Ryan Daniel P, Sunde Margaret, Kwan Ann H-Y, Marianayagam Neelan J, Nancarrow Amy L, Vanden Hoven Rachel N, Thompson Lyndal S, Baca Manuel, Mackay Joel P, Visvader Jane E, Matthews Jacqueline M
School of Molecular and Microbial Biosciences, University of Sydney, NSW, Australia.
J Mol Biol. 2006 May 26;359(1):66-75. doi: 10.1016/j.jmb.2006.02.074. Epub 2006 Mar 20.
The overexpression of LIM-only protein 2 (LMO2) in T-cells, as a result of chromosomal translocations, retroviral insertion during gene therapy, or in transgenic mice models, leads to the onset of T-cell leukemias. LMO2 comprises two protein-binding LIM domains that allow LMO2 to interact with multiple protein partners, including LIM domain-binding protein 1 (Ldb1, also known as CLIM2 and NLI), an essential cofactor for LMO proteins. Sequestration of Ldb1 by LMO2 in T-cells may prevent it binding other key partners, such as LMO4. Here, we show using protein engineering and enzyme-linked immunosorbent assay (ELISA) methodologies that LMO2 binds Ldb1 with a twofold lower affinity than does LMO4. Thus, excess LMO2 rather than an intrinsically higher binding affinity would lead to sequestration of Ldb1. Both LIM domains of LMO2 are required for high-affinity binding to Ldb1 (K(D) = 2.0 x 10(-8) M). However, the first LIM domain of LMO2 is primarily responsible for binding to Ldb1 (K(D) = 2.3 x 10(-7) M), whereas the second LIM domain increases binding by an order of magnitude. We used mutagenesis in combination with yeast two-hybrid analysis, and phage display selection to identify LMO2-binding "hot spots" within Ldb1 that locate to the LIM1-binding region. The delineation of this region reveals some specific differences when compared to the equivalent LMO4:Ldb1 interaction that hold promise for the development of reagents to specifically bind LMO2 in the treatment of leukemia.
由于染色体易位、基因治疗期间的逆转录病毒插入或转基因小鼠模型，T细胞中仅含LIM结构域的蛋白2（LMO2）的过表达会导致T细胞白血病的发生。LMO2包含两个与蛋白质结合的LIM结构域，这使得LMO2能够与多个蛋白质伙伴相互作用，包括LIM结构域结合蛋白1（Ldb1，也称为CLIM2和NLI），它是LMO蛋白的一种必需辅因子。在T细胞中，LMO2对Ldb1的隔离可能会阻止其与其他关键伙伴（如LMO4）结合。在这里，我们使用蛋白质工程和酶联免疫吸附测定（ELISA）方法表明，LMO2与Ldb1结合的亲和力比LMO4低两倍。因此，过量的LMO2而非内在更高的结合亲和力会导致Ldb1的隔离。LMO2的两个LIM结构域对于与Ldb1的高亲和力结合都是必需的（解离常数K(D)=2.0×10^(-8)M）。然而，LMO2的第一个LIM结构域主要负责与Ldb1结合（K(D)=2.3×10^(-7)M），而第二个LIM结构域使结合力提高了一个数量级。我们结合诱变与酵母双杂交分析以及噬菌体展示筛选，以鉴定Ldb1内位于LIM1结合区域的LMO2结合“热点”。与等效的LMO4:Ldb1相互作用相比，该区域的划定揭示了一些特定差异，这为开发在白血病治疗中特异性结合LMO2的试剂带来了希望。
