Interdomain regulation of the ATPase activity of the ABC transporter haemolysin B from Escherichia coli

• Published in: Biochemical journal Vol. 473; no. 16; p. 2471
• Main Authors: Reimann, Sven, Poschmann, Gereon, Kanonenberg, Kerstin, Stühler, Kai, Smits, Sander H J, Schmitt, Lutz
• Format: Journal Article
• Language: English
• Published: England 15.08.2016
• Subjects: Adenosine Triphosphatases - metabolism; Amino Acid Sequence; ATP-Binding Cassette Transporters - chemistry; ATP-Binding Cassette Transporters - genetics; ATP-Binding Cassette Transporters - isolation & purification; ATP-Binding Cassette Transporters - metabolism; Chromatography, Liquid; Cloning, Molecular; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - isolation & purification; Escherichia coli Proteins - metabolism; Hemolysin Proteins - chemistry; Hemolysin Proteins - genetics; Hemolysin Proteins - isolation & purification; Hemolysin Proteins - metabolism; Mass Spectrometry; Protein Folding; ATPase inhibition; type I secretion system; RTX; haemolysin; ABC transporter; enzyme kinetics; protein translocation
• ISSN: 1470-8728
• DOI: 10.1042/bcj20160154

Type 1 secretion systems (T1SS) transport a wide range of substrates across both membranes of Gram-negative bacteria and are composed of an outer membrane protein, a membrane fusion protein and an ABC (ATP-binding cassette) transporter. The ABC transporter HlyB (haemolysin B) is part of a T1SS catalysing the export of the toxin HlyA in E. coli HlyB consists of the canonical transmembrane and nucleotide-binding domains. Additionally, HlyB contains an N-terminal CLD (C39-peptidase-like domain) that interacts with the transport substrate, but its functional relevance is still not precisely defined. In the present paper, we describe the purification and biochemical characterization of detergent-solubilized HlyB in the presence of its transport substrate. Our results exhibit a positive co-operativity in ATP hydrolysis. We characterized further the influence of the CLD on kinetic parameters by using an HlyB variant lacking the CLD (HlyB∆CLD). The biochemical parameters of HlyB∆CLD revealed an increased basal maximum velocity but no change in substrate-binding affinity in comparison with full-length HlyB. We also assigned a distinct interaction of the CLD and a transport substrate (HlyA1), leading to an inhibition of HlyB hydrolytic activity at low HlyA1 concentrations. At higher HlyA1 concentrations, we observed a stimulation of the hydrolytic activities of both HlyB and HlyB∆CLD, which was completely independent of the interaction of HlyA1 with the CLD. Notably, all observed effects on ATPase activity, which were also analysed in detail by mass spectrometry, were independent of the HlyA1 secretion signal. These results assign an interdomain regulatory role for the CLD modulating the hydrolytic activity of HlyB.