Design, production and characterization of FLIN2 and FLIN4: the engineering of intramolecular ldb1 : LMO complexes

The nuclear LIM-only (LMO) transcription factors LMO2 and LMO4 play importa nt roles in both normal and leukemic T-cell development. LIM domains are cy steine/ histidine-rich domains that contain two structural zinc ions and th at function as protein-protein adaptors; members of the LMO family each con tain two closely spaced LIM domains. These LMO proteins all bind with high affinity to the nuclear protein LIM domain binding protein 1 (ldb1). The LM O-ldb1 interaction is mediated through the N-terminal LIM domain (LIM1) of LMO proteins and a 38-residue region towards the C-terminus of ldb1 [ldb1(L ID)]. Unfortunately, recombinant forms of LMO2 and LMO4 have limited solubi lity and stability, effectively preventing structural analysis. Therefore, we have designed and constructed a fusion protein in which Idb1(LID) and LI M1 of LMO2 can form an intramolecular complex. The engineered protein, FLIN 2 (fusion of the LIM interacting domain of ldb1 and the N-terminal LIM doma in of LMO2) has been expressed and purified in milligram quantities. FLIN2 is monomeric, contains significant levels of secondary structure and yields a sharp and well-dispersed one-dimensional H-1 NMR spectrum. The analogous LMO4 protein, FLIN4, has almost identical properties. These data suggest t hat we will be able to obtain high-resolution structural information about the LMO-ldb1 interactions.