Investigations on the substrate binding sites of hemolysin B, an ABC transporter, of a type 1 secretion system

Abstract

The ABC transporter hemolysin B (HlyB) is the key protein of the HlyA secretion system, a paradigm of type 1 secretion systems (T1SS). T1SS catalyze the one-step substrate transport across both membranes of Gram-negative bacteria. The HlyA T1SS is composed of the ABC transporter (HlyB), the membrane fusion protein (HlyD), and the outer membrane protein TolC. HlyA is a member of the RTX (repeats in toxins) family harboring GG repeats that bind Ca²⁺ in the C-terminus upstream of the secretion signal. Beside the GG repeats, the presence of an amphipathic helix (AH) in the C-terminus of HlyA is essential for secretion. Here, we propose that a consensus length between the GG repeats and the AH affects the secretion efficiency of the heterologous RTX secreted by the HlyA T1SS. Our in silico studies along with mutagenesis and biochemical analysis demonstrate that there are two binding pockets in the nucleotide binding domain of HlyB for HlyA. The distances between the domains of HlyB implied to interact with HlyA indicated that simultaneous binding of the substrate to both cytosolic domains of HlyB, the NBD and CLD, is possible and required for efficient substrate secretion.

Keywords: ABC transporter; bacterial secretion systems; protein secretion; putative binding pockets; substrate interaction.

Figure 1

Putative binding pockets (pbp) of HlyB NBD. (A,B) The HlyB NBD monomer (1MT0) is shown in surface representation. The surface was colored with the YRB-script (Hagemans et al., 2015), which highlights carbon atoms that are not bound to oxygen or nitrogen in yellow, the charged oxygens of Glu and Asp in red, the charged nitrogen of Lys and Arg in blue while all other atoms in white. The structural elements of the NBD were labeled. The pbp’s are circled with a black dashed line. From panel A, which shows the pbp-in, to panel B, which shows the pbp-out, the molecules have been rotated toward the reader. (C,D) The residues of pbp-in (C) and pbp-out (D) from the monomeric structure (1MT0) are shown as stick representations. (E,F) Cartoon representation of a model of HlyB based on PCAT1 shown in green (Lin et al., 2015). The identified pbp’s are shown as surface representation with pbp-in in red and pbp-out in orange. The yellow surface maps the HlyA-interaction region on the CLD (Lecher et al., 2012). Pale colors correspond to the same regions in the second monomer. The overall domains of HlyB were labeled.

Figure 2

Secretion of proHlyA through the HlyA T1SS, in which HlyB harbors mutation. (A) SDS-PAGE analysis of the supernatant (unconcentrated) of clones secreting HlyA, in which HlyB is the wild type (control) or contains double mutations in pbp-in. (B) SDS-PAGE analysis of the supernatant (unconcentrated) of clones secreting HlyA, in which HlyB is the wild type (control), triple mutations in the pbp-in, or triple mutations in the pbp-out. Western blotting of E. coli cells (whole cells) demonstrated that (C,E) HlyB and (D,F) HlyD were expressed for all three mutants at levels comparable to the control cells. M, marker proteins; the molecular weight of the marker proteins is given on the left; xh, unconcentrated supernatant of culture, where x denotes the number of hours after induction.

Figure 3

Purification of HlyB NBD mutants. SDS-PAGE of purified HlyB NBD of wild type (1), double-pbp-in (2), and (3) triple-pbp-in (3). M, marker proteins; the molecular weight of the marker proteins is given on the left.

Figure 4

Kinetic measurements of the ATPase activity of HlyB NBD mutants, WT HlyB NBD (circle) and double-pbp-in (triangle). Data are from minimum two independent replicates, error bars were calculated by SEM.

Figure 5

Schematic view of the HlyA and HlyA2. The distance the last C-terminal RTX and the beginning of the AH is 122 residues (122*3.8 Å = 463.6 Å).

Figure 6

Secretion of proHlyA variants through the HlyA T1SS. (A) SDS-PAGE analysis of the supernatant (unconcentrated) of clones secreting wild type proHlyA (control), 30-residues-truncated proHlyA (Δ30 aa), and 50-residues-truncated proHlyA (Δ50 aa) through the HlyA T1SS. (B) Secretion rates of truncated proHlyA. Error bars represent the standard deviation of at least three biological replicates. Western blot analysis of E. coli cells demonstrated that the amounts of (C) HlyB, (D) HlyD or (E) proHlyA were expressed for cells secreting either WT proHlyA, 30-residues-truncated proHlyA, or 50-residues-truncated proHlyA in comparable levels. M, marker proteins; the molecular weight of the marker proteins is given on the left; x h., unconcentrated supernatant of culture, where x denotes the number of hours after induction.

Figure 7

Proposed model of the interaction of HlyA with the NBD of HlyB. Cartoon representation of the trimer of dimers (PDB 7SGR) of the HlyB NBDs (Zhao et al., 2022). The view is from the cytosol toward the membrane. The NBDs of the transport-competent HlyB dimer are colored in orange and light orange, respectively, while the two non-transport competent HlyB dimers are shown in gray. The identified pbp’s are shown as surface representation with pbp-in in red and pbp-out in magenta. The yellow surface maps the HlyA-interaction region of the CLD. The substrate, HlyA, is drawn schematically as a line. The two interacting regions of HlyA are highlighted in green (secretion signal) and red (RTX domain). The arrows emphasis the interaction regions within the NBD of HlyB. Please note that the CLD of NBD 2 was not traced in the single particle cryo EM structure (Zhao et al., 2022). For most efficient secretion, HlyA has to interact simultaneously with both regions of the NBD. In the proposed model, a 1:1 stoichiometry of HlyA and HlyB is assumed. However, one cannot exclude a 2:1 stoichiometry as demonstrated for PCAT1 (Kieuvongngam et al., 2020).