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PDBsum entry 3fzy
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References listed in PDB file
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Key reference
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Title
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Structural and molecular mechanism for autoprocessing of martx toxin of vibrio cholerae at multiple sites.
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Authors
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K.Prochazkova,
L.A.Shuvalova,
G.Minasov,
Z.Voburka,
W.F.Anderson,
K.J.Satchell.
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Ref.
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J Biol Chem, 2009,
284,
26557-26568.
[DOI no: ]
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PubMed id
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Abstract
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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.
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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.
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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.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2009,
284,
26557-26568)
copyright 2009.
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