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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.

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.

BAX is a pro-apoptotic member of the BCL-2 family, which regulates the balance between cellular life and death. During homeostasis, BAX predominantly resides in the cytosol as a latent monomer but, in response to stress, transforms into an oligomeric protein that permeabilizes the mitochondria, leading to apoptosis. Because renegade BAX activation poses a grave risk to the cell, the architecture of BAX must ensure monomeric stability yet enable conformational change upon stress signaling. The specific structural features that afford both stability and dynamic flexibility remain ill-defined and represent a critical control point of BAX regulation. We identify a nexus of interactions involving four residues of the BAX core α5 helix that are individually essential to maintaining the structure and latency of monomeric BAX and are collectively required for dimeric assembly. The dual yet distinct roles of these residues reveals the intricacy of BAX conformational regulation and opportunities for therapeutic modulation., The pro-apoptotic BAX protein is a monomer under homeostatic conditions and, in response to stress, transforms into oligomers that induce apoptosis. Here, the authors characterize structural features of BAX that individually stabilize the monomer while collectively contributing to oligomerization.