Structural basis for defective membrane targeting of mutant enzyme in human VLCAD deficiency

Abstract

Very long-chain acyl-CoA dehydrogenase (VLCAD) is an inner mitochondrial membrane enzyme that catalyzes the first and rate-limiting step of long-chain fatty acid oxidation. Point mutations in human VLCAD can produce an inborn error of metabolism called VLCAD deficiency that can lead to severe pathophysiologic consequences, including cardiomyopathy, hypoglycemia, and rhabdomyolysis. Discrete mutations in a structurally-uncharacterized C-terminal domain region of VLCAD cause enzymatic deficiency by an incompletely defined mechanism. Here, we conducted a structure-function study, incorporating X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, computational modeling, and biochemical analyses, to characterize a specific membrane interaction defect of full-length, human VLCAD bearing the clinically-observed mutations, A450P or L462P. By disrupting a predicted α-helical hairpin, these mutations either partially or completely impair direct interaction with the membrane itself. Thus, our data support a structural basis for VLCAD deficiency in patients with discrete mutations in an α-helical membrane-binding motif, resulting in pathologic enzyme mislocalization.

The assembly of the Mitochondrial Complex I Assembly complex uncovers a redox pathway coordination

“…The comparison of our ACAD9 S191A structure with both the VLCAD-based ACAD9 homology model 7 and a very recently published VLCAD crystal structure (PDB: 7S7G 19 ) revealed a high structural similarity, with RMSD values of 1.16 Å and 1.11 Å, respectively (Fig. 4A ).…”

The structural mechanism of MCIA complex assembly links mitochondrial redox pathways

“…The comparison of our ACAD9S191A structure with both the VLCAD-based ACAD9 homology model 6 and a very recently published VLCAD crystal structure (PDB:7S7G 18 ) revealed a high structural similarity, with RMSD values of 1.16 Å and 1.11 Å, respectively (Fig. S7A).…”

“…S9A ) in contrast to the equivalent residues in ACAD9 ( Fig. S9B-C ) 18 . Notably, this region is also where both proteins show the highest sequence divergence, which could further account for dissimilar conformational abilities.…”

“…The recently reported VLCAD crystal structure models most of those residues (res. 486-499) facing away from the protein and projecting outward 18 . This extended loop mediates trans interactions with the equivalent loop of another symmetry related VLCAD molecule ( Fig.…”

“…This extended loop mediates trans interactions with the equivalent loop of another symmetry related VLCAD molecule ( Fig. S9A ), stabilising the quaternary conformation of a dimer of homodimers 18 . However, superposition with the ACAD9 WT structure reveals that in ACAD9 this loop tucks into the core of the same homodimer instead ( Fig.…”