ジャーナル: EMBO J / 年: 2021 タイトル: Phospho-regulation, nucleotide binding and ion access control in potassium-chloride cotransporters. 著者: Gamma Chi / Rebecca Ebenhoch / Henry Man / Haiping Tang / Laurence E Tremblay / Gabriella Reggiano / Xingyu Qiu / Tina Bohstedt / Idlir Liko / Fernando G Almeida / Alexandre P Garneau / Dong ...著者: Gamma Chi / Rebecca Ebenhoch / Henry Man / Haiping Tang / Laurence E Tremblay / Gabriella Reggiano / Xingyu Qiu / Tina Bohstedt / Idlir Liko / Fernando G Almeida / Alexandre P Garneau / Dong Wang / Gavin McKinley / Christophe P Moreau / Kiran D Bountra / Patrizia Abrusci / Shubhashish M M Mukhopadhyay / Alejandra Fernandez-Cid / Samira Slimani / Julie L Lavoie / Nicola A Burgess-Brown / Ben Tehan / Frank DiMaio / Ali Jazayeri / Paul Isenring / Carol V Robinson / Katharina L Dürr / 要旨: Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions ...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.
電子線照射量: 40 e/Å2 / フィルム・検出器のモデル: GATAN K3 (6k x 4k)
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解析
ソフトウェア
名称
バージョン
分類
phenix.real_space_refine
1.17.1_3660
精密化
PHENIX
1.17.1_3660
精密化
EMソフトウェア
ID
名称
バージョン
カテゴリ
2
EPU
画像取得
7
UCSF Chimera
モデルフィッティング
9
Coot
モデル精密化
13
cryoSPARC
2
3次元再構成
CTF補正
タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION
対称性
点対称性: C2 (2回回転対称)
3次元再構成
解像度: 3.12 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 818813 / 対称性のタイプ: POINT
原子モデル構築
空間: REAL 詳細: Model fitting with UCSF Chimera, sequence replacement with Chainsaw, manual refinement with Coot, and then real-space automated refinement with Phenix was done.