Stapled Voltage-Gated Calcium Channel (Ca_V) α-Interaction Domain (AID) Peptides Act As Selective Protein–Protein Interaction Inhibitors of Ca_V Function

Abstract

For many voltage-gated ion channels (VGICs), creation of a properly functioning ion channel requires the formation of specific protein–protein interactions between the transmembrane pore-forming subunits and cystoplasmic accessory subunits. Despite the importance of such protein–protein interactions in VGIC function and assembly, their potential as sites for VGIC modulator development has been largely overlooked. Here, we develop meta-xylyl (m-xylyl) stapled peptides that target a prototypic VGIC high affinity protein–protein interaction, the interaction between the voltage-gated calcium channel (CaV) pore-forming subunit α-interaction domain (AID) and cytoplasmic β-subunit (CaVβ). We show using circular dichroism spectroscopy, X-ray crystallography, and isothermal titration calorimetry that the m-xylyl staples enhance AID helix formation are structurally compatible with native-like AID:CaVβ interactions and reduce the entropic penalty associated with AID binding to CaVβ. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the CaVα1:CaVβ interaction that modulate CaV function in an CaVβ isoform-selective manner. Together, our studies provide a proof-of-concept demonstration of the use of protein–protein interaction inhibitors to control VGIC function and point to strategies for improved AID-based CaV modulator design.

N‐locking stabilization of covalent helical peptides: Application to Bfl‐1 antagonists

“…These strategies allowed the stabilization of short alpha-helical peptides that in turn (and in principle) render them more cell permeable and resistant to proteases in plasma (Bird, Gavathiotis, LaBelle, Katz, & Walensky, 2014;Cohen et al, 2012;Henchey et al, 2010;LaBelle et al, 2012;Wang et al, 2005). Several studies have been proposed with the hydrocarbon-stapled and lactam-stabilized helices over the past decade Cohen et al, 2012;Findeisen et al, 2017;Huhn et al, 2016;LaBelle et al, 2012;H. Wu, Acharyya, et al, 2018).…”

Translocatable voltage-gated Ca2+channel β subunits in α1–β complexes reveal competitive replacement yet no spontaneous dissociation

“…The apparent α1-β binding affinity depends on the molecular properties of β isoforms; thus, the α1C-β1a complex is more stable than α1C-β2a or α1C-β4 in intact skeletal muscle cells (26). Similarly, intracellular perfusion of degradation-protected AID peptides blocked the interaction of Ca V 1.2 channels with β3 subunits, but not with β2a, indicating that the Ca V 1.2-β3 complex is less stable than the Ca V 1.2-β2a complex inside cells (27). In contrast, in excised inside-out patches, the β2a subunit remains stably bound to the core domains of Ca V 2.1 channels unless the binding affinity of Ca V β GK domain is reduced by mutating the α-binding pocket (ABP) site (28).…”

Design of a Short Thermally Stable α‐Helix Embedded in a Macrocycle

“…For comparison, we also synthesized N-capand C-cap-stabilized macrocyclic peptides (Figure 2A) according to a previously described strategy. [16] In these peptides, key capping residues were retained as in CSP1 and CSP2 , but the macrocyclic link was formed to a side chain near the N or C terminus of the helix, rather than encompassing the entire helix. In each case, the peptides were cyclized by treating the precursor with a bis-benzylic bromide to provide the bis-thioether macrocycle in good to excellent yields.…”