沙門氏菌單股DNA結合蛋白質的表達、純化、晶體成長與結晶結構之解析

Expression, purification, crystallization, and crystal structure of a single-stranded DNA-binding protein from Salmonella enterica serovar Typhimurium LT2

羅仁宏(Ren-Hong Luo)

指導教授 ： 黃晟洋

中山醫學大學/醫學科技學院/生物醫學科學學系/碩士(2022年)

https://doi.org/10.6834/csmu202200059

摘要

鼠傷寒沙門氏菌 (Salmonella enterica serovar Typhimurium) 是人類胃腸炎的主要原因，也作為是人類傷寒的小鼠研究模型。非傷寒沙門氏菌病的發病不斷增加，每年導致了數百萬人感染和許多人的死亡。此外，此細菌的抗生素耐藥性病例正在驚人地增加。因此，迫切需要開發臨床上有用的小分子抗生素或是找到針對此微生物的新標靶，以對抗此日益嚴重的耐藥細菌之威脅。單股DNA結合蛋白質 (SSB) 對於 DNA 複製和細胞存活至關重要，因此SSB可能是潛在的抗病源菌的化學攻擊標靶蛋白質。在此研究中，我們解出了此細菌 SSB 的晶體結構，其解析度為 2.8 Å (PDB ID 7F25)。此結晶結構表明此SSB 單套體在其 N 端具有寡核苷酸/寡糖結合折疊功能區 (OB fold)，而在其 C 端則具有動態的尾端。此結構的OB fold核心是由一6個 β平板的桶狀結構組成，其上有一個 α 螺旋。與其他形成四套體的細菌 SSB 不同，此SSB的晶體結構中每個不對稱單元僅包含兩個單套體。通過MOE對接分析，我們提出了各黃酮醇類，即myricetin、 quercetin、kaempferol與 galangin對此沙門氏菌SSB的結合/抑制模式。在這些對接模式中，主要的結合氨基酸為Gln52、Leu84、Tyr98與 Asn103。這些初步結果將有助於開發針對此沙門氏菌SSB 的新抑製劑，亦可能用於進一步臨床的化學治療。

關鍵字

單股DNA結合蛋白質 ； 寡核苷酸酸摺疊結合域 ； 沙門氏菌

並列摘要

Salmonella enterica serovar Typhimurium is a leading cause of human gastroenteritis and is used as a mouse model of human typhoid fever. The incidence of non-typhoid salmonellosis is increasing worldwide, causing millions of infections and many deaths in the human population each year. In addition, cases of antibiotic-resistant infections from this bacterium are alarmingly increasing. Therefore, developing clinically useful small-molecule antibiotics and identifying new target(s) in this microorganism are urgently needed to fight the growing threat of drug-resistant bacteria. The single-stranded DNA-binding protein (SSB) is essential for DNA replication and cell survival and, thus, is an attractive target for potential antipathogen chemotherapy. In this study, we determined the crystal structure of an SSB from S. enterica serovar Typhimurium LT2 (SeSSB). The crystal structure was solved at a resolution of 2.8 Å (PDB ID 7F25), indicating that the SeSSB monomer possesses an oligonucleotide/oligosaccharide-binding (OB) fold domain at its N-terminus and a flexible tail at its C-terminus. The core of the OB-fold in the SeSSB is made of a six-stranded β-barrel capped by an α-helix. Unlike other bacterial SSBs forming tetramers, the crystal structure of the SeSSB contained two monomers per asymmetric unit. Through MOE analysis, the binding/inhibition mode of the flavonols, namely myricetin, quercetin, kaempferol, and galangin to SeSSB, was suggested. In these docking models, Gln52, Leu84, Tyr98 and Asn103 in SeSSB are predicted to interact with these flavonols. These preliminary findings may facilitate the development of new inhibitors to target SeSSB for further clinical chemotherapies.

並列關鍵字

SSB ； OB fold ； Salmonella enterica

參考文獻

1. Neidhardt, F.C. (ed. in chief) Escherichia coli and Salmonella: Cellular and Molecular Biology (ASM, Washington DC, 1996).

2. McClelland, M.; Sanderson, K.E.; Spieth, J.; Clifton, S.W.; Latreille, P.; Courtney, L.; Porwollik, S.; Ali, J.; Dante, M.; Du, F.; et al. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 2001, 413, 852–856.

3. Kingsley, R.A.; Baumler, A.J. Host adaptation and the emergence of infectious disease: the Salmonella paradigm. Mol. Microbiol. 2000, 36, 1006-1014.

4. Miriagou, V.; Tassios, P.T.; Legakis, N.J.; Tzouvelekis, L.S. Expanded-spectrum cephalosporin resistance in non-typhoid Salmonella. Int. J. Antimicrob. Agents 2004, 23, 547–555.

5. Troha, K.; Ayres, J.S. Cooperative defenses during enteropathogenic infection. Curr. Opin. Microbiol. 2022, 65, 123–130.