high-throughput-expression-purification-human-solute-carriers-for

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, ...

University of Oxford

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the BacMam system. This eukaryotic expression system was selected and a screening process established in 2016 to enable production of highly challenging human integral membrane proteins (IMPs), which are a significant component of our target list. Here, we discuss our recently developed platform for identifying expression and monodispersity of IMPs from 3 mL of HEK293 cells.

Expression Screening of Human Integral Membrane Proteins Using BacMam.

Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho-regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo-EM structures of human KCC3b and KCC1, revealing structural determinants for phospho-regulation in both N- and C-termini. We show that phospho-mimetic KCC3b is arrested in an inward-facing state in which intracellular ion access is blocked by extensive contacts with the N-terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho-regulatory site in the KCC1 N-terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP-binding pocket in the large C-terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.

Phospho-regulation, nucleotide binding and ion access control in potassium-chloride cotransporters.

The integrity of a cell's proteome depends on correct folding of polypeptides by chaperonins. The chaperonin TCP-1 ring complex (TRiC) acts as obligate folder for >10% of cytosolic proteins, including he cytoskeletal proteins actin and tubulin. Although its architecture and how it recognizes folding substrates are emerging from structural studies, the subsequent fate of substrates inside the TRiC chamber is not defined. We trapped endogenous human TRiC with substrates (actin, tubulin) and cochaperone (PhLP2A) at different folding stages, for structure determination by cryo-EM. The already-folded regions of client proteins are anchored at the chamber wall, positioning unstructured regions toward the central space to achieve their native fold. Substrates engage with different sections of the chamber during the folding cycle, coupled to TRiC open-and-close transitions. Further, the cochaperone PhLP2A modulates folding, acting as a molecular strut between substrate and TRiC chamber. Our structural snapshots piece together an emerging model of client protein folding within TRiC.

Snapshots of actin and tubulin folding inside the TRiC chaperonin.

Kv3 channels have distinctive gating kinetics tailored for rapid repolarization in fast-spiking neurons. Malfunction of this process due to genetic variants in the KCNC1 gene causes severe epileptic disorders, yet the structural determinants for the unusual gating properties remain elusive. Here, we present cryo-electron microscopy structures of the human Kv3.1a channel, revealing a unique arrangement of the cytoplasmic tetramerization domain T1 which facilitates interactions with C-terminal axonal targeting motif and key components of the gating machinery. Additional interactions between S1/S2 linker and turret domain strengthen the interface between voltage sensor and pore domain. Supported by molecular dynamics simulations, electrophysiological and mutational analyses, we identify several residues in the S4/S5 linker which influence the gating kinetics and an electrostatic interaction between acidic residues in α6 of T1 and R449 in the pore-flanking S6T helices. These findings provide insights into gating control and disease mechanisms and may guide strategies for the design of pharmaceutical drugs targeting Kv3 channels.

Article	Year
Expression Screening of Human Integral Membrane Proteins Using BacMam. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 33125646	2021
Phospho-regulation, nucleotide binding and ion access control in potassium-chloride cotransporters. The EMBO journal\| PubMed ID: 34031912	2021
Snapshots of actin and tubulin folding inside the TRiC chaperonin. Nature structural & molecular biology\| PubMed ID: 35449234	2022
Cryo-EM structure of the human Kv3.1 channel reveals gating control by the cytoplasmic T1 domain. Nature communications\| PubMed ID: 35840580	2022

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