DNA擬態蛋白MfpA與DNA旋轉酶之交互作用分析

Abstract

DNA分子記錄遺傳資訊，但只有DNA卻並不足以執行生物功能。絕大多數牽涉核酸細胞生理活動，諸如DNA複製、轉錄作用、DNA的同源重組，以及其他DNA修補系統等等，都需要一系列的蛋白質參與，其中也包含許多種DNA結合蛋白。目前已知多種調控給定DNA結合蛋白活性的方法。調控蛋白可以直接結合至目標DNA序列並物理性的佔據此空間，又或者引起DNA構型變化以藉此干擾DNA-蛋白質間的交互作用。有些調控蛋白則是作為可化學修飾目標DNA片段的酵素，經由修飾DNA達成調控生理代謝途徑的目的。DNA擬態蛋白則是另一種不同的DNA結合蛋白活性調控策略。一般來說，一種DNA擬態蛋白可以結合至特定的一或幾種目標DNA結合蛋白，並避免這些DNA結合蛋白與DNA發生交互作用，進而影響生理代謝途徑。 MfpA蛋白被認為是一種DNA旋轉酶 (一種細菌的IIA型DNA拓樸異構酶) 抑制物，並可以使分枝桿菌屬的細菌對quinolones抗生素的最小抗藥濃度略微上升4-8倍。結晶學分析顯示MfpA蛋白是一種桿狀的二聚體、並具有帶高度負電荷的表面，其外觀讓MfpA看起來像是一段30鹼基對長的雙股DNA。在5 μM的高純度MfpA存在時，DNA旋轉酶活性會受到抑制。而Biacore實驗則表明MfpA與大腸桿菌DNA旋轉酶間的解離常數位在次微莫耳濃度的範圍。基於上述實驗結果，目前的分子嵌合模型認為MfpA應是作為G-segment DNA的競爭物。MfpA抑制DNA旋轉酶活性、並強化細胞抗藥性的分子機制推測是經由阻斷G-segment結合位置，進而避免具有細胞毒性的quinolones穩定化DNA切割中間產物累積。 在本研究中，我們試圖探討MfpA與DNA旋轉酶的複合體結構。在實驗前期，我們建立了MfpA蛋白、以及一系列DNA旋轉酶次單元與融合蛋白的表現與純化系統。初步的高通量結晶條件篩選實驗並沒有觀察到蛋白質晶體或類似蛋白質晶體的物質。等溫熱卡計滴定分析也有被用於探測MfpA與DNA旋轉酶的交互作用，然而蛋白質沉澱在此方法中非常嚴重，因此等溫熱卡計滴定有可能並不適合此兩種蛋白質。實驗後期，我們建立了一套基於液相層析系統的MfpA-DNA旋轉酶複合體生成分析方法。我們已測試了幾種可能的複合體生成模型，不過目前尚未有正面結果。

DNA molecule encodes the genetic information, but DNA on its own is not sufficient to exhibit biological functions. Almost all nucleic acid transactions, such as DNA replication, transcription, homologous recombination, and other DNA repair pathways require the involvement of a series of protein components, including numerous DNA-binding proteins. There are many ways to regulate the activity of a given DNA-binding protein. The regulatory proteins may directly bind to the target DNA sequences and occupy the binding sites or introduce conformational change in DNA to interfere with the DNA-protein interactions. In some cases, the regulatory protein functions as an enzyme, which could chemically modify its target DNA to regulate certain biological pathways. DNA mimicry by protein represents another strategy for regulating the activity of DNA-binding proteins. Each DNA-mimic protein can associate with a particular DNA-binding protein to prevent its interaction with DNA. MfpA was reported as a DNA gyrase, a bacterial type IIA topoisomerase inhibitor that mildly increase quinolones resistance of Mycobacteria by four to eight-folds. Crystallographic analysis revealed that MfpA protein exists as a rod-shaped dimer with highly negatively charged surface, which makes the protein similar to a stretch of 30 base pairs long double-stranded DNA. The gyrase activity can be inhibited in the presence of 5 μM of purified MfpA. Further Biacore experiments indicated that the dissociation constant is at sub-micromolar range for the interaction between MfpA and Escherichia coli gyrase. A docking model was constructed based on these experiment results, by treating MfpA as G-segment DNA competitor. Thus it was proposed that MfpA inhibits the gyrase activity by blocking the G-segment binding site and prevents the accumulation of cytotoxic quinolone-stabilized cleavage intermediates. We attempted to elucidate the structure of MfpA-gyrase complex in this study. In the early stage, we established the expression and purification systems of MfpA, gyrase subunits, and gyrase fusion proteins. Preliminary high-throughput crystallization screening was used in this work, but so far no crystal or crystal-like substances were observed. Isothermal calorimetry titration analysis of the MfpA-gyrase interaction suffered from heavy protein precipitation and may not be suitable for studying these two proteins. In the latter stage, a chromatography-based MfpA-gyrase complex formation analysis procedure was established. Several different complex formation models had been tested, despite lack of success.