人源色氨酰tRNA合成酶H130R突变体的酶活和初步晶体学分析（英文）
|
摘要
色氨酰t RNA合成酶(tryptophanyl-t RNA synthetase,Trp RS)催化色氨酸的活化及其特异性t RNA的氨酰化,为蛋白质合成提供原料。在IFN-γ刺激下,人源细胞中的Trp RS通过结合血红素增强其氨酰化活性,以调节吲哚胺2,3-双加氧酶表达水平增高所引起的色氨酸缺失。体外研究发现,锌离子和血红素竞争性结合人源Trp RS以增强其氨酰化活性。然而,由于一个氨基酸位点H130R的突变,牛和鼠的Trp RS以及人的H130R突变体都不再受锌离子和血红素的影响,并具有高氨酰化活性。H130R Trp RS模拟了一种结合着锌离子或血红素的高活性构象,但目前还没有对这种构象的结构描述。为了阐明H130R决定Trp RS氨酰化活性种属特异性的原因,以及锌离子和血红素的结合位点,作者对人H130R Trp RS进行了活性分析和初步晶体学研究,此工作将为锌离子和血红素调节Trp RS氨酰化活性的机制研究奠定基础。
Tryptophanyl-t RNA synthetase(TrpR S) catalyzes the activation of tryptophan and subsequent aminoacylation of its cognate t RNA, providing material for protein synthesis. Upon IFN-γ treatment, TrpR S binds heme to enhance its aminoacylation activity, compensating the tryptophan depletion induced by increased expression of indoleamine 2,3-Dioxygenase(IDO) in many human cell lines. In vitro studies have found that zinc competes with heme in binding and enhancing the aminoacylation activity of TrpR S. However, due to a single amino acid substitution of H130 R, both bovine,mouse and human H130 R TrpR S are no longer affected by zinc and heme, and all display higher aminoacylation activity.H130 R TrpR S represents a constitutively active conformation bound with zinc or heme, but currently there is no structural characterization of this conformation. In order to illustrate the reason of H130 R for determining the species difference of TrpR S aminoacylation activity, and the zinc and heme binding sites, the authors performed enzymatic and preliminary crystallographic studies on human H130 R TrpR S. This work will serve as a template for further investigation of the mechanism of zinc and heme coordination in regulating the aminoacylation activity of TrpR S.
Tryptophanyl-t RNA synthetase(TrpR S) catalyzes the activation of tryptophan and subsequent aminoacylation of its cognate t RNA, providing material for protein synthesis. Upon IFN-γ treatment, TrpR S binds heme to enhance its aminoacylation activity, compensating the tryptophan depletion induced by increased expression of indoleamine 2,3-Dioxygenase(IDO) in many human cell lines. In vitro studies have found that zinc competes with heme in binding and enhancing the aminoacylation activity of TrpR S. However, due to a single amino acid substitution of H130 R, both bovine,mouse and human H130 R TrpR S are no longer affected by zinc and heme, and all display higher aminoacylation activity.H130 R TrpR S represents a constitutively active conformation bound with zinc or heme, but currently there is no structural characterization of this conformation. In order to illustrate the reason of H130 R for determining the species difference of TrpR S aminoacylation activity, and the zinc and heme binding sites, the authors performed enzymatic and preliminary crystallographic studies on human H130 R TrpR S. This work will serve as a template for further investigation of the mechanism of zinc and heme coordination in regulating the aminoacylation activity of TrpR S.
引文
1 .Schimmel P.Aminoacyl tR NA synthetases:General scheme of structure-function relationships in the polypeptides and recognition of transfer RNAs.Annu Rev Biochem,1987,56 :125~158
2 .Yu Y,Liu Y,Shen N,Xu X,Xu F,Jia J,Jin Y,Arnold E,Ding J.Crystal structure of human tryptophanyl-tR NA synthetase catalytic fragment:Insights into substrate recognition,tR NA binding,and angiogenesis activity.J Biol Chem,2004,279(9):8378~8388
3 .Retailleau P,Weinreb V,Hu M,Carter CW Jr.Crystal structure of tryptophanyl-tR NA synthetase complexed with adenosine-5'tetraphosphate:Evidence for distributed use of catalytic binding energy in amino acid activation by class I aminoacyl-tR NA synthetases.J Mol Biol,2007,369(1):108 ~128
4 .Shen N,Zhou M,Yang B,Yu Y,Dong X,Ding J.Catalytic mechanism of the tryptophan activation reaction revealed by crystal structures of human tryptophanyl-tR NA synthetase in different enzymatic states.Nucleic Acids Res,2008,36(4):1288~1299
5 .Guo M,Yang XL,Schimmel P.New functions of aminoacyl-tR NA synthetases beyond translation.Nat Rev Mol Cell Biol,2010,11(9):668~674
6 .Guo M,Schimmel P,Yang XL.Functional expansion of human tR NA synthetases achieved by structural inventions.FEBS Lett,2010,584(2):434~442
7 .Otani A,Slike BM,Dorrell MI,Hood J,Kinder K,Ewalt KL,Cheresh D,Schimmel P,Friedlander M.A fragment of human TrpR S as a potent antagonist of ocular angiogenesis.Proc Natl Acad Sci USA,2002,99(1):178 ~183
8 .Tolstrup AB,Bejder A,Fleckner J,Justesen J.Transcriptional regulation of the interferon-gamma-inducible tryptophanyl-tR NA synthetase includes alternative splicing.J Biol Chem,1995,270(1):397~403
9 .Turpaev KT,Zakhariev VM,Sokolova IV,Narovlyansky AN,Amchenkova AM,Justesen J,Frolova LY.Alternative processing of the tryptophanyl-tR NA synthetase m RNA from interferon-treated human cells.Eur J Biochem,1996,240 (3):732~737
10 .Tzima E,Schimmel P.Inhibition of tumor angiogenesis by a natural fragment of a tR NA synthetase.Trends Biochem Sci,2006,31(1):7~10
11 .Wakasugi K,Slike BM,Hood J,Otani A,Ewalt KL,Friedlander M,Cheresh DA,Schimmel P.A human aminoacyl-tR NA synthetase as a regulator of angiogenesis.Proc Natl Acad Sci USA,2002,99(1):173~177
12 .Kisselev L,Frolova L,Haenni AL.Interferon inducibility of mammalian tryptophanyl-tR NA synthetase:New perspectives.Trends Biochem Sci,1993,18(7):263~267
13 .Yoshida R,Imanishi J,Oku T,Kishida T,Hayaishi O.Induction of pulmonary indoleamine 2,3-dioxygenase by interferon.Proc Natl Acad Sci USA,1981,78(1):129~132
14 .Rubin BY,Anderson SL,Xing L,Powell RJ,Tate WP.Interferon induces tryptophanyl-tR NA synthetase expression in human fibroblasts.J Biol Chem,1991,266(36):24245~24248
15 .Fleckner J,Martensen PM,Tolstrup AB,Kjeldgaard NO,Justesen J.Differential regulation of the human,interferon inducible tryptophanyl-tR NA synthetase by various cytokines in cell lines.Cytokine,1995,7(1):70~77
16 .Boasso A,Herbeuval JP,Hardy AW,Winkler C,Shearer GM.Regulation of indoleamine 2,3-dioxygenase and tryptophanyl-tR NA-synthetase by CTLA-4-Fc in human CD4+T cells.Blood,2005,105(4):1574~1581
17 .Yamamoto S,Hayaishi O.Tryptophan pyrrolase of rabbit intestine.D-and L-tryptophan-cleaving enzyme or enzymes.J Biol Chem,1967,242(22):5260~5266
18 .Knox WE,Mehler AH.The conversion of tryptophan to kynurenine in liver.I.The coupled tryptophan peroxidaseoxidase system forming formylkynurenine.J Biol Chem,1950,187(1):419~430
19 .Efimov I,Basran J,Thackray SJ,Handa S,Mowat CG,Raven EL.Structure and reaction mechanism in the heme dioxygenases.Biochemistry,2011,50(14):2717~2724
20 .Sugimoto H,Oda S,Otsuki T,Hino T,Yoshida T,Shiro Y.Crystal structure of human indoleamine 2,3-dioxygenase:Catalytic mechanism of O2 incorporation by a heme-containing dioxygenase.Proc Natl Acad Sci USA,2006,103(8):2611~2616
21 .Murray MF.Tryptophan depletion and HIV infection:A metabolic link to pathogenesis.Lancet Infect Dis,2003,3 (10):644~652
22 .Thomas SR,Salahifar H,Mashima R,Hunt NH,Richardson DR,Stocker R.Antioxidants inhibit indoleamine2 ,3-dioxygenase in IFN-gamma-activated human macrophages:Posttranslational regulation by pyrrolidine dithiocarbamate.J Immunol,2001,166(10):6332~6340
23 .Wakasugi K.Human tryptophanyl-tR NA synthetase binds with heme to enhance its aminoacylation activity.Biochemistry,2007,46(40):11291~11298
24 .Nurbekov MK,Favorova OO,Dmitrenko SG,Bolotina IA,Kiselev LL.Role of zinc ions in the functioning of bovine tryptophanyl-tR NA-synthetase.Mol Biol(Mosk),1981,15(5):1000~1010
25 .Kisselev LL,Favorova OO,Nurbekov MK,Dmitriyenko SG,Engelhardt WA.Bovine tryptophanyl-tR NA synthetase.A zinc metalloenzyme.Eur J Biochem,1981,120(3):511 ~517
26 .Wakasugi K.Species-specific differences in the regulation of the aminoacylation activity of mammalian tryptophanyltR NA synthetases.FEBS Lett,2010,584(1):229~232
27 .Malkowski MG,Quartley E,Friedman AE,Babulski J,Kon Y,Wolfley J,Said M,Luft JR,Phizicky EM,DeT itta GT,Grayhack EJ.Blocking S-adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing.Proc Natl Acad Sci USA,2007,104(16):6678~6683
28 .Yang XL,Guo M,Kapoor M,Ewalt KL,Otero FJ,Skene RJ,McR ee DE,Schimmel P.Functional and crystal structure analysis of active site adaptations of a potent anti-angiogenic human tR NA synthetase.Structure,2007,15 (7):793~805
29 .Ilyin VA,Temple B,Hu M,Li G,Yin Y,Vachette P,Carter CW Jr.2.9crystal structure of ligand-free tryptophanyltR NA synthetase:Domain movements fragment the adenine nucleotide binding site.Protein Sci,2000,9(2):218 ~231
30 .Buddha MR,Crane BR.Structure and activity of an aminoacyl-tR NA synthetase that charges tR NA with nitro-tryptophan.Nat Struct Mol Biol,2005,12(3):274~275
31 .Zhou M,Dong X,Shen N,Zhong C,Ding J.Crystal structures of Saccharomyces cerevisiae tryptophanyl-tR NA synthetase:New insights into the mechanism of tryptophan activation and implications for anti-fungal drug design.Nucleic Acids Res,2010,38(10):3399~3413
32 .Han GW,Yang XL,McM ullan D,Chong YE,Krishna SS,Rife CL,Weekes D,Brittain SM,Abdubek P,Ambing E,Astakhova T,Axelrod HL,Carlton D,Caruthers J,Chiu HJ,Clayton T,Duan L,Feuerhelm J,Grant JC,Grzechnik SK,Jaroszewski L,Jin KK,Klock HE,Knuth MW,Kumar A,Marciano D,Miller MD,Morse AT,Nigoghossian E,Okach L,Paulsen J,Reyes R,van den Bedem H,White A,Wolf G,Xu Q,Hodgson KO,Wooley J,Deacon AM,Godzik A,Lesley SA,Elsliger MA,Schimmel P,Wilson IA.Structure of a tryptophanyl-tR NA synthetase containing an iron-sulfur cluster.Acta Crystallogr Sect F Struct Biol Cryst Commun,2010,66(Pt 10):1326~1334
33 .Merritt EA,Arakaki TL,Gillespie R,Napuli AJ,Kim JE,Buckner FS,van Voorhis WC,Verlinde CL,Fan E,Zucker F,Hol WG.Crystal structures of three protozoan homologs of tryptophanyl-tR NA synthetase.Mol Biochem Parasitol,2011,177(1):20~28
34 .Khan S,Garg A,Sharma A,Camacho N,Picchioni D,Saint-Leger A,Ribas de Pouplana L,Yogavel M,Sharma A.An appended domain results in an unusual architecture for malaria parasite tryptophanyl-tR NA synthetase.PLoS One,2013,8(6):e66224
35 .Arakaki TL,Carter M,Napuli AJ,Verlinde CL,Fan E,Zucker F,Buckner FS,van Voorhis WC,Hol WG,Merritt EA.The structure of tryptophanyl-tR NA synthetase from Giardia lamblia reveals divergence from eukaryotic homologs.J Struct Biol,2010,171(2):238~243
36 .Kapustina M,Carter CW Jr.Computational studies of tryptophanyl-tR NA synthetase:Activation of ATP by induced-fit.J Mol Biol,2006,362(5):1159~1180
37 .Xu X,Shi Y,Zhang HM,Swindell EC,Marshall AG,Guo M,Kishi S,Yang XL.Unique domain appended to vertebrate tR NA synthetase is essential for vascular development.Nat Commun,2012,3:681.Doi:10 .1038/ncomms1686
38 .Beebe K,Waas W,Druzina Z,Guo M,Schimmel P.A universal plate format for increased throughput of assays that monitor multiple aminoacyl transfer RNA synthetase activities.Anal Biochem,2007,368(1):111~121
39 .Otwinowski Z,Minor W.Processing of X-ray diffraction data collected in oscillation mode//Carter CW Jr,Sweet RM(Ed).Methods in enzymology,Volume 276:Macromolecular crystallography,part A.New York:Academic Press,1997:307~326
40 .Matthews BW.Solvent content of protein crystals.J Mol Biol,1968,33(2):491~497
2 .Yu Y,Liu Y,Shen N,Xu X,Xu F,Jia J,Jin Y,Arnold E,Ding J.Crystal structure of human tryptophanyl-tR NA synthetase catalytic fragment:Insights into substrate recognition,tR NA binding,and angiogenesis activity.J Biol Chem,2004,279(9):8378~8388
3 .Retailleau P,Weinreb V,Hu M,Carter CW Jr.Crystal structure of tryptophanyl-tR NA synthetase complexed with adenosine-5'tetraphosphate:Evidence for distributed use of catalytic binding energy in amino acid activation by class I aminoacyl-tR NA synthetases.J Mol Biol,2007,369(1):108 ~128
4 .Shen N,Zhou M,Yang B,Yu Y,Dong X,Ding J.Catalytic mechanism of the tryptophan activation reaction revealed by crystal structures of human tryptophanyl-tR NA synthetase in different enzymatic states.Nucleic Acids Res,2008,36(4):1288~1299
5 .Guo M,Yang XL,Schimmel P.New functions of aminoacyl-tR NA synthetases beyond translation.Nat Rev Mol Cell Biol,2010,11(9):668~674
6 .Guo M,Schimmel P,Yang XL.Functional expansion of human tR NA synthetases achieved by structural inventions.FEBS Lett,2010,584(2):434~442
7 .Otani A,Slike BM,Dorrell MI,Hood J,Kinder K,Ewalt KL,Cheresh D,Schimmel P,Friedlander M.A fragment of human TrpR S as a potent antagonist of ocular angiogenesis.Proc Natl Acad Sci USA,2002,99(1):178 ~183
8 .Tolstrup AB,Bejder A,Fleckner J,Justesen J.Transcriptional regulation of the interferon-gamma-inducible tryptophanyl-tR NA synthetase includes alternative splicing.J Biol Chem,1995,270(1):397~403
9 .Turpaev KT,Zakhariev VM,Sokolova IV,Narovlyansky AN,Amchenkova AM,Justesen J,Frolova LY.Alternative processing of the tryptophanyl-tR NA synthetase m RNA from interferon-treated human cells.Eur J Biochem,1996,240 (3):732~737
10 .Tzima E,Schimmel P.Inhibition of tumor angiogenesis by a natural fragment of a tR NA synthetase.Trends Biochem Sci,2006,31(1):7~10
11 .Wakasugi K,Slike BM,Hood J,Otani A,Ewalt KL,Friedlander M,Cheresh DA,Schimmel P.A human aminoacyl-tR NA synthetase as a regulator of angiogenesis.Proc Natl Acad Sci USA,2002,99(1):173~177
12 .Kisselev L,Frolova L,Haenni AL.Interferon inducibility of mammalian tryptophanyl-tR NA synthetase:New perspectives.Trends Biochem Sci,1993,18(7):263~267
13 .Yoshida R,Imanishi J,Oku T,Kishida T,Hayaishi O.Induction of pulmonary indoleamine 2,3-dioxygenase by interferon.Proc Natl Acad Sci USA,1981,78(1):129~132
14 .Rubin BY,Anderson SL,Xing L,Powell RJ,Tate WP.Interferon induces tryptophanyl-tR NA synthetase expression in human fibroblasts.J Biol Chem,1991,266(36):24245~24248
15 .Fleckner J,Martensen PM,Tolstrup AB,Kjeldgaard NO,Justesen J.Differential regulation of the human,interferon inducible tryptophanyl-tR NA synthetase by various cytokines in cell lines.Cytokine,1995,7(1):70~77
16 .Boasso A,Herbeuval JP,Hardy AW,Winkler C,Shearer GM.Regulation of indoleamine 2,3-dioxygenase and tryptophanyl-tR NA-synthetase by CTLA-4-Fc in human CD4+T cells.Blood,2005,105(4):1574~1581
17 .Yamamoto S,Hayaishi O.Tryptophan pyrrolase of rabbit intestine.D-and L-tryptophan-cleaving enzyme or enzymes.J Biol Chem,1967,242(22):5260~5266
18 .Knox WE,Mehler AH.The conversion of tryptophan to kynurenine in liver.I.The coupled tryptophan peroxidaseoxidase system forming formylkynurenine.J Biol Chem,1950,187(1):419~430
19 .Efimov I,Basran J,Thackray SJ,Handa S,Mowat CG,Raven EL.Structure and reaction mechanism in the heme dioxygenases.Biochemistry,2011,50(14):2717~2724
20 .Sugimoto H,Oda S,Otsuki T,Hino T,Yoshida T,Shiro Y.Crystal structure of human indoleamine 2,3-dioxygenase:Catalytic mechanism of O2 incorporation by a heme-containing dioxygenase.Proc Natl Acad Sci USA,2006,103(8):2611~2616
21 .Murray MF.Tryptophan depletion and HIV infection:A metabolic link to pathogenesis.Lancet Infect Dis,2003,3 (10):644~652
22 .Thomas SR,Salahifar H,Mashima R,Hunt NH,Richardson DR,Stocker R.Antioxidants inhibit indoleamine2 ,3-dioxygenase in IFN-gamma-activated human macrophages:Posttranslational regulation by pyrrolidine dithiocarbamate.J Immunol,2001,166(10):6332~6340
23 .Wakasugi K.Human tryptophanyl-tR NA synthetase binds with heme to enhance its aminoacylation activity.Biochemistry,2007,46(40):11291~11298
24 .Nurbekov MK,Favorova OO,Dmitrenko SG,Bolotina IA,Kiselev LL.Role of zinc ions in the functioning of bovine tryptophanyl-tR NA-synthetase.Mol Biol(Mosk),1981,15(5):1000~1010
25 .Kisselev LL,Favorova OO,Nurbekov MK,Dmitriyenko SG,Engelhardt WA.Bovine tryptophanyl-tR NA synthetase.A zinc metalloenzyme.Eur J Biochem,1981,120(3):511 ~517
26 .Wakasugi K.Species-specific differences in the regulation of the aminoacylation activity of mammalian tryptophanyltR NA synthetases.FEBS Lett,2010,584(1):229~232
27 .Malkowski MG,Quartley E,Friedman AE,Babulski J,Kon Y,Wolfley J,Said M,Luft JR,Phizicky EM,DeT itta GT,Grayhack EJ.Blocking S-adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing.Proc Natl Acad Sci USA,2007,104(16):6678~6683
28 .Yang XL,Guo M,Kapoor M,Ewalt KL,Otero FJ,Skene RJ,McR ee DE,Schimmel P.Functional and crystal structure analysis of active site adaptations of a potent anti-angiogenic human tR NA synthetase.Structure,2007,15 (7):793~805
29 .Ilyin VA,Temple B,Hu M,Li G,Yin Y,Vachette P,Carter CW Jr.2.9crystal structure of ligand-free tryptophanyltR NA synthetase:Domain movements fragment the adenine nucleotide binding site.Protein Sci,2000,9(2):218 ~231
30 .Buddha MR,Crane BR.Structure and activity of an aminoacyl-tR NA synthetase that charges tR NA with nitro-tryptophan.Nat Struct Mol Biol,2005,12(3):274~275
31 .Zhou M,Dong X,Shen N,Zhong C,Ding J.Crystal structures of Saccharomyces cerevisiae tryptophanyl-tR NA synthetase:New insights into the mechanism of tryptophan activation and implications for anti-fungal drug design.Nucleic Acids Res,2010,38(10):3399~3413
32 .Han GW,Yang XL,McM ullan D,Chong YE,Krishna SS,Rife CL,Weekes D,Brittain SM,Abdubek P,Ambing E,Astakhova T,Axelrod HL,Carlton D,Caruthers J,Chiu HJ,Clayton T,Duan L,Feuerhelm J,Grant JC,Grzechnik SK,Jaroszewski L,Jin KK,Klock HE,Knuth MW,Kumar A,Marciano D,Miller MD,Morse AT,Nigoghossian E,Okach L,Paulsen J,Reyes R,van den Bedem H,White A,Wolf G,Xu Q,Hodgson KO,Wooley J,Deacon AM,Godzik A,Lesley SA,Elsliger MA,Schimmel P,Wilson IA.Structure of a tryptophanyl-tR NA synthetase containing an iron-sulfur cluster.Acta Crystallogr Sect F Struct Biol Cryst Commun,2010,66(Pt 10):1326~1334
33 .Merritt EA,Arakaki TL,Gillespie R,Napuli AJ,Kim JE,Buckner FS,van Voorhis WC,Verlinde CL,Fan E,Zucker F,Hol WG.Crystal structures of three protozoan homologs of tryptophanyl-tR NA synthetase.Mol Biochem Parasitol,2011,177(1):20~28
34 .Khan S,Garg A,Sharma A,Camacho N,Picchioni D,Saint-Leger A,Ribas de Pouplana L,Yogavel M,Sharma A.An appended domain results in an unusual architecture for malaria parasite tryptophanyl-tR NA synthetase.PLoS One,2013,8(6):e66224
35 .Arakaki TL,Carter M,Napuli AJ,Verlinde CL,Fan E,Zucker F,Buckner FS,van Voorhis WC,Hol WG,Merritt EA.The structure of tryptophanyl-tR NA synthetase from Giardia lamblia reveals divergence from eukaryotic homologs.J Struct Biol,2010,171(2):238~243
36 .Kapustina M,Carter CW Jr.Computational studies of tryptophanyl-tR NA synthetase:Activation of ATP by induced-fit.J Mol Biol,2006,362(5):1159~1180
37 .Xu X,Shi Y,Zhang HM,Swindell EC,Marshall AG,Guo M,Kishi S,Yang XL.Unique domain appended to vertebrate tR NA synthetase is essential for vascular development.Nat Commun,2012,3:681.Doi:10 .1038/ncomms1686
38 .Beebe K,Waas W,Druzina Z,Guo M,Schimmel P.A universal plate format for increased throughput of assays that monitor multiple aminoacyl transfer RNA synthetase activities.Anal Biochem,2007,368(1):111~121
39 .Otwinowski Z,Minor W.Processing of X-ray diffraction data collected in oscillation mode//Carter CW Jr,Sweet RM(Ed).Methods in enzymology,Volume 276:Macromolecular crystallography,part A.New York:Academic Press,1997:307~326
40 .Matthews BW.Solvent content of protein crystals.J Mol Biol,1968,33(2):491~497