ELUCIDATING THE INTERACTION BETWEEN CEREBRAL DOPAMINE NEUROTROPHIC FACTOR (CDNF) AND GRP-78 BY NMR: IMPLICATIONS TO ENDOPLASMIC RETICULUM (ER) STRESS RESPONSE

Neurotrophic Factors (NFs) are secreted proteins crucial for developing and maintaining neuronal and other cells. CDNF is an unconventional NF, which is known for its role in mitigating ER stress, particularly to dopaminergic neurons that are damaged in Parkinson's disease (PD). Full-Length CDNF (FL-CDNF) comprises 161 amino acids, organized into 7 alpha-helices and 4 disulfide bonds. The N-terminal domain comprises the first 100 amino acids, including 5 alpha-helices and 3 disulfide bonds. The C-terminal domain contains the remaining 57 amino acids, with 2 alpha-helices, a 310 helix, and a single disulfide bond. These domains are connected by a flexible linker. Our group was the first to present the complete structure of FL-CDNF by NMR and its protective action to neurons in culture as well as to cardiomyocytes. GRP-78 is critical for maintaining ER homeostasis, and its levels tripling under ER stress conditions. Recent studies have suggested the interaction between the C-domain of CDNF and GRP-78. This interaction is protective to the dopaminergic neurons during ER stress induced by α-synuclein aggregation in PD. We aim to elucidate the interactions between GRP-78 and CDNF by NMR, identify the amino acid residues involved in this binding, and how the tertiary structure of CDNF is affected by this interaction. The isolated C-domain of CDNF was used for these experiments. Fluorescence spectroscopy and circular dichroism were also employed to unravel the structural alterations promoted by the formation of the C-domain CDNF-Grp78 complex. Our NMR data revealed significant chemical shift perturbations and changes in peak intensities in the amino acid sequence between residues 129 and 139 of CDNF, as well as in a segment located at the opposite side of the protein. We calculated this interaction's dissociation constant (Kd) and determined the binding stoichiometry. Our findings indicate that the C-terminal of CDNF interacts with the nucleotide-binding domain (NBD) of GRP-78.