Fig 1.
HPLC characterization of the molecule that co-purifies with CST.
A) The aqueous layer of a phenol-chloroform extraction of purified CST was run on a C18 HPLC column (blue trace). The peak representing the unknown molecule is labeled with an ‘*’ and the broad peak representing residual detergent left in the extract is also labeled. A separate sample containing 50 μM each of CMP, UMP, and CMP-Sia was also run (purple trace). Peaks corresponding to each standard are labeled. B) A sample of a phenol-chloroform extract of CST with an extra 2.5 μM CMP added was run on a HPLC C18 column. Absorbance at 274 nm (blue trace) and 254 nm (red trace) was monitored. The ratio of the absorbance at these two wavelengths for a given time is plotted as the open circles on the right y-axis. The black horizontal lines represent the average A274/A254 ratio for the two peaks. C) A sample of a phenol-chloroform extract of CST was run on a C18 HPLC column after it had been incubated for 24 hr at 4°C either with (green trace) or without AnP (blue trace). D) A phenol-chloroform extraction was performed on a CST sample immediately after purification (blue trace), after it had undergone 24 hr dialysis (purple trace), or after it had undergone 24 hr dialysis in the presence of AnP (orange trace). The extracts were then run on C18 HPLC column. The traces in panels C and D are arbitrarily separated by either 0.001 (panel C) or 0.002 (panel D) units along the y-axis to improve clarity.
Fig 2.
Comparison of mass spectra for buffer-only versus CST protein-containing samples.
A) Extracted ion chromatograms for an m/z of 338.0701 ± 0.5 were taken from the LC-MS runs in positive ESI mode (shown in Fig 2 –S1 Fig) and are shown for the buffer-only control (top) or the CST protein-containing sample (bottom). B-E) Mass spectra in either positive (panels B, C, and E) or negative (panel D) ESI modes are shown. Panels B-D show the spectra for the sample eluting from the HPLC column from between 10.7–10.8 min for the buffer-only control (B) or protein-containing samples (C and D). Panel E shows the spectrum for the buffer-only sample eluting from the HPLC column between 9.6–9.8 min. F) Tandem MS/MS was performed on the ion with m/z of 338.0722 from panel C, with a collision energy of 10 eV. A selection filter of 1.3 m/z was used. The black diamond indicates the position of the precursor ion. G) A putative scheme describes the fragmentation of m5CMP ([M+H]+ = 338.0748) to produce the most prominent ion (m/z of 126.0686) in the MS/MS spectrum shown in panel F.
Fig 3.
Comparison of m5CMP with the molecule that co-purifies with CST.
A) The aqueous layer of a phenol-chloroform extraction of purified CST was run on a C18 HPLC column (blue trace). A separate sample containing 5 μM each of CMP and m5CMP was also run (purple trace). B) For the sample containing CMP and m5CMP, shown in panel A, the absorbance was monitored at both 274 nm (blue trace) and 254 nm (red trace) which is shown here. The ratio of the absorbance at these two wavelengths for a given time is plotted as the open circles on the right y-axis. The black horizontal lines represent the average A274/A254 ratio for the two peaks.
Fig 4.
Binding constants and CMP-Sia transport inhibition for m5CMP and CMP.
A) Aliquots of DDM-solubilized GFP-tagged CST protein were either kept at 4°C (black trace) or heated to 41°C, either alone (red trace) or in the presence of the indicated concentration of m5CMP for 15 min. The samples were clarified and then run on a gel filtration column with fluorescence detection. The sample eluting at ~5.4 min represents natively-folded protein. B) The peak heights from the traces in panel A were normalized to the highest (4°C with no ligand added) and lowest (41°C with no ligand added) peak height and are plotted against ligand concentration to determine binding constants. The plotted values represent the mean ± SEM, n = 2. C) Intact Sf9 insect cells expressing CST (diamonds) or uninfected cells (triangles) were incubated with 30nM [3H]CMP-Sia for the indicated amount of time at room temperature. The plotted values represent the mean ± SEM, n = 2. D) Intact Sf9 insect cells expressing CST (diamonds) were incubated with 30 nM [3H]CMP-Sia and the indicated concentration of inhibitor for 5 min at room temperature. The initial rate of uptake is plotted against inhibitor concentration in order to determine inhibition constants. The plotted values represent the mean ± SEM, n = 4.
Fig 5.
Comparison of the m5CMP and CMP binding sites.
A) A close-up of the substrate binding pocket is shown for both the CST-m5CMP (green) and CST-CMP (blue) structures. Only substrate-interacting side chains are shown and portions of TM8 are hidden for clarity. B and C) A slice through the surface representation of the substrate-binding cavity is shown for the CST-m5CMP (B) and CST-CMP (C) structures. The atoms for the pyrimidine ring are numbered. A black line is added as a visual aide to indicate where the cavity volume was sliced. The view is similar to that shown in panel A but is adjusted slightly to better show the part of the substrate-binding cavity that interacts with the C-5 methyl.