Structural And Functional Study Of Pathogenic Mutant γCaMKⅡ R292P

Ca²⁺/calmodulin-dependent protein kinase II（CaMKⅡ）is a member of calmodulin（CaM）-dependent serine/threonine-specific protein kinases（CaMKs）family.CaMKⅡ is regulated by the Ca²⁺/CaM complex and is involved in many cellular signaling cascades.It exists in most tissues,with a high expression of about1%total protein in the brain and is thought to be an important regulator of learning and memory.CaMKⅡ consists of a kinase domain,an autoinhibitory domain,a variable segment and an oligomerization domain.Four different CaMKⅡ subtypes are expressed in human bodies.Theαandβisoforms are brain specific,while theδandγisoforms are expressed in most tissues.Mutation inγCaMKⅡ gene is one of the causes of intellectual disability（ID）and autism.Previous clinical studies from two unrelated pediatric patients by diagnostic exome sequencing identified that mutation Arg292Pro（c.875G>C）onγCaMKⅡ can cause ID.The affected children presented mental retardation with additional minor dysmorphic facial features and severe hypotonia.Current research on the R292P mutation mainly focuses on gene expression,the relevance of nuclear signaling to synaptic plasticity and the behavior of animal individuals.Two prevailing hypotheses currently exist.1.The mutantγCaMKⅡ shows no difference in kinase activity and autophosphorylation ability,but the dissociation rate of CaM from the mutant is1500-fold faster than that from the wild type,resulting in CaM unable to be efficiently transported into the nucleus byγCaMKⅡ.That affects the expression of genes associated with long-term potentiation（LTP）or long-term memory,suggesting that this mutation is considered to be a loss-of-function mutation;2.The R292P mutation does not disrupt the ability of CaM shuttling into the nuclear byγCaMKⅡ,but rather significantly increases the phosphorylation level ofγCaMKⅡ and enhances its kinase activity,resulting in a reduction in nerve length and number of branches,which indicates that this mutation is considered to be a gain-of-function mutation.In order to explore the molecular mechanisms by whichγCaMKⅡ mutation causes ID,the project planned to use a combination of X-ray protein crystallography and biophysical methods to investigate the effects of disease mutation on the structure and function ofγCaMKⅡ and how the mutation affects the interaction betweenγCaMKⅡ and CaM.Ca²⁺/CaM binds differently with wild type and mutantγCaMKⅡ,as shown by biophysical methods.ITC results show that wild typeγCaMKⅡ has a high affinity for Ca²⁺/CaM（K_d=0.2μM）,but the mutant does not bind.Meanwhile,the CaM binding domain of the mutantγCaMKⅡ（amino acids 285-316）binds Ca²⁺/CaM with a 200-fold lower affinity compared to the wild type.By analyzing the crystal structure ofγCaMKⅡ R292P CaM binding domain in complex with Ca²⁺/CaM,we find that there is no direct contact between CaM and mutant CaMKⅡ,suggesting that the mutation does not change their affinity by directly affecting the interaction between the two.Further ITC results show that the autophosphorylation of T306 and T307 could significantly reduce the binding of CaM.Therefore,we speculate that the R292P likely to increase the kinase activity by destabilizing the inhibited state ofγCaMKⅡ,leading to an increased autophosphorylation level ofγCaMKⅡ and finally indirectly alter the binding ability with CaM.At present,protein crystals of mutantγCaMKⅡ kinase domain have been obtained and structural determination is ongoing.Our findings reveal the pathological mechanism of theγCaMKⅡ mutation R292P associated with ID,which provides a theoretical basis for the future development of personalized drug to treat ID-related rare diseases.