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

Abstract

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.

Impact of the nisin modification machinery on the transport kinetics of NisT

“…Thus, ATP hydrolysis is clearly important for ABC transporter mediated substrate translocation, we determined the in vitro activity of NisT in terms of ATPase rate without and with substrate. Here, the basal ATPase activity had a value of 79.9 ± 2.9 nmol•min −1 •mg −1 with a K m value of 0.37 ± 0.04 mM, which is in the range of other ABC transporters [51][52][53][54] (Fig. 3).…”

In vivo quantification of the secretion rates of the hemolysin A Type I secretion system

“…To analyze the importance of ATP hydrolysis during substrate secretion, the H662A mutant of HlyB, which is deficient in ATP hydrolysis, was used. This mutant is able to bind ATP, dimerization of the NBDs but does not catalyze ATP hydrolysis 27 29 . In combination with the concept of a stalled T1SS complex described above, the H662A mutant might help to clarify which step of substrate translocation is coupled to ATP hydrolysis.…”

“…Purification of HlyA and HlyAc was carried out as previously described 16 35 . HlyB was purified as described 29 . The concentration of the purified protein was determined spectrophotometrically (Nanodrop-1000, Thermo Scientific) using the calculated (web.expasy.org/protparam) extinction coefficient at 280 nm.…”

Characterization of the nucleotide-binding domain NsrF from the BceAB-type ABC-transporter NsrFP from the human pathogen Streptococcus agalactiae

“…However, when comparing the data with known NBDs which has been described before in the presence and absence of the transmembrane segment it can be observed that v max might be changed, the k m values however remains very similar. For example the ATP hydrolysis kinetics have been described for the HlyB NBD as well as for the purified full length transporter in detergent solution 26,36,38,43,69 . Here the NBD showed a v max of 200 nmol min −1 mg −1 with a k m value of 0.31 where as the full length transporter displayed a lower v max of 8.1 nmol min −1 mg −1 with a k m value of 0.36.…”