PDBsum entry 3fzy

Toxin

PDB id: 3fzy

Contents

Protein chains

216 a.a. *

Ligands

IHP ×2

UNX-UNX ×2

UNX ×8

Metals

_CL

Waters ×321

* Residue conservation analysis

PDB id:

3fzy

Name:

Toxin

Title:

Crystal structure of pre-cleavage form of cysteine protease domain from vibrio cholerae rtxa toxin

Structure:

Rtx toxin rtxa. Chain: a, b. Fragment: sequence database residues 3440-3650. Engineered: yes. Mutation: yes

Source:

Vibrio cholerae. Organism_taxid: 666. Strain: n16961. Gene: rtxa, vc_1451. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.95Å R-factor: 0.171 R-free: 0.216

Authors:

L.Shuvalova,G.Minasov,K.Prochazkova,K.J.F.Satchell,W.F.Anderson, Center For Structural Genomics Of Infectious Diseases (Csgid)

Key ref:

K.Prochazkova et al. (2009). Structural and molecular mechanism for autoprocessing of MARTX toxin of Vibrio cholerae at multiple sites. J Biol Chem, 284, 26557-26568. PubMed id: 19620709 DOI: 10.1074/jbc.M109.025510

Date:

26-Jan-09 Release date: 17-Feb-09

Protein chains

Q9KS12  (MARTX_VIBCH) -  Multifunctional-autoprocessing repeats-in-toxin from Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)

Seq: 4558 a.a.

Struc: 216 a.a.*

* PDB and UniProt seqs differ at 9 residue positions (black crosses)

Enzyme reactions

• Enzyme class 2: E.C.2.3.1.- - ?????
• Enzyme class 3: E.C.3.4.22.- - ?????
• Enzyme class 4: E.C.6.3.2.- - ?????

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

DOI no: 10.1074/jbc.M109.025510

J Biol Chem 284:26557-26568 (2009)

PubMed id: 19620709

Structural and molecular mechanism for autoprocessing of MARTX toxin of Vibrio cholerae at multiple sites.

K.Prochazkova, L.A.Shuvalova, G.Minasov, Z.Voburka, W.F.Anderson, K.J.Satchell.

ABSTRACT

The multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of Vibrio cholerae causes destruction of the actin cytoskeleton by covalent cross-linking of actin and inactivation of Rho GTPases. The effector domains responsible for these activities are here shown to be independent proteins released from the large toxin by autoproteolysis catalyzed by an embedded cysteine protease domain (CPD). The CPD is activated upon binding inositol hexakisphosphate (InsP(6)). In this study, we demonstrated that InsP(6) is not simply an allosteric cofactor, but rather binding of InsP(6) stabilized the CPD structure, facilitating formation of the enzyme-substrate complex. The 1.95-A crystal structure of this InsP(6)-bound unprocessed form of CPD was determined and revealed the scissile bond Leu(3428)-Ala(3429) captured in the catalytic site. Upon processing at this site, CPD was converted to a form with 500-fold reduced affinity for InsP(6), but was reactivated for high affinity binding of InsP(6) by cooperative binding of both a new substrate and InsP(6). Reactivation of CPD allowed cleavage of the MARTX toxin at other sites, specifically at leucine residues between the effector domains. Processed CPD also cleaved other proteins in trans, including the leucine-rich protein YopM, demonstrating that it is a promiscuous leucine-specific protease.

Selected figure(s)

Figure 1.
Structural model of pro-CPD/C-S reveals enzyme-substrate complex.A, pro-CPD/C-S with N terminus (blue), protease core (green), β-flap (magenta), and InsP[6] (red). Key residues (orange) and catalytic residues (yellow) are labeled according to annotation of Lin et al. (24). B, schematic representation of the Clan CD fold catalytic site with P1 Leu^3428 (magenta) inserted into S1 site. Distances (in angstroms) of key bonds are shown as dashed lines. C, stereo view of the active site of pro-CPD/C-S as a stick model with surrounding 2F[o] − F[c] map contoured at 1 sigma (green) and the N terminus residues, surrounded with omit F[o] − F[c] map contoured at 4 sigma level (blue; omitted residues are Ala-Leu-Ala). For B and C, carbon of the active site, carbon of the substrate, oxygen and nitrogen atoms are colored in green, yellow, red, and blue, respectively.

Figure 3.
Binding of InsP[6] increases pro-CPD/C-S T[m], and the protein becomes trypsin-resistant.A, close-up view of InsP[6] binding pocket shows 12 residues known to contact InsP[6] (red with space-filling dots) derive from the N terminus (blue), the protease core (green), and the β-flap (magenta). B, SYPRO® Orange melting curves of pro-CPD/C-S at different concentrations of InsP[6]. C, Coomassie-stained gel of limited proteolysis of pro-CPD/C-S at varying concentrations of trypsin. Locations of trypsin cleavage in the absence of InsP[6] as determine by FT-MS (supplemental Fig. 4) are shown in orange in D with color scheme used in A except antiparallel β8β9 are highlighted pink and S1 hydrophobic residues are space-filling dots.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2009, 284, 26557-26568) copyright 2009.

Figures were selected by an automated process.