Zhang J, Zhang F, Ebert D, Cobb M H, Goldsmith E J
Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas 75235, USA.
Structure. 1995 Mar 15;3(3):299-307. doi: 10.1016/s0969-2126(01)00160-5.
The mitogen-activated protein (MAP) kinase, ERK2, is a tightly regulated enzyme in the ubiquitous Ras-activated protein kinase cascade. ERK2 is activated by phosphorylation at two sites, Y185 and T183, that lie in the phosphorylation lip at the mouth of the catalytic site. To ascertain the role of these two residues in securing the low-activity conformation of the enzymes we have carried out crystallographic analyses and assays of phosphorylation-site mutants of ERK2.
The crystal structures of four mutants, T183E (threonine at residue 183 is replaced by glutamate), Y185E, Y185F and the double mutant T183E/Y185E, were determined. When T183 is replaced by glutamate, few conformational changes are observed. By contrast, when Y185 is replaced by glutamate, 19 residues become disordered, including the entire phosphorylation lip and an adjacent loop. The conservative substitution of phenylalanine for Y185 also induces relatively large conformational changes. A binding site for phosphotyrosine in the active enzyme is putatively identified on the basis of the high-resolution refinement of the structure of wild-type ERK2.
The remarkable disorder observed throughout the phosphorylation lip when Y185 is mutated shows that the stability of the phosphorylation lip is rather low. Therefore, only modest amounts of binding energy will be required to dislodge the lip for phosphorylation, and it is likely that these residues will be involved in conformational changes associated with both with binding to kinases and phosphatases and with activation. Furthermore, the low-activity structure is specifically dependent on Y185, whereas there is no such dependency on T183. Both residues, however, participate in forming the active enzyme, contributing to its tight control.
丝裂原活化蛋白（MAP）激酶ERK2是普遍存在的Ras激活蛋白激酶级联反应中一种受到严格调控的酶。ERK2通过位于催化位点入口处磷酸化环上的两个位点Y185和T183的磷酸化而被激活。为了确定这两个残基在确保酶的低活性构象中的作用，我们对ERK2的磷酸化位点突变体进行了晶体学分析和磷酸化分析。
测定了四个突变体T183E（第183位的苏氨酸被谷氨酸取代）、Y185E、Y185F和双突变体T183E/Y185E的晶体结构。当T183被谷氨酸取代时，几乎没有观察到构象变化。相比之下，当Y185被谷氨酸取代时，19个残基变得无序，包括整个磷酸化环和一个相邻的环。Y185被苯丙氨酸保守取代也会诱导相对较大的构象变化。基于野生型ERK2结构的高分辨率精修，推测出活性酶中磷酸酪氨酸的一个结合位点。
Y185突变时整个磷酸化环出现的显著无序表明磷酸化环的稳定性相当低。因此，只需适度的结合能就能使环移位进行磷酸化，并且这些残基可能参与了与激酶和磷酸酶结合以及激活相关的构象变化。此外，低活性结构特别依赖于Y185，而对T183没有这种依赖性。然而，这两个残基都参与形成活性酶，有助于对其进行严格调控。
