Table 1.
Data collection and refinement statistics.
Fig 1.
(A) Schematic representation of the domain architecture of human NHLRC2. (B) Ribbon and surface representation of NHLRC2 (9–572) crystal structure with the Trx-like domain colored in cyan and the β-propeller colored in gold. Residues of CCINC motif are shown. Cysteine residues are shown in ball and stick representation and neighboring residues are shown in stick representation. The missing loop connecting the two domains is shown with a dashed line. (C) Two views of NHLRC2 β-propeller are related by 90° rotation around x-axis. Blades are indicated by numbers and strand location is indicated by capital letters. (D) The solvent channel inside of the β-propeller domain contains water molecules (red) and is disrupted by Lys-228 (blue) and Asp-340 (cyan) forming hydrogen bonds with waters and backbone groups of adjacent blades.
Fig 2.
SEC-MALS analysis of NHLRC2 domains interactions and low resolution structure of NHLRC2 determined by SAXS.
(A) SEC-MALS traces (with superimposed calculated molar mass traces) of the Trx-like domain (pink, 25.1 kDa; calculated MW 25.8 kDa), the β-propeller domain (red, 35.7 kDa; calculated MW 39 kDa), the full-length NHLRC2 (blue, 69.4 kDa; calculated MW 80.5), NHLRC2 treated with proteinase GluC (green, 59 kDa and 15 kDa; calculated MW 64.1 kDa and 15.3 kDa, respectively) and co-incubated Trx-like and β-propeller domains (black, 35.2–24.8 kDa). (B) The ab initio model of the full-length NHLRC2 was reconstructed using DAMMIF and shown is the averaged filtered shape from DAMFILT. The model is superimposed with NHLRC2 (9–572) crystal structure.
Fig 3.
Surface properties of NHLRC2 (9–572).
The view is equivalent to that in Fig 1B. (A) Hydrophobic residues are colored in purple. (B) Electrostatic surface potential was calculated using APBS. Negative potential is indicated in red and positive in blue.
Fig 4.
Sequence alignment of NHLRC2 homologs from animalia.
(A) The fully conserved residues are highlighted in red. CCINC motif is indicated by arrowheads and Asp-148 by an asterisk. Secondary structure in the alignment is assigned based on the crystal structure. Mammal sequence is equivalent to human NHLRC2 (UniProt ID: Q8NBF2), bird is Zebra finch (UniProt ID: H0ZKU8), reptile is American chameleon (UniProt ID: H9GFX6), fish is Southern platyfish (UniProt ID: M4A111), mollusk is Pacific oyster (UniProt ID: K1Q646), insect is Red fire ant (UniProt ID: E9IZN5), cnidaria is Sea anemone (UniProt ID: A7RH97), sponge (UniProt ID: A0A1X7VMK9). (B) The majority of conserved residues locate to the cleft between the Trx-like and β-propeller domains. The fully conserved residues are mapped on NHLRC2 (9–572) crystal structure and shown in stick representation in red color.
Fig 5.
NHLRC2 is present across all kingdoms of life.
(A) Trx-like and β-propeller domain sequences from 16 representatives are aligned (the full sequence alignment is in the supplementary material) and conserved residues are indicated in human NHLRC2 (1–590) sequence in red color. The consensus sequence derived from 16 eukaryotic and prokaryotic representatives is provided. CC(I/V)NC motif is indicated by arrowheads and Asp-148 by an asterisk. (B) Domain architecture of NHLRC2 in mammals, plants and bacteria. Plant orthologs contain a transmembrane helix (shown in red) in between HAD and Trx-like domain. Note a small number of bacterial sequences show the mammalian domain architecture. (C) Conserved residues (shown in red) identified in eukaryotic and bacterial orthologs are mapped in human NHLRC2 (9–572) structure.
Fig 6.
D148Y mutation of NHLRC2 causing FINCA disease.
(A) D148 (in red) location in the Trx-like domain of NHLRC2. The CCINC motif is also shown. (B) Close up view of D148 environment in the structure of NHLRC2. The model is colored similarly to that in Fig 1. D148 is shown in ball and stick representation and the neighboring residues are shown in stick representation. Potential hydrogen bonds are shown with black lines. (C) The folding state of the full-length human NHLRC2 and D148Y mutant was assessed by circular dichroism measurements (CD). Visual inspection of the CD spectra indicates that the wild-type NHLRC2 (solid black) and D148Y (grey circles) mutant are folded and contain a mixture of α-helices and β-strands. Both spectra are of similar shape indicating a similar amount of regular secondary structure. (D) The thermal stability of the proteins was analyzed by Thermofluor. Wild-type full-length NHLRC2 (solid black) demonstrated a sharp unfolding transition with average Tm of 61.5°C and the analysis of derivative showed a cooperative unfolding event. The D148Y mutation (grey circles) resulted in a lower Tm of 59.3°C, however, the analysis of derivative still demonstrated a cooperative unfolding, suggesting that the mutation caused small local changes in the protein structure and stability.