Morbiato 2016 Abstract Mito Xmas Meeting Innsbruck

Purification of and characterization of human recombinant COQ4 and its putative Zn binding site mutants.

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Morbiato L, Tonello F, Berto P, Zanotti G, Desbats MA, Salviati L (2016)

Event: Mito Xmas Meeting 2016 Innsbruck AT

Defects in genes involved in coenzyme Q (CoQ) biosynthesis cause primary CoQ deficiency, a clinically heterogeneous disorder with clinical manifestation ranging from fatal neonatal multisytem disorders to adult-onset isolated encephalopathy or nephropathy.

COQ4 codes for an ubiquitously expressed 265 amino acid protein that is peripherally associated with the mitochondrial inner membrane on the matrix side [1,3]; the precise function of human COQ4 is not known, but the yeast ortholog Coq4p seems to play a structural role crucial in the stabilization of a multiheteromeric complex including several, if not all, of the CoQ biosynthetic enzymes [1]. It has also been proposed that yeast Coq4p could be a zinc protease, based on the presence of the zinc binding motif HDxxH [2]. In this view the protein of interest could also play a role in the maturation of other COQ polypeptides.

At the beginning the protein (devoid of the N-terminal mitochondrial targeting sequence) was expressed in E. Coli attached to a 6 His-Tag and a consensus for enterokinase (DDDK). We purified the protein using a Ni affinity cromatografy on an AKTA-FPLC and a gel filtration chromatography, in order to increase the purity. The yield and the purity of recombinant human COQ4 were satisfactory. Biochemical characterization of the protein was carried out using different approaches like: circular dichroism (CD), analysis of zinc content, blue native gel, and electron microscopy.

We found by CD that COQ4 binds zinc, increasing the stability of the recombinant protein. We observed both by BN-PAGE and by gel filtration that our protein forms a high molecular weight complex, a multimeric complex comprised of 5-6 monomers. We would also like to investigate the effective role of the putative Zn binding motif HDxxH; therefore we are going to purify and characterize 3 COQ4 mutants: H155A, D156A, H159A.

Altogether these data provide new insights on the role of COQ4 in CoQ biogenesis. Further work will be aimed at obtaining crystals in order to solve the three-dimensional structure of the protein.

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Event: Poster 

Affiliations

Morbiato L, Tonello F, Berto P, Zanotti G, Desbats MA, Salviati L

1. Dept Biology, Univ Padua, Italy

 

Reference

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2. Marbois B, Gin P, Gulmezian M, Catherine F, Clark CF (2008) The yeast Coq4 polypeptide organizes a mitochondrial protein complex essential for coenzyme Q biosynthesis. BBA 1791:69-75.
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