Figure 4
Structural basis of hTIM-3 binding with Ca++. (a) Overlaid 15N-HSQC spectra showing chemical shift changes of hTIM3 backbone amides peaks induced by Ca++ binding, from 0 mM (red), 2 mM (orange), 5 mM (yellow), 10 mM (green), 20 mM (cyan), 50 mM (blue) to 100 mM (purple) concentrations. Selected residues of hTIM-3 GFCC′ face (Ile114 to Lys122) that show significant chemical shift changes are labeled. Some of peaks of these residues above 10 mM Ca++ concentration are not clear, possibly due to dynamic conformation exchange in solution. (b) 15N-HSQC peak shift monitoring of the hTIM-3 C-C′ and F-G loop aromatic residues Phe61 (F61) and Asn119 (N119) upon binding with 0–100 mM Ca++. (c) Determination of Ca++ binding constant (Kd) of 27.2 mM for hTIM-3 based on peak shifts of residue Phe61 determined by non-linear regression of the chemical shift change index relative to the Ca++concentration. (d) Interactions of hTIM-3 F-G loop residues with bound Ca++ (red sphere) as observed in the crystal structure. Ile114, Gly116, Asn119, and Asp120 residues of hTIM-3 are highlighted in stick representation and these residues make equal distance coordination (2.3 Å) with Ca++. Residues Pro115 and Ile117 of hTIM-3 FG loop are also shown in a stick representation. (e) 2Fo-Fc map at 1.0 σ level of hTIM-3 residues Ile114, Pro115, Gly116, Asn119, and Asp120 with bound Ca++. Map is superimposed on the final refined model.
