Martin J, Geromanos S, Tempst P, Hartl F U
Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York 10021.
Nature. 1993 Nov 18;366(6452):279-82. doi: 10.1038/366279a0.
The chaperonin GroEL, a tetradecameric cylinder consisting of subunits of M(r) approximately 60,000 (60K), and its cofactor GroES, a heptameric ring of 10K subunits, mediate protein folding in the cytosol of Escherichia coli. In the presence of nucleotide, GroES forms a 1:1 complex with GroEL which binds unfolded protein in its central cavity and releases it to allow folding upon ATP hydrolysis. Using labelling with azido-ATP, we have identified a protease-stable nucleotide-binding domain of M(r) 40K in the GroEL subunits (residues 153-531). Azido-ATP is crosslinked to the highly conserved Tyr 477, indicating that this residue is close to the purine ring of the bound nucleotide. Surprisingly, GroES also binds ATP cooperatively and with an affinity comparable to that of GroEL. Azido-nucleotide labelling of GroES subunits occurs at the conserved Tyr 71 in a protease-stable 6.5K domain (starting at residue 33). Proteinase K cleavage at residue 32 is prevented when GroES is bound to GroEL. ATP binding to GroES may be important in charging the seven subunits of the interacting GroEL ring with ATP to facilitate cooperative ATP binding and hydrolysis for substrate protein release.
伴侣蛋白GroEL是一个由约60,000（60K）相对分子质量的亚基组成的十四聚体圆柱体，其辅因子GroES是一个由10K亚基组成的七聚体环，它们在大肠杆菌的细胞质中介导蛋白质折叠。在核苷酸存在的情况下，GroES与GroEL形成1:1复合物，该复合物在其中心腔中结合未折叠的蛋白质，并在ATP水解时将其释放以允许折叠。通过用叠氮基-ATP标记，我们在GroEL亚基中鉴定出一个相对分子质量为40K的蛋白酶稳定的核苷酸结合结构域（第153 - 531位残基）。叠氮基-ATP与高度保守的Tyr 477交联，表明该残基靠近结合核苷酸的嘌呤环。令人惊讶的是，GroES也能协同结合ATP，其亲和力与GroEL相当。GroES亚基的叠氮基核苷酸标记发生在一个蛋白酶稳定的6.5K结构域（从第33位残基开始）中的保守Tyr 71处。当GroES与GroEL结合时，蛋白酶K在第32位残基处的切割受到抑制。ATP与GroES的结合可能在为相互作用的GroEL环的七个亚基加载ATP以促进协同ATP结合和水解以释放底物蛋白方面很重要。
