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. 2016 Feb 18;291(17):9119–9132. doi: 10.1074/jbc.M115.699108

FIGURE 6.

FIGURE 6.

The identification of key residues in tTG that are essential for its ability to adopt the closed conformation. A, cell lysates collected from NIH3T3 cells ectopically expressing the indicated forms of Myc-tagged tTG were assayed for their enzymatic transamidation activity (top panel) as well as immunoblotted (IB) with Myc and actin antibodies (bottom two panels). The lines indicate where a portion of the blot was removed. B, the indicated recombinant forms of tTG were expressed, purified, and then resolved by SDS-PAGE. The gels were stained with Coomassie Blue to visualize the proteins. A protein molecular mass (M.W.) marker was included on the gel to show the sizes of the recombinant proteins. C, purified recombinant forms of tTG WT, tTG W254A, and tTG N681A (600 nm) were incubated with BODIPY-GTP (indicated on the graph as BODIPY-GTP Added), and the resulting changes in fluorescence were determined using a spectrofluorimeter. These experiments were performed at least three separate times, each yielding similar results. D, purified recombinant forms of tTG WT, tTG R580K, tTG W254A, and tTG N681A (3.5 μg of each) were incubated without or with trypsin for 2 h before being resolved by SDS-PAGE and then stained with Coomassie Blue to visualize the proteins. Note that only tTG WT was not efficiently digested by trypsin. E, SAXS was performed on purified recombinant tTG W254A. Two monomeric models derived from a substrate-bound tTG WT crystal structure (PDB code 2Q3Z) were fitted into the calculated SAXS envelope for tTG-Short in a head-to-tail fashion. The N-terminal β-sandwich and the catalytic core domains of the fitted models are colored in blue, and the two C-terminal β-barrel domains are colored in red (left panel). A schematic representation shows the superimposition of one of the monomers in the fitted model (blue and red) (right panel). The experimental scattering profiles from SAXS are shown as blue dots, and the scattering profile for the substrate-bound tTG WT structure (PDB code 2Q3Z) is shown as a green line (bottom panel).

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