A hybrid approach reveals the allosteric regulation of GTP cyclohydrolase I.
Allosteric Regulation
Allosteric Site
/ genetics
Biopterins
/ analogs & derivatives
Cryoelectron Microscopy
Crystallography, X-Ray
GTP Cyclohydrolase
/ genetics
Intracellular Signaling Peptides and Proteins
/ metabolism
Mutagenesis, Site-Directed
Phenylalanine
/ metabolism
Protein Structure, Quaternary
GTP
STD-NMR
X-ray crystallography
allosteric regulation
cryo-EM
Journal
Proceedings of the National Academy of Sciences of the United States of America
ISSN: 1091-6490
Titre abrégé: Proc Natl Acad Sci U S A
Pays: United States
ID NLM: 7505876
Informations de publication
Date de publication:
15 12 2020
15 12 2020
Historique:
pubmed:
25
11
2020
medline:
2
2
2021
entrez:
24
11
2020
Statut:
ppublish
Résumé
Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1-GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.
Identifiants
pubmed: 33229582
pii: 2013473117
doi: 10.1073/pnas.2013473117
pmc: PMC7750480
doi:
Substances chimiques
GCHFR protein, human
0
Intracellular Signaling Peptides and Proteins
0
Biopterins
0
Phenylalanine
47E5O17Y3R
GCH1 protein, human
EC 3.5.4.16
GTP Cyclohydrolase
EC 3.5.4.16
sapropterin
EGX657432I
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
31838-31849Informations de copyright
Copyright © 2020 the Author(s). Published by PNAS.
Déclaration de conflit d'intérêts
Competing interest statement: R.E., R.M., M.R., D.R., M.B., L.W., M.Z., and H.N. were employees of Boehringer Ingelheim at the time of this work.
Références
Protein Sci. 2018 Jan;27(1):293-315
pubmed: 29067766
J Comput Chem. 2004 Oct;25(13):1605-12
pubmed: 15264254
Circ Res. 2007 Oct 12;101(8):830-8
pubmed: 17704208
Curr Pharm Biotechnol. 2011 Oct;12(10):1728-41
pubmed: 21466440
Acta Crystallogr D Struct Biol. 2018 Jun 1;74(Pt 6):531-544
pubmed: 29872004
J Biol Chem. 1998 Aug 7;273(32):20102-8
pubmed: 9685352
Int Immunopharmacol. 2001 Aug;1(8):1397-406
pubmed: 11515807
Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12120-5
pubmed: 8618856
Nat Genet. 1994 Nov;8(3):236-42
pubmed: 7874165
J Am Soc Nephrol. 2013 Jun;24(7):1139-50
pubmed: 23620395
Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1212-7
pubmed: 11818540
Proteins. 2005 Jun 1;59(4):687-96
pubmed: 15815974
J Neural Transm (Vienna). 2019 Apr;126(4):397-409
pubmed: 29995172
J Biol Chem. 1999 Jun 11;274(24):16727-35
pubmed: 10358012
Sci Rep. 2015 Jul 07;5:11998
pubmed: 26149603
Eur J Pediatr. 1984 Feb;141(4):208-14
pubmed: 6734669
J Struct Biol. 1999 Dec 1;128(1):82-97
pubmed: 10600563
Biophys J. 1993 Mar;64(3):754-61
pubmed: 8386014
J Struct Biol. 2015 Nov;192(2):216-21
pubmed: 26278980
Acta Crystallogr D Biol Crystallogr. 2001 Aug;57(Pt 8):1153-6
pubmed: 11468403
J Pharmacol Exp Ther. 2019 Nov;371(2):476-486
pubmed: 31110114
Structure. 2018 Feb 6;26(2):337-344.e4
pubmed: 29395788
J Am Chem Soc. 2010 Dec 29;132(51):18340-50
pubmed: 21138300
Acta Crystallogr D Biol Crystallogr. 2013 Nov;69(Pt 11):2276-86
pubmed: 24189240
J Biol Chem. 1989 Nov 25;264(33):19654-8
pubmed: 2584186
Protein Sci. 2008 Aug;17(8):1295-307
pubmed: 18560010
Front Chem. 2019 Apr 12;7:215
pubmed: 31032246
Nat Methods. 2015 Oct;12(10):943-6
pubmed: 26280328
Biochem J. 2000 Jul 1;349(Pt 1):353-6
pubmed: 10861247
J Am Chem Soc. 2001 Jun 27;123(25):6108-17
pubmed: 11414845
Science. 1993 Jun 4;260(5113):1507-10
pubmed: 8502995
J Biol Chem. 1989 Dec 5;264(34):20496-501
pubmed: 2584226
Nature. 2014 Apr 17;508(7496):331-9
pubmed: 24740064
Arch Biochem Biophys. 2001 Apr 1;388(1):67-73
pubmed: 11361142
J Clin Invest. 1997 Jan 1;99(1):41-6
pubmed: 9011574
Ernst Schering Res Found Workshop. 2004;(44):149-67
pubmed: 14579779
J Biol Chem. 1989 Dec 25;264(36):21660-4
pubmed: 2557335
Am J Physiol Endocrinol Metab. 2014 May 15;306(10):E1120-31
pubmed: 24644242
Biol Chem. 1997 Mar-Apr;378(3-4):185-92
pubmed: 9165069
Biochemistry. 2011 Oct 4;50(39):8508-18
pubmed: 21870820
Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
pubmed: 20383002
J Biol Chem. 2004 Dec 3;279(49):51534-40
pubmed: 15448133
Chemistry. 2010 Jul 12;16(26):7803-12
pubmed: 20496354
Nat Methods. 2013 Jun;10(6):584-90
pubmed: 23644547
Biochemistry. 1966 Jan;5(1):365-85
pubmed: 5938952
Structure. 1995 May 15;3(5):459-66
pubmed: 7663943
Nat Methods. 2015 Sep;12(9):859-65
pubmed: 26237227
Proc Natl Acad Sci U S A. 2001 Mar 27;98(7):3773-7
pubmed: 11259676
Eur Biophys J. 1984;11(2):103-9
pubmed: 6544679
Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13567-72
pubmed: 11087827
Elife. 2018 Nov 09;7:
pubmed: 30412051
Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):125-32
pubmed: 20124692
Circ Res. 2010 Feb 5;106(2):328-36
pubmed: 19926872
Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):293-302
pubmed: 21460447
Angew Chem Int Ed Engl. 2003 Feb 24;42(8):864-90
pubmed: 12596167
Angew Chem Int Ed Engl. 1999 Jun 14;38(12):1784-1788
pubmed: 29711196
Annu Rev Biochem. 1985;54:729-64
pubmed: 2862841
J Mol Biol. 2003 Feb 14;326(2):503-16
pubmed: 12559918
J Mol Biol. 1965 May;12:88-118
pubmed: 14343300
Nat Med. 2006 Nov;12(11):1269-77
pubmed: 17057711
Cell. 2010 Jul 9;142(1):101-11
pubmed: 20603017
Chem Rev. 2004 Aug;104(8):3641-76
pubmed: 15303832