Compared effects of missense mutations in Very-Long-Chain Acyl-CoA Dehydrogenase deficiency: Combined analysis by structural, functional and pharmacological approaches

Abstract

Very-Long-Chain Acyl-CoA Dehydrogenase deficiency (VLCADD) is an autosomal recessive disorder considered as one of the more common ss-oxidation defects, possibly associated with neonatal cardiomyopathy, infantile hepatic coma, or adult-onset myopathy. Numerous gene missense mutations have been described in these VLCADD phenotypes, but only few of them have been structurally and functionally analyzed, and the molecular basis of disease variability is still poorly understood. To address this question, we first analyzed fourteen disease-causing amino acid changes using the recently described crystal structure of VLCAD. The predicted effects varied from the replacement of amino acid residues lining the substrate binding cavity, involved in holoenzyme-FAD interactions or in enzyme dimerisation, predicted to have severe functional consequences, up to amino acid substitutions outside key enzyme domains or lying on near enzyme surface, with predicted milder consequences. These data were combined with functional analysis of residual fatty acid oxidation (FAO) and VLCAD protein levels in patient cells harboring these mutations, before and after pharmacological stimulation by bezafibrate. Mutations identified as detrimental to the protein structure in the 3-D model were generally associated to profound FAO and VLCAD protein deficiencies in the patient cells, however, some mutations affecting FAD binding or monomer-monomer interactions allowed a partial response to bezafibrate. On the other hand, bezafibrate restored near-normal FAO rates in some mutations predicted to have milder consequences on enzyme structure. Overall, combination of structural, biochemical, and pharmacological analysis allowed assessment of the relative severity of individual mutations, with possible applications for disease management and therapeutic approach.

Figure 1. Ribbon diagram of the human VLCAD dimer (PDB 3B96) with various mutation sites

Each subunit is represented in gray or blue. Stick representations of the FAD cofactors have CPK coloring with yellow carbons. Mutation sites are shown in only one subunit.

Figure 2. Comparison of mild and severe VLCAD human mutation sites

Wild-type amino acid residues (magenta) are overlayed with the corresponding residues in the clinical variant (cyan). Potential steric hindrances are depicted with red dashes. The cartoon carbon backbone representation is colored either grey or blue depending on the location of the mutation site in Figure 1. A) The severe mutation R453Q would most certainly directly affect substrate binding and catalysis. The arginine to glutamine mutation would break a salt bridge between residue Arg453 on helix J and Asp123 on helix A. The positioning of the catalytic glutamate (Glu462, not shown in the Figure), which is on a loop between helices J and K, would likely be affected by the loss of this salt bridge. B) The severe clinical variant R286G is also not near FAD or substrate binding. However, the mutation would lead to the breaking of hydrogen bonds with the main chain carbonyl oxygens of Glu253 and Ala256. This would lead to major tertiary structural rearrangement and protein instability. C) V174M (mild) mutation site. No hydrogen bonds would be broken by this mutation. However, potential steric hindrances with Thr132 and Leu136 would lead to some structural instability. D) The A304T (mild) mutation site also results potentially in only minor steric hindrance. The distal location of this mutation from FAD and substrate binding combined with only slight structural perturbation result in a mild clinical phenotype.

Figure 3. Three dimensional dimeric structure of the VLCAD: ribbon diagram (PDB 3B96) with mutation sites

The two monomers are colored gray and blue. Helices and beta-strands are labeled as described [7] and those in the blue monomer are labeled with asterisks Stick representations of the FAD cofactors have CPK coloring with yellow carbons. The boxes indicate mutation sites associated with FAD binding, whose vicinities are shown in greater detail in panels A–D. In each panel, amino acid residues that are mutated in patients are shown with CPK coloring with magenta carbons and are labeled in magenta. Other neighboring residues depicted with CPK coloring grey carbons and labeled in black. Hydrogen bonds and salt bridges are marked with dotted lines. A) G222R mutation site. Gly222 is located on the loop between β-strands 1 and 2 close to the FAD phosphate and thesubstitution for a bulky charged arginine may change the loop conformation, and disrupt binding of the cofactor, including a hydrogen bond between the FAD pyrophosphate and the neighboring residue Ser223. B) G439D and G441D mutation sites are part of a conserved Gly-Gly-x-Gly motif on the loop between helices I and J of neighboring monomer in the VLCAD dimeric molecule. Again, substitution of either Gly to an acidic residue would be detrimental to the interaction with the acidic pyrophosphate moiety of FAD in the neighboring monomer, resulting in a weakening of the dimer interaction. C) R366H mutation site. Arg366 forms salt bridges with the FAD pyrophosphate of the other monomer. Substitution of positively charged arginine 366 for a neutral histidine could lose this ionic interaction, instead forming a hydrogen bond between histidine 366 and the pyrophosphate, weakening FAD binding and the association between enzyme monomers. D) K382Q mutation site. Lys382 on helix H forms a salt bridge with Glu432 on helix I, and another salt bridge with Asp466 on helix K of the other monomer, which hydrogen bonds with the adenosine 2′-OH of the FAD. Since glutamine is almost isosteric as lysine, Gln382 can make hydrogen bonds with both Glu432 and Asp466, which are much weaker interactions compared to the salt bridges. The VLCAD catalytic glutamate (Glu462) is proximal to the site of this salt bridge, and thereforethe K382Q mutation may also affect its positioning

Figure 4. VLCAD protein levels in untreated (DMSO) or bezafibrate-treated (BZ) patient fibroblasts

Western-blot of protein extracts from patient or control cells were incubated with anti-VLCAD antibody, and re-probed with anti-β-actin antibody.