The N‐ and C‐terminal ends of RPGR can bind to PDE 6δ

Abstract

L ee and Seo propose in their article [1] that RPGR binds to PDE6d not with the N-terminal RCC1-like propeller domain but solely with the C-terminus. They show, using an immunoprecipitation experi-ment, that FLAG-tagged fragments missing the C-terminal CaaX motif of RPGR fail to co-immunoprecipitate together with myc-tagged PDE6d. We have previously shown that the N-terminal 400 residues of RPGR form a stable RCC1-like propeller domain and that this protein forms a complex with PDE6d. This can be demonstrated by (untagged) pull-down and gel permeation chromato-graphy experiments. Additionally, the equilibrium dissociation constant was determined to be 500 nM by fluorescence polarization which also agrees with previous results published by Linari et al in which they report the affinity of RPGR (aa 1–392), using surface plasmon resonance, to PDE6d to be 100 nM [2,3]. Finally, we have solved the structure of the complex PDE6d–RPGR (aa 8–368) by X-ray crystallography and veri-fied the interaction interface by mutational analysis [3]. Since immunoprecipitation experiments reflect dissociation kinetics rates rather than equilibrium dissociation, which are in turn very much dependent on many aspects of the experiments (see discus-sion below), we feel confident about the results by Wätzlich et al [3].