CRYSTAL STRUCTURE OF FLINC4, AN INTRAMOLECULAR lmo4:LDB1 COMPLEX

Janet E. Deane, Megan Maher, J. Mitchell Guss, and Jacqueline M. Matthews

School of Molecular and Microbial Biosciences, University of Sydney, NSW 2006, Australia (J.Deane@mmb.usyd.edu.au)

LMO4 is a member of a small family of nuclear transcriptional regulators that is important for both normal development and disease processes. The LMO family comprises four members, LMO1–4 of which two, LMO1 and LMO2, are known oncogenes. LMO4 was originally identified as a breast cancer autoantigen [1] and has recently been shown to be overexpressed in over 50% of primary breast cancers [2]. LMO4 is comprised primarily of two tandemly repeated LIM domains. These domains each contain two structural zinc ions and have been identified as important motifs that mediate specific protein:protein interactions. In particular, LMO4 has been shown to interact directly and simultaneously with BRCA1, CtIP and the ubiquitous nuclear adaptor protein LIM domain binding protein 1 (ldb1) to form a stable complex in vivo [3]. LMO4 interacts with ldb1 via a 39-residue region towards the C-terminus known as the LIM interaction domain (LID). Ldb1 is of particular interest as it binds LMOs and related LIM homeodomain (LHX) proteins with high affinity, and contains an N-terminal homodimerization domain that may allow the formation of higher order functional complexes [4].

An intramolecular complex consisting of the two LIM domains from LMO4 linked to the LID domain of ldb1 has been engineered; FLINC4 (fusion of the LID domain of ldb1 and the N- and C-terminal LIM domains of LMO4). FLINC4 was purified and crystals belonging to space group P312 with unit-cell parameters a = 61.3 Å and c = 93.2 Å were grown. There is one molecule of FLINC4 per asymmetric unit (ASU). Native and multiple-wavelength anomalous dispersion (MAD) data at the zinc edge have been recorded to resolutions of 1.3 Å and 1.7 Å, respectively. Anomalous Patterson maps calculated from data collected at the peak wavelength and showed strong peaks sufficient to determine the positions of the four zinc atoms per ASU. Initial phases calculated to a resolution of 1.7 Å allowed 154 out of 188 residues of FLINC4 to be traced. Refinement of this structure to 1.3 Å resolution is underway with current values of R = 20.4 % and R_free = 23.8 %. Analysis of the structure of FLINC4 suggests a mechanism by which ldb1 can bind LMO4 specifically, and LIM domains from LMO and LHX proteins in general. Important residues at the interaction interface have been identified and may help in the design of specific inhibitors of this interaction as potential treatments for some forms of breast cancer.

References

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