REVIEW: Studies on ligand binding to histidine triad nucleotide binding protein 1
Guoyun Bai, Bo Feng, Jia Bei Wang, Edwin Pozharski, Michael Shapiro *

Introduction

This review provides a summary of the NMR studies conducted by G Bai et al on the ligand binding interactions of the histidine triad nucleotide binding protein 1 (HINT1). As its name suggest the HINT1 has a conserved sequence of three polar histidine amino acids which create a binding site for an α-phosphate. Although the exact function of HINT1 remains elusive the α-phosphate binding site suggests it can bind to purine nucleotides. In vitro studies showed that HINT1 also bind to purine nucleosides which are absent of the phosphate group. Bai et al aimed to use NMR to identify chemicals compounds that bind to HINT1 and therefore allow for HINT1 structure based drug design.
Method
Previous NMR and protein crystallography studies recognised two ligands that bound to HINT1 to be GMP and 5-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR). Bai et al 2009 have previously reported the backbone assignments of HINT1 using isotopically labelled (C13 N15) and expressed and purified the labelled HINT1 protein.
Saturation transfer difference (STD)-NMR spectroscopy was used to check for the binding of the different ligands to isotopically unlabelled HINT1. GMP is known as a HINT1 nucleotide ligand was used to validate the binding of AICAR and AICAR phosphate. AICAR is a known nucleoside mimetic and X-ray crystallography results suggest that it can bind to HINT1. An off resonance ligand reference spectrum was registered at -10ppm. A second experiment was then recorded in which the HINT1 was selectively saturated at 0.8ppm. Gaussian shaped pulses showed the regions of saturation from the protein resonances for a specific 3s period. The intensity of the selective Gauss pulses corresponds to a field