A yeast 2‐hybrid analysis of human GTP cyclohydrolase I protein interactions

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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 α‐helices are not the only domains involved in the formation of dimers from monomers and also suggest an important role for the C‐terminal α‐helix in the assembly of dimers to form decamers. Moreover, a previously unknown role of the extended N‐terminal α–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.