Molecular basis for dimer formation of TRbeta variant D355R.
Protein quality and stability are critical during protein purification for X-ray crystallography. A target protein that is easy to manipulate and crystallize becomes a valuable product useful for high-throughput crystallography for drug design and discovery. In this work, a single surface mutation, D355R, was shown to be crucial for converting the modestly stable monomeric ligand binding domain of the human thyroid hormone receptor (TR LBD) into a stable dimer. The structure of D335R TR LBD mutant was solved using X-ray crystallography and refined to 2.2 A resolution with R(free)/R values of 24.5/21.7. The crystal asymmetric unit reveals the TR dimer with two molecules of the hormone-bound LBD related by twofold symmetry. The ionic interface between the two LBDs comprises residues within loop H10-H11 and loop H6-H7 as well as the C-terminal halves of helices 8 of both protomers. Direct intermolecular contacts formed between the introduced residue Arg 355 of one TR molecule and Glu 324 of the second molecule become a part of the extended dimerization interface of 1330 A(2) characteristic for a strong complex assembly that is additionally strengthened by buffer solutes.
Jouravel
Natalia
N
Department of Biochemistry and Biophysics, University of California San Francisco (UCSF), San Francisco, California 94158, USA. njouravel@msg.ucsf.edu
Sablin
Elena
E
Togashi
Marie
M
Baxter
John D
JD
Webb
Paul
P
Fletterick
Robert J
RJ
D018360:Crystallography, X-Ray; D006801:Humans; D008024:Ligands; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D055550:Protein Stability; D011988:Receptors, Thyroid Hormone / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D008024:Ligands; D050505:Mutant Proteins; D011988:Receptors, Thyroid Hormone
Nat Rev Drug Discov. 2004 Nov;3(11):950-64
15520817
Protein Sci. 2004 Oct;13(10):2814-8
15340174
Science. 2008 Jan 11;319(5860):206-9
18187656
J Steroid Biochem Mol Biol. 2001 Jan-Mar;76(1-5):31-42
11384861
Nature. 1995 Dec 14;378(6558):690-7
7501015
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Jul 1;64(Pt 7):625-8
18607092
Nature. 1997 Oct 16;389(6652):753-8
9338790
Proc Natl Acad Sci U S A. 2003 Dec 23;100(26):15358-63
14673100
Mol Endocrinol. 2005 Jan;19(1):35-51
15459250
J Biol Chem. 1997 May 16;272(20):13060-5
9148917
J Biol Chem. 2005 Jul 8;280(27):25665-73
15886199
Mol Endocrinol. 2007 Dec;21(12):2919-28
17823305
Thyroid. 1998 Aug;8(8):703-13
9737367
Methods Enzymol. 1997;276:307-26
27754618
Clin Exp Pharmacol Physiol Suppl. 1998 Nov;25:S2-11
9809185
Biochem Biophys Res Commun. 1993 Aug 31;195(1):385-92
8363616
Proc Natl Acad Sci U S A. 2007 Oct 9;104(41):16074-9
17911242
Genes Dev. 1998 Nov 1;12(21):3343-56
9808622
Nature. 1992 Jan 30;355(6359):441-6
1310350
Nat Struct Biol. 1995 Dec;2(12):1062-7
8846217
Nature. 1995 Jun 1;375(6530):377-82
7760929
J Biol Chem. 1991 Oct 15;266(29):19636-44
1918070
Mol Cell Biol. 2002 Aug;22(16):5782-92
12138189
J Biol Chem. 2001 May 4;276(18):14987-95
11145963
Mol Endocrinol. 1991 Aug;5(8):1049-61
1944303
Proc Natl Acad Sci U S A. 2006 Oct 31;103(44):16230-5
17050682
Neuroreport. 1994 Sep 8;5(14):1805-8
7827337
EMBO J. 1999 Sep 1;18(17):4608-18
10469641