A yeast 2-hybrid analysis of human GTP cyclohydrolase I protein interactions.
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.
Mesh Terms:
Amino Acid Sequence, Binding Sites, Biopterin, Chaperonins, Crystallography, X-Ray, Endothelium, Vascular, Enzyme Activation, GTP Cyclohydrolase, Gene Library, Guanosine Triphosphate, HSP90 Heat-Shock Proteins, Humans, Intracellular Signaling Peptides and Proteins, Molecular Sequence Data, Nitric Oxide, Nitric Oxide Synthase Type III, Polymers, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Two-Hybrid System Techniques
Amino Acid Sequence, Binding Sites, Biopterin, Chaperonins, Crystallography, X-Ray, Endothelium, Vascular, Enzyme Activation, GTP Cyclohydrolase, Gene Library, Guanosine Triphosphate, HSP90 Heat-Shock Proteins, Humans, Intracellular Signaling Peptides and Proteins, Molecular Sequence Data, Nitric Oxide, Nitric Oxide Synthase Type III, Polymers, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Two-Hybrid System Techniques
J. Neurochem.
Date: Jun. 01, 2006
PubMed ID: 16696853
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