Conformation as the determinant of saccharide binding in concanavalin A: Ca2+-concanavalin A complexes.
Conformation as the determinant of saccharide binding in concanavalin A: Ca2+-concanavalin A complexes.
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构象作为刀豆球蛋白 A 中糖结合的决定因素：Ca2 -刀豆球蛋白 A 复合物。
DOI：
10.1021/bi00613a022
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发表时间：
1978
期刊：
影响因子：
2.9
通讯作者：
R. Brown
中科院分区：
文献类型：
--
作者：
S. H. Koenig;C. Brewer;R. Brown
最近已证明存在具有不同金属离子结合特性的刀豆球蛋白 A (Con A) 的两种构象状态 (Brown, R. D., Brewer, C. F., & Koenig, S. H. (1977) Biochemistry 16, 3883)。将 Mn2+ 引入到 apo-Con A 的 S1 位点，将 Ca2+ 引入到 apo-Con A 的 S2 位点，可诱导蛋白质构象变化，这是由于二级结构中肽键的顺反异构化，从而导致金属离子的极其紧密的结合。这种诱导的构象被称为“锁定”，而初始构象被称为“解锁”。锁定的三元复合物与天然蛋白质相同。在本文中，我们报告了形成相对稳定、锁定的三元 Ca2+-Con A 复合物的证据，该复合物具有与天然 Ca2+-Mn2+Con A 相似的性质。实验技术包括在添加缓慢与蛋白质结合的 Mn2+ 离子后，测量 Ca2+-Con A 溶液中溶剂水质子的核磁弛豫率 (1/T1) 的磁场和时间依赖性。动力学数据可以通过 Ca2+ 与 Con A 相互作用的模型进行拟合，该模型表明，在没有 Mn2+ 的情况下，Ca2+ 可以结合在蛋白质的 S1 和 S2 位点上，此外，可以诱导蛋白质经历解锁到锁定构象的转变。根据该模型，Mn2+ 离子的时间依赖性结合是由于锁定蛋白中 S1 位点处 Ca2+ 离子的替换所致。 Ca2+ 从锁定三元 Ca2+-Con A 复合物的 S2 位点的解离速率远大于从锁定 Ca2+-Mn2+-Con A 复合物的解离速率。根据添加的 α-甲基 D-吡喃甘露糖苷对锁定三元 Ca2+-Con A 复合物 S1 位点处 Ca2+ 被 Mn2+ 取代速率的影响，得出结论，后者复合物与锁定 Ca2+-Mn2+-Con A 复合物一样牢固地结合糖类。此外，数据分析表明，锁定构象的apo-Con A 与α-甲基D-吡喃甘露糖苷的结合亲和力约为完全金属化锁定形式蛋白质的7%。与解锁的 apo-Con A 相比，锁定的 apo-Con A 的这种强糖结合活性通过解锁的 apo-Con A 与 α-甲基 D-吡喃甘露糖苷的平衡得到了进一步证明，这导致了锁定的 apo-Con A-糖复合物的形成。这些结果表明，Con A 的锁定构象主要负责糖结合活性，并且结合金属的功能主要是维持蛋白质处于锁定构象。
The existence of two conformational states of concanavalin A (Con A) with different metal ion binding properties has been recently demonstrated (Brown, R. D., Brewer, C. F., & Koenig, S. H. (1977) Biochemistry 16, 3883). Introduction of Mn2+ to the S1 site and Ca2+ to the S2 site of apo-Con A was shown to induce a conformational change in the protein, ascribed to a cis-trans isomerization of a peptide bond in the secondary structure, which results in extremely tight binding of the metal ions. This induced conformation is referred to as "locked" and the initial conformation as "unlocked". The locked ternary complex is identical with the native protein. In the present paper, we report evidence for the formation of a relatively stable, locked, ternary Ca2+-Con A complex that possesses properties similar to those of native Ca2+-Mn2+Con A. The experimental technique involves measurement of the magnetic field and time dependence of the nuclear magnetic relaxation rate (1/T1) of solvent water protons in solutions of Ca2+-Con A, after the addition of Mn2+ ion which slowly bind to the protein. The kinetic data can be fit by a model for Ca2+ interactions with Con A which indicates that Ca2+, in the absence of Mn2+, can bind at both the S1 and S2 sites of the protein and, furthermore, can induce the protein to undergo the unlocked to locked conformational transition. In terms of this model, the time-dependent binding of the Mn2+ ions is due to replacement of Ca2+ ions at the S1 sites in the locked protein. The off-rate of Ca2+ from the S2 site of the locked ternary Ca2+-Con A complex is much greater than that from the locked Ca2+-Mn2+-Con A complex. From the effects of added alpha-methyl D-mannopyranoside on the rate of replacement of Ca2+ by Mn2+ at the S1 site of the locked ternary Ca2+-Con A complex, it is concluded that the latter complex binds saccharides as strongly as the locked Ca2+-Mn2+-Con A complex. In addition, analysis of the data indicates that apo-Con A in the locked conformation binds alpha -methyl D-mannopyranoside with approximately 7% of the affinity of the fully metallized locked form of the protein. This strong saccharide-binding activity of locked apo-Con A, compared with that of the unlocked apo-Con A, was further demonstrated by equilibration of unlocked apo-Con A with alpha-methyl D-mannopyranoside, which resulted in the formation of the locked apo-Con A-saccharide complex. These results demonstrate that it is the locked conformation of Con A that is primarily responsible for saccharide-binding activity, and that the function of the bound metals is primarily to maintain the protein in the locked conformation.