Fig. 3: INTS10 forms a module with INTS13–INTS14 in cells and binds to the MIDAS pocket of the INTS14 VWA domain. | Nature Communications

Fig. 3: INTS10 forms a module with INTS13–INTS14 in cells and binds to the MIDAS pocket of the INTS14 VWA domain.

From: INTS10–INTS13–INTS14 form a functional module of Integrator that binds nucleic acids and the cleavage module

Fig. 3: INTS10 forms a module with INTS13–INTS14 in cells and binds to the MIDAS pocket of the INTS14 VWA domain.

a Size exclusion chromatography of nuclear extract from HEK293T cells. All even fractions were analyzed by Western blotting. INTS1 and INTS7 served as marker proteins for the INT core, while INTS4–9–11 form the cleavage module. Elution points of molecular weight markers are indicated on the top. In addition, elution volumes of purified INTS13–INTS14 and INTS10–INTS13–INTS14 are indicated. b, c Coprecipitation of HA-INTS10 from HEK293T cells with V5-SBP-INTS13 b or V5-SBP-INTS14 c. Wild-type and binding patch double mutants were compared. Coprecipitation of endogenous INTS14 or INTS13 is detected as control for complex disruption. Inputs (αV5-blots 1%, αHA-blots 0.38% and 0.75%, αINTS14-blot 0.38%, αINTS13-blot 0.03%) and bound fractions (αV5-blots 3%, αHA-blots 20 and 15%, αINTS14/αINTS13-blots 20%) were analyzed by Western blotting. d Deletion constructs of V5-SBP-INTS14 were tested for copurification of HA-INTS10 and endogenous INTS13. Inputs (αV5-blot 1%, αHA-blot 1%, αINTS13-blot 0.1%) and bound fractions (αV5-blot 4%, αHA-blot 5%, αINTS13-blot 20%) were characterized by Western blotting. e Copurification of MBP-INTS10 with GST-INTS14-VWA from E. coli lysates. GST served as negative control. f Structure superposition (r.m.s.d. 3.6 Å over 160 residues) of the VWA domain of INTS14 (green) with the VWA domain of integrin-αL (ITGAL, gray, PDB-ID: 1T0P73) in complex with intercellular adhesion molecule 3 (ICAM3, purple). The black square highlights the position of the MIDAS pocket, residues lining the pocket are shown as sticks. g Focused view of the MIDAS pockets of both VWA domains highlights conservation of the site in INTS14. The Mg2+-ion in the ITGAL pocket that coordinates a Glu of the interaction partner ICAM3 is shown as a gray sphere. Ion coordination is indicated by black dotted lines. Coloring as in f. Residues mutated in INTS14 are underlined. h Coprecipitation of HA-INTS10 from HEK293T cells with V5-SBP-INTS14 wt or MIDAS pocket mutants (D8A, S10A, S12A [DA2SA] and L11E, R15A [LERA]). V5-SBP-MBP served as control. Inputs (αV5-blots 1%, αHA-blots 0.75%) and bound fractions (αV5-blots 3%, αHA-blots 15%) were analyzed by Western blotting.

Back to article page