Fundamental studies on development of chemotherapeutics targeting cell surface proteins which are essential for growth in Escherichia coli

(1) ~(8)取得了丰硕的成果。(1)确定了参与外膜主要成分脂蛋白定位的LolA和Lo1B的晶体结构。(2)我们发现Lol回避信号需要一个临界长度的负电荷在第二残基，磷脂酰乙醇胺是Lol回避功能的重要组成部分。(3)革兰氏阴性菌Lo1B同源物中保守的5个Trp残基对受体活性有重要影响。(4)我们验证了脂蛋白从LolA转移到Lo1B的机制是单向的、非常高效的，但不需要能量输入。(5) LolA上14个残基的随机突变揭示了疏水腔和疏水盖在脂蛋白结合和转移中的重要作用。(6)基于一些LolD突变降低Lo1CDE的atp酶活性而不影响LolD的结果，LolD基序在与Lo1C / LolE的功能相互作用中起关键作用。(7)利用新开发的靶向Lol系统的新型抗菌药物筛选方法，筛选出2个抑制LolA功能的化合物。(8)利用体外分析系统，我们发现一种质周蛋白YbiS是一种主要的转肽酶，参与了Lpp和肽聚糖之间的共价键。上述结果有助于开发针对大肠杆菌生长所必需的细胞表面蛋白的化疗药物

Fruitful results described in (1)〜(8) were obtained during the period of this research.(1) The crystal structures of LolA and Lo1B which are involved in the localization of lipoproteins, a major component of the outer membrane, were determined.(2) We showed that the Lol avoidance signal requires a critical length of negative charge at the second residue, and that phosphatidylethanolamine is important for the Lol avoidance function.(3) Five Trp residues which are conserved among Lo1B homologs in Gram-negative bacteria were shown to be important for the receptor activity.(4) We verified the mechanism underlying lipoprotein transfer from LolA to Lo1B is unidirectional and very efficient, but requires no energy input.(5) The random mutagenesis of 14 residues on LolA revealed crucial roles of the hydrophobic cavity and lid in the binding of lipoproteins and their transfer.(6) On the basis of the result that some LolD mutations lowered the ATPase activity of Lo1CDE without affecting that of LolD, the LolD motif was found to be critical for functional interplay with Lo1C / LolE.(7) Using a new screening method which was developed to identify new antibacterial agents targeting the Lol system, we screened for 2 compounds which inhibit the LolA function.(8) We found that a periplasmic protein, YbiS, is a major transpeptidase involved in the covalent linkage between the Lpp and peptidoglycan, using in vitro assay system. The results described above contribute to development of chemotherapeutics targeting cell surface proteins which are essential for growth in Escherichia coli