Wu Jian, Bruystens Jessica G H, Sahoo Puspashree, Bubis José, Maillard Rodrigo A, Taylor Susan S, Ilouz Ronit
Department of Pharmacology, University of California at San Diego, San Diego, California, USA.
The Azrieli Faculty of Medicine, Bar Ilan University, Safed, Israel.
Protein Sci. 2025 Nov;34(11):e70332. doi: 10.1002/pro.70332.
The RIβ subunit of cAMP-dependent protein kinase (PKA) is highly expressed in the brain, yet it remains the least studied of the PKA regulatory subunits (R). As pathologic variants of its gene are increasingly implicated in neurodevelopmental disorders, neurodegeneration, and cancer, gaining more information about the structure/function of RIβ, and how it differs from RIα, has become increasingly important. We previously reported the structure of the RIβC holoenzyme, which revealed a novel conformation where ATP binding was stabilized by a head-to-head anti-parallel packing of the C-tail wrapped around the N-lobe of the catalytic subunit (C). Although visible, the Dimerization/Docking Domain was poorly folded and reduced. Since RIβ is oxidized in brain tissues, we asked if oxidation or binding of an A Kinase Anchoring Protein (AKAP) would affect the holoenzyme structure. Oxidation or addition of an AKAP peptide to crystals led to the release of nucleotide. To capture this at higher resolution we crystallized RIβC in the presence of an AKAP peptide. This new structure represents an RIβ:C heterodimer. Density for the D/D domain was missing; ATP was absent, the kinase adopted an open conformation, and the C-terminus of the RIβ subunit was no longer resolved. Because the crosstalk between ATP and cAMP in the R:C complex appears to be mediated by the two N3A motifs (N3A and N3A) as well as by the linker, which in free RIβ is intrinsically disordered, we describe the conserved features of these two motifs as well as the linker and show how each contributes in a unique but coordinated way to allosteric activation of RIβ holoenzymes by cAMP. A key difference in our RIβ:C structure is the rotation of the side chain of W260 at the N-terminus of the αA Helix in N3A. W260, at the R:C interface in the holoenzyme, is also the capping residue for cAMP bound to CNB-A, so we may have actually captured the first step in cAMP activation.
环磷酸腺苷依赖性蛋白激酶（PKA）的RIβ亚基在大脑中高度表达，但它仍是PKA调节亚基（R）中研究最少的。由于其基因的病理变异越来越多地与神经发育障碍、神经退行性变和癌症有关，因此获取更多关于RIβ的结构/功能以及它与RIα的差异的信息变得越来越重要。我们之前报道了RIβC全酶的结构，该结构揭示了一种新的构象，其中ATP结合通过C末端围绕催化亚基（C）的N叶的头对头反平行堆积而得以稳定。虽然二聚化/对接结构域可见，但折叠不佳且减少。由于RIβ在脑组织中被氧化，我们询问A激酶锚定蛋白（AKAP）的氧化或结合是否会影响全酶结构。向晶体中添加AKAP肽或进行氧化会导致核苷酸释放。为了在更高分辨率下捕捉这一过程，我们在存在AKAP肽的情况下使RIβC结晶。这个新结构代表了RIβ:C异二聚体。D/D结构域的密度缺失；ATP不存在，激酶呈现开放构象，并且RIβ亚基的C末端不再解析。由于R:C复合物中ATP和环磷酸腺苷之间的串扰似乎由两个N3A基序（N3A和N3A）以及连接子介导，在游离的RIβ中连接子是内在无序的，我们描述了这两个基序以及连接子的保守特征，并展示了它们如何以独特但协调的方式对环磷酸腺苷对RIβ全酶的变构激活做出贡献。我们的RIβ:C结构中的一个关键差异是N3A中αA螺旋N末端的W260侧链的旋转。在全酶的R:C界面处的W260也是与CNB - A结合的环磷酸腺苷的封端残基，所以我们实际上可能捕捉到了环磷酸腺苷激活的第一步。
