Southerland W M
Department of Biochemistry and Molecular Biology, Howard University, College of Medicine, Washington, DC 20059.
J Comput Aided Mol Des. 1994 Apr;8(2):113-22. doi: 10.1007/BF00119862.
The inhibition of Pneumocystis carinii dihydrofolate reductase (DHFR) continues to be the major treatment strategy for P. carinii pneumonia (PCP). The design of new anti-pneumocystis agents would be significantly enhanced by the availability of a 3D model of the methotrexate (MTX) binding site of the P. carinii DHFR. However, an X-ray crystal structure of the P. carinii DHFR is not yet available. Alignment of the amino acid sequences of P. carinii and Lactobacillus casei DHFRs indicates that the two proteins show approximately 80% homology among MTX binding-site residues. This high level of homology suggests that the L. casei DHFR MTX binding-site structure could serve as a structural template in developing a model of the P. carinii DHFR MTX binding site. Therefore, the X-ray crystal structure of L. casei DHFR was used to develop a 3D model of the methotrexate binding site of P. carinii DHFR. The molecular modeling and dynamics software QUANTA/CHARMm was used. Amino acid residue mutations and deletions were performed using QUANTA and macromolecular minimizations were achieved with CHARMm. The MTX binding-site residues of L. casei DHFR were mutated to the corresponding residues of the P. carinii DHFR sequence. The resulting structure was extensively minimized. The resulting P. carinii MTX binding-site model showed significant differences in hydrogen-bonding patterns from the L. casei MTX binding site. Also, the P. carinii site is more hydrophobic than the corresponding L. casei site. Analysis of atom-to-atom close contacts between methotrexate and protein binding-site residues indicates that the P. carinii MTX binding-site complex is primarily stabilized by hydrophobic interactions, while the L. casei complex is mostly stabilized by electrostatic interactions. The model is consistent with the observed increased sensitivity of P. carinii DHFR to lipid-soluble inhibitors and provides a rational basis for the design of new anti-pneumocystis agents.
抑制卡氏肺孢子虫二氢叶酸还原酶（DHFR）仍然是治疗卡氏肺孢子虫肺炎（PCP）的主要策略。如果能获得卡氏肺孢子虫DHFR的甲氨蝶呤（MTX）结合位点的三维模型，新的抗肺孢子虫药物的设计将得到显著加强。然而，卡氏肺孢子虫DHFR的X射线晶体结构尚未可得。卡氏肺孢子虫和干酪乳杆菌DHFR的氨基酸序列比对表明，这两种蛋白质在MTX结合位点残基之间显示出约80%的同源性。这种高度同源性表明，干酪乳杆菌DHFR的MTX结合位点结构可以作为构建卡氏肺孢子虫DHFR的MTX结合位点模型的结构模板。因此，利用干酪乳杆菌DHFR的X射线晶体结构构建了卡氏肺孢子虫DHFR的MTX结合位点的三维模型。使用了分子建模和动力学软件QUANTA/CHARMm。使用QUANTA进行氨基酸残基的突变和缺失，并使用CHARMm实现大分子的最小化。将干酪乳杆菌DHFR的MTX结合位点残基突变为卡氏肺孢子虫DHFR序列的相应残基。对所得结构进行了广泛的最小化。所得的卡氏肺孢子虫MTX结合位点模型在氢键模式上与干酪乳杆菌MTX结合位点有显著差异。此外，卡氏肺孢子虫的位点比相应的干酪乳杆菌位点更疏水。对甲氨蝶呤与蛋白质结合位点残基之间的原子间紧密接触的分析表明，卡氏肺孢子虫MTX结合位点复合物主要通过疏水相互作用稳定，而干酪乳杆菌复合物大多通过静电相互作用稳定。该模型与观察到的卡氏肺孢子虫DHFR对脂溶性抑制剂敏感性增加一致，并为设计新的抗肺孢子虫药物提供了合理依据。
