Structure-Function Relationships of the Repeat Domains of RTX Toxins

Abstract

RTX proteins are a large family of polypeptides of mainly Gram-negative origin that are secreted into the extracellular medium by a type I secretion system featuring a non-cleavable C-terminal secretion signal, which is preceded by a variable number of nine-residue tandem repeats. The three-dimensional structure forms a parallel β-roll, where β-strands of two parallel sheets are connected by calcium-binding linkers in such a way that a right-handed spiral is built. The Ca²⁺ ions are an integral part of the structure, which cannot form without them. The structural determinants of this unique architecture will be reviewed with its conservations and variations together with the implication for secretion and folding of these proteins. The general purpose of the RTX domains appears to act as an internal chaperone that keeps the polypeptide unfolded in the calcium-deprived cytosol and triggers folding in the calcium-rich extracellular medium. A rather recent addition to the structural biology of the RTX toxin is a variant occurring in a large RTX adhesin, where this non-canonical β-roll binds to ice and diatoms.

Keywords: RTX toxin; calcium; internal chaperone; protein folding; tertiary structure; type I secretion.

Figure 1

(A) Cartoon of AprA (PDB entry 1KAP). Ca²⁺ ions are shown as golden spheres. The N-terminal helix and the protease domain are depicted in grey with the active-site Zn²⁺ as cyan sphere together with the residues mentioned in the text. The β-roll is depicted in crimson and purple with green calcium-binding loops. The intermediate/N-cap and C-cap subdomains are depicted in different shades of light blue. (B) Close-up of the parallel β-roll. (C) Side-view of one turn of the parallel β-roll depicting 2 tandem nonapeptides of sequence XLXGGXGXD with X being variable amino acids. (D) Details of the octahedral Ca²⁺ coordination by the GGXGXD turn. The left Ca²⁺ ion is the most accessible one and the only that can be exchanged by Pb²⁺, for example. Its coordination sphere is completed by water molecules depicted as small red spheres. All figures were prepared with ChimeraX [42].

Figure 2

Two views on LipA rotated by about 180 deg. around the vertical (PDB entry 2QUA). (A): The color coding is as in Figure 1. This time, there are two C-caps, one for each RTX β-roll. Water molecules occupying one edge of the second roll are shown as red spheres. Roll 2 has only at one side calcium ions bound. (B): Same as in A but rotated by about 180 degrees around the vertical. The interaction between the two β-roll domains is clearly visible.

Figure 3

(A) the parallel β-roll of the MpIBP is colored as rainbow from N-terminus (blue) to C-terminus (red). Oxygen atoms are painted in red, Ca²⁺ and Mg²⁺ ions are shown as golden and green spheres, respectively. Waters are shown as small red spheres. (B) Side-view of the roll depicting the motif XGTGND-XUXU-GGXUXG-XUX where U stands for a non-polar and X for a polar amino acid. Carbon atoms are shown in grey, nitrogen and oxygen in blue and red, respectively, and Ca²⁺ ions as golden spheres.