Winkler Herbert H, Daugherty Robin M, Audia Jonathon P
Laboratory of Molecular Biology, Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, Alabama 36688, USA.
Biochemistry. 2003 Nov 4;42(43):12562-9. doi: 10.1021/bi035254m.
The contribution of transmembrane region VIII of the Rickettsia prowazekii ATP/ADP translocase to the structure of the water-filled channel through which ATP is transported was evaluated from the accessibility of three hydrophilic, thiol reactive, methanethiosulfonate reagents to a library of 21 single-cysteine substitution mutants expressed in Escherichia coli. A negatively charged reagent (MTSES) and two positively charged reagents (MTSET and MTSEA) were used. Mutants Q323C and G327C did not tolerate cysteine substitution and were almost completely deficient in ATP transport. The remaining mutants exhibited 25-226% of the cysteine-less parent's transport activity. Five patterns of inhibition of ATP transport by the MTS reagents were observed. (i) ATP transport was not inhibited by any of the three MTS reagents in mutants Q321C, F324C, A332C, and L335C and only marginally in F333C. (ii) Transport activity of mutants F322C, Q326C, and A330C was markedly inhibited by all three reagents. (iii) ATP transport was inhibited by MTSEA in only the largest group of mutants (M334C, I336C, G337C, S338C, N339C, I340C, and I341C). (iv) Transport activity was inhibited by MTSET and MTSEA, whereas high concentrations of MTSES were required to inhibit mutants W328C, V329C, and I331C. However, mutant W328C could be inhibited by MTSES in the presence of sub-K(m) concentrations of the substrate. (v) ATP transport by mutant Y325C was unaffected by MTSEA, but inhibited approximately 50% by MTSET and MTSES. Transport of ATP protected mutants (F322C, W328C, V329C, A330C, and I331C) from MTS inhibition. Mutants in the half of TM VIII that is closest to the cytoplasm were not inhibited well by MTSES or MTSET in either whole cells or inside-out vesicles. The results indicate that TM VIII makes a major contribution to the structure of the aqueous translocation pathway, that the accessibility to impermeant thiol reagents is influenced (blocked or stimulated) by substrate, and that there is great variation in accessibility to MTS reagents along the length of TM VIII.
通过三种亲水性、硫醇反应性甲硫基磺酸盐试剂对在大肠杆菌中表达的21个单半胱氨酸取代突变体文库的可及性，评估了普氏立克次氏体ATP/ADP转位酶跨膜区VIII对ATP运输所通过的充满水的通道结构的贡献。使用了一种带负电荷的试剂（MTSES）和两种带正电荷的试剂（MTSET和MTSEA）。突变体Q323C和G327C不能耐受半胱氨酸取代，并且在ATP运输方面几乎完全缺陷。其余突变体表现出无半胱氨酸亲本运输活性的25%-226%。观察到MTS试剂对ATP运输的五种抑制模式。（i）在突变体Q321C、F324C、A332C和L335C中，三种MTS试剂均未抑制ATP运输，在F333C中仅轻微抑制。（ii）所有三种试剂均显著抑制突变体F322C、Q326C和A330C的运输活性。（iii）仅在最大的一组突变体（M334C、I336C、G337C、S338C、N339C、I340C和I341C）中，MTSEA抑制ATP运输。（iv）MTSET和MTSEA抑制运输活性，而抑制突变体W328C、V329C和I331C需要高浓度的MTSES。然而，在底物浓度低于K(m)时，MTSES可抑制突变体W328C。（v）突变体Y325C的ATP运输不受MTSEA影响，但被MTSET和MTSES抑制约50%。ATP的运输保护突变体（F322C、W328C、V329C、A330C和I331C）免受MTS抑制。在整个细胞或内翻囊泡中，最靠近细胞质的跨膜区VIII一半中的突变体不受MTSES或MTSET的良好抑制。结果表明，跨膜区VIII对水相转运途径的结构有主要贡献，非渗透性硫醇试剂的可及性受底物影响（阻断或刺激），并且沿跨膜区VIII的长度，对MTS试剂的可及性有很大差异。
