Ghrelin O-acyltransferase (GOAT) is an integral membrane acyltransferase responsible for catalyzing a serine-octanoylation posttranslational modification within the peptide hormone ghrelin. Ghrelin requires this octanoylation for its biological activity in stimulating appetite and in regulating other physiological pathways involved in energy balance. Blocking ghrelin acylation using GOAT inhibitors is a new potential avenue to treat health conditions impacted by ghrelin signaling, such as obesity and diabetes. Designing novel and potent GOAT inhibitors as potential therapeutics requires insight into the interactions between the ghrelin and octanoyl coenzyme A substrates and the GOAT active site. Through structure-activity investigation of ghrelin-mimetic peptide substrates and inhibitors, we have analyzed the amino acid selectivity of the enzyme as well as the functional groups involved in substrate recognition by human GOAT (hGOAT). This analysis reveals that hGOAT both prefers and tolerates a distinct set of chemical properties at each position within the N-terminal sequence of ghrelin and that sequence elements downstream of the ghrelin N-terminal sequence contribute to ghrelin binding to hGOAT. We also found that the hGOAT active site exhibits a marked preference for binding an eight-carbon acyl chain, which potentially explains the biological observation of ghrelin octanoylation in light of the acyl donor promiscuity reported for GOAT. Bioinformatics analysis, guided by our reactivity data, supports the conclusion that ghrelin is a unique substrate for hGOAT within the human proteome, providing further justification for the ghrelin-hGOAT system as a desirable drug target. By defining an array of substrate-enzyme interactions used by hGOAT to bind, recognize, and acylate ghrelin, this study yields novel insight into the character of the hGOAT active site that can serve as a guide toward the rational design of hGOAT inhibitors.

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Malaria parasites use the RhopH complex for erythrocyte invasion and channel-mediated nutrient uptake. As the member proteins are unique to Plasmodium spp., how they interact and traffic through subcellular sites to serve these essential functions is unknown. We show that RhopH is synthesized as a soluble complex of CLAG3, RhopH2, and RhopH3 with 1:1:1 stoichiometry. After transfer to a new host cell, the complex crosses a vacuolar membrane surrounding the intracellular parasite and becomes integral to the erythrocyte membrane through a PTEX translocon-dependent process. We present a 2.9 Å single-particle cryo-electron microscopy structure of the trafficking complex, revealing that CLAG3 interacts with the other subunits over large surface areas. This soluble complex is tightly assembled with extensive disulfide bonding and predicted transmembrane helices shielded. We propose a large protein complex stabilized for trafficking but poised for host membrane insertion through large-scale rearrangements, paralleling smaller two-state pore-forming proteins in other organisms.