Biophysical and structural characterization of DCC 3-5 and Netrin-1 binding interaction
Loading...
Date
2019-08
Authors
Gabir, Haben
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Deleted in Colorectal Cancer (DCC) is a transmembrane receptor that belongs to the immunoglobulin superfamily. Its function as a dependence receptor makes DCC a tumour suppressor in colorectal cancer. Its primary ligand is Netrin-1, a chemotropic soluble protein responsible for attractive and repulsive migration of axon growth tips during neural development. Netrin -1 and DCC are also play significant roles in cell migration, proliferation, differentiation and apoptosis in other tissues.
DCC has two splice variant whose functional difference or binding sites with netrin-1 are not fully known. In this project, to better understand the mechanism of interaction between DCC and Netrin-1, We focused on using an integrated approach of biophysical and structural biology techniques to characterize DCC and DCC with Netrin-1 bound in solution. Both DCC and Netrin-1 were expressed in mammalian cells and purified by affinity chromatography and Size exclusion chromatography (SEC). The proteins were then characterized by Analytical Ultracentrifugation to determine the stability, dispersion, and behaviour in solution, as well as to confirm complex formation. Further characterization was done by SEC-Small Angle X-ray Scattering (SEC-SAXS) to generate low resolution models. The SEC-SAXS models were made Ab initio using the ATSAS data analysis software package and Density in Solution Scattering electron density determination algorithm. The models showed that the DCC variants to be long flexible proteins that when bound to Netrin-1 make a stable complex. When comparing the Netrin-1 complexes with the DCC variants, the larger and longer DCC was more stable, suggesting the additional components had an effect of binding. Final DCC alone and Netrin-1 with DCC were crystallized to build high resolution models. A 2.6A crystal structure of DCC was built that corroborated the SEC-SAXS models.
Description
Keywords
Netrin-1