Structural and Biophysical Analysis of the Calcium-Activated Chloride Channel Regulator 1 (CLCA1) VWA Domain Suggests Mode of TMEM16A Engagement

Abstract

The secreted protein calcium-activated chloride channel regulator 1 (CLCA1) utilizes a von Willebrand Factor type A domain (VWA) to bind to and potentiate the calcium activated chloride channel TMEM16A. To gain insight into this unique potentiation mechanism, we determined the 2.0 Å crystal structure of human CLCA1 VWA bound to Ca²⁺, the first CLCA family structure reported. The structure reveals the metal ion dependent adhesion site (MIDAS) in a high-affinity "open" conformation, engaging in crystal contacts that likely mimic how CLCA1 engages TMEM16A. The CLCA1 VWA contains a disulfide bond between α3 and α4 in close proximity to the MIDAS that is invariant in the CLCA family and unique in VWA structures. Further biophysical studies indicate that CLCA1 VWA is preferably stabilized by Mg²⁺ over Ca²⁺ and that a6 atypically extends from the VWA core. Finally, an analysis of TMEM16A structures suggests residues likely to mediate interaction with CLCA1 VWA.

Keywords: von Willebrand factor A (VWA) domain, metal ion dependent adhesion site (MIDAS) motif, crystal structure, TMEM16A, calcium-activated chloride channel, small-angle X-ray scattering (SAXS), calcium-activated chloride channel regulator (CLCA), cystic fibrosis, airway disease