Bhunia Anirban, Domadia Prerna N, Mohanram Harini, Bhattacharjya Surajit
Biomolecular NMR and Drug Discovery Laboratory, Division of Structural and Computational Biology, School of Biological Sciences, Nanyang Technological University, Singapore.
Proteins. 2009 Feb 1;74(2):328-43. doi: 10.1002/prot.22166.
The sterile alpha-motif (SAM), a relatively small ( approximately 70 amino acids) interaction domain, is found in a variety of proteins involved in cell signaling, transcription regulation, and scaffolding. The Ste11 protein kinase from the mitogen activated protein kinase (MAPK) signaling cascades of the budding yeast is regulated by a SAM domain located at the N-terminus of full-length protein. In solution, the Ste11 SAM domain exists as a well-folded dimeric structure that is involved in interaction with the cognate SAM domain from an adaptor protein Ste50. In this work, we show that the Ste11 SAM domain has an intrinsic affinity towards the lipid membranes. The solution conformation of the Ste11 SAM determined in perdeuterated DPC micelle, using NMR spectroscopy, is defined by five helices of different lengths connected by a number of loops. In the micelle bound state, the non-polar and aromatic residues of the Ste11 SAM lack a native-like packing and are presumably engaged in interactions with the micelle. Using two different paramagnetic doxyl-lipids; we have mapped out localization of Ste11 SAM residues at the micelle surface. Most of the residues appear to localize at the interfacial region of the micelle. However, a number of non-polar residues from the central region of the domain are found to be located inside the core of the micelle including residues from the helix 4 and a loop between helix 2 and helix 3. Isothermal titration calorimetry studies demonstrate that a facile insertion of the Ste11 SAM into the DPC micelle is primarily driven by a large change in enthalpy, -50 kcal/mol with an apparent equilibrium association constant (Ka) of 7.86 x 10(6) M(-1). Interestingly, an interfacial mutant L60R of the Ste11 SAM lacking the dimeric structure does not show detectable interactions with the lipid micelle. The micelle-bound structure of the Ste11 SAM domain described in this work may have potential implications in the regulation of MAPK signaling whereby positioning of the Ste11 protein in close proximity to the membrane may facilitate efficient phosphorylation of the Ste11 kinase by the membrane attached upstream Ste20/pak kinase.
无菌α-基序（SAM）是一个相对较小（约70个氨基酸）的相互作用结构域，存在于多种参与细胞信号传导、转录调控和支架作用的蛋白质中。来自芽殖酵母丝裂原活化蛋白激酶（MAPK）信号级联的Ste11蛋白激酶受位于全长蛋白N端的一个SAM结构域调控。在溶液中，Ste11 SAM结构域以一种折叠良好的二聚体结构存在，该结构参与与衔接蛋白Ste50的同源SAM结构域相互作用。在这项研究中，我们表明Ste11 SAM结构域对脂质膜具有内在亲和力。使用核磁共振光谱法在全氘代DPC胶束中测定的Ste11 SAM的溶液构象由五个不同长度的螺旋通过多个环连接而成。在胶束结合状态下，Ste11 SAM的非极性和芳香族残基缺乏类似天然的堆积，可能参与与胶束的相互作用。使用两种不同的顺磁性多聚体脂质；我们绘制出了Ste11 SAM残基在胶束表面的定位。大多数残基似乎定位在胶束的界面区域。然而，发现该结构域中心区域的一些非极性残基位于胶束核心内部，包括来自螺旋4以及螺旋2和螺旋3之间一个环的残基。等温滴定量热法研究表明，Ste11 SAM轻松插入DPC胶束主要由焓的大幅变化驱动，焓变为-50千卡/摩尔，表观平衡缔合常数（Ka）为7.86×10⁶ M⁻¹。有趣的是，缺乏二聚体结构的Ste11 SAM的界面突变体L60R与脂质胶束未显示出可检测到的相互作用。这项工作中描述的Ste11 SAM结构域的胶束结合结构可能对MAPK信号传导的调控具有潜在意义，即Ste11蛋白靠近膜的定位可能有助于膜附着的上游Ste20/pak激酶对Ste11激酶进行高效磷酸化。
