PMID:16696853

Citation	Swick, L and Kapatos, G (2006) A yeast 2-hybrid analysis of human GTP cyclohydrolase I protein interactions. J. Neurochem. 97:1447-55
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.
Links	PubMed PMC2239266 Online version:10.1111/j.1471-4159.2006.03836.x
Keywords	Amino Acid Sequence/physiology; Binding Sites/physiology; Biopterin/analogs & derivatives; Biopterin/biosynthesis; Chaperonins; Crystallography, X-Ray; Endothelium, Vascular/enzymology; Enzyme Activation/physiology; GTP Cyclohydrolase/chemistry; GTP Cyclohydrolase/genetics; GTP Cyclohydrolase/metabolism; Gene Library; Guanosine Triphosphate/metabolism; HSP90 Heat-Shock Proteins/metabolism; Humans; Intracellular Signaling Peptides and Proteins/metabolism; Molecular Sequence Data; Nitric Oxide/biosynthesis; Nitric Oxide Synthase Type III/metabolism; Polymers/metabolism; Protein Binding/physiology; Protein Structure, Secondary/physiology; Protein Structure, Tertiary/physiology; Saccharomyces cerevisiae/metabolism; Saccharomyces cerevisiae Proteins/metabolism; Two-Hybrid System Techniques
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