In silico interaction analysis of cannabinoid receptor interacting protein 1b (CRIP1b) - CB1 cannabinoid receptor

Abstract

Cannabinoid Receptor Interacting Protein isoform 1b (CRIP1b) is known to interact with the CB₁ receptor. Alternative splicing of the CNRIP1 gene produces CRIP1a and CRIP1b with a difference in the third exon only. Exons 1 and 2 encode for a functional domain in both proteins. CRIP1a is involved in regulating CB₁ receptor internalization, but the function of CRIP1b is not very well characterized. Since there are significant identities in functional domains of these proteins, CRIP1b is a potential target for drug discovery. We report here predicted structure of CRIP1b followed by its interaction analysis with CB₁ receptor by in-silico methods A number of complementary computational techniques, including, homology modeling, ab-initio and protein threading, were applied to generate three-dimensional molecular models for CRIP1b. The computed model of CRIP1b was refined, followed by docking with C terminus of CB₁ receptor to generate a model for the CRIP1b- CB₁ receptor interaction. The structure of CRIP1b obtained by homology modelling using RHO_GDI-2 as template is a sandwich fold structure having beta sheets connected by loops, similar to predicted CRIP1a structure. The best scoring refined model of CRIP1b in complex with the CB₁ receptor C terminus peptide showed favourable polar interactions. The overall binding pocket of CRIP1b was found to be overlapping to that of CRIP1a. The Arg82 and Cys126 of CRIP1b are involved in the majority of hydrogen bond interactions with the CB₁ receptor and are possible key residues required for interactions between the CB₁ receptor and CRIP1b.

Keywords: CB(1) Receptor; CRIP1a; CRIP1b; Coupled receptor; G Protein; Molecular modeling.

Figure 1

Schematic outline of the general workflow for CRIP1b modeling and its interaction analysis withCB₁ receptor.

Figure 2

The de novo model of peptide from distal tail of CB₁ receptor C-terminus

Figure 3

The secondary structure of CRIP1b predicted by various protocols, the consensus of results was taken into account to estimate secondary structural content in CRIP1b. The beta sheets are represented by ‘E’ and alpha helices by ‘H’.

Figure 4. Sequence alignment of Rho-GDI 2/1DS6_B (template) and CRIP1b sequence

The ‘1DS6_b(PDB)’ represents secondary structure of Rho-GDI2 in PDB, and consensus secondary structure represents predicted secondary structure of CRIP1b.

Figure 5

The CRIP1b model (A) and its alignment with the template Rho-GDI 2, cyan colored (B)

Figure 6. Docked poses of CB1 receptor C terminus peptide on CRIP1b model

Docking studies yielded various docked complexes with peptide on distinct positions. All the poses were refined and scored. On the left side of figure, the colored spheres show the positions of peptide on CRIP1b in different poses. While on the right side, binding pockets of top scoring poses from three different clusters are written with differently colored residue fonts. Also, the chemical properties of residues are represented by oval background of different colors i.e., negative charged by pink, positive charged by violet, hydrophobic by green, polar by light blue and glycine by cream.

Figure 7. Interaction between CRIP1b (wheat colored) and the CB₁-receptor 9-mer peptide (magenta stick model)

The CRIP1b residues involved in the binding domain are represented by wheat colored sticks; those involved in hydrogen bonds are connected to the CB₁ receptor peptide by blue dashed lines.

Figure 8. Binding pockets of CRIP1a (green) and CRIP1b (cyan) for CB₁ receptor peptide (magenta)

The above portion of image represents positions of CB₁ receptor peptide on CRIP1a and 1b, while lower portion shows their respective binding pockets.