Key Residues Characteristic of GATA N-fingers Are Recognized By FOG

Archa H Fox,Kasper Kowalski,Glenn F King,Joel P Mackay,Merlin Crossley

doi:10.1074/jbc.273.50.33595

Abstract

Protein-protein interactions play significant roles in the control of gene expression. These interactions often occur between small, discrete domains within different transcription factors. In particular, zinc fingers, usually regarded as DNA-binding domains, are now also known to be involved in mediating contacts between proteins. We have investigated the interaction between the erythroid transcription factor GATA-1 and its partner, the 9 zinc finger protein, FOG (Friend Of GATA). We demonstrate that this interaction represents a genuine finger-finger contact, which is dependent on zinc-coordinating residues within each protein. We map the contact domains to the core of the N-terminal zinc finger of GATA-1 and the 6th zinc finger of FOG. Using a scanning substitution strategy we identify key residues within the GATA-1 N-finger which are required for FOG binding. These residues are conserved in the N-fingers of all GATA proteins known to bind FOG, but are not found in the respective C-fingers. This observation may, therefore, account for the particular specificity of FOG for N-fingers. Interestingly, the key N-finger residues are seen to form a contiguous surface, when mapped onto the structure of the N-finger of GATA-1.

Highlights

Individual eukaryotic transcription factors rarely work alone to activate gene expression
FOG is a zinc finger protein, which was originally isolated in a screen for proteins that could interact with the zinc finger domain of GATA-1 [7]
We sought to determine whether fingers 5 and 7 could bind GATA-1 and to delineate the minimal region of finger 6 required for the interaction

Summary

EXPERIMENTAL PROCEDURES

Metal Binding Studies on FOG-Finger 6 —FOG-finger 6 (residues 694 –723) was obtained as a crude product from a solid-phase synthesis carried out by Chiron Mimotopes (Clayton, Victoria, Australia), and was purified using reversed phase HPLC (rpHPLC) on a Vydac analytical C18 column (5 ␮m), employing linear water/acetonitrile gradients. GST Fusion Protein Binding Assays—The N-finger (residues 200 – 254, 200 –248, 200 –243, 200 –239, 200 –235, 200 –231, and 200 –227), C-finger (residues 249 –318), and mutants of the N-finger of GATA-1 (residues 200 –248) were generated by polymerase chain reaction and subcloned in-frame into the expression vector pGEX2T. The expression of both GST fusion proteins and GST alone was performed using Escherichia coli strain DH5␣ and purification was carried out as described previously [36]. All cell culture data is the result of three separate experiments and has been normalized to LacZ levels derived from a co-transfected ␤-galactosidase-encoding plasmid, EF1␣-LacZ

RESULTS

GST pull downsb ϩ ϩ ϩ ϩ

DISCUSSION