1H, 15N and 13³C resonance assignment of the N-terminal C39 peptidase-like domain of the ABC transporter Haemolysin B (HlyB)

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins, which catalyze the translocation of molecules across biological membranes in an ATP-dependent manner. Despite the diversity in the transported substrates, they all share the same architecture, comprised of two transmembrane (TMD) and two nucleotide-binding domains (NBD). Members of the bacteriocin ABC transporter subfamily feature a special domain, belonging to the C39 (cystein protease family 39) peptidase protein family. These domains are assumed to cleave a C-terminal signal sequence from the protein or peptide substrate before or during the transport process. Although the C39 peptidase-like domain of the ABC transporter haemolysin B from E. coli shows no proteolytic activity, it is essential for the function of this transporter. In order to elucidate the contribution of the isolated C39 peptidase-like domain in the whole transport process, the backbone and side chain (1)H, (15)N and (13)C-NMR chemical shifts have been assigned

An RTX toxin transporters tether ist substrate prior to secretion via the unique function of ist N- terminal domain

Type 1 secretion systems (T1SS) catalyze the one step protein transport across the membranes of Gram-negative bacteria and are composed of an outer membrane protein, a membrane fusion protein and an ABC transporter. The ABC transporter consists of the canonical nucleotide binding and transmembrane domains. For the toxin hemolysin A (HlyA), the ABC transporter HlyB carries an additional, N-terminal domain sharing about 40% homology to C39 peptidases, but this "C39-like domain" (CLD) is suggested to feature another, yet unknown function. Our functional and structural analysis demonstrates that the CLD is essential for secretion and that it specifically interacts with the unfolded state of HlyA. We determined the nuclear magnetic resonance structure of the CLD as well as the substrate-binding region within the CLD. This mode of action, represents a mechanism within T1SS and answers the question, how a large and unfolded substrate is protected inside the cells during secretion

Crystallisation and preliminary X-ray crystallographic studies of an oligomeric species of a refolded C39 peptidase-like domain of the Escherichia coli ABC transporter Haemolysin B

The ABC transporter haemolysin B (HlyB) from Escherichia coli is part of a type I secretion system that translocates a 110 kDa toxin in one step across both membranes of this Gram-negative bacterium in an ATP-dependent manner. Sequence analysis indicates that HlyB contains a C39 peptidase-like domain at its N-terminus. C39 domains are thiol-dependent peptidases that cleave their substrates after a GG motif. Interestingly, the catalytically invariant cysteine is replaced by a tyrosine in the C39-like domain of HlyB. Here, the overexpression, purification and crystallization of the isolated C39-like domain are described as a first step towards obtaining structural insights into this domain and eventually answering the question concerning the function of a degenerated C39 domain in the ABC transporter HlyB