Doctor of Philosophy
Dr. Gary S. Shaw
Membrane repair can be modulated by the association of an S100A10 dimer with the calcium- and phospholipid-binding protein annexin A2. This heterotetrameric complex has the ability to form larger multiprotein assemblies such as those with the enlargeosome protein AHNAK and members of the transmembrane ferlin family. The main goals of this thesis were to design, synthesize and characterize a molecule that would facilitate assembly of larger S100 multiprotein complexes, investigate the arrangement of the proteins, stoichiometry and affinity of AHNAK for the S100A10-annexin A2 complex and identify structural details of the ternary complex formed between S100A10, annexin A2 and AHNAK.
Successful expression, purification and characterization of two S100-target peptide hybrid proteins comprised of S100A10 and S100B linked in tandem to annexin A2 (residues 1-15) (referred to as A10A2) and CapZ (TRTK12) (referred to as BT12), respectively, was achieved. Different protease cleavage sites were incorporated into the linkers of the hybrid proteins. Since in situ proteolytic cleavage showed the linker did not perturb the structures of the S100A10-annexin A2 or S100B-TRTK12 complexes, this approach was used as a scaffold for larger S100 complexes.
Peptide array experiments identified the regions of interaction within the C-terminus of AHNAK. Of the eight consensus regions observed, one of the sequences of AHNAK (AHNAK5) which showed the strongest interaction was used as a synthetic peptide to identify the binding region(s), stoichiometry and affinity with the A10A2 complex. Using NMR, fluorescence and non-denaturing electrospray mass spectrometries, a novel asymmetric arrangement between a single AHNAK5 peptide and the A10A2 dimer having an affinity near 3 nM was identified.
The binding region of AHNAK5 on A10A2 was determined by NMR spectroscopy and X-ray crystallography. X-ray crystallography of the A10A2-AHNAK5 complex showed that the backbone structure of A10A2-AHNAK5 is similar to that of S100A10 bound to the N-terminal peptide of annexin A2 (Rety et al. 1999). From the structure, it was clear that only one AHNAK5 was bound to a dimeric A10A2 through helix IV in S100A10 and the C-terminal portion from the annexin A2 peptide. These results provide a novel mode of interaction for AHNAK5 in an S100-target complex.
Rezvanpour, Atoosa, "Structural Role of S100 Proteins in Membrane Repair" (2011). Electronic Thesis and Dissertation Repository. 203.