PHOSPHORYLATING THE POSTSYNAPTIC SCAFFOLD: CDKL5 ORCHESTRATES SHANK1–HOMER1BC ASSEMBLY TO SHAPE EXCITATORY SYNAPSES

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a severe X-linked neurodevelopmental encephalopathy characterized by early-onset seizures and profound cognitive impairment. CDKL5 has been implicated in the regulation of excitatory synapse development, through interactions with PSD-95 and phosphorylation of NGL1. However, the molecular mechanisms by which CDKL5 orchestrates the organization of the postsynaptic compartment remain poorly understood.
Here, we identify a previously unrecognized mechanism by which CDKL5 controls excitatory synapse maturation by stabilizing the postsynaptic scaffold protein Shank1 and promoting its interaction with Homer1bc. Analysis of the somatosensory cortex of CDKL5 knockout mice revealed a significant reduction of both Shank1 and Homer1bc protein levels, accompanied by a marked disruption of their colocalization at postsynaptic sites, indicating impaired assembly of the postsynaptic scaffold.
Biochemical assays in HEK-293T cells demonstrated that CDKL5 directly interacts with Shank1 via its C-terminal PDZ-binding domain and enhances the formation of the Shank1–Homer1bc complex in a kinase-dependent manner. To elucidate the underlying structural basis, we combined AlphaFold3-based structural modelling with molecular dynamics simulations. These analyses suggested that CDKL5-mediated phosphorylation of Shank1 induces a conformational rearrangement of the Shank1–Homer1bc complex, increasing the buried interaction surface and stabilizing the protein–protein interface.
Collectively, our results provide functional, biochemical, and structural evidence that CDKL5 regulates the assembly of the postsynaptic density by modulating Shank1–Homer1bc interactions through phosphorylation. This mechanism offers new insight into the synaptic dysfunction underlying CDD and identifies the Shank1–Homer1bc complex as a critical downstream effector of CDKL5 signalling at excitatory synapses.