Goulas et al. 1 Crystallization and preliminary X-Ray diffraction analysis of eukaryotic α 2 - 1 macroglobulin family members modified by methylamine, proteases and 2 glycosidases. 3 4 Theodoros Goulas 1,* , Irene Garcia-Ferrer 1 , Sonia García-Piqué 1 , Lars Sottrup-Jensen 2 & F. 5 Xavier Gomis-Rüth 1,* 6 7 8 9 10 1 Proteolysis Lab, Molecular Biology Institute of Barcelona, CSIC, Barcelona Science Park, 11 Helix Building, c/ Baldiri Reixac, 15-21, E-08028 Barcelona (Spain). 12 2 Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus 13 (Denmark). 14 * To whom correspondence should be addressed. Tel: (+34) 934 020 186; Fax: (+34) 934 15 034 979; E-mail addresses: [email protected](F.X.Gomis-Rüth) or [email protected]16 (T. Goulas). 17 18 19 The authors state they have no competing financial interest. 20 21 22 23 Email addresses 24 IGF: [email protected]25 SGP: [email protected]26 LSJ: [email protected]27 28 29 ABBREVIATIONS 30 α 2 M: α 2 -macroglobulin; hα 2 M: human α 2 -macroglobulin; MA: methylamine; PNGase F: 31 peptide-N-glycosidase F; Endo F: endo-β-N-acetyl-glucosaminidase F; Tm: melting 32 temperature. 33 34
24
Embed
a2M Crystallisation manuscript def (2) - Digital.CSICdigital.csic.es/bitstream/10261/124241/4/a2M_Crystallisation_manuscript_def.pdfHowever, in order to understand its mechanism of
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Goulasetal.
1
Crystallization and preliminary X-Ray diffraction analysis of eukaryotic α2-1
macroglobulin family members modified by methylamine, proteases and 2
glycosidases. 3
4
Theodoros Goulas1,*, Irene Garcia-Ferrer1, Sonia García-Piqué1, Lars Sottrup-Jensen2 & F. 5
Xavier Gomis-Rüth1,* 6
7
8
9
101Proteolysis Lab, Molecular Biology Institute of Barcelona, CSIC, Barcelona Science Park, 11
Helix Building, c/ Baldiri Reixac, 15-21, E-08028 Barcelona (Spain). 122Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus 13
(Denmark). 14*To whom correspondence should be addressed. Tel: (+34) 934 020 186; Fax: (+34) 934 15
2“TRIGGER”; BFU2012-32862; CSD2006-00015; Fundació “La Marató de TV3” grant 416
2009-100732; and 2009SGR1036). We acknowledge the help provided by ESRF 417
synchrotron local contacts. Funding for travelling and synchrotron data collection was 418
provided in part by ESRF. 419
420
421
LITERATURE 422
Andersen, G.R., Jacobsen, L., Thirup, S., Nyborg, J., and Sottrup-Jensen, L. (1991) 423Crystallization and preliminary X-ray analysis of methylamine-treated alpha-2-424macroglobulin and 3 alpha-2-macroglobulin-proteinase complexes. FEBS 292: 267–270. 425
Andersen, G.R., Koch, T., Sorensen, A., et al. (1994) Crystallization of proteins of the 426alpha-2-macroglobulin superfamily. Ann New York Acad Sci 10: 444–446. 427
Armstrong, P.B. (2001) The contribution of proteinase inhibitors to immune defense. 428Trends Immunol 22: 47–52. 429
Baker, H., Day, C., Norris, G., and Baker, E. (1994) Enzymatic deglycosylation as a tool 430for crystallization of mammalian binding proteins. Acta Crystallogr D 50: 380–384. 431
Barrett, A.J., Brown, M.A., and Sayers, C.A. (1979) The electrophoretically “slow” and 432“fast” forms of the alpha-2-macroglobulin molecule. Biochem J 181: 401–418. 433
Baxter, R.H.G., Chang, C., Chelliah, Y., Levashina, E.A., and Deisenhofer, J. (2007) 434Structural basis for conserved complement factor-like function in the antimalarial protein 435TEP1. Proc Natl Acad Sci 104: 11615–11620. 436
Budd, A., Blandin, S., Levashina, E.A., and Gibson, T.J. (2004) Bacterial alpha-2-437macroglobulins: Colonization factors acquired by horizontal gene transfer from the 438metazoan genome? Genome Biol 5: R38.1–R38.13. 439
Goulasetal.
14
Chang, V.T., Crispin, M., Aricescu, A R., et al. (2007) Glycoprotein structural genomics: 440Solving the glycosylation problem. Structure 15: 267–73. 441
Cohn, E.J., Strong, L.E., Hughes, W.L., et al. (1946) Preparation and properties of serum 442and plasma proteins. IV. A system for the separation into fractions of the protein and 443lipoprotein components of biological tissues and fluids. J Am Chem Soc 68: 459–475. 444
Doan, N., and Gettins, P.G.W. (2008) alpha-Macroglobulins are present in some Gram-445negative bacteria: Characterization of the alpha-2-macroglobulin from Escherichia coli. J 446Biol Chem 283: 28747–28756. 447
Dunn, J., and Spiro, G.R. (1967) The alpha-2-macroglobulin of human plasma. J Biol 448Chem 242: 5556–5563. 449
Ericsson, U.B., Hallberg, B.M., DeTitta, G.T., Dekker, N., and Nordlund, P. (2006) 450Thermofluor-based high-throughput stability optimization of proteins for structural studies. 451Anal Biochem 357: 289–298. 452
Fredslund, F., Laursen, N.S., Roversi, P., et al. (2008) Structure and influence of a tick 453complement inhibitor on human complement component 5. Nat Immunol 9: 753–760. 454
Goulas, T., Arolas, J.L., and Gomis-Rüth, F.X. (2011) Structure, function and latency 455regulation of a bacterial enterotoxin potentially derived from a mammalian 456adamalysin/ADAM xenolog. Proc Natl Acad Sci 108: 1856–1861. 457
Grueninger-Leitch, F., D’Arcy, A., D’Arcy, B., and Chène, C. (1996) Deglycosylation of 458proteins for crystallization using recombinant fusion protein glycosidases. Protein Sci 5: 4592617–2622. 460
Haider, S.R., Sharp, B.L., and Reid, H.J. (2011) A comparison of Tris-glycine and Tris-461tricine buffers for the electrophoretic separation of major serum proteins. J Sep Sci 34: 4622463–2467. 463
Heras, B., and Martin, J.L. (2005) Post-crystallization treatments for improving diffraction 464quality of protein crystals. Acta Crystallogr D Biol Crystallogr 61: 1173–1180. 465
Janssen, B.J.C., Huizinga, E.G., Raaijmakers, H.C. A, et al. (2005) Structures of 466complement component C3 provide insights into the function and evolution of immunity. 467Nature 437: 505–511. 468
Kabsch, W. (2010) XDS. Acta Crystallogr D Biol Crystallogr 66: 125–32. 469
Kantyka, T., Rawlings, N.D., and Potempa, J. (2010) Prokaryote-derived protein inhibitors 470of peptidases: A sketchy occurrence and mostly unknown function. Biochimie 92: 1644–4711656. 472
Kolodziej, S., and Schroeter, J. (1996) The novel three-dimensional structure of native 473human alpha-2-macroglobulin and comparisons with the structure of the methylamine 474derivative. J Struct Biol 116: 366–376. 475
Goulasetal.
15
Marrero, A., Duquerro, S., Trapani, S., et al. (2012) The crystal structure of human alpha-4762-macroglobulin reveals a unique molecular cage. Angew Chem Int Ed Engl 51: 3340–4773344. 478
Meyer, C., Hinrichs, W., and Hahn, U. (2012) Human alpha-2-macroglobulin-another 479variation on the venus flytrap. Angew Chem Int Ed Engl 51: 5045–5047. 480
Nagase, H., Harris, E.D., Woessner, J.F., and Brew, K. (1983) Ovostatin: A novel 481proteinase inhibitor from chicken egg white. I. Purification, physicochemical properties and 482tissue distribution of ovostatin. J Biol Chem 258: 7481–7489. 483
Nielsen, K., Sottrup-Jensen, L., Nagase, H., Thorgensen, H., and Etzerodt, M. (1994) 484Amino acid sequence of hen ovomacroglobulin (ovostatin) deduced from cloned cDNA. 485DNA Seq 5: 111–119. 486
Paiva, M.M., Soeiro, M.N.C., Barbosa, H.S., Meirelles, M.N.L., Delain, E., and Araújo-487Jorge, T.C. (2010) Glycosylation patterns of human alpha-2-macroglobulin: Analysis of 488lectin binding by electron microscopy. Micron 41: 666–673. 489
Reddy, A., Grimwood, B.G., Plummer, T.H., and Tarentino, A.L. (1998) High-level 490expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E. coli: One step 491purification to homogeneity. Glycobiology 8: 633–636. 492
Rehman, A.A., Ahsan, H., and Khan, F. (2013) alpha-2-Macroglobulin: A physiological 493guardian. J Cell Physiol 228: 1665–1675. 494
Robert-Genthon, M., Casabona, M.G., Neves, D., et al. (2013) Unique features of a 495Pseudomonas aeruginosa alpha-2-macroglobulin homolog. MBio 4: e00309–13. 496
Saunders, A.J., and Tanzi, R.E. (2003) Welcome to the complex disease world. alpha-2-497Macroglobulin and Alzheimer’s disease. Exp Neurol 184: 50–53. 498
Schägger, H. (2006) Tricine-SDS-PAGE. Nat Protoc 1: 16–22. 499
Schaller, J., and Gerber, S.S. (2011) The plasmin-antiplasmin system: Structural and 500functional aspects. Cell Mol Life Sci 68: 785–801. 501
Sottrup-Jensen, L. (1989) alpha-Macroglobulins: Structure, shape, and mechanism of 502proteinase complex formation. J Biol Chem 264: 11539–11542. 503
Sottrup-Jensen, L., Petersen, T.E., and Magnusson, S. (1980) A thiol-ester in alpha-2-504macroglobulin cleaved during proteinase complex formation. FEBS Lett 121: 275–279. 505
Sottrup-Jensen, L., Petersen, T.E., and Magnusson, S. (1981) Mechanism of proteinase 506complex formation with alpha 2-macroglobulin. Three modes of trypsin binding. FEBS Lett 507128: 127–132. 508
Sottrup-Jensen, L., Sand, O., Kristensens, L., and Fey, G.H. (1989) The alpha-509macroglobulin bait region. J Biol Chem 264: 15781–15789. 510
Goulasetal.
16
Stoops, J., Schroeter, J., Bretaudiere, J.-P., Olson, N., Baker, T., and Strickland, D. (1991) 511Structural studies of human alpha-2-macroglobulin : Concordance between projected 512views obtained by negative-stain and cryoelectron microscopy. J Struct Biol 106: 172–178. 513
Varki, A., and Schauer, R. (2009) Sialic acids. In Essentials of Glycobiology. 2nd edition. 514Varki, A., Cummings, R., and Esko, J. (eds). Cold Spring Harbor (NY), Chapter 14. 515
Waddling, C., Plummer, T., Tarentino, A., and Roey, P. Van (2000) Structural basis for the 516substrate specificity of endo-beta-N-acetylglucosaminidase F(3). Biochemistry 39: 7878–5177885. 518
Walsh, C.T., Garneau-Tsodikova, S., and Gatto, G.J. (2005) Protein posttranslational 519modifications: the chemistry of proteome diversifications. Angew Chem Int Ed Engl 44: 5207342–7372. 521
Williams, S.E., Kounnas, M.Z., Argraves, K.M., Argraves, W.S., and Strickland, D.K. 522(1994) The alpha-2-macroglobulin receptor/low density lipoprotein receptor-related protein 523and the receptor-associated protein. An overview. Ann N Y Acad Sci 737: 1–13. 524
Woessner, J.F. (1999) Matrix metalloproteinase inhibition from the Jurassic to the third 525millennium. Ann New York Acad Sceinces 878: 388–403. 526
527
528
529
530
531
532
533
534
535
536
537
538
539
Goulasetal.
17
540
541
Table 1: Crystallization and X-ray diffraction of crystals. 542
543
1MIB buffer is produced by mixing sodium malonate, imidazole, and boric acid in the molar ratios 2:3:3. 544Ha2M, human a2-macrogobulin. 545 546
Protein Crystallization conditions Crystal morphology Diffraction resolution
Average intensity (<[<I> / σ(<I>)]>) 19.3 (3.2) 15.9 (4.08)
B-Factor (Wilson) (Å2) / Average multiplicity 293.5/ 6.4 (6.6) 335.3/ 13.6 (13.8) aValues in parentheses refer to the outermost resolution shell. bRr.i.m.= Σhkl(nhkl /[nhkl-1]1/2)Σi |Ii(hkl) - <I(hkl)>| / ΣhklΣi Ii(hkl) where Ii(hkl) is the i-th intensity measurement and nhkl the number of observations of reflection hkl,including symmetry-related reflections, and <I(hkl)> its average intensity. Rmerge = ΣhklΣi |Ii(hkl) - <I(hkl)>| / ΣhklΣiIi(hkl).
Goulasetal.
20
[Figure Legends]
Figure 1. Modification of native hα2M and ovostatin by deglycosylation or activation to the
induced form. (A) SDS-PAGE of hα2M and ovostatin after isolation and purification from
individual sources. (B)(C) Desialylation of ovostatin and hα2M, respectively. Protein
samples were incubated in the absence (lane 1) or presence (lane 2) of sialidase and
analyzed by native PAGE. (D) Deglycosylation of hα2M by PNGase F. Purified protein
(lane 1) was digested with PNGase F (lane 2) in a weight ratio of 10:1 and analyzed by
native PAGE. (E) Activation of hα2M by methylamine. Deglycosylated hα2M (lane 1) was
induced with methylamine (lane 2) and analyzed by native PAGE. (F)(G) Deglycosylation
of hα2M and ovostatin by glycosidases. Protein samples were incubated in the absence
(lanes 1 and 7) or presence of either PNGase F (lane2) or different Endo Fs (F1, F2 and
F3) individually (lanes 3-5) or in a single reaction (lane 6) and subsequently analyzed by
SDS-PAGE or native PAGE, respectively. (H) Thermal shift curves of native hα2M and
ovostatin before (black lines) and after deglycosylation (gray lines). Values are
represented as means of three experiments. (I)(J) Activation of hα2M and ovostatin by
peptidases. Proteins were incubated with thermolysin or chymotrypsin at various ratios
and analyzed by SDS-PAGE. The residual proteolytic activity (in box) against BODIPY FL-
casein is expressed as percentage of the activity in absence of α2M. Reactions were kept
30min at room temperature before fluorescence measurements.
Figure 2. Protein crystals of native and induced hα2M and ovostatin. (A) Irregularly-
shaped crystals of native hα2M. (B) Spherulites formed in crystallization drops with
deglycosylated native hα2M. (C)(D) Prism-shaped crystals and two dimensional plates of
methylamine induced hα2M. (E)(F) Prism-shaped crystals of deglycosylated hα2M in
complex with thermolysin. (G) Pyramid-shaped crystals of native ovostatin. (H) Needle-
shaped crystals of ovostatin in complex with thermolysin. (I) Pyramid-shaped crystals of
ovostatin in complex with chymotrypsin.
Figure 3. Images of the diffraction pattern of (A) hα2M and (B) ovostatin crystals after
exposure to synchrotron radiation. Both images were obtained after 1o rotation and
Goulasetal.
21
exposure for 0.5sec or 20sec at 100% transmission in ID23-1 or ID29, respectively. Black