title: Small-angle X-ray scattering of calpain-5 reveals a highly open conformation among calpains.
abstract: Calpain-5 is a calcium-activated protease expressed in the retina. Mutations in calpain-5 cause autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM#193235). The structure of calpain-5 has not been determined, thus hindering the investigation of its proteolytic targets and pathological role in ADNIV. Herein, we report models of the proteolytic core of calpain-5 (mini-calpain-5) containing two globular domains (termed DIIa-IIb) connected by a short, flexible linker, consistent with small-angle X-ray scattering (SAXS) data. Structural modeling in the absence of calcium suggests that mini-calpain-5 adopts a more open conformation when compared to previously determined structures of other calpain cores. This open conformation, achieved by a rotation of DIIa and DIIb with respect to each other, prevents formation of the active site and constrains the enzyme in an inactivated form. The relative domain rotation of 60-100° we found for mini-calpain-5 (a non-classical calpain) is significantly greater than the largest rotation previously observed for a classical calpain (i.e., 55.0° for mini-calpain-9). Together with our prediction that, in the full-length form, a long loop in DIIb (loop C1), a few residues downstream of the inter-domain linker, likely interacts with the shorter, acidic, inactivating loop on domain-III (DIII), these structural insights illuminate the complexity of calpain regulation. Moreover, our studies argue that pursuing higher resolution structural studies are necessary to understand the complex activity regulation prevalent in the calpain family and for the design of specific calpain inhibitors.
authors.affiliation: Department of Biochemistry, University of Iowa, Iowa City, IA, USA; Protein Crystallography Facility, University of Iowa, Iowa City, IA, USA.
authors.forename: Lokesh
authors.initials: L
authors.lastname: Gakhar
authors.affiliation: Department of Pediatrics, University of Iowa, Iowa City, IA, USA; Omics Lab, University of Iowa, Iowa City, IA, USA.
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Bassuk
authors.affiliation: Omics Lab, University of Iowa, Iowa City, IA, USA; Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, USA; Medical Scientist Training Program, University of Iowa, Iowa City, IA, USA.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Velez
authors.affiliation: Protein Crystallography Facility, University of Iowa, Iowa City, IA, USA.
authors.forename: Saif
authors.initials: S
authors.lastname: Khan
authors.affiliation: Protein Crystallography Facility, University of Iowa, Iowa City, IA, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.affiliation: Barbara and Donald Jonas Laboratory of Stem Cells and Regenerative Medicine and Bernard & Shirlee Brown Glaucoma Laboratory, Edward S. Harkness Eye Institute, Columbia University, New York, NY, USA; Department of Pathology & Cell Biology, College of Physicians & Surgeons, Columbia University, New York, NY, USA.
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Tsang
authors.affiliation: Omics Lab, University of Iowa, Iowa City, IA, USA; Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, USA. Electronic address: mahajanlab@gmail.com.
authors.forename: Vinit B
authors.initials: VB
authors.lastname: Mahajan
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D002154:Calpain / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D020134:Catalytic Domain; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012160:Retina / Q000737:chemistry* / Q000473:pathology; D053838:Scattering, Small Angle; D018630:Vitreoretinopathy, Proliferative / Q000235:genetics / Q000473:pathology; D014965:X-Rays
chemical_list: D002154:Calpain; C494426:Capn5 protein, human; D002118:Calcium
keywords: ADNIV; CAPN5; Protease; SAXS; Uveitis