Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

A split EGFP complementation system for finding novel α-helical peptides that bind to LMO4 (#211)

Gabrielle McClymont 1 , Jacqui Matthews 1 , Philippa Stokes 1
  1. School of Life and Environment Sciences, University of Sydney, Sydney, NSW, Australia


LMO protein 4 (LMO4) is a transcriptional co-regulatory protein that contributes to development, but also to the progression of breast cancer (1), although the mechanism or mechanisms involved are only partially known. LMO4 functions solely through protein-protein interactions(2), generating inhibitors of LMO4 could form useful research tools for revealing these mechanisms of action and might form the basis of new breast cancer therapies. Previously, the laboratory has worked with β-strand peptide inhibitors of LMO4 based on natural binding partners of LMO4, or de novo designed peptides, but those peptides tend to lack specificity for LMO4 over related proteins.


Other studies have shown that α-helical peptides can be engineered for be more specific binding(3). Our hypothesis is that we can engineer a helical peptide to bind to the plastic peptide-binding face of LMO4 that could have more specificity than β-strand peptides. To test this hypothesis we have designed a split EGFP complementation system that will be used to screen for novel α-helical peptides that bind to LMO4. Fragments of EGFP were fused to the N- and C-terminal ends of LMO4-peptide tethered constructs and the proteins expressed in E. coli. In the positive control LMO4 interacts with the LIM interaction domain (LID) of its native binding partner LDB1, bringing the two fragments of EGFP together, allowing them to fold and become fluorescent. In the negative control LMO4 and a nonsense peptide fail to interact and significantly less fluorescence was detected by eye, in solution fluorescence and flow cytometry. Experiments to optimize this screening methodology including fluorescence activated cell sorting.

  1. 1. Sum, E. Y. M., Segara, D., Duscio, B., Bath, M. L., Field, A. S., Sutherland, R. L., Lindeman, G. J., and Visvader, J. E. (2005) Overexpression of LMO4 induces mammary hyperplasia, promotes cell invasion, and is a predictor of poor outcome in breast cancer. Proc. Natl. Acad. Sci. U. S. A. 102, 7659-7664
  2. 2.  Matthews, J. M., Bhati, M., Lehtomaki, E., Mansfield, R. E., Cubeddu, L., and Mackay, J. P. (2009) It Takes Two to Tango: The Structure and Function of LIM, RING, PHD and MYND Domains. Curr. Pharm. Des. 15, 3681-3696
  3. 3.  Dutta, S., Gulla, S., Chen, T. S., Fire, E., Grant, R. A., and Keating, A. E. (2010) Determinants of BH3 Binding Specificity for Mcl-1 versus BcI-x(L). J. Mol. Biol. 398, 747-762