Structure of the human heterodimeric transporter 4F2hc-LAT2 in complex with Anticalin, an alternative binding protein for applications in single-particle cryo-EM

Abstract

Cryo-EM structure determination of relatively small and flexible membrane proteins at high resolution is challenging. Increasing the size and structural features by binding of high affinity proteins to the biomolecular target allows for better particle alignment and may result in structural models of higher resolution and quality. Anticalins are alternative binding proteins to antibodies, which are based on the lipocalin scaffold and show potential for theranostic applications. The human heterodimeric amino acid transporter 4F2hc-LAT2 is a membrane protein complex that mediates transport of certain amino acids and derivatives thereof across the plasma membrane. Here, we present and discuss the cryo-EM structure of human 4F2hc-LAT2 in complex with the anticalin D11vs at 3.2 Å resolution. Relative high local map resolution (2.8–3.0 Å) in the LAT2 substrate binding site together with molecular dynamics simulations indicated the presence of fixed water molecules potentially involved in shaping and stabilizing this region. Finally, the presented work expands the application portfolio of anticalins and widens the toolset of binding proteins to promote high-resolution structure solution by single-particle cryo-EM.

Conserved Lysine in transmembrane helix 5 is key for the inner gating of the LAT transporter BasC

“…Relevant to this work, smFRET could help to show that the LeuT transporter, a bacterial homolog of the neurotransmitter sodium symporter (NSS) family with APC fold(Yamashita et al , 2005), undergoes a rocking motion and switch from an outward-facing to an inward-facing conformation (Zhao et al , 2010, 2011; Tavoulari et al , 2016; Malinauskaite et al , 2014; Terry et al , 2018; Khan et al , 2020). The proposed rocking motion of the bundle domain (TM1, TM2, TM6 and TM7) versus the scaffold domain (TM3, TM4, TM8 and TM9) between outward-facing to inward-facing conformations is consistent with the general mechanism observed in other members of the APC transporter superfamily, including LAT structures (Errasti-Murugarren et al , 2019; Yan et al , 2019; Lee et al , 2019; Rodriguez et al , 2021; Parker et al , 2021; Yan et al , 2020a; Wu et al , 2020; Lee et al , 2022; Jeckelmann et al , 2022; Yan et al , 2020b, 2021; Lee et al , 2023).…”

“…The proposed rocking motion of the bundle domain (TM1, TM2, TM6 and TM7) versus the scaffold domain (TM3, TM4, TM8 and TM9) between outward-facing to inwardfacing conformations is consistent with the general mechanism observed in other members of the APC transporter superfamily, including LAT structures (Errasti-Murugarren et al, 2019;Yan et al, 2019;Lee et al, 2019;Rodriguez et al, 2021;Parker et al, 2021;Yan et al, 2020a;Wu et al, 2020;Lee et al, 2022;Jeckelmann et al, 2022;Yan et al, 2020bYan et al, , 2021Lee et al, 2023).…”

Improving Speed and Affordability without Compromising Accuracy: Standard Binding Free-Energy Calculations Using an Enhanced Sampling Algorithm, Multiple-Time Stepping, and Hydrogen Mass Repartitioning

“…The evaluation of the force-field terms and the CVs are particularly time-consuming in PMF calculations. To reduce the computational cost of the simulation, these force-field terms could be integrated with a longer time step, given that fast degrees of freedom are suitably handled, on the one hand, by the Shake/Rattle/Settle algorithms, − and, on the other hand, by the hydrogen-mass repartitioning (HMR) trick, , which is broadly used in MD-based investigations. − Concomitantly, the CVs and the corresponding biasing forces could be computed at a lower frequency in lieu of every single MD step, turning to a multiple-time-stepping (MTS) strategy . Put together, to assuage the computational burden of binding-affinity calculations, we propose to meld an MTS scheme for CV and biasing-force evaluation with HMR in order to accelerate PMF calculations, while tuning the WTM-eABF parameters to preserve reliability of our simulations.…”

Characterization of substrates and inhibitors of the human heterodimeric transporter 4F2hc-LAT1 using purified protein and the scintillation proximity radioligand binding assay

“…The methylotrophic yeast Pichia pastoris is a well-established cellular factory for the production of recombinant mammalian membrane proteins ( Byrne, 2015 ; Looser et al, 2015 ; Pochini and Galluccio, 2022 ). Over the years, different human LATs and HATs were successfully overexpressed in P. pastoris for functional and structural studies ( Costa et al, 2013 ; Meury et al, 2014 ; Rosell et al, 2014 ; Jeckelmann and Fotiadis, 2019 ; 2020 ; Kantipudi et al, 2020 ; Kantipudi et al, 2021 ; Kantipudi and Fotiadis, 2021 ; Jeckelmann et al, 2022 ). For example, cell-based transport assays using P. pastoris overexpressing 4F2hc-LAT1 were established and used for the characterization of the specificity for substrates and inhibitors, and their kinetic parameters ( Kantipudi et al, 2020 ; Kantipudi and Fotiadis, 2021 ).…”