Kwan Jamie J, Warner Neil, Maini Joban, Chan Tung Kelvin W, Zakaria Hoshang, Pawson Tony, Donaldson Logan W
Department of Biology, York University, 4700 Keele Street, Toronto, Ont., Canada M3J 1P3.
J Mol Biol. 2006 Feb 10;356(1):142-54. doi: 10.1016/j.jmb.2005.11.012. Epub 2005 Nov 28.
In Saccharomyces cerevisiae, signal transduction through pathways governing mating, osmoregulation, and nitrogen starvation depends upon a direct interaction between the sterile alpha motif (SAM) domains of the Ste11 mitogen-activated protein kinase kinase kinase (MAPKKK) and its regulator Ste50. Previously, we solved the NMR structure of the SAM domain from Ste11 and identified two mutants that diminished binding to the Ste50 SAM domain. Building upon the Ste11 study, we present the NMR structure of the monomeric Ste50 SAM domain and a series of mutants bearing substitutions at surface-exposed hydrophobic amino acid residues. The mid-loop (ML) region of Ste11-SAM, defined by helices H3 and H4 and the end-helix (EH) region of Ste50-SAM, defined by helix H5, were sensitive to substitution, indicating that these two surfaces contribute to the high-affinity interaction. The combination of two mutants, Ste11-SAM-L72R and Ste50-SAM-L69R, formed a high-affinity heterodimer unencumbered by competing homotypic interactions that had prevented earlier NMR studies of the wild-type complex. Yeast bearing mutations that prevented the heterotypic Ste11-Ste50 association in vitro presented signaling defects in the mating and high-osmolarity growth pathways.
在酿酒酵母中，通过控制交配、渗透调节和氮饥饿的信号转导途径取决于丝裂原活化蛋白激酶激酶激酶（MAPKKK）Ste11的无菌α基序（SAM）结构域与其调节因子Ste50之间的直接相互作用。此前，我们解析了Ste11的SAM结构域的核磁共振结构，并鉴定出两个降低与Ste50的SAM结构域结合能力的突变体。基于对Ste11的研究，我们展示了单体Ste50的SAM结构域的核磁共振结构以及一系列在表面暴露的疏水氨基酸残基处有取代的突变体。由螺旋H3和H4定义的Ste11-SAM的中环（ML）区域以及由螺旋H5定义的Ste50-SAM的末端螺旋（EH）区域对取代敏感，表明这两个表面有助于高亲和力相互作用。两个突变体Ste11-SAM-L72R和Ste50-SAM-L69R的组合形成了一种高亲和力异二聚体，不受竞争性同型相互作用的阻碍，而这种相互作用此前妨碍了对野生型复合物的核磁共振研究。在体外携带阻止异型Ste11-Ste50缔合的突变的酵母在交配和高渗生长途径中表现出信号缺陷。
