Thioredoxin as a fusion tag for carrier-driven crystallization.
Structural investigations are frequently hindered by difficulties in obtaining diffracting crystals of the target protein. Here, we report the crystallization and structure solution of the U2AF homology motif (UHM) domain of splicing factor Puf60 fused to Escherichia coli thioredoxin A. Both modules make extensive crystallographic contacts, contributing to a well-defined crystal lattice with clear electron density for both the thioredoxin and the Puf60-UHM module. We compare two short linker sequences between the two fusion domains, GSAM and GSPPM, for which only the GSAM-linked fusion protein yielded diffracting crystals. While specific interdomain contacts are not observed for both fusion proteins, NMR relaxation data in solution indicate reduced interdomain mobility between the Trx and Puf60-UHM modules. The GSPPM-linked fusion protein is significantly more flexible, albeit both linker sequences have the same number of degrees of torsional freedom. Our analysis provides a rationale for the crystallization of the GSAM-linked fusion protein and indicates that in this case, a four-residue linker between thioredoxin A and the fused target may represent the maximal length for crystallization purposes. Our data provide an experimental basis for the rational design of linker sequences in carrier-driven crystallization and identify thioredoxin A as a powerful fusion partner that can aid crystallization of difficult targets.
Corsini
Lorenzo
L
Structural and Computational Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
Hothorn
Michael
M
Scheffzek
Klaus
K
Sattler
Michael
M
Stier
Gunter
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization / Q000379:methods*; D018360:Crystallography, X-Ray / Q000379:methods; D019281:Dimerization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics
D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D013879:Thioredoxins
Acta Crystallogr D Biol Crystallogr. 1998 May 1;54(Pt 3):423-6
9761916
J Biomol NMR. 1994 Sep;4(5):603-14
22911360
Proc Natl Acad Sci U S A. 2001 Jun 5;98(12):6877-82
11381122
Protein Sci. 2003 Jul;12(7):1313-22
12824478
J Biomol NMR. 1995 Nov;6(3):277-93
8520220
Protein Sci. 2003 Feb;12(2):306-12
12538894
Acta Crystallogr D Biol Crystallogr. 2002 Nov;58(Pt 11):1948-54
12393927
Eur J Biochem. 2000 Jul;267(13):4002-10
10866799
RNA. 1999 Dec;5(12):1548-60
10606266
Gene. 2001 Dec 27;281(1-2):1-9
11750122
Protein Sci. 1994 Dec;3(12):2233-44
7538846
EMBO J. 2001 Jan 15;20(1-2):40-6
11226153
J Mol Biol. 1993 Dec 5;234(3):779-815
8254673
Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
15299926
J Biol Chem. 1999 Nov 19;274(47):33580-6
10559245
J Biomol NMR. 1994 Jan;4(1):61-78
22911159
Proc Natl Acad Sci U S A. 1999 Apr 13;96(8):4319-24
10200260
Proc Natl Acad Sci U S A. 1975 Jun;72(6):2305-9
1094461
Science. 2007 Nov 23;318(5854):1258-65
17962520
EMBO J. 2001 Nov 1;20(21):5813-21
11689422
Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12178-82
7527555
J Mol Biol. 2007 Sep 21;372(3):774-97
17681537
Science. 2007 Nov 23;318(5854):1266-73
17962519
Protein Sci. 1999 Dec;8(12):2663-71
10631982
Protein Sci. 1998 May;7(5):1147-55
9605319
Mol Cell. 2000 Feb;5(2):331-41
10882074
Biochemistry. 1994 May 17;33(19):5984-6003
7514039
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765
Biochemistry. 1989 Nov 14;28(23):8972-9
2690953
PLoS One. 2007 Jun 20;2(6):e538
17579712
J Mol Biol. 1990 Mar 5;212(1):167-84
2181145
J Biol Chem. 1998 Jul 24;273(30):18681-4
9668035
Structure. 1994 Sep 15;2(9):853-68
7812718
Protein Sci. 1997 Aug;6(8):1783-6
9260294
Protein Sci. 2007 Nov;16(11):2542-51
17962407
Dev Cell. 2002 Mar;2(3):343-53
11879639
Nature. 2005 Oct 20;437(7062):1173-8
16189514
Nat Struct Mol Biol. 2007 Jul;14(7):620-9
17589525
J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
19461840