Figure 3.
Fig. 3. β-Flap mutations decouple CPD autocatalysis and RTX
activity from InsP[6] binding. (A) Comparison of autocleavage
efficiency (AC[50]) versus InsP[6] binding (K[d]) measured by
SPR for mutations in the InsP[6]-binding site (left table) and
β-flap (right tables, top and bottom). The β-flap region of
the CPD is rainbow-colored, starting with blue at the N-terminal
end. The β-flap, catalytic site, and visible
InsP[6]-interacting side chains are shown as sticks. Data are
expressed as mean ± SD. ND, not determinable. (B) Western
blot analysis of RTX in supernatant harvested from log-phase V.
cholerae cultures. Supernatants from V. cholerae strains
harboring either an intact rtxA gene (wt), a null mutation in
rtxA ( rtxA), or point
mutations in the region encoding the CPD domain of RTX (C140A is
catalytic-dead; R182Q/K183N is mutated at two InsP[6]-binding
residues; and W192A is a β-flap mutation) were blotted using an
anti-CPD antibody. (C) Actin crosslinking induced upon
incubation of V. cholerae with HFF cells. V. cholerae strains
used in (A) were incubated with HFFs for 90 min, then the HFF
cells were lysed. Actin crosslinking was visualized by SDS-PAGE
and Western blotting by using an actin-specific antibody. The
crosslinked forms of actin are labeled to the right.
The above figure is reprinted
by permission from the AAAs:
Science
(2008,
322,
265-268)
copyright 2008.
rtxA), or point
mutations in the region encoding the CPD domain of RTX (C140A is
catalytic-dead; R182Q/K183N is mutated at two InsP[6]-binding
residues; and W192A is a β-flap mutation) were blotted using an
anti-CPD antibody. (C) Actin crosslinking induced upon
incubation of V. cholerae with HFF cells. V. cholerae strains
used in (A) were incubated with HFFs for 90 min, then the HFF
cells were lysed. Actin crosslinking was visualized by SDS-PAGE
and Western blotting by using an actin-specific antibody. The
crosslinked forms of actin are labeled to the right.