Structure And Function Of CaMKII Protein And Their Relationship

CaMKII (Ca2+/calmodulin dependent protein kinaseⅡ) has been considered to relate to neuron memory, immune cell memory and events of egg activation. Many studies indicate that CaMKII autonomous activity code stimulation and remember stimulation frequency and amplitude and THR286 phosphorylation lead to CaMKII autonomous activity. Why dose CaMKII have autonomous activity after THR286 is phosphorylated? How can CaMKII autonomous activity code stimulation frequency and amplitude? This study tries to explain these questions, the results are as follows.1. A program Glocate was developed to predicte pig CaMK2B gene, this software can be used to annotate new released genome sequence based on gene homology comparison. Compare to CaMK2A, CaMK2B and CaMK2G gene, vertebrate CaMK2D gene is more similar to fruit fly and C. elegans CaMKII gene, and CaMK2D gene contain many segments which are similar to CaMK2A, CaMK2B and CaMK2G. Therefore CaMK2D can be considered as the earliest emergence gene. Taking the protein sequence of human CaMK2A as template, we divided CaMKII protein sequence to four functional domains and a binding site, which are kinase domain (1-271 amino acid), regulatory domain (272-315 amino acid), variable domain (316-374 amino acid), association domain (375-489 amino acid) and calmodulin binding site (293-315 amino acid) which is located at regulatory domain. By comparison of four isoforms of CaMKII, NGI peptide (130-132 amino acids) is considered to be responsible for deviation of enzymatic activity between four isoforms. On calmodulin binding site, R292, V310 and S311 of CaMK2G are different to other three isoforms; it is considred to be responsible for CaMK2G has maximum affinity to calmodulin.2. CaMKII protein 3D structure was analyzed by comparison of crystal structures. The process of CaMKII activation is illustrated as:Ca2+/calmodulin bind to regulatory domain of CaMKII, then regulatory domain release from kinase domain, Lip structure of kinase domain spontaneously changes to close conformation, the active center and substrate binding site is released, and then CaMKII has activity. When Ca2+/calmodulin release from regulatory domain, regulatory domain restores to close kinase domain and occupies the positon of substrate binding site, Lip structure changes to open conformation, CaMKII is inhibited. If two activated CaMKII subunits bind each other to form enzyme-substrate complex, ATP deliver its phosphate group to THR286 of substrate CaMKII subunit, THR286 changes to THP286. Even Ca2+/calmodulin released from regulatory domain, THP286 state regulatory domain can not restore to inhibited conformation, and therefore, THP286 state CaMKII has autonomous activity.3. Spontaneous allosteric transition of CaMKII kinase domain was simulated by molecular dynamics method. Molecular dynamics trajectory of CaMKII kinase domain was classified to a series of local stabilized conformation by autocorrelation function. There is a key residue PHE106 which is pushed to close kinase active center by hydrophobic force. The Lip structure of 96-108 residues moves to close kinase active center. This process is induced by PHE106. The conformation transition of regulatory domain was simulated by improved steered molecular dynamics (SMD). Results indicate that the external force at 0-10 A is larger than at 10-25 A. Spontaneous molecular dynamics simulation indicate that THR286 state regulatory domain restores to inhibited conformation at 0-10 A, but THP286 state regulatory domain trends to far away from kinase domain by THP286 dragging. Water around THP286 is increasing during simulation, but decreasing around THR286. Therefore, hydrophobic force induces the regulatory domain spontaneously close to kinase domain at 0-10 A. THP286’s phosphate groups formed hydrogen bond with nearby water, stabilized hydration shell, thus, THP286 is excluded by hydrophobic cave of kinase domain, regulatory domain can not close to kinase domain. Therefore, hydrophilic force is the reason for regulatory domain retains activated conformation. THR286 phosphorylation in CaMKII leads to autonomous activity by dragging regulatory domain far away from kinase domain.4. According to auto-phosphorylation and auto-dephosphorylation reaction cycle of THR286 in CaMKII, a mathematic model was constructed to illustrate CaMKII autonomous activity response to stimulation. Simulation results indicate that CaMKII autonomous activity is bidirectional response, gradient activity level and rememberable activity for stimulation frequency and amplitude. Therefore, CaMKII autonomous activity codes a series of stimulation, these different stimulations of frequency and amplitude is called operate code. Transcription factor activity and gene production response to CaMKII autonomous activity in different stimulation frequency and amplitude was simulated by a mathematic model. Simulation results indicate that transcription factor activity and gene production are bidirectional response, ultrasensitive response and rememberable activity for stimulation frequency and amplitude. Therefore, stimulation controls the switch of transcription factor activity and gene production by writing and erasing CaMKII autonomous activity.