9BE8

Alkalihalobacillus halodurans (Aha) trp RNA binding attenuation protein (TRAP) mutant T49A/T52A dTRAP with Trp

9BE8 の概要

• エントリーDOI: 10.2210/pdb9be8/pdb
• EMDBエントリー: 44473
• 分子名称: Transcription attenuation protein MtrB, TRYPTOPHAN (2 entities in total)
• 機能のキーワード: hexamer of t49a/t52a dtrap with trp, rna binding protein
• 由来する生物種: Halalkalibacterium halodurans; 詳細
• タンパク質・核酸の鎖数: 6
• 化学式量合計: 111375.87

構造登録者

Yang, H.,Stachowski, K.,Foster, M. (登録日: 2024-04-15, 公開日: 2025-01-22, 最終更新日: 2025-05-21)

主引用文献

Li, W.,Yang, H.,Stachowski, K.,Norris, A.S.,Lichtenthal, K.,Kelly, S.,Gollnick, P.,Wysocki, V.H.,Foster, M.P.
Structural basis of nearest-neighbor cooperativity in the ring-shaped gene regulatory protein TRAP from protein engineering and cryo-EM.
Proc.Natl.Acad.Sci.USA, 122:e2409030121-e2409030121, 2025

PubMed Abstract: The homo-dodecameric ring-shaped RNA binding attenuation protein (TRAP) from binds up to twelve tryptophan ligands (Trp) and becomes activated to bind a specific sequence in the 5' leader region of the operon mRNA, thereby downregulating biosynthesis of Trp. Thermodynamic measurements of Trp binding have revealed a range of cooperative behavior for different TRAP variants, even if the averaged apparent affinities for Trp have been found to be similar. Proximity between the ligand binding sites, and the ligand-coupled disorder-to-order transition has implicated nearest-neighbor interactions in cooperativity. To establish a solid basis for describing nearest-neighbor cooperativity in TRAP, we engineered variants constructed with two subunits connected by a flexible linker (dTRAP). We mutated the binding sites of alternating protomers such that only every other site was competent for Trp binding (WT-Mut dTRAP). Ligand binding monitored by NMR, calorimetry, and native mass spectrometry revealed strong cooperativity in dTRAP containing adjacent binding-competent sites, but a severe binding defect when the wild-type sites were separated by mutated sites. Cryo-EM experiments of dTRAP in its ligand-free apo state, and both dTRAP and WT-Mut dTRAP in the presence of Trp, revealed progressive stabilization of loops that gate the Trp binding site and participate in RNA binding. These studies provide important insights into the thermodynamic and structural basis for the observed ligand binding cooperativity in TRAP. Such insights can be useful for understanding allosteric control networks and for the development of those with defined ligand sensitivity and regulatory control.

PubMed: 39793047
DOI: 10.1073/pnas.2409030121

実験手法

ELECTRON MICROSCOPY (4.14 Å)