A hybrid approach reveals the allosteric regulation of GTP cyclohydrolase I

Abstract

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.

GTP Cyclohydrolase I as a Potential Drug Target: New Insights into Its Allosteric Modulation via Normal Mode Analysis

“…Previous attempts to obtain a 3D structure of a mammalian GCH1 could not define the structure of the first 47 N-terminal amino acids of the R. norvegicus GCH1 and the first 67 N-terminal amino acids of the human GCH1, probable due to their high mobility. 4,72 Furthermore, deletions in the N-terminal region resulted in increased catalytic activity relative to the full-length region. 73 As a result, it was proposed that the N-terminal domain does not contribute directly to catalysis but may exert an allosteric effect on GCH1 activity.…”

“…Notably, in their singleparticle electron cryo-electron microscopy (cryo-EM) derived structures of the GCH1−GFRP inhibitory and stimulatory complexes, Ebenhoch and colleagues reported the movement of the GCH1 helices to approach each other by 1 Å. 72 This finding was deduced by an observed increase in the density of the electron microscopy map at the GCH1 core of the inhibitory complex. The atomic MSF is related to the experimentally determined fluctuation, the so-called B-factor.…”

“…4 Furthermore, it was suggested that the binding of Phe induces the association of the GFRP with GCH1 by filling the surface space between the two proteins, hence increasing the contact. 4 Based on recent experimental studies, Ebenhoch and colleagues reported drastic changes at the GCH1 central five-helix bundle and its barrel, 72 in which the barrel/tunnel was found to be more contracted in the inhibitory form.…”

“…D118 and E119 showed a relatively higher fluctuation in the inhibitory form than the stimulatory one; these residues were reported to trigger structural changes of residues located on the path to the active site. 72 In a study by Ebenhoch and colleagues, two key residues of the human GCH1 homolog, R235 and R241, were reported to change position to enable the binding of BH4, resulting in the shrinking of the central five-helix bundle and conformational rearrangement of the GCH1 β-barrel curvature. 72 The two residues corresponded to R226 and R232 of the R. norvegicus GCH1 and exhibited a slight fluctuation in the inhibitory form (Figure 8C).…”

“…72 In a study by Ebenhoch and colleagues, two key residues of the human GCH1 homolog, R235 and R241, were reported to change position to enable the binding of BH4, resulting in the shrinking of the central five-helix bundle and conformational rearrangement of the GCH1 β-barrel curvature. 72 The two residues corresponded to R226 and R232 of the R. norvegicus GCH1 and exhibited a slight fluctuation in the inhibitory form (Figure 8C). Furthermore, site-directed mutagenesis involving BH4 inhibitor-binding residues H126, D127, E128, corresponding to H117, D118, E119 in the R. norvegicus structure as well as R235, and R241, showed that all GCH1 mutants except for R235 and R241 were still functional or with a slight reduction in activity relative to the wild type.…”