Finer-Moore Janet S, Anderson Amy C, O'Neil Robert H, Costi M Paola, Ferrari Stefania, Krucinski Jolanta, Stroud Robert M
Department of Biochemistry and Biophysics, University of California, San Francisco, 600 16th Street, Room S412B, San Francisco, CA 94143-2240, USA.
Acta Crystallogr D Biol Crystallogr. 2005 Oct;61(Pt 10):1320-34. doi: 10.1107/S0907444905022638. Epub 2005 Sep 28.
The ternary complex crystal structures of Cryptococcus neoformans and Escherichia coli thymidylate synthase (TS) suggest mechanisms of species-specific inhibition of a highly conserved protein. The 2.1 Angstrom structure of C. neoformans TS cocrystallized with substrate and the cofactor analog CB3717 shows that the binding sites for substrate and cofactor are highly conserved with respect to human TS, but that the structure of the cofactor-binding site of C. neoformans TS is less constrained by surrounding residues. This feature might allow C. neoformans TS to form TS-dUMP-inhibitor complexes with a greater range of antifolates than human TS. 3',3''-Dibromophenol-4-chloro-1,8-naphthalein (GA9) selectively inhibits both E. coli TS and C. neoformans TS (K(i) = 4 microM) over human TS (K(i) >> 245 microM). The E. coli TS-dUMP-GA9 complex is in an open conformation, similar to that of the apoenzyme crystal structure. The GA9-binding site overlaps the binding site of the pABA-glutamyl moiety of the cofactor. The fact that human apoTS can adopt an unusual fold in which the GA9-binding site is disordered may explain the poor affinity of GA9 for the human enzyme. These observations highlight the critical need to incorporate multiple target conformations in any computational attempt to facilitate drug discovery.
新型隐球菌和大肠杆菌胸苷酸合成酶（TS）的三元复合晶体结构揭示了对一种高度保守蛋白质进行物种特异性抑制的机制。与底物和辅因子类似物CB3717共结晶的新型隐球菌TS的2.1埃结构表明，底物和辅因子的结合位点相对于人类TS高度保守，但新型隐球菌TS辅因子结合位点的结构受周围残基的限制较小。这一特征可能使新型隐球菌TS比人类TS能与更广泛的抗叶酸药物形成TS-dUMP-抑制剂复合物。3',3''-二溴苯酚-4-氯-1,8-萘啶（GA9）对大肠杆菌TS和新型隐球菌TS均有选择性抑制作用（K(i)=4 microM），而对人类TS的抑制作用较弱（K(i)>>245 microM）。大肠杆菌TS-dUMP-GA9复合物呈开放构象，类似于无酶晶体结构。GA9结合位点与辅因子对氨基苯甲酸-谷氨酰部分的结合位点重叠。人类无酶TS可呈现一种异常折叠，其中GA9结合位点无序，这一事实可能解释了GA9对人类酶的亲和力较差的原因。这些观察结果突出了在任何促进药物发现的计算尝试中纳入多个靶点构象的迫切需求。
