METAL-ION BINDING AND CONFORMATIONAL TRANSITIONS IN CONCANAVALIN-A - A STRUCTURE-FUNCTION STUDY
METAL-ION BINDING AND CONFORMATIONAL TRANSITIONS IN CONCANAVALIN-A - A STRUCTURE-FUNCTION STUDY
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DOI：
10.1080/07391102.1983.10507497
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发表时间：
1983-01-01
影响因子：
4.4
通讯作者：
KOENIG, SH
中科院分区：
文献类型：
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作者：
BREWER, CF;BROWN, RD;KOENIG, SH
刀豆蛋白A(ConA)以两种构象状态的混合物存在：锁定状态和非锁定状态。蛋白质的解锁形式与金属离子和糖类弱结合，是平衡状态下脱金属刀豆蛋白A(apo-conA)的主要构象。锁定形式与每个单体结合2个金属离子，生成的络合物具有完全的糖结合活性。测定了含Mn2+和Ca2+的解锁构象向完全金属化的锁定构象转变的动力学。单独的Mn2+可以与ConA形成锁定的三元络合物；快速去除这些离子导致锁定构象中的apo-conA以亚稳态的形式存在，在25℃下缓慢(小时)。C)恢复到(主要)未锁定的构象。因此，在没有或存在金属离子的情况下形成构象的能力使我们能够探索金属离子结合和ConA构象转变之间的关系，作为凝集素糖结合活性的决定因素。未锁定的apo-conA向完全金属化的conA转变的动力学和X射线晶体数据表明，conA的两种构象之间的转变涉及蛋白质骨架中靠近两个金属离子结合部位之一的Ala-Asp多肽键的顺反异构化。相对较大的转变活化能(.apprx。22千卡M-1)导致构象之间的相互转化相对缓慢(从几分钟到几天)，而与金属离子和糖的平衡是快速的。因此，可以形成许多亚稳态络合物，并研究了两种构象之间的各种过渡途径。讨论了含Mn2+和糖的两种构象的ConA的二元、三元和四元配合物，它们的金属离子和糖的离解常数以及它们之间的构象转变的平衡值和动态值。糖结合力较弱(Kd.apprx.2M)解锁的apo-conA，并与锁定的三元Mn2+-conA络合物紧密结合(Kd.apprx.10-4M)。糖结合沿连接这两个物种的不同途径以非相加的方式增加。构象和金属离子结合都决定了每个复合体的糖亲和力，尽管不同物种的糖结合的特异性始终保持不变。
Concanavalin A (Con A) exists as a mixture of 2 conformational states: a locked form an an unlocked form. The unlocked form of the protein weakly binds metal ions and saccharide, is the predominate conformation of demetallized Con A (apo-Con A) at equilibrium. The locked form binds 2 metal ions per monomer with the resulting complex(es) possessing full saccharide binding activity. The kinetics of the transition of the unlocked form to the fully metallized locked conformation containing Mn2+ and Ca2+ was measured. Mn2+ alone could form a locked ternary complex with Con A; rapid removal of the ions resulted in a metastable form of apo-Con A in the locked conformation which slowly (hours at 25.degree. C) reverted back to (predominantly) the unlocked conformation. The ability to form either conformation in the absence or presence of metal ions has thus allowed us to explore the relationship between metal ion binding and conformational transitions in Con A as determinants of the saccharide binding activity of the lectin. The kinetics of the transition of unlocked apo-Con A to fully metallized locked Con A, and X-ray crystallographic data indicated that the transition between the 2 conformations of Con A involves a cis-trans isomerization of an Ala-Asp peptide bond in the backbone of the protein, near 1 of the 2 metal ion binding sites. The relatively large activation energy for the transition (.apprx. 22 kcal M-1) results in relatively slow interconversions between the conformations (from minutes to days), whereas the equilibria with metal ions and saccharide are rapid. Thus, many metastable complexes can be formed and a variety of transition pathways between the 2 conformations studied. Binary, ternary and quaternary complexes of both conformations of Con A containing Mn2+ and saccharide are discussed, both metal ion and saccharide dissociation constants for all of them as well as equilibrium and kinetic values for the conformational transitions between them. Saccharide binds weakly (Kd .apprx. 2 M) to unlocked apo-Con A and tightly to the locked ternary Mn2+-Con A complex (Kd .apprx. 10-4 M). Saccharide binding increases along the various pathways connecting these 2 species in a nonadditive fashion. Both conformation and metal ion binding determine the saccharide affinity of each complex, although the specificity of saccharide binding of the various species is maintained throughout.