Disordered Peptides Looking for Their Native Environment: Structural Basis of CB1 Endocannabinoid Receptor Binding to Pepcans

Abstract

Endocannabinoid peptides, or "pepcans," are endogenous ligands of the CB1 cannabinoid receptor. Depending on their length, they display diverse activity: For instance, the nona-peptide Pepcan-9, also known as hemopressin, is a powerful inhibitor of CB1, whereas the longer variant Pepcan-12, which extends by only three amino acid residues at the N-terminus, acts on both CB1 and CB2 as an allosteric modulator, although with diverse effects. Despite active research on their pharmacological applications, very little is known about structure-activity relationships of pepcans. Different structures have been proposed for the nona-peptide, which has also been reported to form fibrillar aggregates. This might have affected the outcome and reproducibility of bioactivity studies. In an attempt of elucidating the determinants of both biological activity and aggregation propensity of Pepcan-9 and Pepcan-12, we have performed their structure characterization in solvent systems characterized by different polarity and pH. We have found that, while disordered in aqueous environment, both peptides display helical structure in less polar environment, mimicking the proteic receptor milieu. In the case of Pepcan-9, this structure is fully consistent with the observed modulation of the CB1. For Pepcan-12, whose allosteric binding site is still unknown, the presented structure is compatible with the binding at one of the previously proposed allosteric sites on CB1. These findings open the way to structure-driven design of selective peptide modulators of CB1.

Keywords: CB1 endocannabinoid receptor; endocannabinoid system; hemopressin; intrinsically unfolded peptides; pepcans; structure-activity relationships.

Figure 1

CD spectra of Pepcan-12 in different HFIP/ NaP mixtures, pH 7.4. The appearance of a signal diagnostic of helical secondary structure is visible in the presence of as little as 10% HFIP.

Figure 2

Molar ellipticity per residue measured at 222 nm (canonical minimum of α-helix conformation) as function of HFIP content in the mixture, for all conditions studied.

Figure 3

Backbone overlay of the 10 lowest energy NMR-conformations of Pepcan-9 (A) and Pepcan-12 (B) in 50/50 v/v HFIP/NaP, pH 3.0, and Pepcan-9 (C) and Pepcan-12 (D) in 50/50 v/v HFIP/NaP, pH 7.4.

Figure 4

Superimposition of the structures of (A) Pepcan-9 (orange) and (B) Pepcan-12 (blue) on the α-chain of hemoglobin (PDB code 2DN1).

Figure 5

Binding of (A) Pepcan-9 (pink) and (B) taranabant (purple) to the CB1 receptor (grey ribbon) (PDB code 5U09). The residues of the receptor in close proximity to the ligand are displayed as blue sticks.

Figure 6

Possible binding modes of Pepcan-12 (yellow and green surfaces) to the CB1 receptor (blue ribbon) (PDB code 5U09). The orthosteric binding site is highlighted by the presence of a taranabant molecule (purple stick and surface).