Calf spleen purine-nucleoside phosphorylase: crystal structure of the binary complex with a potent multisubstrate analogue inhibitor.

Luic M; Koellner G; Yokomatsu TShibuya SBzowska A

摘要

Purine-nucleoside phosphorylase (PNP) deficiency in humans leads to inhibition of the T-cell response. Potent membrane-permeable inhibitors of this enzyme are therefore considered to be potential immunosuppressive agents. The binary complex of the trimeric calf spleen phosphorylase, which is highly homologous to human PNP, with the potent ground-state analogue inhibitor 9-(5,5-difluoro-5-phosphonopentyl)guanine (DFPP-G) was crystallized in the cubic space group P2(1)3, with unit-cell parameter a = 93.183 A and one monomer per asymmetric unit. High-resolution X-ray diffraction data were collected using synchrotron radiation (EMBL Outstation, DESY, Hamburg, station X13). The crystal structure was refined to a resolution of 2.2 A and R and R(free) values of 19.1 and 24.2%, respectively. The crystal structure confirms that DFPP-G acts as a multisubstrate analogue inhibitor as it binds to both nucleoside- and phosphate-binding sites. The structure also provides the answers to some questions regarding the substrate specificity and molecular mechanism of trimeric PNPs. The wide access to the active-site pocket that was observed in the reported structure as a result of the flexibility or disorder of two loops (residues 60-65 and 251-266) strongly supports the random binding of substrates. The putative hydrogen bonds identified in the base-binding site indicate that N(1)-H and not O(6) of the purine base defines the specificity of trimeric PNPs. This is confirmed by the fact that the contact of guanine O(6) with Asn243 O(delta1) is not a direct contact but is mediated by a water molecule. Participation of Arg84 in the binding of the phosphonate group experimentally verifies the previous suggestion [Blackburn & Kent (1986), J. Chem. Soc. Perkin Trans. I, pp. 913-917; Halazy et al. (1991), J. Am. Chem. Soc. 113, 315-317] that fluorination of alkylphosphonates yields compounds with properties that suitably resemble those of phosphate esters and in turn leads to optimized interactions of such analogues with the phosphate-binding site residues. DFPP-G shows a K(i)(app) in the nanomolar range towards calf and human PNPs. To date, no high-resolution X-ray structures of these enzymes with such potent ground-state analogue inhibitors have been available in the Protein Data Bank. The present structure may thus be used in the rational structure-based design of new PNP inhibitors with potential medical applications.

机译：Purine-nucleoside磷酸化酶(PNP)不足在人类导致t细胞的抑制作用响应。因此,这种酶被认为是潜在的免疫抑制药物。复杂的三聚物的小腿脾磷酸化酶,这是高度同源人类PNP型,强有力的极化子模拟抑制剂(9) - 5 5-difluoro-5-phosphonopentyl鸟嘌呤(DFPP-G)结晶的立方空间集团P2(1) 3、晶胞参数= 93.183每不对称单元和一个单体。高分辨率x射线衍射数据使用同步辐射(EMBL的收集边防哨,谜底,汉堡,车站* 13)。晶体结构是精制的决议2.2 R和R(免费的)值为19.1和24.2%,分别。DFPP-G充当multisubstrate模拟结合这两种核苷和抑制剂phosphate-binding网站。提供了一些问题的答案底物特异性和分子机制的三聚物的pnp型。观察到的活性部位的口袋里报告结构的灵活性或两个循环障碍(60 - 65和残留物251 - 266年)强烈支持随机绑定的基板。中标识表明base-binding网站N (1) - h,而不是O(6)嘌呤的基本定义三聚物的特异性的pnp型。得到了证实,鸟嘌呤的联系O(6)与Asn243 O (delta1)不是直接的但由水分子联系。Arg84参与绑定的膦酸酯组实验验证先前的建议(布莱克本和肯特(1986),J。化学。et al . (1991), J . Am。氟化alkylphosphonates收益率化合物的性质相似的磷酯,反过来导致这些类似物的相互作用进行了优化phosphate-binding网站残留。K(我)(app)摩尔对小腿和范围人类pnp型。这些酶结构如此强大极化子的抑制剂在蛋白质数据银行。结构可能因此被用于理性基于结构的新PNP型抑制剂的设计潜在的医学应用。

Calf spleen purine-nucleoside phosphorylase: crystal structure of the binary complex with a potent multisubstrate analogue inhibitor.

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