The N- and C-terminal ends of RPGR can bind to PDE6δ.

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 experiment, that FLAG-tagged fragments missing the C-terminal CaaX motif of RPGR fail to coimmunoprecipitate 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 chromatography 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 verified 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 discussion below), we feel confident about the results by Wätzlich et al [3]. Lee and Seo then show that RPGR interacts with the C-terminal end of RPGR which contains a CaaX motif. This finding may not be too surprising since we and others have previously shown that PDE6d is a general prenyl-binding protein that is required to shuttle lipidated proteins between membranes and that the binding is released by Arl2/3•GTP [4–7]. Our earlier structural studies revealed molecular details of the binding of farnesylated peptides/proteins to PDE6d, showing how the farnesyl moiety is inserted into the hydrophobic cavity of PDE6d and that in addition to the farnesyl group, only the last three residues of the farnesylated cargo make contacts with the binding pocket of PDE6d [5]. Zhang et al have shown that geranylgeranylated proteins such as rhodopsin kinase (GRK7) which carries a CaaX motif signifying geranylgeranylation, as does the CTIL motif of RPGR, bind to PDE6d. Also localization of geranylgeranylated ciliary proteins such as the alpha’ subunit of cone phosphodiesterase 6 (PDE6a’) is dependent on PDE6d [6,7]. However, so far no structural information exists that would explain how the PDE6d hydrophobic pocket is able to accommodate an additional five carbon atoms (prenyl group) of a geranylgeranylated peptide. In order to investigate this structural plasticity, we solved the crystal structure of PDE6d in complex with a geranylgeranylated and carboxy-methylated peptide derived from the C-terminus of PDE6a’ at resolution of 2.1 Å. This peptide resembles the RPGR C-terminal sequence. Solid phase synthesis of the PDE6a’ peptide (DDKKSKT-C(gerger)-OMe) was carried out on an Activotec P-11 peptide synthesizer, on H-Cys(Trt)-ClTrt resin, using N-Fmoc amino acids and HCTU as the coupling reagent (Merck Chemicals). The N-terminal Asp was incorporated as Boc-Asp (OtBu)-OH. Methylation was as previously described [8]. Following chain assembly and methylation, the peptidyl resin was cleaved, purified, geranylgeranylated, and purified as previously described [9]. The geranylgeranylated peptide from PDE6a’ was dissolved in DMSO and mixed with PDE6d at 1:1 molar ratio with final Table 1. Data collection and refinement statistics (molecular replacement).