Substrate binding in the bile acid transporter ASBT_YffromYersinia frederiksenii

Abstract

Apical sodium-dependent bile acid transporter (ASBT) retrieves bile acids from the small intestine and plays a pivotal role in enterohepatic circulation. Currently, high-resolution structures are available for two bacterial ASBT homologs (ASBTNM from Neisseria meningitides and ASBTYf from Yersinia frederiksenii), from which an elevator-style alternating-access mechanism has been proposed for substrate transport. A key concept in this model is that the substrate binds to the central cavity of the transporter so that the elevator-like motion can expose the bound substrate alternatingly to either side of the membrane during a transport cycle. However, no structure of an ASBT has been solved with a substrate bound in its central cavity, so how a substrate binds to ASBT remains to be defined. In this study, molecular docking, structure determination and functional analysis were combined to define and validate the details of substrate binding in ASBTYf. The findings provide coherent evidence to provide a clearer picture of how the substrate binds in the central cavity of ASBTYf that fits the alternating-access model.

Multitasking Na+/Taurocholate Cotransporting Polypeptide (NTCP) as a Drug Target for HBV Infection: From Protein Engineering to Drug Discovery

“…Apical sodium-dependent bile acid transporter (ASBT, gene symbol SLC10A2 ), also known as ileal bile acid transporter (IBAT), is the second relevant bile acid transporter of the SLC10 family expressed in the apical domain of ileal enterocytes [ 21 ]. In the past two ASBT-related crystal structures were obtained from Neisseria meningitidis (Asbt Nm ) [ 22 , 23 ] and from Yersinia frederiksenii (Asbt Yf ) [ 24 ]. They both share approximately 25% protein sequence homology with ASBT as well as with NTCP [ 22 , 24 , 25 ].…”

“…In the past two ASBT-related crystal structures were obtained from Neisseria meningitidis (Asbt Nm ) [ 22 , 23 ] and from Yersinia frederiksenii (Asbt Yf ) [ 24 ]. They both share approximately 25% protein sequence homology with ASBT as well as with NTCP [ 22 , 24 , 25 ]. Based on these structures, numerous homology models have been generated for hASBT and hNTCP, giving a first impression about their membrane topologies, conformation, and underlying mechanisms of the bile acid transport process [ 24 , 26 , 27 ].…”

“…They both share approximately 25% protein sequence homology with ASBT as well as with NTCP [ 22 , 24 , 25 ]. Based on these structures, numerous homology models have been generated for hASBT and hNTCP, giving a first impression about their membrane topologies, conformation, and underlying mechanisms of the bile acid transport process [ 24 , 26 , 27 ]. During the last years, bioinformatics and computational tools for protein structure prediction have made great progress and are being increasingly used to analyze and to make predictions about the structure of proteins and protein complexes [ 28 , 29 ].…”

“…As the structure generally dictates protein function, this AlphaFold-based model may not only help to study NTCP‘s transporter activity, ligand/receptor interaction or oligomerization, but might also provide valuable information on dynamic interactions in living cells required for NTCP-dependent HBV/HDV entry into human hepatocytes. Over the last years, NTCP topology has been widely discussed and several structures of NTCP have been proposed [ 24 , 26 , 27 ]. According to most recent studies (based on the bacterial Asbt crystal structures and the AlphaFold prediction), human NTCP possess nine transmembrane (TMD) helices [ 18 , 23 ] with an extracellular glycosylated amino terminus and a C-terminal arm localized intracellularly ( Figure 2 A) [ 18 , 23 , 33 , 34 ].…”

“…However, all above-mentioned methods require time-consuming and lab-intense purification of stable, correctly folded and functionally active proteins, which, in the case of membrane receptors/transporters such as NTCP, is very challenging. Apart from the predicted models based on the protein structure homology or highly accurate computational approaches such as AlphaFold [ 22 , 23 , 24 , 30 ], the exact structure of human NTCP has thus far not been reported. Hence, instead of analyzing hypothetical 3-D protein models, introducing missense mutations into target protein is an alternative option for studying the role of individual amino acids in protein folding, stability, and molecular function [ 33 , 57 , 58 , 59 , 60 , 61 ].…”