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 important roles in both normal and leukemic T-cell development. LIM domains are cysteine/histidine-rich domains that contain two structural zinc ions and that function as protein-protein adaptors; members of the LMO family each contain two closely spaced LIM domains. These LMO proteins all bind with high affinity to the nuclear protein LIM domain binding protein 1 (ldb1). The LMO-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(LID)]. Unfortunately, recombinant forms of LMO2 and LMO4 have limited solubility and stability, effectively preventing structural analysis. Therefore, we have designed and constructed a fusion protein in which ldb1(LID) and LIM1 of LMO2 can form an intramolecular complex. The engineered protein, FLIN2 (fusion of the LIM interacting domain of ldb1 and the N-terminal LIM domain 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 (1)H NMR spectrum. The analogous LMO4 protein, FLIN4, has almost identical properties. These data suggest that we will be able to obtain high-resolution structural information about the LMO-ldb1 interactions.
Deane
J E
JE
Department of Biochemistry, University of Sydney, Sydney, NSW 2006, Australia.
Sum
E
E
Mackay
J P
JP
Lindeman
G J
GJ
Visvader
J E
JE
Matthews
J M
JM
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000302:isolation & purification; D060588:LIM Domain Proteins; D009682:Magnetic Resonance Spectroscopy; D008667:Metalloproteins / Q000737:chemistry* / Q000302:isolation & purification; D051379:Mice; D009687:Nuclear Proteins / Q000737:chemistry; D011485:Protein Binding; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors / Q000737:chemistry* / Q000302:isolation & purification; D015032:Zinc / Q000737:chemistry
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C486312:LDB1 protein, human; D060588:LIM Domain Proteins; C102084:Ldb1 protein, mouse; C513188:Lmo2 protein, mouse; C491976:Lmo4 protein, mouse; D008667:Metalloproteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D015032:Zinc