Structural and functional comparison of fumarylacetoacetate domain containing protein 1 in human and mouse

Abstract

FAH domain containing protein 1 (FAHD1) is a mammalian mitochondrial protein, displaying bifunctionality as acylpyruvate hydrolase (ApH) and oxaloacetate decarboxylase (ODx) activity. We report the crystal structure of mouse FAHD1 and structural mapping of the active site of mouse FAHD1. Despite high structural similarity with human FAHD1, a rabbit monoclonal antibody (RabMab) could be produced that is able to recognize mouse FAHD1, but not the human form, whereas a polyclonal antibody recognized both proteins. Epitope mapping in combination with our deposited crystal structures revealed that the epitope overlaps with a reported SIRT3 deacetylation site in mouse FAHD1.culture was grown in 1000 ml NZCYM medium, containing the respective selective antibiotics. At 37 • C the bacteria were amplified to an optical density of 0.4 at 600 nm. Protein expression was induced by the addition of 500 μM isopropyl-1-thio-β-d-galactopyranoside (IPTG) and incubation was continued for 4 h at 37 • C. Bacteria were harvested via centrifugation and stored at −70 • C. Enzyme purificationEnzyme purification of His6/S-double-tagged proteins followed a defined protocol [7]. Recombinant protein was extracted via a three-step purification strategy involving metal affinity chromatography (Ni-NTA), anion exchange chromatography, and gel filtration (SEC). Fractions containing FAHD1 were pooled, concentrated and stored at −70 • C. Gel electrophoresis of two preparations followed by silver staining verified the protein's homogeneity; contaminations were barely visible (<<100 ng/μl).Recombinant mouse FAHD1 (mFAHD1) of concentration in between 1.5 and 2.0 mg/ml (Vivaspin 10 MWCO) was sent for high throughput screening at the HTX laboratory (EMBL, Grenoble). Native mFAHD1 and mFAHD1 supplemented with 1 mM oxalate were screened for crystallization, and hits were obtained in various conditions. A total of 68 crystals were harvested by laser photoablation, cryo-cooled using the CrystalDirect Robot [8,9], and screened for diffraction. Best diffracting crystals of mFAHD1 grew from 25% (w/v) PEG 3350, 0.2 M MgCl 2 and 0.1 M Bis/Tris pH 5.5 and for the oxalate bound protein from 20% (w/v) PEG 4000, 0.05 M MgCl 2 and 0.1 M MES pH 5.5. As the crystals were small needles with volumes between 10 −6 and 10 −5 mm 3 a beam diameter of 15 μm was selected [10]. X-ray diffraction data were collected by the autonomous European Synchrotron Radiation Facility (ESRF) beamline using automatic protocols for the location and optimal centring of crystals [14]. Strategy calculations accounted for flux and crystal volume in the parameter prediction for complete datasets. All data were processed using the automatic pipelines at the ESRF [15]. Statistical analysisKinetic profiles of recombinant protein were obtained with three independent batches of protein preparations (n=3). Data analysis was performed using GraphPad Prism version 5 (www.graphpad.com). Presented data are the mean and standard deviation within 95% confidence interval.

Regulation of cellular senescence by eukaryotic members of the FAH superfamily – A role in calcium homeostasis?

“…A putative interaction with FAHD1 may complement our recently hypothesized model of senescence ( Etemad et al, 2019 ) due to the inactivation of genes required for mitochondrial function (such as SIRT3 ( Hallows et al, 2011 ) and FAHD1 ( Etemad et al, 2019 )), thus explaining how in some cellular models the inactivation of either ETC complex I (by metformin) or ETC complex II (by FAHD1 knockdown) has the potential to increase p21 gene expression in the absence of AMPK ( Etemad et al, 2019 ). In agreement with results obtained from a high-throughput proteomics study ( Dittenhafer-Reed et al, 2015 ), we recently provided circumstantial evidence for a SIRT3 deacetylation site ( Dittenhafer-Reed et al, 2015 ) in mouse FAHD1 ( Weiss et al, 2020 ), which further supports this model.…”

Inhibitors of Fumarylacetoacetate Hydrolase Domain Containing Protein 1 (FAHD1)

“…The information obtained by comparing the unliganded with the liganded protein models enabled the structural definition of the FAHD1 catalytic center in high resolution. X-ray data suggests a bidentate-binding mode of the 1,2-dicarbonyl motif of the inhibitor to the Mg cofactor and provides a rationale for closure of the lid domain and induction of a catalytically competent dyad (His30-Glu33) [ 7 , 8 ]. However, the structure model of FAHD1 liganded by oxalate does not tell us which residues are key in the enzymatic process, and the essential residues involved in driving the enzymatic reaction remain to be elucidated.…”