*Article* **Transmembrane Polar Relay Drives the Allosteric Regulation for ABCG5**/**G8 Sterol Transporter**

**Bala M. Xavier 1,**†**,**‡ **, Aiman A. Zein 1,**†**,**‡ **, Angelica Venes 1,2 , Junmei Wang 3, \* and Jyh-Yeuan Lee 1, \***


Received: 7 October 2020; Accepted: 17 November 2020; Published: 19 November 2020

**Abstract:** The heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the biliary and transintestinal secretion of cholesterol and dietary plant sterols. Missense mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis. A new molecular framework was recently established by a crystal structure of human ABCG5/G8 and reveals a network of polar and charged amino acids in the core of the transmembrane domains, namely, a polar relay. In this study, we utilize genetic variants to dissect the mechanistic role of this transmembrane polar relay in controlling ABCG5/G8 function. We demonstrated a sterol-coupled ATPase activity of ABCG5/G8 by cholesteryl hemisuccinate (CHS), a relatively water-soluble cholesterol memetic, and characterized CHS-coupled ATPase activity of three loss-of-function missense variants, R543S, E146Q, and A540F, which are respectively within, in contact with, and distant from the polar relay. The results established an in vitro phenotype of the loss-of-function and missense mutations of ABCG5/G8, showing significantly impaired ATPase activity and loss of energy sufficient to weaken the signal transmission from the transmembrane domains. Our data provide a biochemical evidence underlying the importance of the polar relay and its network in regulating the catalytic activity of ABCG5/G8 sterol transporter.

**Keywords:** ABCG5; ABCG8; ATP-binding cassette transporter; cholesterol; polar relay; sitosterolemia
