Very long chain fatty acids (VLCFAs) are essential building blocks for synthesis of the ceramides and sphingolipids required for nerve, skin and retina function and 3-keto acyl-CoA synthases (ELOVL elongases) perform the first step in the FA elongation cycle. Although ELOVLs are implicated in common diseases including insulin resistance, hepatic steatosis and Parkinson's, their underlying molecular mechanisms are unknown. Here we report the structure of the human ELOVL7 elongase, which includes an inverted transmembrane barrel structure surrounding a 35 Å long tunnel containing a covalently-attached product analogue. The structure reveals the substrate binding sites in the tunnel and an active site deep in the membrane including the canonical ELOVL HxxHH sequence. This indicates a ping-pong mechanism for catalysis, involving unexpected covalent histidine adducts. The unusual substrate-binding arrangement and chemistry suggest mechanisms for selective ELOVL inhibition, relevant for diseases where VLCFAs accumulate such as X-linked adrenoleukodystrophy. MainThe seven human 3-keto acyl-CoA synthases (elongation of very long chain fatty acids proteins: ELOVL1-7 elongases) catalyse the first, rate-limiting step in the cycle that adds two carbon units to the acyl chains of fatty acids (FAs) with 12 or more carbons per chain (Fig. 1a).These long and very long chain FAs (LCFAs: 12C:20C and VLCFAs: >20C) 1,2 are the precursors for synthesis of ceramides, sphingolipids and sphingolipid signalling molecules 3 .VLCFAs are essential for the myelin sheaths of nerves 4,5 , the skin permeability barrier 6,7 , retina 8 and liver function 4,9 . Mutations in ELOVL elongases cause severe genetic diseases

Nanopore-based sensors for nucleic acid sequencing and single-molecule detection typically employ pore-forming membrane proteins with hydrophobic external surfaces, suitable for insertion into a lipid bilayer. In contrast, hydrophilic pore-containing molecules, such as DNA origami, have been shown to require chemical modification to favor insertion into a lipid environment. In this work, we describe a strategy for inserting polar proteins with an inner pore into lipid membranes, focusing here on a circular 12-subunit assembly of the thermophage G20c portal protein. X-ray crystallography, electron microscopy, molecular dynamics, and thermal/chaotrope denaturation experiments all find the G20c portal protein to have a highly stable structure, favorable for nanopore sensing applications. Porphyrin conjugation to a cysteine mutant in the protein facilitates the protein’s insertion into lipid bilayers, allowing us to probe ion transport through the pore. Finally, we probed the portal interior size and shape using a series of cyclodextrins of varying sizes, revealing asymmetric transport that possibly originates from the portal’s DNA-ratchet function.

Very long chain fatty acids (VLCFAs) are essential building blocks for synthesis of the ceramides and sphingolipids required for nerve, skin and retina function and 3-keto acyl-CoA synthases (ELOVL elongases) perform the first step in the FA elongation cycle. Although ELOVLs are implicated in common diseases including insulin resistance, hepatic steatosis and Parkinson’s, their underlying molecular mechanisms are unknown. Here we report the structure of the human ELOVL7 elongase, which includes an inverted transmembrane barrel structure surrounding a 35 Å long tunnel containing a covalently-attached product analogue. The structure reveals the substrate binding sites in the tunnel and an active site deep in the membrane including the canonical ELOVL HxxHH sequence. This indicates a ping-pong mechanism for catalysis, involving unexpected covalent histidine adducts. The unusual substrate-binding arrangement and chemistry suggest mechanisms for selective ELOVL inhibition, relevant for diseases where VLCFAs accumulate such as X-linked adrenoleukodystrophy.

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Very long chain fatty acids (VLCFAs) are essential building blocks for synthesis of the ceramides and sphingolipids required for nerve, skin and retina function and 3-keto acyl-CoA synthases (ELOVL elongases) perform the first step in the FA elongation cycle. Although ELOVLs are implicated in common diseases including insulin resistance, hepatic steatosis and Parkinson’s, their underlying molecular mechanisms are unknown. Here we report the structure of the human ELOVL7 elongase, which includes an inverted transmembrane barrel structure surrounding a 35 Å long tunnel containing a covalently-attached product analogue. The structure reveals the substrate binding sites in the tunnel and an active site deep in the membrane including the canonical ELOVL HxxHH sequence. This indicates a ping-pong mechanism for catalysis, involving unexpected covalent histidine adducts. The unusual substrate-binding arrangement and chemistry suggest mechanisms for selective ELOVL inhibition, relevant for diseases where VLCFAs accumulate such as X-linked adrenoleukodystrophy.

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