Structural basis of the activation of MARTX cysteine protease domain from Vibrio vulnificus

Abstract

The multifunctional autoprocessing repeat-in-toxin (MARTX) toxin is the primary virulence factor of Vibrio vulnificus displaying cytotoxic and hemolytic properties. The cysteine protease domain (CPD) is responsible for activating the MARTX toxin by cleaving the toxin precursor and releasing the mature toxin fragments. To investigate the structural determinants for inositol hexakisphosphate (InsP6)-mediated activation of the CPD, we determined the crystal structures of unprocessed and β-flap truncated MARTX CPDs of Vibrio vulnificus strain MO6-24/O in complex with InsP6 at 1.3 and 2.2Å resolution, respectively. The CPD displays a conserved domain with a central seven-stranded β-sheet flanked by three α-helices. The scissile bond Leu3587-Ala3588 is bound in the catalytic site of the InsP6-loaded form of the Cys3727Ala mutant. InsP6 interacts with the conserved basic cleft and the β-flap inducing the active conformation of catalytic residues. The β-flap of the post-CPD is flexible in the InsP6-unbound state. The structure of the CPD Δβ-flap showed an inactive conformation of the catalytic residues due to the absence of interaction between the active site and the β-flap. This study confirms the InsP6-mediated activation of the MARTX CPDs in which InsP6-binding induces conformational changes of the catalytic residues and the β-flap that holds the N terminus of the CPD in the active site, facilitating hydrolysis of the scissile bond.

Fig 1. The overall structure of the V. vulnificus MARTX CPD.

(A) Schematic representation of the domain structures of the MARTX toxin of Vibrio vulnificus strain MO6-24/O. The MARTX toxin contains a CPD (residue 3578–3796) and four effector domains including DUF1, RID, ABH, and MCF. (B) The purified CPD wild type (residue 3570–3796) shows autocleavage activity upon the addition of 0.5 mM InsP₆. The active site mutant C3727A has no autocleavage activity. To examine the effect of InsP₆ concentration on autocleavage, 20 μM of CPD was incubated with a series of InsP₆ concentrations ranging from 1 μM to 200 μM for one hour. (C) The 2Fo-Fc electron density maps of the pre-CPD C3727A crystal with the final models superimposed. The three panels of density maps show the InsP₆-binding site, N3686 of the β5-β6 loop, and the N-terminal five residues, respectively. (D) The overall structure of the pre-CPD C3727A bound with InsP₆. The N-terminal leader, protease core, and β-flap are colored blue, green, and magenta, respectively, according to the annotation of Prochazkova et al [12]. The scissile residue, Leu3587 is shown as sticks with transparent spheres. (E) The N-terminal autocleavage sequence in the substrate-binding cleft. Leu3587 and the catalytic residues, His3678 and Cys3727Ala are shown with dotted sticks. Hydrogen bonds are shown in dotted lines.

Fig 2. Structural comparison of the MARTX CPDs from different Vibrio strains.

(A) Multiple sequence alignment of cysteine protease domains from MARTX toxins of various Vibrio strains including V. vulnificus MO6-24/O (WP_015728045.1), V. cholerae N16961 (AAD21057.1), V. vulnificus FORC_009 (WP_060534095.1), and V. vulnificus BAA87 (WP_039507922.1). The autocleavage sites in the N-termini of CPDs were indicated by yellow shades with red arrows. The dotted lines indicate the residues invisible in the electron density maps. (B) Structural comparison of V. vulnificus pre-CPD C3727A (this study) and V. cholerae pre-CPD (PDB code: 3FZY) [12]. The catalytic residues (Cys3727 and His3678) and the scissile residue (Leu3587) were shown for each CPD with stick models. (C) Structural comparison of the N-termini of V. vulnificus pre-CPD C3727A (this study) and the recombinant V. cholerae pre-CPD (PDB code: 3FZY). (D) The surface representation of the N-terminal autocleavage site. The N-terminal cleavage sequence is shown in green stick models. The active site residues, Cys3727Ala and His3678, are colored yellow and cyan, respectively.

Fig 3. InsP₆-binding to the basic pocket of the pre-CPD.

(A) The InsP₆-binding to the pre-CPD C3727A. The electrostatic interactions between InsP₆ and the basic residues of the CPD are shown in dotted lines. The N-terminal cleavage sequence is colored in pale blue. The scissile residue Leu3587 is shown by dot representation. (B) Electrostatic surface representation of the basic InsP₆-binding site of pre-CPD C3727A. The active conformation of the β-flap induced by InsP₆-binding. The dotted lines indicate the hydrogen-bond networks of β-flap with the substrate, β7- α3 loop, and InsP₆. (C) Measurement of binding affinities of InsP₆ to the pre-CPD C3727A (3570–3796) and CPD ΔNt wt (3592–3796) by ITC. The CPDs of 0.1 mM were titrated with 1 mM of InsP₆. The removal of the β-flap abolishes InsP₆-binding and autocleavage activities.

Fig 4. The structure of the CPD Δβ-flap in inactive conformation.

(A) SDS-PAGE analysis of the crystallized CPD constructs. Lane 1, molecular weight marker; Lane 2, the purified CPD (residue 3592–3796) incubated with InsP₆ was used for crystallization; Lane 3, the purified CPD protein (residue 3592–3796); Lane 4, purified pre-CPD C3727A (3578–3796). The purified proteins were incubated at room temperature for 48 h before SDS-PAGE analysis. The lane 1 and the other lanes were cut from the identical gel. (B) The 2Fo-Fc electron density maps of the InsP₆ binding site in the post-CPD structure. (C) The structure of a dimeric CPD lacking the β-flap. InsP₆ binds to the CPD in a 1:2 ratio. One of the protomers shown in gray is related by a noncrystallographic 2-fold axis. The disordered N-terminal loop (3611–3613) is shown in dotted lines. (D) Structural comparison of the InsP₆-binding modes in the post-CPD Δβ-flap dimer. The identical InsP₆ molecules bound to each promotor are overlayed to show the orientation of InsP₆ to each protomer. The ionic interactions of InsP₆ with the four basic residues were indicated by yellow dotted lines. (E) Structural comparison of the InsP₆-binding to pre-CPD C3727A and the post-CPD wt Δβ-flap. (F) Structural comparison of pre-CPD C3727A and the proteolyzed CPD Δβ-flap. The equivalent catalytic residues, Cys3727, are indicated by red arrows. (G) Structure of the post-CPD Δβ-flap with the inactive conformation. The catalytic dyad residues, His3678 and Cys3727, are shown in dotted stick models.