PDBsum entry 3eeb

Toxin

PDB id: 3eeb

Contents

Protein chains

205 a.a.

Ligands

IHP ×2

Metals

_NA ×2

Waters ×224

References listed in PDB file

Key reference

Title

Small molecule-Induced allosteric activation of the vibrio cholerae rtx cysteine protease domain.

Authors

P.J.Lupardus, A.Shen, M.Bogyo, K.C.Garcia.

Ref.

Science, 2008, 322, 265-268. [DOI no: 10.1126/science.1162403]

PubMed id

18845756

Abstract

Vibrio cholerae RTX (repeats in toxin) is an actin-disrupting toxin that is autoprocessed by an internal cysteine protease domain (CPD). The RTX CPD is efficiently activated by the eukaryote-specific small molecule inositol hexakisphosphate (InsP6), and we present the 2.1 angstrom structure of the RTX CPD in complex with InsP6. InsP6 binds to a conserved basic cleft that is distant from the protease active site. Biochemical and kinetic analyses of CPD mutants indicate that InsP6 binding induces an allosteric switch that leads to the autoprocessing and intracellular release of toxin-effector domains.

Figure 2.
Fig. 2. The InsP[6]-binding and active sites. (A) Electrostatic surface potential of the CPD as viewed from above the InsP[6]-binding site. Blue denotes a positively charged surface; red denotes a negatively charged surface. InsP[6] is shown in the binding site as a stick model. (B) Close-up view of the InsP[6]-binding site. Side chains that directly interact with InsP[6] are labeled and shown as yellow sticks. The electron density for InsP[6] (2F[obs] – F[calc]) is contoured at 2 . (C) Surface topology of the CPD active site. The P1 substrate pocket, C140, and H91 are highlighted in orange, yellow, and blue, respectively. The N terminus is shown as a yellow ribbon, terminating at Ile5 and highlighting the threading of this region along the surface of the core domain. The remaining residues not visible at the N terminus are depicted as a yellow dashed line to illustrate the approximate positioning of the chain during catalysis. (D) Close-up view of the P1 substrate pocket. Amino acids that line the pocket are labeled and colored orange. InsP[6] is shown as in (B) to demonstrate the position of the catalytic site with respect to the InsP[6]-binding site.

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 figures are reprinted by permission from the AAAs: Science (2008, 322, 265-268) copyright 2008.