FKBP12 dimerization mutations effect FK506 binding and differentially alter calcineurin inhibition in the human pathogen Aspergillus fumigatus.
FKBP12 dimerization mutations effect FK506 binding and differentially alter calcineurin inhibition in the human pathogen Aspergillus fumigatus.
复制标题
FKBP12 二聚化突变影响 FK506 结合并差异改变人类病原体烟曲霉中钙调神经磷酸酶的抑制作用。
DOI：
10.1016/j.bbrc.2020.03.062
复制
发表时间：
2020
影响因子：
3.1
通讯作者：
Steinbach,WilliamJ
中科院分区：
文献类型：
--
作者：
Juvvadi,PraveenR;Bobay,BenjaminG;Gobeil,SophieMC;Cole,DChristopher;Venters,RonaldA;Heitman,Joseph;Spicer,LeonardD;Steinbach,WilliamJ
12 kDa FK 506结合蛋白（FKBP 12）是常用免疫抑制药物FK 506的靶点。FKBP 12-FK 506复合物与钙调磷酸酶结合并抑制其活性，导致免疫抑制并防止器官移植排斥。我们最近对病原真菌中FKBP 12蛋白的晶体结构进行了表征，揭示了在自底物相互作用中活性位点口袋中的80 ′环残基（Pro 90）的参与，为FKBP 12在体内的二聚化提供了新的证据。40 ′环残基还显示参与哺乳动物和酵母系统中FKBP 12的可逆二聚化。为了了解FKBP 12二聚化如何影响FK 506结合和影响钙调磷酸酶功能，我们在40和50环中产生了烟曲霉FKBP 12突变（F37 M/L; W 60 V）。有趣的是，突变体表现出可变的FK 506体内亲和力，表明不同的二聚体强度。与80年代的环P90 G和V91 C突变体相比，F37 M/L和W 60 V突变体表现出更大的FK 506抗性，其中F37 M突变体在体内显示钙调磷酸酶结合完全丧失。对每个二聚体FKBP 12蛋白的分子动力学和拉动模拟显示，F37 M、F37 L和W 60 V突变的二聚体强度增加了两倍，接触次数显著增加，进一步证实了它们对体内FK 506结合和钙调磷酸酶抑制的不同程度的影响。
The 12-kDa FK506-binding protein (FKBP12) is the target of the commonly used immunosuppressive drug FK506. The FKBP12-FK506 complex binds to calcineurin and inhibits its activity, leading to immunosuppression and preventing organ transplant rejection. Our recent characterization of crystal structures of FKBP12 proteins in pathogenic fungi revealed the involvement of the 80’s loop residue (Pro90) in the active site pocket in self-substrate interaction providing novel evidence on FKBP12 dimerizationin vivo. The 40’s loop residues have also been shown to be involved in reversible dimerization of FKBP12 in the mammalian and yeast systems. To understand how FKBP12 dimerization affects FK506 binding and influences calcineurin function, we generatedAspergillus fumigatusFKBP12 mutations in the 40’s and 50’s loop (F37 M/L; W60V). Interestingly, the mutants exhibited variable FK506 susceptibilityin vivoindicating differing dimer strengths. In comparison to the 80’s loop P90G and V91C mutants, the F37 M/L and W60V mutants exhibited greater FK506 resistance, with the F37M mutation showing complete loss in calcineurin bindingin vivo. Molecular dynamics and pulling simulations for each dimeric FKBP12 protein revealed a two-fold increase in dimer strength and significantly higher number of contacts for the F37M, F37L, and W60V mutations, further confirming their varying degree of impact on FK506 binding and calcineurin inhibitionin vivo.