Sluchanko Nikolai N, Uversky Vladimir N
A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prospect 33, Moscow 119071, Russian Federation.
Department of Molecular Medicine and USF Health Byrd Alzheimer's Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL, USA; Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow Region, Russian Federation; Biology Department, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia; Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russian Federation.
Biochim Biophys Acta. 2015 May;1854(5):492-504. doi: 10.1016/j.bbapap.2015.02.017. Epub 2015 Mar 5.
The multiplicity of functions of 14-3-3 proteins, integrated into many cellular interactions and signaling networks, is primarily based upon their dimeric α-helical structure that is capable of binding phosphorylated protein partners as well as displaying a "moonlighting" chaperone-like activity. The structure and functions of 14-3-3 proteins are regulated in different ways, including Ser58 phosphorylation in the interface, which shifts equilibrium towards the formation of protein monomers whose role is poorly understood. While modification of Ser58 induced only partial dissociation, the engineered triple mutation of human 14-3-3ζ located in the first α-helix deeply monomerized the protein, allowing for a structural analysis of the monomeric form. Dimer-incapable 14-3-3 proteins retained binding capacity and specificity towards some phosphopartners, and also demonstrated increased chaperone-like activity on various substrates. Here, we found a substantial propensity of the N-terminal segment (~40 residues) of 14-3-3 proteins to intrinsic disorder, showing remarkable conservation across different isoforms and organisms. We hypothesized that this intrinsic disorder propensity, hidden in the α-helical 14-3-3 dimer, can be manifested upon its dissociation and interrogated novel monomeric 14-3-3ζ carrying both monomerizing and S58E mutations (14-3-3ζmS58E). CD spectroscopy showed that, at physiological temperatures, this protein has ~10-15% reduced helicity relative to the wild type protein, corresponding to roughly 40 residues. Along with the known flexibility of C-terminus, SAXS-based modeling of the 14-3-3ζmS58E structure strongly suggested pliability of its N-terminus. The unraveled disorder propensity of the N-terminal tails of 14-3-3 proteins provides new clues for better understanding of the molecular mechanisms of dimerization and multifunctionality of these universal adapter proteins.
14-3-3蛋白具有多种功能，融入了许多细胞相互作用和信号网络中，其主要基于其二聚体α-螺旋结构，该结构能够结合磷酸化的蛋白质伙伴，并表现出“兼职”的伴侣样活性。14-3-3蛋白的结构和功能以不同方式受到调节，包括界面处的Ser58磷酸化，这会使平衡向蛋白质单体的形成转移，而其作用尚不清楚。虽然Ser58的修饰仅诱导了部分解离，但位于第一个α-螺旋中的人14-3-3ζ的工程化三重突变使该蛋白深度单体化，从而能够对单体形式进行结构分析。无法形成二聚体的14-3-3蛋白保留了对某些磷酸化伙伴的结合能力和特异性，并且在各种底物上也表现出增强的伴侣样活性。在这里，我们发现14-3-3蛋白的N端片段（约40个残基）具有显著的内在无序倾向，在不同的异构体和生物体中表现出显著的保守性。我们推测，隐藏在α-螺旋14-3-3二聚体中的这种内在无序倾向，在其解离后可能会表现出来，并对携带单体化和S58E突变的新型单体14-3-3ζ（14-3-3ζmS58E）进行了研究。圆二色光谱表明，在生理温度下，该蛋白相对于野生型蛋白的螺旋度降低了约10-15%，相当于大约40个残基。连同已知的C端灵活性，基于小角X射线散射的14-3-3ζmS58E结构建模强烈表明其N端具有柔韧性。14-3-3蛋白N端尾巴解开的无序倾向为更好地理解这些通用衔接蛋白的二聚化和多功能性的分子机制提供了新线索。
