Benabdelhak Houssain, Schmitt Lutz, Horn Carsten, Jumel Kornelia, Blight Mark A, Holland I Barry
Institut de Génétique et Microbiologie, Bâtiment 409, Université Paris-Sud, 91405 Orsay Cedex, France.
Biochem J. 2005 Mar 15;386(Pt 3):489-95. doi: 10.1042/BJ20041282.
The ATPase activity of the ABC (ATP-binding cassette) ATPase domain of the HlyB (haemolysin B) transporter is required for secretion of Escherichia coli haemolysin via the type I pathway. Although ABC transporters are generally presumed to function as dimers, the precise role of dimerization remains unclear. In the present study, we have analysed the HlyB ABC domain, purified separately from the membrane domain, with respect to its activity and capacity to form physically detectable dimers. The ATPase activity of the isolated ABC domain clearly demonstrated positive co-operativity, with a Hill coefficient of 1.7. Furthermore, the activity is (reversibly) inhibited by salt concentrations in the physiological range accompanied by proportionately decreased binding of 8-azido-ATP. Inhibition of activity with increasing salt concentration resulted in a change in flexibility as detected by intrinsic tryptophan fluorescence. Finally, ATPase activity was sensitive towards orthovanadate, with an IC50 of 16 microM, consistent with the presence of transient dimers during ATP hydrolysis. Nevertheless, over a wide range of protein or of NaCl or KCl concentrations, the ABC ATPase was only detected as a monomer, as measured by ultracentrifugation or gel filtration. In contrast, in the absence of salt, the sedimentation velocity determined by analytical ultracentrifugation suggested a rapid equilibrium between monomers and dimers. Small amounts of dimers, but apparently only when stabilized by 8-azido-ATP, were also detected by gel filtration, even in the presence of salt. These data are consistent with the fact that monomers can interact at least transiently and are the important species during ATP hydrolysis.
HlyB（溶血素B）转运蛋白的ABC（ATP结合盒）ATP酶结构域的ATP酶活性是大肠杆菌溶血素通过I型途径分泌所必需的。尽管通常认为ABC转运蛋白以二聚体形式发挥作用，但二聚化的确切作用仍不清楚。在本研究中，我们分析了从膜结构域中单独纯化出来的HlyB ABC结构域的活性及其形成可物理检测到的二聚体的能力。分离出的ABC结构域的ATP酶活性明显表现出正协同性，希尔系数为1.7。此外，该活性在生理范围内的盐浓度下（可逆地）受到抑制，同时8-叠氮基ATP的结合也相应减少。随着盐浓度增加对活性的抑制导致通过内在色氨酸荧光检测到的柔韧性发生变化。最后，ATP酶活性对原钒酸盐敏感，IC50为16微摩尔，这与ATP水解过程中存在瞬时二聚体一致。然而，在广泛的蛋白质浓度、NaCl或KCl浓度范围内，通过超速离心或凝胶过滤测量，ABC ATP酶仅被检测为单体。相反，在无盐条件下，分析超速离心测定的沉降速度表明单体和二聚体之间存在快速平衡。即使在有盐存在的情况下，通过凝胶过滤也检测到少量二聚体，但显然只有在被8-叠氮基ATP稳定时才会出现。这些数据与单体至少可以瞬时相互作用且是ATP水解过程中的重要形式这一事实一致。
