Báo cáo Y học: Probing the rotor subunit interface of the ATP synthase from Ilyobacter tartaricus

Probing the rotor subunit interface of the ATP synthase from Ilyobacter tartaricus

Tác giả: Denys Pogoryelov, Yaroslav Nikolaev, Uwe Schlattner, Konstantin Pervushin, Peter Dimroth, Thomas Meier

Lĩnh vực: Sinh hóa, Sinh học cấu trúc

Nội dung tài liệu: Nghiên cứu này khám phá sự tương tác giữa vòng c₁₁ và phức hợp γɛ, tạo nên rotor của ATP synthase từ Ilyobacter tartaricus. Sử dụng phương pháp phổ cộng hưởng plasmon bề mặt (SPR) và phân tích tái tổ hợp trong ống nghiệm, các nhà nghiên cứu đã đánh giá trực tiếp và định lượng độ ổn định của rotor. Kết quả cho thấy sự gắn kết chặt chẽ giữa vòng c₁₁ và phức hợp γɛ, với hằng số phân ly biểu kiến (Ka) xấp xỉ 7.4 nm. Quá trình lắp ráp rotor chủ yếu phụ thuộc vào tương tác giữa vòng c và tiểu đơn vị γ, không quan sát thấy sự gắn kết của vòng c với tiểu đơn vị ɛ tự do. Đột biến ở các axit amin bảo tồn đã ảnh hưởng nghiêm trọng đến quá trình lắp ráp rotor. Các thí nghiệm NMR cho thấy sự gia tăng trật tự cấu trúc trong các vùng đầu cuối của tiểu đơn vị γ khi rotor được lắp ráp. Nghiên cứu này đề xuất rằng sự liên kết của trục trung tâm của phức hợp F₁ với vòng rotor của F₀, tạo ra các tiếp xúc chặt chẽ nhưng có thể đảo ngược, giải thích cho sự phân ly và tái tổ hợp tương đối dễ dàng của các phức hợp F₁F₀.

Mục lục chi tiết:

• Probing the rotor subunit interface of the ATP synthase from Ilyobacter tartaricus
• Keywords
• Correspondence
• Present addresses
• The interaction between the c₁₁ ring and the γɛ complex, forming the rotor of the Ilyobacter tartaricus ATP synthase, was probed by surface plasmon resonance spectroscopy and in vitro reconstitution analysis.
• F-ATP synthases convert the energy of an electrochemical proton or sodium ion gradient into ATP, the universal chemical energy source of living cells.
• Abbreviations
• Rotor interactions of the F-ATP synthase
• y subunit within the hexameric assembly of alternating α and β subunits elicits conformational changes in the catalytic β subunit sites, resulting in ATP synthesis, consistent with the ‘binding change model’ [5], the crystal structure of F₁ [6] and single-molecule video microscopy [7].
• In an ATP synthase at work, drag is imposed by the F₁ motor components; this has been proposed to cause elastic energy storage within the α-helical domain of the y subunit [8], the peripheral stalk [9] and the rotating c ring [10].
• In this article, we have used surface plasmon resonance (SPR) [23] and NMR spectroscopy [transverse relaxation-optimized NMR spectroscopy (TROSY) and NOE-TROSY [24]] to obtain a greater understanding of the interaction sites and affinities between the γɛ complex and the c₁₁ ring during the assembly of the I. tartaricus ATP synthase.
• Results
• In vitro rotor assembly from the c₁₁ ring and subunits γ and ɛ
• The aim of this study was to obtain a better understanding of the binding processes of the rotor subunits (C₁₁, γ and ɛ) from I. tartaricus ATP synthase.
• Binding characteristics of the c₁₁ ring to the γɛ complex studied by SPR
• The kinetic characteristics of the interaction between the γɛ complex (as a ligand) and the isolated c ring (as an analyte) were studied in detail by SPR spectroscopy with a Biacore instrument.
• Mutations affecting the interaction of C₁₁ with γɛ
• In order to assess the interaction of the γɛ complex with selected amino acids in the loop region of the isolated cring [amino acids RQPE(D)], we introduced point mutations at position cR45 or cQ46 and isolated the corresponding c rings (Fig. 1B, lanes 2 and 4).
• NMR analysis of the interaction between the γɛ complex and the c₁₁ ring
• To investigate the interaction between the isolated γɛ complex and the detergent-solubilized c₁₁ ring by NMR spectroscopy, we employed conventional stable isotope (²H/¹⁵N/¹³C) labelling techniques, as well as TROSY-heteronuclear single quantum correlation (HSQC) and three-dimensional (3D)-TROSY-HNCA/HNCACB pulse schemes [32] (for assignment and data interpretation, see Supporting information).
• Discussion
• Two F₁-Fo binding affinities during rotor assembly
• We have shown that the interaction between the c ring and the central stalk subunits γ and ɛ of the rotor of I. tartaricus ATP synthase comprises high-affinity
• Structural considerations in the assembly and interaction of rotor components
• Figure S1 shows a model for the location of the residues involved in the high-affinity interaction (γE204 and ɛH38), which is based on the structure of the corresponding complex from E. coli [15].
• Experimental procedures
• The construction of the plasmids, the synthesis and purification of the subunits (γ´, ɛ and c rings) and NMR methods are described in Supporting information.
• In vitro reconstitution of the rotor complex
• The whole reconstitution procedure was performed at 20 °C.
• SPR binding assays
• Binding of the cring to immobilized His-tagged γɛ complexes, or to separate γ and ɛ subunits, was studied quantitatively using a BIACORE 2000 and nitrilotriacetic acid sensor chip from Biacore AB (Uppsala, Sweden).
• Kinetic analysis
• On and off kinetics were analysed with BIAEVALUATION 4.1 software.
• Acknowledgements
• We thank Gregory Cook for reading the manuscript.
• References