the calculation of accurate geometric parameters for this important coordination complex. Purification of Myoglobin from Blackfin Tuna—Myoglobin was purified from the red muscle of blackfin tuna (Thunnus atlanticus) caught near Rincón, Puerto Rico. The red muscle was dissected, homogenized, and centrifuged, and myoglobin was precipitated from the supernatant between 65 and 95% ammonium sulfate. Following resuspension of the precipitated myoglobin and dialysis against phosphate-buffered saline, the protein was separated by size exclusion chromatography on a Sephadex G-75 column. Myoglobin-containing fractions were concentrated to ∼1.2 mm and stored at -80 °C. Myoglobin isolated by this method was nearly 100% ferric aquo (met)myoglobin as judged by UV-visible spectroscopy. Trans-S-nitrosylation of Myoglobin—Purified myoglobin was S-nitrosylated by reaction of 300 μm myoglobin with 3 mm S-nitrosocysteine in borax buffer containing 500 μm diethylene-triamine pentaacetate at pH 9. S-Nitrosocysteine was removed by passing the sample through two sequential Micro Bio-Spin 6 size exclusion columns (Bio-Rad). The amount of S-nitrosylation in purified myoglobin samples was quantitated using the Saville (20Saville B. Analyst. 1958; 83: 670Crossref Google Scholar) and Griess (21Greiss J.P. Chem. Ber. 1879; 12: 426Crossref Scopus (729) Google Scholar) reactions in acidified aqueous solution as described previously (22Nims R.W. Darbyshire J.F. Saavedra J.E. Christodoulou D. Hanbauer I. Cox G.W. Grisham M.B. Laval F. Cook J.A. Krishna M.C. Wink D.A. Methods: A Companion to Methods Enzymol. 1995; 7: 48-54Crossref Scopus (105) Google Scholar, 23Cook J.A. Kim S.Y. Teague D. Krishna M.C. Pacelli R. Mitchell J.B. Vodovotz Y. Nims R.W. Christodoulou D. Miles A.M. Grisham M.B. Wink D.A. Anal. Biochem. 1996; 238: 150-158Crossref PubMed Scopus (151) Google Scholar, 24Park J.K. Kostka P. Anal. Biochem. 1997; 249: 61-66Crossref PubMed Scopus (53) Google Scholar), which results in the production of an azo dye product that can be quantitated spectrophotometrically using an extinction coefficient of 50,000 m-1 cm-1 at 545 nm. A calibration curve was generated for this assay using S-nitrosoglutathione, which was quantitated using an extinction coefficient of 767 m-1 cm-1 at 337 nm (25Mathews W.R. Kerr S.W. J. Pharmacol. Exp. Ther. 1993; 267: 1529-1537PubMed Google Scholar). A chemiluminescence nitric oxide analyzer (Sievers) was also used to verify production of S-nitrosylated myoglobin, again using S-nitrosoglutathione or S-nitrosocysteine for calibration. Crystallization and Data Collection—Purified myoglobin, at a concentration of 1.2 mm in phosphate-buffered saline, was crystallized by hanging-drop vapor diffusion at room temperature by mixing of protein solution with of a solution containing and ammonium sulfate. as of in were by for in the solution containing or and to in a for data of were and in the as unmodified myoglobin. of ferrous nitrosyl myoglobin were by of in solution containing ammonium pH and mm dithionite for by to solution with 3 mm nitric oxide donor by the were transferred to ammonium pH and in Data were on a x-ray with a or at the in data were and using J.W. Biol. 1999; PubMed Scopus Google Scholar) from within the or Methods Enzymol. 1997; 276: PubMed Scopus Google Scholar). Data are provided in and in is for the resolution in is for the resolution is the and is the of the for the in is for the resolution in is for the resolution in is for the resolution of for of in of The in is for the resolution is the and is the of the for the for of in in a and blackfin tuna myoglobin structure was by molecular using the A.J. R.W. Biol. 2005; PubMed Scopus Google Scholar) with tuna myoglobin as a Data M. Biol. PubMed Scopus Google Scholar). The structure was and using P. K. Biol. PubMed Scopus Google Scholar) and within the Biol. PubMed Scopus Google Scholar), Although the of blackfin tuna myoglobin is in data the was on the and to be to and tuna for a at position which is also in other tuna at the was as an that this modification is known to in from other tuna species D.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). at position in of myoglobin with S-nitrosocysteine was as two of S–NO A and and one of unmodified Cys-10 for the of the Cys-10 S–NO group were generated on a crystal structure of J. M.A. J. Nitric Oxide. 2005; 12: PubMed Scopus Google Scholar). the ferrous nitrosyl myoglobin no were on the bond or the of the of of the atomic resolution were for and were in their are provided in of Blackfin structure of myoglobin isolated from blackfin tuna was to resolution by molecular The structure is to that of from other species, tuna M. Biol. PubMed Scopus Google Scholar), and will be of various heme and of blackfin tuna myoglobin from high-resolution crystal structures will be published and of tuna myoglobin was S-nitrosylated at Cys-10 via reaction with S-nitrosocysteine or Although the of at nm coefficient m-1 was by the heme at that coefficient m-1 the of was using the assay as well as chemiluminescence extensive of the sample to S-nitrosocysteine and of the myoglobin Cys-10 was S-nitrosylated under these reaction conditions, with an of The amount of S-nitrosothiol in the sample the of a at room temperature when stored in the in a of by reaction with S-nitrosocysteine crystallized in the form as unmodified myoglobin. Data were to resolution using a x-ray to data to resolution using a to of the S–NO group. The x-ray of protein S–NO has been in other as well A. W.R. PubMed Scopus Google Scholar). of the S–NO group was also using a when were were to of the The position a modification of the sulfur of Cys-10 as well as of the Cys-10 The of two that been S-nitrosylated A and and one that unmodified the to the and was of the resolution of the The unmodified Cys-10 that seen in the unmodified myoglobin and was at The S-nitrosylation of Cys-10 in the crystal of formation in The of S-nitrosylated Cys-10 is by the from the unmodified The second has a by but the NO moiety nearly the as that of A. Cys-10 are as the with to upon of the structure of S-nitrosylated myoglobin with the unmodified structure that there are in several of the structure. The are to the structural including helix the of helix and of the of S-nitrosylated and unmodified myoglobin a of 1.2 for of the structure Cys-10 and the of the structure a of and The NO group of S-nitrosylated Cys-10 between the of within and the of from helix A of the unmodified myoglobin structure that the S-nitrosothiol group with and these of the structure Although two of S-nitrosylated Cys-10 in these for helix A and were in or as a direct consequence of S-nitrosylation at We also effects of the of helix A. The of helix which was against helix to the by the of helix A. was from helix A. Although both and in both structures and to be at one of this in the unmodified structure was by upon of helix A following In to the structural changes Cys-10 following S-nitrosylation, no changes were in the of the heme the site of binding in myoglobin. NO from Cys-10 and to with S-nitrosocysteine and a UV-visible that the heme group of isolated blackfin tuna myoglobin in the ferric aquo state The of the dithionite to this sample in a of the and of the heme to of the ferrous nitrosyl red that of the sample was to the ferrous nitrosyl ferrous and ferric Furthermore, the of dithionite to of or in the of nitric oxide that be by a nitric oxide R. M. M. A. R. and J. to that, upon reaction of with dithionite, the NO group is from the Cys-10 sulfur and can to the reduced heme of unmodified with dithionite in the ferrous form of the heme ferrous ferrous and ferric We were also to this NO group in two structures from a single data to the x-ray on a crystal of a structure to that described and with Cys-10 by NO and the heme in the ferric aquo state of the crystal in a solution containing mm dithionite and the of that Cys-10 was no and that the heme was by NO structure a of the ferrous ferrous and ferric upon dithionite reduction in as described with ferrous nitrosyl as the conformational changes in the structure dithionite that the myoglobin to the it to It is that this conformational change is within the context of a single We to that the reactions of with dithionite light on the that may take place in was used in these as a to demonstrate the reversibility of the S-nitrosylation of at Cys-10 and was for the of it is to that a were in the myoglobin heme the of of the form of blackfin tuna myoglobin were by reduction and with the nitric oxide donor and a was to resolution using this of the complex were in the accurate bond and to be parameters of the heme from this structure are in The heme group was nearly as demonstrated by the parameters in using structural as described previously J.A. J. Chem. B. 1997; Scopus Google and heme geometry in the 0.95-Å ferrous nitrosyl myoglobin 0.95-Å ferrous nitrosyl blackfin tuna myoglobin structure described in this structure of the crystal following a in a mm dithionite demonstrated by UV-visible in solution this structure a of and myoglobin, with as the crystal structure of in are for a of NO in the crystal structure from functional on the The 0.95-Å ferrous nitrosyl blackfin tuna myoglobin structure described in this The structure of the crystal following a in a mm dithionite demonstrated by UV-visible in solution this structure a of and myoglobin, with as the crystal structure of W.R. Chem. 2003; PubMed Scopus Google Scholar). in are for a of NO in the crystal structure from functional on the C. K. J. P. M. J. Chem. A. 1997; Scopus Google Scholar) in a and of Blackfin were to myoglobin isolated from the red muscle of blackfin tuna by in vitro using S-nitrosocysteine or S-Nitrosylation of the myoglobin was using the and by of the NO group from Cys-10 and at the heme iron following dithionite reduction by UV-visible and x-ray The group at Cys-10 was in the and in the of and an for the of the effects of S-nitrosylation on protein structure. previously to the formation or of the the thiol (25Mathews W.R. Kerr S.W. J. Pharmacol. Exp. Ther. 1993; 267: 1529-1537PubMed Google Scholar), redox J.S. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J.S. Cell. Full Text PDF PubMed Scopus Google Scholar), of the and The sulfur of Cys-10 in blackfin tuna myoglobin is and is between the of an and a at the of the It has been that be an important of cysteine S-nitrosylation D.T. Matsumoto A. Kim S.O. Marshall H.E. Stamler J.S. Nat. Rev. Mol. Cell Biol. 2005; 6: 150-166Crossref PubMed Scopus (1731) Google Scholar). The functional of the and are from the Cys-10 and may have a influence its In the S-nitrosylated the NO group is also surface-exposed and is by from the protein and were to the NO group. of proteins were also to S-nitrosylation by the formation of a A. R. N. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). may a role in the formation of the that S-nitrosylated Cys-10 the protein in structure. the atomic resolution structure of S-nitrosylated blackfin tuna myoglobin, we two of the cysteine were as the with of two of this group were in the crystal structure of J. M.A. J. Nitric Oxide. 2005; 12: PubMed Scopus Google Scholar), that of may be an of this demonstrate that two for the at and to of the the bond that may be from the bond within the of results in a bond and a for of the between and J. Am. Chem. 2006; PubMed Scopus Google Scholar). of that we in myoglobin the and geometric parameters for structures of and S-nitrosylation at both and The to the with of the at one site A. W.R. PubMed Scopus Google Scholar). A structure of the protein from S-nitrosylation of a heme also as the with a to A. J.F. J.G. T. W.R. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar). two other are as containing a protein A structure of hemoglobin with NO modification of Cysβ93 A. PubMed Scopus Google Scholar) but was to be the on between the and of several oxide species J. Am. Chem. 2006; PubMed Scopus Google Scholar, J.S. A. PubMed Scopus Google Scholar). Finally, an atomic resolution structure of a with a modification of the surface-exposed A of was the that no the group were with a of the and bond from in crystal structures J. M.A. J. Nitric Oxide. 2005; 12: PubMed Scopus Google Scholar) and from J. Am. Chem. 2006; PubMed Scopus Google Scholar), that it may also be a more reduced oxide Protein the evidence that S-nitrosylation the function of a variety of there is little data directly that S-nitrosylation can alter the structure of a with demonstrated that S-nitrosylation at results in changes in the of in J.G. Pappu K. Campbell S.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6376-6381Crossref PubMed Scopus (84) Google Scholar). The structural of these changes were S-Nitrosylation of the transcription at to but changes in its a conformational change (6Kim S.O. Merchant K. Nudelman R. Beyer Jr., W.F. Keng T. DeAngelo J. Hausladen A. Stamler J.S. Cell. 2002; 109: 383-396Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). In other systems, there has been that S-nitrosylation may conformational changes in a protein to the of its function. it was that S-nitrosylation of of the its and results in H. D. A. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), but no direct evidence is to the of these conformational changes. Myoglobin crystal structures of the of from whale and have been previously and in the in Ref. J. Biochem. 2006; 100: PubMed Scopus Google Scholar). the bond the for the NO group accurate of the NO in the are for the and which more in their The geometry of the heme in the ferrous nitrosyl blackfin tuna myoglobin structure is to that in both crystal structures of W.R. Chem. 2003; PubMed Scopus Google Scholar) and in C. K. J. P. M. J. Chem. A. 1997; Scopus Google Scholar), with a bond of and a of The other atomic resolution protein crystal structures that a nitric and heme are of the protein from a heme The coordination geometry of the ferrous form of this protein is to described in but it a more heme a consequence of the protein and a that is important for its function A. Y. J.A. W.R. 2001; PubMed Scopus Google Scholar, A. W.R. 2005; PubMed Scopus Google Scholar). A detailed understanding of nitric oxide by heme has for the mechanism of guanylate cyclase, which a heme to and by little structural information is about guanylate cyclase, and several NO binding to the heme group within this protein changes its activity Biol. 2006; Scopus Google Scholar). x-ray we were to changes in the structure of blackfin tuna myoglobin as a direct result of S-nitrosylation at a surface-exposed cysteine Although there are to the of conformational such as the and x-ray of we this will as a to to the molecular effects of this modification on protein structure and function. with blackfin tuna myoglobin will S-nitrosylation at Cys-10 has a functional effect on the properties of this It will also be important to S-nitrosylation of myoglobin as a physiological in the muscle tissue of the blackfin We and for blackfin and for with the sample and for with data In addition, we for data and

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