ジャーナル: Nature / 年: 2024 タイトル: De novo design of allosterically switchable protein assemblies. 著者: Arvind Pillai / Abbas Idris / Annika Philomin / Connor Weidle / Rebecca Skotheim / Philip J Y Leung / Adam Broerman / Cullen Demakis / Andrew J Borst / Florian Praetorius / David Baker / 要旨: Allosteric modulation of protein function, wherein the binding of an effector to a protein triggers conformational changes at distant functional sites, plays a central part in the control of ...Allosteric modulation of protein function, wherein the binding of an effector to a protein triggers conformational changes at distant functional sites, plays a central part in the control of metabolism and cell signalling. There has been considerable interest in designing allosteric systems, both to gain insight into the mechanisms underlying such 'action at a distance' modulation and to create synthetic proteins whose functions can be regulated by effectors. However, emulating the subtle conformational changes distributed across many residues, characteristic of natural allosteric proteins, is a significant challenge. Here, inspired by the classic Monod-Wyman-Changeux model of cooperativity, we investigate the de novo design of allostery through rigid-body coupling of peptide-switchable hinge modules to protein interfaces that direct the formation of alternative oligomeric states. We find that this approach can be used to generate a wide variety of allosterically switchable systems, including cyclic rings that incorporate or eject subunits in response to peptide binding and dihedral cages that undergo effector-induced disassembly. Size-exclusion chromatography, mass photometry and electron microscopy reveal that these designed allosteric protein assemblies closely resemble the design models in both the presence and absence of peptide effectors and can have ligand-binding cooperativity comparable to classic natural systems such as haemoglobin. Our results indicate that allostery can arise from global coupling of the energetics of protein substructures without optimized side-chain-side-chain allosteric communication pathways and provide a roadmap for generating allosterically triggerable delivery systems, protein nanomachines and cellular feedback control circuitry.
A: de novo protein sr322 B: de novo protein sr322 C: de novo protein sr322 D: de novo protein sr322 E: de novo protein sr322 F: de novo protein sr322 G: de novo protein sr322 H: de novo protein sr322
名称: sr322 / タイプ: COMPLEX 詳細: Complex is made up of 4 monomeric proteins, and is purified as a 4 sided multimer sr322. In this structure two copies of sr322 interact in an off target way. Entity ID: all / 由来: RECOMBINANT
分子量
値: 0.326592 MDa / 実験値: NO
由来(天然)
生物種: synthetic construct (人工物)
由来(組換発現)
生物種: Escherichia coli (大腸菌) / 株: BL21(DE3)
緩衝液
pH: 8 / 詳細: 150 mM NaCl, 40 mM Tris pH 8.0
緩衝液成分
ID
濃度
名称
式
Buffer-ID
1
150mM
sodiumchloride
NaCl
1
2
40mM
Trishydrochloricacid
Tris-HCL
1
試料
濃度: 0.971 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES
急速凍結
装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE / 湿度: 100 % / 凍結前の試料温度: 295.15 K
平均露光時間: 5 sec. / 電子線照射量: 52 e/Å2 / フィルム・検出器のモデル: GATAN K3 (6k x 4k) / 撮影したグリッド数: 1 / 実像数: 4213
-
解析
EMソフトウェア
ID
名称
カテゴリ
7
UCSF ChimeraX
モデルフィッティング
8
ISOLDE
モデルフィッティング
10
Coot
モデル精密化
11
PyMOL
モデル精密化
12
PHENIX
モデル精密化
15
cryoSPARC
分類
16
cryoSPARC
3次元再構成
CTF補正
タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION
粒子像の選択
選択した粒子像数: 699357
対称性
点対称性: C2 (2回回転対称)
3次元再構成
解像度: 4.32 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 144551 / アルゴリズム: FOURIER SPACE / クラス平均像の数: 1 / 対称性のタイプ: POINT
原子モデル構築
プロトコル: FLEXIBLE FIT 詳細: Initial fit was done using Chimera using Rigid body from In silico model. Then Isolde and Namdinator were used. Coot and Phenix were also used.