Cryo-EM of AKAP350/AKAP9 Reveals Fibrillar Clusters and an Association With DNA

Abstract

AKAP350 is a massive human scaffolding protein, encoded by AKAP9, that anchors protein kinase A (PKA) to the Golgi apparatus and the centrosome. AKAP9 is among the most frequently mutated genes in various human cancers, and dysregulation of AKAP350 function is strongly linked to metastasis. However, the molecular mechanisms underlying these disease processes remain poorly understood due to the challenges of studying its large and unstructured protein sequence. To learn more about AKAP350 basic function and architecture, we successfully expressed and purified full-length AKAP350 from human cells. Its functional state was validated based on the scaffolding protein’s ability to pulldown endogenous CEP170 and CDK5RAP2, and to co-purify with endogenous PKA. Cryo-electron microscopy (cryo-EM) revealed that AKAP350 appears as polydisperse clusters ∼50 nm in diameter, characterized by fibrous outgrowths. Surprisingly, these fibers reconstructed into double-stranded DNA. DNA sequencing and mass spectrometry confirmed that AKAP350 co-purified with human DNA and endogenous DNA-binding proteins, including nuclear factor 1B (NFIB) and nucleolin (NCL). Interestingly, the pull-down of NFIB and NCL, but not the centrosomal proteins CEP290, CDK5RAP2, or CEP170, was reduced in the presence of DNase-I, suggesting that AKAP350’s interaction with these DNA-binding proteins is mediated by DNA. Furthermore, cryo-electron tomography (cryo-ET) revealed that AKAP350 fibrillar clusters form a three-dimensional network of entangled filaments, a structure significantly altered by DNase-I treatment. Overall, our findings suggest AKAP350 forms fibrillar clusters that serve as nucleoprotein scaffolding complexes in human cells.