Repeats-in-Toxins (RTX) protein family is a heterogeneous group of proteins
that are secreted by Gram-negative bacteria. The polypeptides vary in size including molecular weights from about 10 kDa
to larger than 1000 kDa and exhibit a wide range of biological and
biochemical activities. While many of classical RTX proteins function as
toxins/cytolysins involved in bacterial pathogenesis, the others comprise
secreted proteases, lipases, bacteriocins, or play a role in plant nodulation,
motility, adhesion and biofilm formation. Here, we examine the
structure-function aspects of the RTX domain of the Bordetella pertussis
adenylate cyclase toxin (CyaA) and its role in the T1SS-mediated secretion of
the toxin polypeptide. We show that unfolded RTX
repeats keep the stability of CyaA polypeptide in the Ca2+-depleted
bacterial cytosol and demonstrate that secretion levels of CyaA are
proportional to the number of RTX motifs retained in the different CyaA
constructs. Furthermore, we describe the crystal structure of the RTX
block IV-V of CyaA (CyaA1372-1681) containing a continuous assembly
of two parallel β-roll motifs not previously seen in RTX lipases or other RTX proteins.
Our data provide structural insight into the architecture of the RTX
motifs of large RTX proteins and support the push-ratchet mechanism for the
T1SS-mediated secretion of very large RTX proteins1.