Identification of the key LMO2-binding determinants on Ldb1.
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.
Ryan
Daniel P
DP
School of Molecular and Microbial Biosciences, University of Sydney, NSW, Australia.
Sunde
Margaret
M
Kwan
Ann H-Y
AH
Marianayagam
Neelan J
NJ
Nancarrow
Amy L
AL
Vanden Hoven
Rachel N
RN
Thompson
Lyndal S
LS
Baca
Manuel
M
Mackay
Joel P
JP
Visvader
Jane E
JE
Matthews
Jacqueline M
JM
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D060588:LIM Domain Proteins; D060850:LIM-Homeodomain Proteins; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C486312:LDB1 protein, human; C085473:LHX1 protein, human; D060588:LIM Domain Proteins; D060850:LIM-Homeodomain Proteins; C085117:LMO2 protein, human; C114905:LMO4 protein, human; C513188:Lmo2 protein, mouse; D008667:Metalloproteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors