PMC4391918_01
Clinical Imaging.. 
A. The pedigree of a family with clinical features of ADNIV did not show a specific pattern of inheritance. Black symbols represent clinically affected subjects. Open symbols represent unaffected subjects. B. A color fundus photo of the proband's (II:1) left macula. C. A fluorescein angiogram reveals CME (arrow) and temporal window defects. D. Fundus autofluorescence showed a central hyperreflective ring in the macula and hypofluorescence temporal to the macula (arrows). E, F. Optical coherence tomography (OCT) shows CME greater in the left eye. G. A color fundus photo of the sister's (II:2) right macula shows a dense epiretinal membrane (ERM, arrow) over the nerve and macula. H, I. OCT shows an ERM and CME OU. (ADNIV, autosomal dominant neovascular inflammatory vitreoretinopathy; CME, cystoid macular edema; OCT, optical coherence tomography; OD, right eye; OS left eye; OU, both eyes.)
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Dilated funduscopic exam showed 2+ vitreous cell OU, 1+ vitreous haze OS with cystoid macular edema OU (Fig 1), but no bone-spicule pigmentation or pigment clumping, and the periphery was well vascularized. Dilated funduscopic examination showed 1+ vitreous cells and a recurrent epiretinal membrane with cystoid macular edema OD (Fig 1). Their father and two uncles were not affected (Fig 1).
The CAPN5 gene harbors a novel mutation in exon-6.. 
A. Chromatogram of proband showed a heterozygous c.750G>T DNA sequence mutation (arrow) in exon 6 of CAPN5. B. Calpain-5 is composed of four domains, and the mutation was located in catalytic domain IIb (green). C. Primary protein sequence alignment of calpain-5 orthologs shows high evolutionary conservation of the new mutated p.Lys250Asn residue, along with the two prior ADNIV mutations, p.Arg243Leu and p.Leu244Pro (red arrow/box) in catalytic domain IIb. The two previously identified ADNIV mutations and the newly identified ADNIV mutation are 2 to 9 amino acids upstream of the histidine catalytic residue (blue box). Amino acid mismatches are color-highlighted. D. Twelve human calpain family paralogs show the p.Lys250Asn is highly conserved. (Black, 100% similarity; dark grey, 80 to 100% similarity; light grey, 60 to 80% similarity; white, less than 60% similarity).
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ADNIV cases, the CAPN5 coding region of the family members was sequenced. A heterozygous guanine to thymine nucleotide variant, resulting in a lysine to asparagine substitution (at genomic position Chr11:77115445, NM_004055.4:c.750G>T, p.Lys250Asn), was found in both affected subjects but none of the unaffected (Fig 2). Among the 13 human paralogs, Lys250 is conserved in seven, and in two other calpains the protein has an identically charged arginine (Fig 2). Interestingly, eight of the nine calpain-5 paralogs with a conserved positively charged residue at the Lys250 position (Lys252 for calpain-9; Fig 2) also have a conserved negatively charged residue (Asp239 for calpain-5, Glu241 for calpain-9) on a short alpha-helix with which it forms a salt bridge (Fig 5).
Protein structure modeling of calpain-5 and ADNIV mutants.. A three dimensional model of the calpain-5 catalytic domain II was generated using calpain-9 (the closest sequence match) as a template. The residues of the catalytic triad (yellow sticks) are located in the catalytic groove (outlined in blue) between domains IIa and IIb. Although the primary sequence (Fig 2) showed the ADNIV Lys250Asn mutation was several residues away from the two known ADNIV mutations, the three-dimensional model reveals all three ADNIV mutations (magenta sticks) are adjacent to one another on a flexible gating loop (green) that regulates enzymatic activity by controlling access to the catalytic groove. This loop samples many conformations, including a short beta sheet harboring the ADNIV residues as shown here. The mutations alter charged or hydrophobic residues.
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Homology modeling to calpain-9 generated a three-dimensional structure for the calpain-5 catalytic core (Fig 3). The two previously identified calpain-5 mutations (p.Arg243Leu, p.Leu244Pro) were modeled as part of a short beta sheet in a flexible gating loop that undergoes calcium-induced conformation changes during enzyme activation (Fig 3). (This location is in contrast to the original homology model that placed these residues in the loop's alpha helix.) This loop is located on the 5-prime side of the catalytic groove and controls access to substrates. In the primary sequence, the prior two ADNIV mutations are separated from the newly identified mutation, but when modeled in three-dimensions, the three mutations lie very near one another (Fig 3).
Structure and modeling of the Calpain-5 gating loop.. The flexible gating loop (colored differently in each panel) can be observed in different conformations across the known structures of the calpain family. The loops shown here are from the crystal structures of calpain-1 (A-E; pdb ids 2ARY, 1ZCM, 1TL9, 1KXR, 1KFX), calpain-2 (F-H; 1U5I, 1MDW, 3DF0), calpain-8 (I; 2NQA), calpain-9 (J, K; 1ZIV, 2P0R) and the homology model of calpain-5 (L). The loop in its most ordered form tends to have a short alpha helix (C, G, I, J, K, L) and a short beta sheet (D, L). Often the loop is disordered and cannot be located in the crystal structure due to lack of sufficient electron density (E, F, H). The catalytic residues are shown in yellow and the residues at the site of the calpain-5 ADNIV mutations are shown in magenta.
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The gating loop has been found in variable conformations in almost all other calpain crystal structures or left unmodeled due to poor electron density, indicating that it is highly flexible (Fig 4). These mutations likely affect the formation of the alpha helix and beta sheet within this gating loop also observed in other calpain crystal structures and the homology model of calpain-5 (Fig 4).
Hydrogen bonding/salt bridge network at the site of p.Lys250Asn ADNIV mutation.. 
A. In the crystal structure of calpain-9, Lys252 forms a salt bridge with Glu241, which in turn hydrogen bonds with the backbone nitrogen of Arg237 and locks the flexible gating loop in place. B. The same set of interactions are observed in the homology model of calpain-5, where Lys250 is well positioned to form a salt bridge with Asp239, which in turn can form a hydrogen bond with the backbone nitrogen of Val235. C. The p.Lys250Asn (purple) mutation modeled here indicates that Asn250, with altered hydrogen bonding capability, will interact differently with Asp239 and affect the conformation and flexibility that this catalytically important gating loop adopts. Changes in the conformation of the gating loop would subsequently alter enzymatic activity.
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Therefore, in the context of the catalytic site formation and maintenance, we believe the homology model based on calpain-9 (Fig 5) provides a better perspective on the effect of all the ADNIV mutations identified so far. Interestingly, eight of the nine calpain-5 paralogs with a conserved positively charged residue at the Lys250 position (Lys252 for calpain-9; Fig 2) also have a conserved negatively charged residue (Asp239 for calpain-5, Glu241 for calpain-9) on a short alpha-helix with which it forms a salt bridge (Fig 5). This Asp/Glu in turn hydrogen bonds with the backbone nitrogen of Val235 (Arg237 for calpain-9; Fig 5), suggesting a hydrogen bond network locks the loop in place. The hydrogen bonding capability of the shorter, polar mutant asparagine is quite different from the longer, positively charged lysine it replaces (Fig 5).