Structural analysis of human NHLRC2, mutations of which are associated with FINCA disease

Abstract

NHLRC2 (NHL repeat-containing protein 2) is an essential protein. Mutations of NHLRC2, including Asp148Tyr, have been recently associated with a novel FINCA disease (fibrosis, neurodegeneration, cerebral angiomatosis), which is fatal in early childhood. To gain insight into the mechanisms of action of this essential protein, we determined the crystal structure of the Trx-like and NHL repeat β-propeller domains of human NHLRC2 to a resolution of 2.7 Å. The structure reveals two domains adjacent to each other that form a cleft containing a conserved CCINC motif. A SAXS structure of full-length NHLRC2 reveals that the non-conserved C-terminal domain does not pack against the N-terminal domains. Analysis of the surface properties of the protein identifies an extended negative electrostatic potential in the surface of the cleft formed by the two domains, which likely forms a binding site for a ligand or interaction partner(s). Bioinformatics analysis discovers homologs across a range of eukaryotic and prokaryotic species and conserved residues map mostly to the adjacent surfaces of the Trx-like and β-propeller domains that form the cleft, suggesting both that this forms the potential functional site of NHLRC2 and that the function is conserved across species. Asp148 is located in the Trx-like domain and is not conserved across species. The Asp148Tyr mutation destabilizes the structure of the protein by 2°C. The NHLRC2 structure, the first of any of its homologs, provides an important step towards more focused structure-function studies of this essential protein.

A Genome-Wide Knockout Screen in Human Macrophages Identified Host Factors Modulating Salmonella Infection

“…Whole-exome sequencing (WES) of samples from children with FINCA (fibrosis neurodegeration cerebral angiomatosis) identified mutations in NHLRC2 (compound heterozygous mutations p.Asp148Tyr and p.Arg201GlyfsTer6 [37]) associated with fatality from FINCA in early childhood. The NHLRC2 gene is present across all kingdoms of life from mammals to prokaryotes, and its sequences are highly conserved across evolution (38).…”

Extension of the taxonomic coverage of the family GH126 outside Firmicutes and in silico characterization of its non-catalytic terminal domains

“…While the Rv2874 motif Cys437-Ile-Asn-Cys440 (Goldstone et al 2016 ) has no correspondence in any of the three GH126 proteins from C. butyricum , the equivalent motif Cys103-Pro-Asp-Cys106 is present in the potential family GH126 member from B. xylanolyticus (not shown) indicating the function might have been preserved in this protein. The two further reliable structural templates for the thioredoxin-like fold in the family GH126 members have been identified in the human NHL repeat-containing protein 2 (Biterova et al 2018 ) and the mouse selenocysteine-dependent iodothyronine deiodinase (Schweizer et al 2014 ).…”

“…xylanolyticus (not shown) indicating the function might have been preserved in this protein. The two further reliable structural templates for the thioredoxin-like fold in the family GH126 members have been identified in the human NHL repeat-containing protein 2 (Biterova et al 2018) and the mouse selenocysteine-dependent iodothyronine deiodinase (Schweizer et al 2014).…”

Variant in NHLRC2 leads to increased hnRNP C2 in developing neurons and the hippocampus of a mouse model of FINCA disease

“…NHLRC2 consists of an N-terminal thioredoxin (Trx)-like domain, a six-bladed β-propeller domain, and a C-terminal β-stranded region (Biterova et al 2018 ). Structural analysis of the protein has revealed a highly conserved cleft between the Trx-like and β-propeller domains that forms a possible binding site for currently unknown substrates or interaction partners (Biterova et al 2018 ). Recent in vitro studies have shed some light on the possible functions of NHLRC2.…”

“…The mouse ortholog of human NHLRC2 has 84% protein sequence similarity (Uusimaa et al 2018 ), and it has been proposed that the function of NHLRC2 is conserved across species (Biterova et al 2018 ). The complete loss of Nhlrc2 leads to early embryonic lethality, highlighting its essential role in embryonic development (Uusimaa et al 2018 ; Perez-Garcia et al 2018 ; Delhotal 2016 ).…”