title: A yeast 2-hybrid analysis of human GTP cyclohydrolase I protein interactions.
abstract: The yeast 2-hybrid system was used to identify protein domains involved in the oligomerization of human guanosine 5'-triphosphate (GTP) Cyclohydrolase I (GCH1) and the interaction of GCH1 with its regulatory partner, GCH1 feedback regulatory protein (GFRP). When interpreted within the structural framework derived from crystallography, our results indicate that the GCH1 N-terminal alpha-helices are not the only domains involved in the formation of dimers from monomers and also suggest an important role for the C-terminal alpha-helix in the assembly of dimers to form decamers. Moreover, a previously unknown role of the extended N-terminal alpha-helix in the interaction of GCH1 and GFRP was revealed. To discover novel GCH1 protein binding partners, we used the yeast 2-hybrid system to screen a human brain library with GCH1 N-terminal amino acids 1-96 as prey. This protruding extension of GCH1 contains two canonical Type-I Src homology-3 (SH3) ligand domains located within amino acids 1-42. Our screen yielded seven unique clones that were subsequently shown to require amino acids 1-42 for binding to GCH1. The interaction of one of these clones, Activator of Heat Shock 90 kDa Protein (Aha1), with GCH1 was validated by glutathione-s-transferase (GST) pull-down assay. Although the physiological relevance of the Aha1-GCH1 interaction requires further study, Aha1 may recruit GCH1 into the endothelial nitric oxide synthase/heat shock protein (eNOS/Hsp90) complex to support changes in endothelial nitric oxide production through the local synthesis of BH4.
authors.affiliation: Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
authors.forename: Lance
authors.initials: L
authors.lastname: Swick
authors.forename: Gregory
authors.initials: G
authors.lastname: Kapatos
mesh_terms: D000595:Amino Acid Sequence / Q000502:physiology; D001665:Binding Sites / Q000502:physiology; D001708:Biopterins / Q000031:analogs & derivatives / Q000096:biosynthesis; D018833:Chaperonins; D018360:Crystallography, X-Ray; D004730:Endothelium, Vascular / Q000201:enzymology; D004789:Enzyme Activation / Q000502:physiology; D006136:GTP Cyclohydrolase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D006160:Guanosine Triphosphate / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000096:biosynthesis; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D011108:Polymers / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: C475092:AHA1 protein, S cerevisiae; C101092:GCHFR protein, human; D018841:HSP90 Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011108:Polymers; D029701:Saccharomyces cerevisiae Proteins; D001708:Biopterins; D009569:Nitric Oxide; D006160:Guanosine Triphosphate; D052250:Nitric Oxide Synthase Type III; D006136:GTP Cyclohydrolase; D018833:Chaperonins; C003402:sapropterin