Institute of Biomedical Sciences, Academia Sinica, Taipei, 11529, Taiwan.
Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, Rhode Island, 02903, USA.
Sci Rep. 2017 Dec 12;7(1):17391. doi: 10.1038/s41598-017-17465-0.
The negatively charged amino acid-dependent sumoylation motif (NDSM) carries an additional stretch of acidic residues downstream of the consensus Ψ-K-x-E/D sumoylation motif. We have previously shown that acetylation of the SUMO E2 conjugase enzyme, Ubc9, at K65 downregulates its binding to the NDSM and renders a selective decrease in sumoylation of substrates with the NDSM motif. Here, we provide detailed structural, thermodynamic, and kinetics results of the interactions between Ubc9 and its K65 acetylated variant (Ac-Ubc9) with three NDSMs derived from Elk1, CBP, and Calpain2 to rationalize the mechanism beneath this reduced binding. Our nuclear magnetic resonance (NMR) data rule out a direct interaction between the NDSM and the K65 residue of Ubc9. Similarly, we found that NDSM binding was entropy-driven and unlikely to be affected by the negative charge by K65 acetylation. Moreover our NMR, mutagenesis and molecular dynamics simulation studies defined the sequence of the NDSM as Ψ-K-x-E/D-x-x-(x/E/D)-(x/E/D)-x and determined that K74 and K76 were critical Ubc9 residues interacting with the negatively charged residues of the NDSM.
带负电荷的氨基酸依赖的 SUMO 化模体（NDSM）在公认的 Ψ-K-x-E/D SUMO 化模体下游带有额外的酸性残基延伸。我们之前已经表明，SUMO E2 连接酶 Ubc9 的 K65 乙酰化会下调其与 NDSM 的结合，并导致具有 NDSM 模体的底物的 SUMO 化选择性降低。在这里，我们提供了 Ubc9 与其 K65 乙酰化变体（Ac-Ubc9）与三个源自 Elk1、CBP 和 Calpain2 的 NDSM 之间相互作用的详细结构、热力学和动力学结果，以合理化这种结合减少的机制。我们的核磁共振（NMR）数据排除了 NDSM 和 Ubc9 的 K65 残基之间的直接相互作用。同样，我们发现 NDSM 结合是熵驱动的，不太可能受到 K65 乙酰化的负电荷的影响。此外，我们的 NMR、突变和分子动力学模拟研究定义了 NDSM 的序列为 Ψ-K-x-E/D-x-x-(x/E/D)-(x/E/D)-x，并确定 K74 和 K76 是与 NDSM 的负电荷残基相互作用的关键 Ubc9 残基。
