Harris JR, Adrian M, Bhakdi S, Palmer M
Institute of Zoology, University of Mainz, Mainz, D-55099, Germany
J Struct Biol. 1998;121(3):343-55. doi: 10.1006/jsbi.1998.3989.
We present transmission electron microscopical data from negatively stained specimens of cholesterol following interaction with the thiol-activated bacterial toxin streptolysin O (SLO) (wild-type and a number of cysteine substitution mutants), with and without chemical modification of the cysteine residues. Two experimental systems were used, one with an aqueous suspension of cholesterol microcrystals and the other with immobilized thin planar cholesterol crystals attached to a carbon film. In both systems the wild-type SLO and two cytolytically active mutants, Cys 530 --> Ala (C530A) and Ser 101 --> Cys (S101C), readily generated the characteristic SLO arc- and ring-like oligomers on the surface of cholesterol microcrystals and immobilized planar cholesterol crystals. An underlying array of bound toxin can sometimes be detected. In the presence of high concentrations of SLO monomer, extensive sheet-like networks of linked oligomers extend from the microcrystals. The SLO mutant Thr250 --> Cys (T250C), which also possesses a relatively high cytolytic activity, has been found to create ring-like toxin oligomers somewhat more slowly than wild-type SLO, but the linear monomolecular layer array of cholesterol-bound toxin is more readily detected. With mutant Asn402 --> Cys (N402C), which has approximately 10% cytolytic activity compared to wild-type SLO, the formation of ring-like oligomers is markedly reduced, with incomplete arcs and the parallel arrays predominating. Chemical modification of the functional cysteine groups of SLO mutants T250C and N402C completely inhibits the formation of toxin oligomers, but does not prevent the ability of these mutants to bind to cholesterol as a linear array. Such chemical modification is also known to abolish hemolysis/cytolysis. For both mutant T250C and N402C the parallel array of bound SLO adopts an orientation that appears to be determined by the underlying lattice of the crystalline cholesterol. The cholesterol-binding of biotinylated SLO mutant N402C was confirmed by labeling in suspension with 5-nm streptavidin-conjugated colloidal gold particles. Removal of the maltose-binding protein from the SLO fusion products increases the order of the monolayer array of biotinylated SLO bound to cholesterol crystals. Overall, our data support the concept that there is sterospecific binding of the SLO monomer to crystalline cholesterol bilayers, prior to oligomer formation. With the mutants tested, cysteine modification does not prevent binding to cholesterol, but subsequent release and oligomer formation are blocked. Copyright 1998 Academic Press.
我们展示了胆固醇与硫醇激活的细菌毒素链球菌溶血素O（SLO）（野生型及多个半胱氨酸替代突变体）相互作用后，经负染色标本的透射电子显微镜数据，其中半胱氨酸残基有或没有化学修饰。使用了两个实验系统，一个是胆固醇微晶的水悬浮液，另一个是附着在碳膜上的固定化薄平面胆固醇晶体。在这两个系统中，野生型SLO以及两个具有细胞溶解活性的突变体，即半胱氨酸530突变为丙氨酸（C530A）和丝氨酸101突变为半胱氨酸（S101C），都能在胆固醇微晶和固定化平面胆固醇晶体表面轻易产生特征性的SLO弧形和环形寡聚体。有时可以检测到一层潜在的结合毒素。在高浓度SLO单体存在的情况下，连接的寡聚体形成广泛的片状网络，从微晶延伸出来。已发现SLO突变体苏氨酸250突变为半胱氨酸（T250C），其也具有相对较高的细胞溶解活性，产生环形毒素寡聚体的速度比野生型SLO稍慢，但更容易检测到胆固醇结合毒素的线性单分子层阵列。对于与野生型SLO相比具有约10%细胞溶解活性的突变体天冬酰胺402突变为半胱氨酸（N402C），环形寡聚体的形成明显减少，以不完整的弧形和平行阵列为主。对SLO突变体T250C和N402C的功能性半胱氨酸基团进行化学修饰完全抑制毒素寡聚体的形成，但不阻止这些突变体以线性阵列形式结合胆固醇的能力。已知这种化学修饰也会消除溶血/细胞溶解作用。对于突变体T250C和N402C，结合的SLO平行阵列采用的取向似乎由结晶胆固醇的底层晶格决定。通过用5纳米链霉亲和素偶联的胶体金颗粒在悬浮液中标记，证实了生物素化SLO突变体N402C与胆固醇的结合。从SLO融合产物中去除麦芽糖结合蛋白可增加与胆固醇晶体结合的生物素化SLO单分子层阵列的有序性。总体而言，我们的数据支持这样的概念，即在寡聚体形成之前，SLO单体与结晶胆固醇双层存在立体特异性结合。在所测试的突变体中，半胱氨酸修饰并不阻止与胆固醇的结合，但随后的释放和寡聚体形成受到阻碍。版权所有1998年学术出版社。
