Krysztofinska, E. | bioRxiv Science

bioRxiv2021

Mapping of a N-terminal α-helix domain required for human PINK1 stabilisation, Serine228 autophosphorylation and activation in cells

Human autosomal recessive mutations in the PINK1 gene are causal for Parkinsons disease (PD). PINK1 encodes a mitochondrial localised protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function, however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain are unknown. We have employed mutagenesis studies of human PINK1 in cells to define the minimal region of PINK1, required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Bioinformatic analysis of the region spanning Ile111 to the kinase domain and inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal -helical domain extension (NTE domain) within this region corroborated by hydrogen/deuterium exchange mass spectrometry (HDX-MS) of recombinant insect PINK1 protein. The AlphaFold structure also predicts the NTE domain forms an intramolecular interaction with the C-terminal extension (CTE). Cell-based analysis of human PINK1 reveals that PD-associated mutations (e.g. Q126P), located within the NTE:CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228); and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE domain mutants do not affect intrinsic catalytic kinase activity but do disrupt PINK1 stabilisation at the mitochondrial Translocase of outer membrane (TOM) complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE:CTE interface towards PINK1 stabilisation and activation and show that loss of NTE:CTE interactions is a major mechanism of PINK1-associated mutations linked to PD.

biochemistry↗

bioRxiv2018

Genetic, structural, and functional analysis of mutations causing methylmalonyl-CoA epimerase deficiency

Human methylmalonyl-CoA epimerase (MCEE) catalyzes the interconversion of D-methylmalonyl-CoA and L-methylmalonyl-CoA in propionate catabolism. Autosomal recessive mutations in MCEE reportedly cause methylmalonic aciduria (MMAuria) in eleven patients. We investigated a cohort of 150 individuals suffering from MMAuria of unknown origin, identifying ten new patients with mutations in MCEE. Nine patients were homozygous for the known nonsense mutation p.Arg47* (c.139C>T), and one for the novel missense mutation p.Ile53Arg (c.158T>G). To understand better the molecular basis of MCEE deficiency, we mapped p.Ile53Arg, and two previously described patient mutations p.Lys60Gln and p.Arg143Cys, onto our 1.8 [A] structure of wild-type (wt) human MCEE. This revealed potential dimeric assembly disruption by p.Ile53Arg, but no clear defects from p.Lys60Gln or p.Arg143Cys. Functional analysis of MCEE-Ile53Arg expressed in a bacterial recombinant system as well as patient-derived fibroblasts revealed nearly undetectable soluble protein levels, defective globular protein behavior, and using a newly developed assay, lack of enzymatic activity - consistent with misfolded protein. By contrast, soluble protein levels, unfolding characteristics and activity of MCEE-Lys60Gln were comparable to wt, leaving unclear how this mutation may cause disease. MCEE-Arg143Cys was detectable at comparable levels to wt MCEE, but had slightly altered unfolding kinetics and greatly reduced activity. We solved the structure of MCEE-Arg143Cys to 1.9 [A] and found significant disruption of two important loop structures, potentially impacting surface features as well as the active-site pocket. These studies reveal ten new patients with MCEE deficiency and rationalize misfolding and loss of activity as molecular defects in MCEE-type MMAuria.

biochemistry↗