Ligand binding to pentraxins

Kolstoe, Simon Erik (2005) Ligand binding to pentraxins. University of Southampton, Doctoral Thesis.

Abstract

The human Pentraxins are a class of pentameric blood proteins including C reactive protein (CRP) and serum amyloid P component (SAP).

SAP is thought to contribute to the pathology of amyloid by protecting aggregated amyloid proteins from degradation by normal cellular scavenging pathways. The crystal structures of SAP complexed to two peptide analogues, N-Acety-L-Proline and O-Phospho-L-Threonine, have been solved to 1.55Å and 1.8Å resolution respectively, in order to try and identify how SAP recognizes and binds to amyloidogenic peptides.

SAP ligand-binding has been shown to be dependent on two calcium atoms coordinated within the proteins binding pocket, however purified SAP rapidly aggregates in the presence of physiological calcium levels. In vivo SAP appears to be solubilised by interactions with serum albumin and in vitro using low pH or high salt. Two structures of SAP in the presence of high salt have been solved to 1.9Å and 2.6Å resolution, and one structure at pH 4 to 2.4Å resolution.  These structures have confirmed previous suggestions that SAP aggregation is caused by calcium dependent interactions between the residue Glu167 and neighbouring SAP promoters, and may be inhibited by competition with chloride ions.

In addition to the crystallographic studies, Isothermal Titration Calorimetry has been used to experimentally measure the K_a, ΔH, ΔS and ΔG for the binding of SAP and CRP with their known ligands. These values have been compared to results obtained from in silico docking experiments using the program of AUTODOCK in order to gain further insights into pentraxin binding.

In light of the above experiments, a number of longer peptide ligands have been synthesized and screened, with the sequence GEVFTKPQLWP identified as binding to SAP in solution with a μmolar binding affinity. In addition a solid phase assay has been developed in order to identify a fragment of the Alzheimer’s Aβ peptide that binds to SAP.

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