Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México México City, México.
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México México City, México.
Front Plant Sci. 2014 May 16;5:199. doi: 10.3389/fpls.2014.00199. eCollection 2014.
Specialized carbohydrate-binding domains, the Starch-Binding Domain (SBD) and the Glycogen Binding Domain (GBD), are motifs of approximately 100 amino acids directly or indirectly associated with starch or glycogen metabolism. Members of the regulatory β subunit of the heterotrimeric complex AMPK/SNF1/SnRK1 contain an SBD or GBD. In Arabidopsis thaliana, the β regulatory subunit AKINβ2 and a γ-type subunit, AKINβγ, also have an SBD. In this work, we compared the SBD of AKINβ2 and AKINβγ with the GBD present in rat AMPKβ1 and demonstrated that they conserved the same overall topology. The majority of the amino acids identified in the protein-carbohydrate interactions in the rat AMPKβ1 are conserved in the two plant proteins. In AKINβγ, there is an insertion of three amino acids that creates a loop adjacent to one of the conserved tryptophan residues. Functionally, the SBD from AKINβγ and AKINβ2 could bind starch, but there was an important difference in the association when an amylose/amylopectin (A/A) mixture was used. The physiological relevance of binding to starch was clear for AKINβγ, because immunolocalization experiments identified this protein inside the chloroplast. SnRK1 activity was not affected by the addition of A/A to the reaction mixture. However, addition of starch inhibited the activity 85%. Furthermore, proteins associated with A/A and starch in an in vitro-binding assay accounted for 10-20% of total SnRK1 kinase activity. Interestingly, the identification of the SnRK1 subunits associated to the protein-carbohydrate complex indicated that only the catalytic subunits, AKIN10 and AKIN11, and the regulatory subunit AKINβγ were present. These results suggest that a dimer formed between either catalytic subunit and AKINβγ could be associated with the A/A mixture in its active form but the same subunits are inactivated when binding to starch.
特异性碳水化合物结合结构域，即淀粉结合结构域（SBD）和糖原结合结构域（GBD），是与淀粉或糖原代谢直接或间接相关的约 100 个氨基酸的基序。异三聚体复合物 AMPK/SNF1/SnRK1 的调节β亚基的成员包含 SBD 或 GBD。在拟南芥中，β调节亚基 AKINβ2 和 γ 型亚基 AKINβγ 也具有 SBD。在这项工作中，我们比较了 AKINβ2 和 AKINβγ 的 SBD 与大鼠 AMPKβ1 中的 GBD，并证明它们保持了相同的整体拓扑结构。在大鼠 AMPKβ1 中鉴定的与蛋白质-碳水化合物相互作用有关的大多数氨基酸在两种植物蛋白中都得到了保守。在 AKINβγ 中，存在三个氨基酸的插入，形成了与一个保守色氨酸残基相邻的环。功能上，AKINβγ 和 AKINβ2 的 SBD 可以结合淀粉，但当使用直链淀粉/支链淀粉（A/A）混合物时，其结合方式存在重要差异。AKINβγ 与淀粉结合的生理相关性是明确的，因为免疫定位实验在叶绿体内部鉴定了这种蛋白质。SnRK1 活性不受向反应混合物中添加 A/A 的影响。然而，添加淀粉可抑制 85%的活性。此外，在体外结合测定中与 A/A 和淀粉结合的蛋白质占总 SnRK1 激酶活性的 10-20%。有趣的是，与蛋白质-碳水化合物复合物相关的 SnRK1 亚基的鉴定表明，只有催化亚基 AKIN10 和 AKIN11 以及调节亚基 AKINβγ 存在。这些结果表明，与 A/A 混合物以其活性形式形成的二聚体可以与 A/A 混合物结合，但当与淀粉结合时，相同的亚基失活。
