Bagdiya Priyanka, Soni Neelesh, Jaiswal Damini, Dalvi Somavally, Kanitkar Tejashree, Madhusudhan M S, Venkatraman Prasanna
Protein Interactome Lab for Structural and Functional Biology, Advanced Centre for Treatment Research and Education in Cancer (ACTREC), tata Memorial Centre, Navi Mumbai, India.
Homi Bhabha National Institute, Mumbai, India.
FASEB J. 2025 Sep 30;39(18):e70985. doi: 10.1096/fj.202500445R.
Many 14-3-3 paralogs, except sigma, could bind and hydrolyze ATP. However, the catalytic residues and the significance of ATP binding or hydrolysis remain unknown. Here we confirm that there are two binding pockets for ATP, one at the peptide binding amphipathic pocket and the other at the dimer interface. As predicted by a new computational method, CLICK, and by limited proteolysis coupled to mass spectroscopy, we identify E131 and E180 as the catalytic residues. We further confirm that ATP hydrolysis is an inherent property of 14-3-3, and mutations result in either gain or loss of ATPase activity. The dimeric fold of the protein is mandatory for ATP hydrolysis but not for peptide binding. While ATP at the dimer interface acts as an allosteric activator of ATP hydrolysis, it acts as a selective negative regulator of a nonphosphopeptide, originating from ExoS, a pathogenic Pseudomonas protein. This study for the first time, unveils the hidden allosteric properties of the 14-3-3 proteins and its role in excluding specific ligands of disease relevance.
除了σ之外，许多14-3-3旁系同源物都能结合并水解ATP。然而，催化残基以及ATP结合或水解的意义仍然未知。在此我们证实，ATP存在两个结合口袋，一个位于肽结合两亲口袋，另一个位于二聚体界面。正如一种新的计算方法CLICK以及有限蛋白酶解结合质谱所预测的那样，我们确定E131和E180为催化残基。我们进一步证实，ATP水解是14-3-3的固有特性，突变会导致ATP酶活性的增加或丧失。蛋白质的二聚体折叠对于ATP水解是必需的，但对于肽结合则不是。虽然二聚体界面处的ATP作为ATP水解的变构激活剂，但它作为一种非磷酸化肽（源自致病性假单胞菌蛋白ExoS）的选择性负调节剂。这项研究首次揭示了14-3-3蛋白隐藏的变构特性及其在排除与疾病相关的特定配体中的作用。
