Figure 5. Comparison of substrate accessibility for DMT protein structures, and characterization of mCST’s intrinsic tryptophan fluorescence.
(A) The surface representation of the mCST-CMP-Sia structure from Figure 2D is reproduced. The front half of the protein that was removed to make the sliced view is put back on top of the plane of the slice with the alpha-helical TMs represented as cylinders. The TMs are numbered. (B and C) The structures of Vrg4 (B, green), and YddG (C, cyan) are superimposed with the mCST-CMP-Sia structure (orange). These side view orientations are very similar to what is shown in (A) and identical to the view in Figure 2A and B. In (A–C), the top and bottom are the lumenal and cytosolic sides, respectively. (D–F) Top-down views from the lumenal side of the Golgi membrane are shown for the mCST-CMP-Sia structure (D, orange, Vrg4 (E, green), and YddG (F, cyan). This orientation is the same as shown in Figure 2C and F. The CMP-Sia binding site is highlighted in panel (D) and the equivalent substrate-binding sites are indicated in panels (E) and (F). In panels (B–F), all TMs are also represented as cylinders and TMs are numbered. (G) Quenching of the intrinsic tryptophan fluorescence of wild-type and mutant mCST is shown as a function of CMP concentration. The lack of any quenching for either Trp207 mutant indicates that it is sensing the CMP-induced conformational changes. (H) Left axis: a SPA assay was used to determine the amount of CMP bound to wild-type and mutant mCST to give an indication as to the relative CMP affinity for the tryptophan mutants compared to wild-type mCST. The black bars represent binding in the presence of 30 nM [3H]CMP whereas the gray bars represent binding with an additional 5 µM unlabeled CMP added. CMP affinities, which were all very similar, were estimated by fitting the data to a one-site model and are listed in Table 1. These results show that the Trp207 mutants still bind CMP. Right axis: Tryptophan fluorescence in the absence of any ligand is shown for wild-type and mutant mCST. These results show that the initial, ligand-free fluorescence of the Trp207 mutants is not significantly different from wild-type. The symbols and bars in (G) and (H) represent the mean ± SEM for n = 2. (I) The location and local environment of Trp207 of mCST is shown (orange) and compared to the structure of Vrg4 (green). In (B and C) the location of Trp207 is also indicated with the ‘W’ label.