A β‐propeller domain within TolB

We read with interest recent reports that the periplasmic protein TolB interacts with the outer membrane peptidoglycan-associated proteins Pal, Lpp and OmpA, with several porins and with the N-terminal domain of colicin E3 (Bouveret et al., 1997, Mol Microbiol 23: 909–920; Rigal et al., 1997, J Bacteriol 179: 7274–7279; Clavel et al., 1998, Mol Microbiol 29: 359–368). TolB has also received attention recently as it is required for mouse lethal infection by Salmonella typhimurium (Bowe et al., 1998, Infect Immun 66: 3372–3377). Here we present evidence that the C-terminal region of TolB contains six tandem repeats similar to those found in b-propeller structures. This observation leads us to predict the regions of TolB that interact with its ligands. Crystallographic determinations of protein structures have recently revealed several enzymes that contain repeats arranged radially about a central axis, similar in appearance to the blades of a propeller (Murzin, 1992, Proteins 14: 191–201). Each repeat represents a single blade and is folded into a four-stranded antiparallel twisted b-sheet. The number of blades in a propeller can vary: from four in hemopexin-like enzymes (Faber et al., 1995, Structure 3: 551–559; Libson et al., 1995, Nature Struct Biol 2: 938–942), to six in neuraminidase (Varghese et al., 1983, Nature 303: 35–40), to seven in galactose oxidase (Ito et al., 1991, Nature 350: 87–90), methylamine dehydrogenase H chain (Vellieux et al., 1989, EMBO J., 8: 2171–2178) and prolyl oligopeptidase (Fulop et al., 1998, Cell 94: 161–170), and eight in methanol dehydrogenase (Xia et al., 1996, J Mol Biol 259: 480–501) and the C-terminal (haem d1) domain of cytochrome cd1-nitrite reductase (Baker et al., 1997, J Mol Biol 269: 440–455). Not all b-propeller proteins are enzymes. The b-subunit of heterotrimeric G proteins is folded into a seven-bladed propeller (Wall et al., 1995, Cell 83: 1047–1058; Sondek et al., 1996, Nature 379: 369–374) that is used as a scaffold to bind the GDP-bound a-subunit. Homologues of these G b-subunits possess between 4 and 10 repeats that often contain a conserved Trp–Asp (WD) dyad; hence their common description as WD repeats (Neer et al., 1994, Nature 371: 297–300). WD repeats have been identified in dozens of eukaryotic proteins involved in a range of cellular activities (signal transduction, transcription, splicing, cytoskeletal organization and vesicular fusion) (reviewed in Neer et al., 1994, Nature 371: 297–300). Prokaryotic WD repeats have been proposed in a serine/ threonine protein kinase PkwA from Thermomonospora curvata (Janda et al., 1996, J Bacteriol 178: 1487–1489) and in the HatA and HatR proteins from Synechocystis sp. PCC6803 (Bedu et al., 1997, Mol Microbiol 24: 230; Dagnall BH et al., 1997, Mol Microbiol 24: 229–230). Recent annotation of the Synechocystis sp. PCC6803 genome sequence (Kaneko et al., 1996, DNA Res 3: 109–136) suggests that HatA and HatR in fact represent different parts of a single, much larger protein sequence, slr0143 (SWISSPROT ID: Y143_SYNY3). In a search for additional WD repeats in eubacterial proteins, the SpTrembl non-redundant sequence database was queried using the gapped BLAST algorithm (Altschul et al., 1997, Nucleic Acids Res 25: 3389–3402) and the WD repeat region from PkwA (residues 455–742). Two regions of Haemophilus influenzae TolB were aligned with PkwA with significance (E 1⁄4 3 ×10 and 2 ×10). Moreover, the TolB sequence appeared to contain numerous tandem repeats judging by the presence of several AxSPD motifs (‘x’ represents any amino acid). Subsequent multiple alignments revealed six tandem repeats in the C-terminal region of TolB (Fig. 1). These repeats are also apparent in a previously identified TolB homologue, the GA1 antigen from a parasitic nematode (Jasmer et al., 1996, Proc Natl Acad Sci USA 93: 8642–8647) and a related Caenorhabditis elegans protein, C31C9.1. A reciprocal PSI-BLAST (Altschul et al., 1997, Nucleic Acids Res 25: 3389–3402) search using E. coli TolB as query, highlighted the significance of these findings: for example, TolB was aligned with a Bacillus subtilis prolyl oligopeptidase homologue (YuxL), T. curvata PkwA and human transducin G b-subunit with significance by iterations one (E 1⁄4 4 ×10), two (E 1⁄4 2 ×10) and three (E 1⁄4 4 ×10) respectively (Fig. 1). Alignment of TolB repeats with similar sequences in GA1 and PkwA (Fig. 1) required foreknowledge that b-propeller structural repeats need not represent complete sequence repeats because of their circular permutation (Russell and Ponting, 1998, Curr Opin Struct Biol 8: 364–371) (see legend to Fig. 1). We predict from these observations that the C-terminal region of TolB (residues 165–430) contains repeats folded into a b-propeller structure with sixfold pseudosymmetry. This prediction is amenable to investigation by protein crystallography and we note that Abergel et al. (1998, Acta Cryst D54: 102–104) are currently attempting to determine the structure of TolB. Xia et al. (1992, J Biol Chem 267: 22289–22297) and Russell et al. (1998, J Mol Biol, in press), among others, Molecular Microbiology (1999) 31(2), 739–740