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Protein-protein interactions and inhibition of the ADP-ribosyl transferase reaction of Pseudomonas aeruginosa exotoxin A.

機譯：銅綠假單胞菌外毒素A的蛋白-蛋白相互作用和對ADP-核糖基轉(zhuǎn)移酶反應的抑制。

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摘要

Pseudomonas aeruginosa exotoxin A (ETA) catalyzes the transfer of the ADP-ribose moiety from NAD+ to its target protein, eukaryotic elongation factor 2 (eEF2). The objective of this research was to improve the understanding of the interactions between the catalytic domain of ETA and both of its substrates, eEF2 and NAD +. Through a study of water-soluble compounds, the inhibition of the catalytic domain of ETA was evaluated. These compounds mimic nicotinamide and the more potent inhibitors contained planar ring systems. PJ34 was further characterized and shown to act as a competitive inhibitor and was co-crystallized with the toxin to 2.1 A resolution, whereby important hydrogen bonds and van der Waals interactions were identified. An HPLC-based NAD+ -glycohydrolase assay was developed and showed that the NAD +-analogue, 2'-F-ribo-NAD+, was a competitive inhibitor and not a competing substrate, as it was hydrolyzed at 0.2% of the rate for NAD+. Next, the binding between ETA and eEF2 was investigated. The binding of eEF2 to the toxin is pH-dependent and correlates with the pH profile for catalytic function. Also, eEF2 retained native binding affinity for the toxin when it was ADP-ribosylated or when it bound a guanyl nucleotide, illustrating that eEF2 does not undergo large structural changes that would disrupt the eEF2-toxin binding site. A fluorescence study identified the molecular contacts between the catalytic domain of ETA and eEF2. The change in wavelength emission maxima, lifetime and acrylamide quenching for the IAEDANS fluorophore conjugated to the toxin was determined before and after eEF2 bound. The contact between these proteins is minimal and occurs primarily near the active site and a loop that modulates the transferase activity of the toxin. A fluorescence resonance energy transfer (FRET) technique was used to measure the energy transfer efficiencies between the acceptor fluorophore, fluorescein, on eEF2 at position 574 and the various positions of the donor fluorophore, IAEDANS, on the toxin. Distances between the donor-acceptor pair were calculated from the efficiency values and used to develop a FRET model for this apo-toxin-eEF2 complex. Moreover, the addition of beta-TAD, an NAD+ analogue, results in closer association of these proteins as indicated by increased efficiency values.
機譯：銅綠假單胞菌外毒素A（ETA）催化ADP核糖部分從NAD +轉(zhuǎn)移到其靶蛋白真核延伸因子2（eEF2）。這項研究的目的是增進對ETA催化結(jié)構(gòu)域與其兩種底物eEF2和NAD +之間相互作用的理解。通過對水溶性化合物的研究，評估了對ETA催化域的抑制作用。這些化合物模擬煙酰胺，更有效的抑制劑包含平面環(huán)系統(tǒng)。 PJ34被進一步表征并顯示出它是競爭性抑制劑的作用，并與毒素共結(jié)晶至2.1 A分辨率，從而鑒定出重要的氫鍵和范德華相互作用。進行了基于HPLC的NAD +-糖基水解酶測定，結(jié)果表明NAD +-類似物2'-F-ribo-NAD +是競爭性抑制劑而不是競爭性底物，因為它以NAD +的0.2％的速率水解。接下來，研究了ETA和eEF2之間的結(jié)合。 eEF2與毒素的結(jié)合是pH依賴性的，并且與用于催化功能的pH譜相關(guān)。同樣，當eEF2被ADP核糖基化或結(jié)合胍基核苷酸時，它仍保留了對毒素的天然結(jié)合親和力，這說明eEF2并未經(jīng)歷會破壞eEF2-毒素結(jié)合位點的大結(jié)構(gòu)變化。熒光研究確定了ETA和eEF2催化域之間的分子接觸。在結(jié)合eEF2之前和之后，確定與毒素綴合的IAEDANS熒光團的最大波長發(fā)射，壽命和丙烯酰胺猝滅的變化。這些蛋白質(zhì)之間的接觸極少，主要發(fā)生在活性位點和調(diào)節(jié)毒素轉(zhuǎn)移酶活性的環(huán)附近。熒光共振能量轉(zhuǎn)移（FRET）技術(shù)用于測量eEF2上受體熒光團574上的熒光素與毒素上供體熒光團IAEDANS各個位置之間的能量轉(zhuǎn)移效率。從效率值計算供體-受體對之間的距離，并用于建立該載脂蛋白-eEF2復合物的FRET模型。此外，添加β-TAD（一種NAD +類似物）會導致這些蛋白質(zhì)之間的緊密結(jié)合，如效率值的提高所表明。

著錄項

  • 作者

    Yates, Susan Pamela.;

  • 作者單位

    University of Guelph (Canada).;

  • 授予單位 University of Guelph (Canada).;
  • 學科 Chemistry Biochemistry.
  • 學位 Ph.D.
  • 年度 2005
  • 頁碼 218 p.
  • 總頁數(shù) 218
  • 原文格式 PDF
  • 正文語種 eng
  • 中圖分類 生物化學;
  • 關(guān)鍵詞

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