Summary
The aim of this practical is to detect the chemical shift of HINT1 if the ligands interact with the protein. HINT1 gene is associated with schizophrenia and bipolar disorders, and therefore the binding ligand might be a potential drug target for mental disorders. Through the understanding of interaction increases the identification for drug design. Two ligands, GMP and 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) are used in the practical. GMP is a well-known binding ligand to HINT1, and it will be a validation for determination of AICAR phosphate and AICAR. AICAR is known as a nucleoside mimetic and a potential candidate.

The NMR samples for ligand titration contain 0.2 mM 15N labelled, HINT1, 50 mM sodium phosphate, 50 mM NaCl, pH 7.3, in 90% H2O and 10% D2O. The HINT1-ligand reaction occurs by titrating 50 mMsolution for GMP or 20 mMfor AICAR to a 0.2 mM HINT1, respectively. 1H–15N two dimensional (2D) heteronuclear single quantum coherence (HSQC) spectra can be obtained to assess the structural change of HINT1. NMR spectra were recorded at 25℃, and 1H–15N HSQC spectra were recorded on a Bruker 600 using 1024 points and 128 increments. The software programmes named NMRPipe and NMRDraw are responsible for analysis.

The results of saturation-transfer difference (STD) NMR are shown in figure 1. Section A represents the STD data for GMP as well as two other ligands AICAR phosphate (B) and AICAR (C). The presence of the residual NMR signal is observed in the STD experiment, namely the ligands might interact with HINT1. For instance there is a peak at d = 8.18 for GMP. Similar STD results were obtained for the AICAR ligands. The similarity of chemical shifts checks for the binding of these ligands. The AICAR share similar pattern with phosphorylated AICAR, namely the phosphorylation on AICAR results insignificant chemical shift.

Figure 1. STD-NMR results of binding ligands. (Bai et al 2010)
Section A stands for the STD data for GMP, B and C represents AICAR and AICAR phosphate respectively. D is a control with the mixture of GMP and AICAR in the absence of the protein.

HSQC is a two dimensional spectra to detect correlations between nuclei of two different types which are separated by one bond. The backbone resonance of HINT1 is detected by previous study (Bai et al 2009). It is a comparison of the different chemical shift to confirm ligand-binding site on HINT1. In this case, 1H and 15N are used to detect the presence of HINT1-GMP and HINT1-AICAR (Fig 2).

Figure 2. 1H–15N HSQC spectra of HINT1 (Bai et al 2010)
The apo HINT1 (black) and HINT1 interacts with GMP (red) and AICAR (green) is revealed to determine the interaction and binding site.

The pattern of figure 2 represents a typical of beta-sheet like proteins by observing well resolved peaks. Although the peaks appear broader than normal, this pattern is consistent with the protein being a dimer, observed from X-ray data. The chemical shift of GMP is larger than AICAR (more of the buried peaks of GMP become visible) to provide more information about ligand-protein binding. In addition, HNCACB spectra helps to confirm the sequences in figure are correct, and the HSQC chemical shift changes with ligands GMP and AICAR are plotted against the linear sequence of HINT (Fig 3).
Figure 3. The linear sequence of HINT (Bai et al 2010)
The chemical shift perturbation of HINT1-AICAR complex is showed in the A. B is the chemical shift perturbation of HINT1-GMP complex.

The highly similarity of A and B form figure 3 proves that GMP and AICAR might interact with HINT1 at same binding site. Residues 30 and 43–45 (α1, β1 and β2) with large chemical shift perturbations are revealed because the binding pocket where the guanine and sugar residues of GMP are located. The presence of 5’-phosphate being able to affect the chemical shift perturbations of residues 99,106. 109-114 of GMP is observed. Therefore, it can be concluded that the effect of phosphate makes GMP and other derivatives become poor candidates for drug. AICAR and analogues, however, interact with the same binding site of HINT1 without the influence of phosphate may be a better candidates for mental disorder medication.
Reference
Bai, G., Feng, B. Wang, J.B., Pozharski, E., Shapiro, M. (2010) ‘Studies on ligand binding to histidine triad nucleotide binding protein1’ Bioorganic & Medicinal Chemistry. 18, 6756–6762.
Bai, G., Feng, B., Wang, J.B., Varney, K.M., Shapiro, M. (2009) ‘Backbone assignment of HINT1 protein, a mouse histidine triad nucleotide binding protein.’ Biomol NMR Assign 3(1), 57-59.