透過蛋白質工程學設計高ATP水解活性之OLA1突變

Engineering an Obg-like ATPase 1 (OLA1) Mutant with Higher Activity

施柏仰(Po-Yang Shih)

指導教授 ： 鄭惠春

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2020年)

摘要

Obg 家族蛋白是GTPase中的一大成員，並且和蛋白轉譯的執行功能有關。其中YcHF為此大家族的一分支，然而其水解ATP的效率卻比GTP高很多。在人類中與YcHF同為同源蛋白被稱為obg-like ATPase 1 (OLA1)，OLA1是由coiled-coil domain、TGS domain 和 G domain所組成的，其中G domain被認為和GTP 水解息息相關。在G domain中有五個motif在序列上是非常高度保守的，其中G1 motif 和 phosphate binding有關，G2與G3 motif則會隨著nucleotide的水解而產生結構的變化，G4和G5 motif則與鹼基的辨認及結合相關。在過去的研究中發現BARD1會藉由它的BRCT domain與OLA1產生作用並藉此調控中心粒的複製。在這裡我們想要創造人工改造OLA1，使其突變後能夠更有效率地水解ATP。未來希望這些突變的OLA1能夠與BRCT更穩定的結合，以利之後利用X-ray結晶繞射實驗了解BRCT是如何調控OLA1的。

關鍵字

三磷酸腺苷水解活性 ； 三磷酸鳥苷水解活性 ； 三磷酸腺苷水解酶 ； 三磷酸鳥苷水解酶

並列摘要

The Obg family proteins are a large group of GTPases which are responsible for protein translation. One subfamily of this large group of proteins is YchF which has been considered as GTPase, but not ATPase, for a long time. However, it was found that human homologue of YchF could hydrolyze ATP more efficiently than GTP. Thus this protein is termed as Obg-like ATPase 1 (OLA1) [1] [2]. OLA1 contains a coil-coiled domain, TGS domain and G domain, which is important for GTP hydrolysis. In the G domain, there are five motifs, which are conserved on sequence level in all GTPases. The G1 motif (P-loop) is a loop responsible for phosphate binding. The G2 motif (switch I) and G3 motif (switch II) display nucleotide-dependent conformations. The G4 and G5 motifs are involved in base recognition and binding (Figure 4.a.). Previous studies found that BARD1 can interact with OLA1 by its BRCT domain, which is critical to DNA repair and centrosome regulation [2]. Here, we engineered OLA1 mutants having hyper ATPase-activity. This finding may be useful for biochemical and cellular studies of OLA1 and BARD1 in the future.

並列關鍵字

OLA1 ； Ras ； GTPase hydrolysis ； ATPase hydrolysis

參考文獻

[1] Koller-Eichhorn R, Marquardt T, Gail R, Wittinghofer A, Kostrewa D, Kutay U, et al. Human OLA1 defines an ATPase subfamily in the Obg family of GTP-binding proteins. J Biol Chem. 2007;282:19928-37.

[2] Matsuzawa A, Kanno S, Nakayama M, Mochiduki H, Wei L, Shimaoka T, et al. The BRCA1/BARD1-interacting protein OLA1 functions in centrosome regulation. Mol Cell. 2014;53:101-14.

[3] Wu Q, Paul A, Su D, Mehmood S, Foo TK, Ochi T, et al. Structure of BRCA1-BRCT/Abraxas Complex Reveals Phosphorylation-Dependent BRCT Dimerization at DNA Damage Sites. Mol Cell. 2016;61:434-48.

[4] Brzovic PS, Rajagopal P, Hoyt DW, King MC, Klevit RE. Structure of a BRCA1-BARD1 heterodimeric RING-RING complex. Nat Struct Biol. 2001;8:833-7.

[5] Birrane G, Varma AK, Soni A, Ladias JA. Crystal structure of the BARD1 BRCT domains. Biochemistry. 2007;46:7706-12.