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A Hybrid Approach Reveals the Allosteric Regulation of GTP Cyclohydrolase I

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A Hybrid Approach Reveals the Allosteric Regulation of GTP Cyclohydrolase I A hybrid approach reveals the allosteric regulation of GTP cyclohydrolase I Rebecca Ebenhocha, Simone Prinzb, Susann Kaltwasserb, Deryck J. Millsb, Robert Meineckea, Martin Rübbelkea, Dirk Reinerta, Margit Bauera, Lisa Weixlera, Markus Zeeba, Janet Vonckb, and Herbert Nara,1 aMedicinal Chemistry, Boehringer Ingelheim Pharma GmbH & Co. KG, 88397 Biberach an der Riss, Germany; and bStructural Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany Edited by Robert Huber, Max Planck Institute of Biochemistry, Planegg-Martinsried, Germany, and approved October 16, 2020 (received for review July 7, 2020) Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes levels of BH4. Nature therefore evolved a highly regulated the conversion of GTP to dihydroneopterin triphosphate (H2NTP), mechanism of BH4 homeostasis. the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). In a seminal paper (17) by Harada et al., the molecular basis of Besides other roles, BH4 functions as cofactor in neurotransmitter BH4 homeostasis was uncovered and shown to involve GCH1 and a biosynthesis. The BH4 biosynthetic pathway and GCH1 have been regulatory protein, now known as GTP-cyclohydrolase-I-feedback- identified as promising targets to treat pain disorders in patients. regulatory protein (GFRP), which simultaneously functions as a The function of mammalian GCH1s is regulated by a metabolic positive and negative regulator of GCH1 (17). The effects of GFRP sensing mechanism involving a regulator protein, GCH1 feedback on GCH1 occur via formation of heteromeric protein complexes regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or between GCH1 and GFRP, which are dependent on the intracel- activated complexes dependent on availability of cofactor ligands, lular concentrations of the effector molecules phenylalanine or BH4 and phenylalanine, respectively. We determined high- BH4. Elevated phenylalanine levels lead to stimulation of GCH1 resolution structures of human GCH1−GFRP complexes by cryoe- activity, whereas BH4, the end product of the biosynthesis pathway, lectron microscopy (cryo-EM). Cryo-EM revealed structural flexibil- inhibits GCH1 in a feedback inhibition type mode (18). Mammalian ity of specific and relevant surface lining loops, which previously GCH1 shows cooperative enzymatic activity. Complex formation was not detected by X-ray crystallography due to crystal packing with GFRP-Phe leads to increased activity at lower substrate con- effects. Further, we studied allosteric regulation of isolated GCH1 centrations and eliminates substrate cooperativity. Conversely, BIOPHYSICS AND COMPUTATIONAL BIOLOGY by X-ray crystallography. Using the combined structural informa- GCH1 alone is allosterically inhibited by BH4. In the presence of tion, we are able to obtain a comprehensive picture of the mecha- GFRP, the inhibitory effect of BH4 is boosted and occurs at lower, nism of allosteric regulation. Local rearrangements in the allosteric physiologically relevant BH4 concentrations. The GCH1−GFRP pocket upon BH4 binding result in drastic changes in the quaternary system can therefore be regarded as a metabolic sensor that es- structure of the enzyme, leading to a more compact, tense form of tablishes BH4 and aromatic amino acid homeostasis. the inhibited protein, and translocate to the active site, leading to The human GCH1 sequence comprises 250 amino acids and an open, more flexible structure of its surroundings. Inhibition of forms a 270-kDa, D5-symmetric homodecameric functional the enzymatic activity is not a result of hindrance of substrate bind- ing, but rather a consequence of accelerated substrate binding ki- netics as shown by saturation transfer difference NMR (STD-NMR) Significance and site-directed mutagenesis. We propose a dissociation rate con- trolled mechanism of allosteric, noncompetitive inhibition. We present a comprehensive structural study, which shows the human GCH1 and GCH1−GFRP complexes in all states (apo, li- allosteric regulation | GTP | cryo-EM | X-ray crystallography | STD-NMR gand bound, partially and fully inhibited). We observed local rearrangements in the allosteric pocket upon BH4 binding, which result in drastic changes in the quaternary structure of uanosine triphosphate (GTP) cyclohydrolase I (GCH1) the enzyme leading to a more compact, tense form in the (EC:3.5.4.16) catalyzes the conversion of GTP to dihy- G inhibited protein. Inhibition of the enzymatic activity is not a droneopterin triphosphate (H2NTP). This reaction is the first result of hindrance of substrate binding, but rather a conse- and rate-limiting step involved in the de novo synthesis of tet- quence of accelerated substrate binding kinetics as shown by rahydrobiopterin (BH4) (1). BH4 plays key roles in phenylalanine STD-NMR and site-directed mutagenesis. We propose a disso- catabolism and the biosynthesis of serotonin and catecholamine- ciation rate-controlled mechanism of allosteric, noncompetitive type neurotransmitters like dopamine or norepinephrine by inhibition, which could stimulate further research toward the functioning as an essential cofactor for hydroxylases of the aro- development of GCH1 inhibitors to treat neuropathic and matic amino acids phenylalanine, tyrosine, and tryptophan (2, 3). inflammatory pain disorders. Further, BH4 is cofactor for the family of nitric oxide synthases (4), which produce the cellular signaling molecule nitric oxide that Author contributions: R.E., R.M., M.R., M.Z., J.V., and H.N. designed research; R.E., S.P., helps to modulate vascular tone and insulin secretion and affects S.K., D.J.M., R.M., M.R., D.R., and L.W. performed research; M.B. contributed new re- inflammation as well as the regulation of immune responses (5). agents/analytic tools; R.E., R.M., M.R., D.R., M.Z., J.V., and H.N. analyzed data; and R.E. and H.N. wrote the paper. Several lines of evidence, including human genetic data that Competing interest statement: R.E., R.M., M.R., D.R., M.B., L.W., M.Z., and H.N. were show that a GCH1-deficient haplotype is pain resistant, suggest employees of Boehringer Ingelheim at the time of this work. that selective inhibition of GCH1 is an attractive target to treat This article is a PNAS Direct Submission. neuropathic and inflammatory pain disorders (6–8). Abnormal- This open access article is distributed under Creative Commons Attribution License 4.0 ities in the control mechanisms of GCH1 or the activities in other (CC BY). enzymes of its biosynthetic pathway leads to BH4 deficiency, 1To whom correspondence may be addressed. Email: herbert.nar@boehringer- which is linked to a variety of vascular diseases such as diabetes, ingelheim.com. atherosclerosis, and hypertension (9–14) and to neurological This article contains supporting information online at https://www.pnas.org/lookup/suppl/ disorders, including Parkinson’s disease (15, 16). These examples doi:10.1073/pnas.2013473117/-/DCSupplemental. impressively show the serious consequences of nonphysiological www.pnas.org/cgi/doi/10.1073/pnas.2013473117 PNAS Latest Articles | 1of12 Downloaded by guest on September 28, 2021 enzyme complex in solution (19, 20). GFRP occurs as a pen- regions are predominantly located at the periphery of GCH1 tamer of 50 kDa (5 × 10 kDa). GCH1−GFRP complexes consist monomers, forming the interface between the individual of one GCH1 decamer flanked by two pentameric GFRP com- protomers. plexes. Association is along the particle fivefold axes, and the complexes are ∼370 kDa in size (21). Cryo-EM Structures of Human GCH1−GFRP Complexes Reveal Structural information on GCH1 was first obtained on the Dramatic Quaternary Conformational Changes and Order−Disorder Escherichia coli enzyme (19, 22, 23); later structures of the hu- Transitions. Due to their size and symmetry, hGCH1−hGFRP man GCH1 (24) and rat GCH1−GFRP complexes (18, 25) were complexes are well suited for high-resolution studies using cryo- determined. The structures revealed the subunit fold and qua- EM. Structures of the inhibitory (EM_hGCH1-hGFRP+BH4) ternary structure of the functional complex and established and the stimulatory (EM_hGCH1-hGFRP+Phe+active) GCH1 as a Zn(II)-dependent hydrolase. The X-ray structures of hGCH1−hGFRP complexes were generated using single-particle stimulatory and inhibitory rat GCH1−GFRP complexes show cryo-EM. Suitable protein material for the cryo-EM studies was that phenylalanine binds to a surface pocket on GFRP close to generated by complex formation in the presence of effector the protein−protein interaction interface with GCH1, whereas molecules, after separate expression and purification of both BH4 binds to an allosteric pocket on GCH1 close to the GFRP protein components, GFRP and GCH1. For the inhibitory interface (18, 25, 26). Structural differences between stimulatory complex, an hGCH1−hGFRP−BH4 ternary complex was pro- and inhibitory complexes were found to be minor. The medium duced by mixing the components (GCH1, GFRP, BH4) and resolution (2.8 Å) of the studies and the circumstance that, for purification via size exclusion chromatography (SEC). The this particular case, folding and unfolding events play a major stimulatory hGCH1−GRFP complex was formed in the presence role, and are impacted by crystal packing artifacts, did not allow of phenylalanine and 8-oxo-GTP, a tightly binding, substrate for detailed insights into the structural basis of allosteric control analog GCH1 inhibitor. The identification of suitable buffer mechanisms. conditions for both complexes
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