scholarly journals Crystal structure of a ligand-free stable TSH receptor leucine-rich repeat domain

2019 ◽  
Vol 62 (3) ◽  
pp. 117-128 ◽  
Author(s):  
Jennifer Miller-Gallacher ◽  
Paul Sanders ◽  
Stuart Young ◽  
Andrew Sullivan ◽  
Stuart Baker ◽  
...  

The crystal structures of the thyroid-stimulating hormone receptor (TSHR) leucine-rich repeat domain (amino acids 22–260; TSHR260) in complex with a stimulating human monoclonal autoantibody (M22TM) and in complex with a blocking human autoantibody (K1-70™) have been solved. However, attempts to purify and crystallise free TSHR260, that is not bound to an autoantibody, have been unsuccessful due to the poor stability of free TSHR260. We now describe a TSHR260 mutant that has been stabilised by the introduction of six mutations (H63C, R112P, D143P, D151E, V169R and I253R) to form TSHR260-JMG55TM, which is approximately 900 times more thermostable than wild-type TSHR260. These six mutations did not affect the binding of human TSHR monoclonal autoantibodies or patient serum TSHR autoantibodies to the TSHR260. Furthermore, the response of full-length TSHR to stimulation by TSH or human TSHR monoclonal autoantibodies was not affected by the six mutations. Thermostable TSHR260-JMG55TM has been purified and crystallised without ligand and the structure solved at 2.83 Å resolution. This is the first reported structure of a glycoprotein hormone receptor crystallised without ligand. The unbound TSHR260-JMG55TM structure and the M22 and K1-70 bound TSHR260 structures are remarkably similar except for small changes in side chain conformations. This suggests that neither the mutations nor the binding of M22TM or K1-70TM change the rigid leucine-rich repeat domain structure of TSHR260. The solved TSHR260-JMG55TM structure provides a rationale as to why the six mutations have a thermostabilising effect and provides helpful guidelines for thermostabilisation strategies of other soluble protein domains.

2006 ◽  
Vol 20 (8) ◽  
pp. 1880-1893 ◽  
Author(s):  
Henry F. Vischer ◽  
Joke C. M. Granneman ◽  
Jan Bogerd

Abstract Glycoprotein hormone receptors contain large N-terminal extracellular domains (ECDs) that distinguish these receptors from most other G protein-coupled receptors. Each glycoprotein hormone receptor ECD consists of a curved leucine-rich repeat domain flanked by N- and C-terminal cysteine-rich regions. Selectivity of the different glycoprotein hormone receptors for their cognate hormones is exclusively determined by their ECDs and, in particular, their leucine-rich repeat domain. To identify human (h)FSH-selective determinants we used a gain-of-function mutagenesis strategy in which β-strands of the hLH receptor (hLH-R) were substituted with their hFSH receptor (hFSH-R) counterparts. Introduction of hFSH-R β-strand 1 into hLH-R conferred responsiveness to hFSH, whereas hLH-R mutants harboring one of the other hFSH-R β-strands displayed none or very limited sensitivity to hFSH. However, combined substitution of hFSH-R β-strand 1 and some of the other hFSH-R β-strands further increased the sensitivity of the mutant hLH-R to hFSH. The apparent contribution of multiple hFSH-R β-strands in providing a selective hormone binding interface corresponds well with their position in relation to hFSH as recently determined in the crystal structure of hFSH in complex with part of the hFSH-R ECD.


2002 ◽  
Vol 109 (11) ◽  
pp. 1445-1452 ◽  
Author(s):  
Koji Nakabayashi ◽  
Hirotaka Matsumi ◽  
Alka Bhalla ◽  
Jeehyeon Bae ◽  
Sietse Mosselman ◽  
...  

2016 ◽  
Vol 25 (17) ◽  
pp. 3824-3835 ◽  
Author(s):  
Marialetizia Motta ◽  
Giovanni Chillemi ◽  
Valentina Fodale ◽  
Serena Cecchetti ◽  
Simona Coppola ◽  
...  

2015 ◽  
Vol 114 (5) ◽  
pp. 1793-1802 ◽  
Author(s):  
Hiroki Maeda ◽  
Koshi Kurisu ◽  
Takeshi Miyata ◽  
Kodai Kusakisako ◽  
Remil Linggatong Galay ◽  
...  

2006 ◽  
Vol 20 (2) ◽  
pp. 414-425 ◽  
Author(s):  
Shannon L. Okada ◽  
Jeff L. Ellsworth ◽  
Diane M. Durnam ◽  
Harald S. Haugen ◽  
James L. Holloway ◽  
...  

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