The crystal structure of filamentous hemagglutinin secretion domain and its implications for the two-partner secretion pathway

Abstract

Filamentous hemagglutinin (FHA), the major 230-kDa adhesin of the whooping cough agent Bordetella pertussis, is one of the most efficiently secreted proteins in Gram-negative bacteria. FHA is secreted by means of the two-partner secretion (TPS) pathway. Several important human, animal, and plant pathogens also secrete adhesins and other virulence factors by using this mode of secretion. A TPS system is composed of two separate proteins, with TpsA the secreted protein and TpsB its associated specific outermembrane transporter. All TPS-secreted proteins contain a distinctive N-proximal module essential for secretion, the TPS domain. We report here the 1.7- A structure of a functionally secreted 30-kDa N-terminal fragment of FHA. It reveals that the TPS domain folds into a beta-helix, with three extrahelical motifs, a beta-hairpin, a four-stranded beta-sheet, and an N-terminal capping, mostly formed by the nonconserved regions of the TPS domain. The structure thus explains why the TPS domain is able to initiate folding of the beta-helical motifs that form the central domain of the adhesin, because it is itself a beta-helical scaffold. It also contains less conserved extrahelical regions most likely involved in specific properties, such as the recognition of the outer-membrane transporter. This structure is representative of the TPS domains found so far in >100 secreted proteins from pathogenic bacteria. It also provides a mechanistic insight into how protein folding may be linked to secretion in the TPS pathway.

Fig. 1.

Ribbon representation of the overall structure of Fha30. The helix β-sheets PB1, PB2, and PB3, the β-hairpin β7/β8, and the β-sheet β16/β25 are shown. The three β-strands, β1, β2, and β3, involved in the N-terminal β-helix capping are represented in red. N- and C-terminal extremities are also indicated.

Fig. 2.

Diagram illustrating the topology of Fha30. PB1, PB2, PB3, and extra β-helical motifs are represented.

Fig. 3.

Ball-and-stick view of the coil formed by β-strands β31, β32, and β33 that belong to PB1, PB2, and PB3, respectively. This coil corresponds to the first R1 repeat and displays an isosceles-triangular-shaped section. The hydrogen bonds between the coil's main chain and the side chain of Ser-257 are illustrated.

Fig. 4.

Ribbon representation of the N-terminal capping that shields the β-helix hydrophobic interior from the solvent. The helix β-sheets PB1, PB2, and PB3, the β-hairpin β7/β8, and the β-sheet β16/β25 are shown. The three β-strands β1, β2, and β3 involved in the capping are represented in red.

Fig. 5.

Sequence alignment of representative TpsA proteins. Secondary structure elements observed in the Fha30 structure are indicated. The conserved residues are boxed in black (cut off at 60% conservation), and the homologous residues are boxed in gray. The conserved regions C1 and C2 are delimited.

Fig. 6.

Ribbon view of Fha30 illustrating the LC1, C1, LC2, and C2 regions of the TPS domain. The R1 repeats that belong to the central right-handed β-helix domain of the full-length adhesin are also shown.