Folding of proteins with WD-repeats: Comparison of six members of the WD-repeat superfamily to the G protein beta subunit
Folding of proteins with WD-repeats: Comparison of six members of the WD-repeat superfamily to the G protein beta subunit
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DOI：
10.1021/bi9612879
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发表时间：
1996-11-05
期刊：
影响因子：
2.9
通讯作者：
Neer, EJ
中科院分区：
文献类型：
--
作者：
GarciaHiguera, I;Fenoglio, J;Neer, EJ
WD-Repeat蛋白家族由30多种不同的蛋白组成，它们共享一个高度保守的重复基序[Neer，E.J.，Schmidt，C.J.，Nambudripad，R.，&Smith，T.F.(1994)Nature 371,297-300]。这个家族的成员包括信号转导的G蛋白β亚基，以及调节信号转导、转录、前mRNA剪接、细胞骨架组织和囊泡融合的其他蛋白质。一个WD-重复蛋白(Gβ)的晶体结构现在已经被解决(Wall等人，1995；Sondek等人，1996)，并揭示了7个重复单元形成了一个圆形的螺旋桨状结构，每个叶片由4条β链组成。很可能所有的WD-Repeat蛋白都形成了类似的结构。如果是这样的话，就有可能利用一个家庭成员重要表面区的信息来预测另一个家庭成员的属性。如果WD蛋白形成类似于Gβ的结构，它们的流体动力学性质应该是致密的球状蛋白的那些，并且它们应该抵抗胰酶的切割。然而，唯一被研究的WD-Repeat蛋白Gβ，在体外合成的兔网织红细胞裂解物中，如果没有它的伙伴蛋白G Gamma，就不能折叠成天然结构。Gβ的非WD-重复氨基末端a螺旋不会抑制折叠，因为即使去除这个区域，Gβ也不会折叠。目前尚不清楚是否所有的WD-Repeat蛋白在体外合成时都不能折叠。我们在兔网织红细胞裂解液中合成了该家族的7个成员，测定了它们的斯托克斯半径、沉降系数和摩擦比，并测定了它们对胰酶的稳定性。我们对折叠的工作定义是，这些蛋白质形成球状的、抗胰酶的结构，因为除了Gβ伽马，它们的功能是未知的，或者不能在网织红细胞裂解物中进行检测。我们选择了包括氨基和羧基延伸的蛋白质，以及完全由WD-重复组成的蛋白质。我们发现，与Gβ不同的是，几种带有WD重复序列的蛋白质能够在兔网织红细胞裂解液中折叠成球状蛋白质。一种名为βTrcD的蛋白质形成了像Gβ一样的大聚集体，这表明它可能也需要伴侣蛋白。尽管存在许多潜在的胰酶切割位点，但根据Gβ的结构预测，所有折叠的蛋白质都能产生稳定的胰酶蛋白水解物。这些研究表明，其他WD-重复蛋白很可能形成类似于Gβ的螺旋桨结构。
The family of WD-repeat proteins comprises over 30 different proteins that share a highly conserved repeating motif [Neer, E. J., Schmidt, C. J., Nambudripad, R., & Smith, T. F. (1994) Nature 371, 297-300]. Members of this family include the signal-transducing G protein beta subunit, as well as other proteins that regulate signal transduction, transcription, pre-mRNA splicing, cytoskeletal organization, and vesicular fusion. The crystal structure of one WD-repeat protein (G beta) has now been solved (Wall et al., 1995; Sondek et al., 1996) and reveals that the seven repeating units form a circular, propeller-like structure with seven blades each made up of four beta strands. It is very likely that all WD-repeat proteins form a similar structure. If so, it will be possible to use information about important surface regions of one family member to predict proper-ties of another. If WD proteins form structures similar to G beta, their hydrodynamic properties should be those of compact, globular proteins, and they should be resistant to cleavage by trypsin. However, the only studied example of a WD-repeat protein, G beta, synthesized in vitro in a rabbit reticulocyte lysate, is unable to fold into a native structure without its partner protein G gamma. The non-WD-repeat amino terminal a helix of G beta does not inhibit folding because G beta does not fold even when this region is removed. It is not known whether all WD-repeat proteins are unable to fold when synthesized in an in vitro system. We synthesized seven members of the family in a rabbit reticulocyte lysate, determined their Stokes radius, sedimentation coefficient, and frictional ratio, and assayed their stability to trypsin. Our working definition of folding was that the proteins form globular, trypsin-resistant structures because, except for G beta gamma, their functions are not known or cannot be assayed in reticulocyte lysates. We chose proteins that include amino and carboxyl extensions as well as proteins that are made up entirely of WD-repeats. We show that unlike G beta, several proteins with WD-repeats are able to fold into globular proteins in a rabbit reticulocyte lysate. One protein, beta TrcD, formed large aggregates like G beta, suggesting that it may also require a partner protein. Despite the presence of many potential tryptic cleavage sites, all of the proteins that did fold gave stable large products on tryptic proteolysis, as predicted on the basis of the structure of G beta. These studies suggest that other WD-repeat proteins are likely to form propeller structures similar to G beta.