通过冷冻电子显微镜确定的酿酒酵母磷酸果糖激酶ATP结合状态的结构。
The structure of the ATP-bound state of S. cerevisiae phosphofructokinase determined by cryo-electron microscopy.
作者信息
Bárcena Montserrat, Radermacher Michael, Bär Jörg, Kopperschläger Gerhard, Ruiz Teresa
机构信息
University of Vermont, College of Medicine, Department of Molecular Physiology and Biophysics, Burlington, VT 05405, USA.
出版信息
J Struct Biol. 2007 Jul;159(1):135-43. doi: 10.1016/j.jsb.2007.03.004. Epub 2007 Mar 31.
Phosphofructokinase (Pfk1, EC 2.7.1.11) plays a key regulatory role in the glycolytic pathway. The combination of X-ray crystallographic and biochemical data has provided an understanding of the different conformational changes that occur between the active and inhibited states of the bacterial enzyme, and of the role of the two bacterial effectors. Eukaryotic phosphofructokinases exhibit a far more sophisticated regulatory mechanism, they are more complex structures regulated by a large number of effectors (around 20). Saccharomyces cerevisiae Pfk1 is an 835 kDa hetero-octamer which shows cooperative binding for fructose-6-phosphate (F6P) and non-cooperative binding for ATP. The 3D structure of the F6P-bound state was obtained by cryo-electron microscopy to 1.1 nm resolution. This electron microscopy structure, in combination with molecular replacement using the bacterial enzyme has helped provide initial phases to solve the X-ray structure of the F6P-bound state 12S yeast truncated-tetramer. Biochemical and small-angle X-ray scattering (SAXS) studies had indicated that Pfk1 underwent a large conformational change upon Mg-ATP binding. We have calculated a reconstruction using reference-based 3D projection alignment methods from 0 degrees images acquired from frozen-hydrated preparations of the enzyme in the presence of Mg-ATP. The ATP-bound structure is more extended or open, and the calculated radius of gyration of 7.33 nm (7.0 nm for F6P) is in good agreement with the SAXS data. There is a substantial decrease in the rotational angle between the top and bottom tetramers. Interestingly, all these changes have arisen from a reorientation of the alpha- and beta-subunits in the dimers. The interface region between the alpha- and beta-subunits is now approximately half the size of the one in the F6P-bound structure. This is the first time that the 3D structure of a eukaryotic Pfk1 has been visualized in its T-state (inhibited-state).
磷酸果糖激酶（Pfk1，EC 2.7.1.11）在糖酵解途径中起关键调节作用。X射线晶体学和生化数据相结合，使人们了解了细菌酶活性状态和抑制状态之间发生的不同构象变化，以及两种细菌效应物的作用。真核磷酸果糖激酶表现出更为复杂的调节机制，它们是由大量效应物（约20种）调节的更为复杂的结构。酿酒酵母Pfk1是一种835 kDa的异源八聚体，对6-磷酸果糖（F6P）表现出协同结合，对ATP表现出非协同结合。通过冷冻电子显微镜获得了F6P结合状态的三维结构，分辨率为1.1 nm。这种电子显微镜结构，结合使用细菌酶的分子置换，有助于提供初始相位，以解析F6P结合状态的12S酵母截短四聚体的X射线结构。生化和小角X射线散射（SAXS）研究表明，Pfk1在结合Mg-ATP后发生了大的构象变化。我们使用基于参考的三维投影对齐方法，从在Mg-ATP存在下酶的冷冻水合制剂获得的0度图像中计算出一个重建结构。ATP结合结构更伸展或开放，计算出的回转半径为7.33 nm（F6P为7.0 nm），与SAXS数据吻合良好。顶部和底部四聚体之间的旋转角度大幅减小。有趣的是，所有这些变化都源于二聚体中α亚基和β亚基的重新定向。α亚基和β亚基之间的界面区域现在大约是F6P结合结构中界面区域大小的一半。这是首次在其T态（抑制态）下可视化真核Pfk1的三维结构。
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