STRUCTURAL AND FUNCTIONAL INSIGHTS INTO CAMKIIΓ AND DISEASE-LINKED VARIANTS

Neuronal growth and plasticity are fundamental processes that shape brain structure and function, enabling learning, memory, and adaptation. A key regulator of these processes is Calcium/Calmodulin-Dependent-Protein-Kinase-II (CaMKII), a serine/threonine kinase that plays a central role in calcium signaling and activity-dependent neuronal plasticity.
Among the CaMKII isoforms, the role of CaMKIIγ in the brain and during neurodevelopment remains poorly understood. Recently, de novo CAMK2G mutations have been identified in patients with neurodevelopmental disorders, highlighting its potential significance. While it is known that CaMKII forms oligomeric assemblies, typically dodecamers, consisting of a rigid central hub and flexible kinase domains, full-length structural and biophysical information for CaMKIIγ is still missing.
To address this gap, we established a mammalian expression and purification pipeline for CaMKIIγ using STREP-tag affinity purification. Initial SAXS and negative-stain EM analyses reveal higher-order assemblies consistent with the expected dodecameric architecture, and kinase assays confirm enzymatic activity. We are currently purifying patient-derived variants, and preliminary thermostability assays indicate reduced stability. Further structural and biophysical characterization will assess how these mutations alter holoenzyme properties.
To investigate functional roles in neuronal development, we use a CAMK2G knockout mouse model and measure neuronal activity in primary cortical neurons using multi-electrode array recordings. At early stages, knockout neurons seem to show faster activity development, although this difference is not seen at later time points, where activity appears similar across genotypes.
By combining structural and neuronal biology approaches, we aim to advance our understanding of CaMKIIγ function in brain development and its relevance to disease-associated variants.