Connector enhancer of kinase suppressor of Ras 2 (CNKSR2) is a scaffold protein expressed in the neuronal system which is known for organizing proteins involved in Rac GTPase signaling (1). Intriguingly, the CNKSR2 truncations are related with non-syndromic, X-linked mental retardation (MRX) (2,3,75), which shows deficits in cognition and abnormal dendritic spine morphology (4). Recent mass spectrum-based investigations identified ARHGAP39 (Rho GTPase Activating Protein 39) as a binding partner of CNKSR2 (5,6). Further biochemical assays indicate that ARHGAP39 N-terminal tandem WW domains mediate this association with the CNKSR2 P1 polyP ligand. Depletion of ARHGAP39 and eliminating its interaction to CNKSR2 both would induce malformation of dendritic spines in cultured neurons...[ Read more ]

Connector enhancer of kinase suppressor of Ras 2 (CNKSR2) is a scaffold protein expressed in the neuronal system which is known for organizing proteins involved in Rac GTPase signaling (1). Intriguingly, the CNKSR2 truncations are related with non-syndromic, X-linked mental retardation (MRX) (2,3,75), which shows deficits in cognition and abnormal dendritic spine morphology (4). Recent mass spectrum-based investigations identified ARHGAP39 (Rho GTPase Activating Protein 39) as a binding partner of CNKSR2 (5,6). Further biochemical assays indicate that ARHGAP39 N-terminal tandem WW domains mediate this association with the CNKSR2 P1 polyP ligand. Depletion of ARHGAP39 and eliminating its interaction to CNKSR2 both would induce malformation of dendritic spines in cultured neurons, reminiscent of the MRX symptoms. Therefore, ARHGAP39 is regarded as an essential mediator for CNKSR2 to regulate the RacGDP/GTP balance and dendritic spine development.

Although the function is partially revealed (3,21,23), the molecular mechanism governing the interaction between CNKSR2 and ARHGAP39 remains unclear. From sequence alignment, the 1st WW domain of ARHGAP39 is noncanonical (7), while the 2nd WW is in type II which should be too weak to fulfill ARHGAP39's function (8). To get insight into the particular binding mode, the crystal structure of ARHGAP39 WW tandem fused with the CNKSR2 peptide is solved. Besides a canonical docking pocket on WW2, multiple additional binding sites have been identified. Three cation-π interactions out site the type II WW core efficiently enhance this specific binding. Also, the C-ter extension following ARHGAP39 WW2 functions to tether the two WW motifs together, thus more interface on the WW1 domain and the linker is exposed to the ligand. In addition, CNKSR2 contains a positive charged N-ter extension which recognizes a negatively charged surface on ARHGAP39, further strengthening the association for 3 times. Due to all the above contributions, the binding affinity ascends to 30 nanomolar, hundreds of times stronger than the other Type II WW interactions (8). Importantly, this super strong type II WW binding mode is also applied to other ARHGAP39 ligands such as CNKSR3 and ROBO1, implying the wide adoption of this specific mode during vertebrate development.