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

J Neurochem. 2006 Jun;97(5):1447-55. doi: 10.1111/j.1471-4159.2006.03836.x.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence / physiology
  • Binding Sites / physiology
  • Biopterins / analogs & derivatives
  • Biopterins / 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

Substances

  • AHA1 protein, S cerevisiae
  • GCHFR protein, human
  • HSP90 Heat-Shock Proteins
  • Intracellular Signaling Peptides and Proteins
  • Polymers
  • Saccharomyces cerevisiae Proteins
  • Biopterins
  • Nitric Oxide
  • Guanosine Triphosphate
  • Nitric Oxide Synthase Type III
  • GTP Cyclohydrolase
  • Chaperonins
  • sapropterin