Department of Chemistry, Xavier University of Louisiana, 1 Drexel Drive, New Orleans, Louisiana 70125-1098, United States.
Biochemistry. 2021 Aug 24;60(33):2524-2536. doi: 10.1021/acs.biochem.1c00384. Epub 2021 Aug 6.
Lysine acetylation and deacetylation are critical for regulation of many cellular proteins. Despite the importance of this cycle, it is unclear how lysine deacetylase (KDAC) family members discriminate between acetylated proteins to react with a discrete set of substrates. Potential short-range interactions between KDAC8 and a known biologically relevant peptide substrate were identified using molecular dynamics (MD) simulations. Activity assays with a panel of peptides derived from this substrate supported a putative ionic interaction between arginine at the -1 substrate position and KDAC8 D101. Additional assays and MD simulations confirmed this novel interaction, which promotes deacetylation of substrates. Verification that a negatively charged residue at the 101 position is necessary for the ionic interaction and observed reactivity with the substrates was performed using KDAC8 derivatives. Notably, this interaction is specific to KDAC8, as KDAC1 and KDAC6 do not form this interaction and each KDAC has a different specificity profile with the peptide substrates, even though all KDACs could potentially form ionic interactions. When reacted with a panel of putative human KDAC substrates, KDAC8 preferentially deacetylated substrates containing an arginine at the -1 position. KDAC8 D101-R(-1) is a specific enzyme-substrate interaction that begins to explain how KDACs discriminate between potential substrates and how different KDAC family members can react with different subsets of acetylated proteins in cells. This multi-pronged approach will be extended to identify other critical interactions for KDAC8 substrate binding and determine critical interactions for other KDACs.
赖氨酸乙酰化和去乙酰化对于许多细胞蛋白的调节至关重要。尽管这个循环很重要，但不清楚赖氨酸去乙酰化酶 (KDAC) 家族成员如何区分乙酰化蛋白，以与一组离散的底物发生反应。使用分子动力学 (MD) 模拟鉴定了 KDAC8 与已知具有生物学相关性的肽底物之间的潜在短程相互作用。用来自该底物的肽进行的活性测定支持 -1 底物位置的精氨酸与 KDAC8 D101 之间存在假定的离子相互作用。其他测定和 MD 模拟证实了这种新的相互作用，促进了底物的去乙酰化。使用 KDAC8 衍生物验证了 101 位带负电荷的残基对于离子相互作用和与底物的观察到的反应性是必需的。值得注意的是，这种相互作用是 KDAC8 特有的，因为 KDAC1 和 KDAC6 不会形成这种相互作用，并且每个 KDAC 与肽底物都有不同的特异性谱，尽管所有 KDAC 都有可能形成离子相互作用。当与一组假定的人类 KDAC 底物反应时，KDAC8 优先去乙酰化 -1 位含有精氨酸的底物。KDAC8 D101-R(-1) 是一种特异性的酶-底物相互作用，它开始解释 KDAC 如何区分潜在的底物，以及不同的 KDAC 家族成员如何在细胞中与不同的乙酰化蛋白亚群发生反应。这种多管齐下的方法将扩展到识别 KDAC8 底物结合的其他关键相互作用，并确定其他 KDAC 的关键相互作用。
