STRUCTURAL STUDIES OF LIGAND-RECEPTOR COMPLEXES

PhD student project 4
(Early Stage Researcher 4, ESR4)

STRUCTURAL STUDIES OF LIGAND-RECEPTOR COMPLEXES

Supervision: Piet Gros

Early Stage Researcher: Camilla de Nardis

   

Aim:
Understanding the ligand binding of the LRP1 receptor

Methodology:
The wide variety of ligand binding of the LDLR family is likely due to the highly modular domain built-up of LRP1. Extracellularly, LRP1 contains four clusters of complement of LDL-A (A), β-propeller (B) and epidermal (E) growth factor domain repeats; with most ligand-binding sites mapped to cluster II. Based on successful expression of large plasma-proteins in HEK-293 EBNA GNTI- (suitable for crystallization) ESR4 will be taught to express constructs of LRP1 (first focusing on cluster II: A3-10-E5-6-B3E7-B4E8-B5E9-B6E10) containing extended domain stretches (collaboration UPE). Expression experiments will include the use of chaperones (collaboration Braakman). Purified constructs will be used for binding assays with complement components and protease complexes (collaboration Lambris). Assays will include SPR (collaboration Janssen) and native and H/D Mass Spectrometry (collaboration Heck). Where successful ESR4 will start crystallization trials for crystal-structure determination of the large protein complexes.

Collaborators:
Braakman, Janssen, Heck; Lambris (UPenn, USA); UPE

Key publications:

  1. F. Forneris, D. Ricklin, J. Wu, A. Tzekou, R.S. Wallace, J. Lambris and P. Gros (2010) 'Structures of C3b in complex with factors B and D give insight into complement convertase formation', Science 330, 1816-1820.
  2. J. Wu, Y.-Q. Wu, D. Ricklin, B.J.C. Janssen, J.D. Lambris and P. Gros (2009) 'Structure of complement fragment C3b-factor H and implications for host protection by complement regulators', Nature Immunology 10, 728-733.
  3. L. Rutten, J.P.B.A. Mannie, C.M. Stead, C.R.H. Raetz, C.M. Reynolds, AM.J.J. Bonvin, J.P. Tommassen, M.R. Egmond, M.S. Trent and P. Gros (2009) 'Active site architecture and catalytic mechanism of the lipid A deacylase LpxR of Salmonella typhimurium', Proc Natl Acad Sci USA 106, 1960-1964.