Involvement of the "occluded nucleotide conformation" of P-glycoprotein in the catalytic pathway.
Involvement of the "occluded nucleotide conformation" of P-glycoprotein in the catalytic pathway.
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DOI：
10.1021/bi0509797
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发表时间：
2005-09
期刊：
影响因子：
2.9
通讯作者：
G. Tombline;Alma Muharemagić;L. White;A. E. Senior
中科院分区：
文献类型：
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作者：
G. Tombline;Alma Muharemagić;L. White;A. E. Senior
我们最近发现，小鼠p -糖蛋白（Pgp）中两个“催化羧酸”残基（E552A/E1197A）的组合突变使该蛋白处于“封闭的核苷酸构象”中，可能是一种稳定的核苷酸结合结构域（NBDs）二聚体，以1mol /mol Pgp的化学量紧密结合MgATP [Tombline， G., Bartholomew， L., Urbatsch， I. L., and Senior, a . E. (2004) J. Biol]。化学通报，2003,26(3):326 - 326。在这里，我们进一步研究了这种构象在催化途径中的潜在参与。药物可促进封闭的核苷酸构象。维拉帕米明显加快了MgATP的紧密结合速度，而对解离速度没有影响。“q环”残基的突变被认为会干扰药物和催化位点之间的通信，从而阻止核苷酸构象的闭合，共价试剂n -乙基马来酰亚胺和7-氯-4-硝基苯-2-氧-1,3-二唑也会这样做，它们已知会通过在催化位点反应来抑制ATP水解。Walker A Ser和Lys残基与E552A/E1197A结合的突变具有相同的效果，表明这些保守残基与MgATP的相互作用需要稳定被封闭的核苷酸构象。我们提出了一种结合这种构象的酶方案。我们提出，在两个核苷酸结合结构域（NBDs）的初始松散结合以及药物结合时，NBDs二聚化形成封闭的构象，其中一个紧密结合的MgATP致力于水解。该途径的进展使得紧密结合的MgATP进入过渡状态并被水解。这项工作表明，在NBD二聚体界面上相互作用的小分子或肽可能有效地禁用Pgp催化。
We found recently that the combined mutation of both "catalytic carboxylate" residues (E552A/E1197A) in mouse P-glycoprotein (Pgp) arrested the protein in an "occluded nucleotide conformation", possibly a stabilized dimer of nucleotide-binding domains (NBDs), that binds MgATP tightly at stoichiometry of 1 mol/mol Pgp [Tombline, G., Bartholomew, L., Urbatsch, I. L., and Senior, A. E. (2004) J. Biol. Chem. 279, 31212-31220]. Here, we further examine this conformation in respect to its potential involvement in the catalytic pathway. The occluded nucleotide conformation is promoted by drugs. Verapamil markedly accelerated the rate of tight binding of MgATP, whereas it did not effect the rate of dissociation. Mutations in "Q-loop" residues that are thought to interfere with communication between drug and catalytic sites prevented the occluded nucleotide conformation, as did covalent reagents N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, which are known to inhibit ATP hydrolysis by reacting in catalytic sites. Mutations of Walker A Ser and Lys residues in combination with E552A/E1197A had the same effect, showing that interaction of these conserved residues with MgATP is required to stabilize the occluded nucleotide conformation. We present an enzymatic scheme that incorporates this conformation. We propose that upon initial loose binding of MgATP at two nucleotide-binding domains (NBDs), together with drug binding, the NBDs dimerize to form the occluded conformation, with one tightly bound MgATP committed to hydrolysis. The pathway progresses such that the tightly bound MgATP enters the transition state and is hydrolyzed. This work suggests that small molecules or peptides that interact at the NBD dimer interface might effectively disable Pgp catalysis.