Calmodulin-like protein 19 (CML19) is an Arabidopsis centrin that modulates nucleotide excision repair (NER) by binding to RAD4 protein， the Arabidopsis homolog of human Xeroderma pigmentosum complementation group C protein. Although the necessity of CML19 as a part of the RAD4 plant recognition complex for functional NER is known at a cellular level， little is known at a molecular level. Herein， we used a combination of biophysical and biochemical approaches to investigate the structural and ion and target-peptide binding properties of CML19. We found that CML19 possesses four Ca-specific binding sites， two of high affinity in the N-terminal domain and two of low affinity in the C-terminal domain. Bind... More
Calmodulin-like protein 19 (CML19) is an Arabidopsis centrin that modulates nucleotide excision repair (NER) by binding to RAD4 protein， the Arabidopsis homolog of human Xeroderma pigmentosum complementation group C protein. Although the necessity of CML19 as a part of the RAD4 plant recognition complex for functional NER is known at a cellular level， little is known at a molecular level. Herein， we used a combination of biophysical and biochemical approaches to investigate the structural and ion and target-peptide binding properties of CML19. We found that CML19 possesses four Ca-specific binding sites， two of high affinity in the N-terminal domain and two of low affinity in the C-terminal domain. Binding of Ca to CML19 increases its alpha-helix content， stabilizes the tertiary structure， and triggers a conformational change， resulting in the exposure of a hydrophobic patch instrumental for target protein recognition. Using bioinformatics tools we identified a CML19-binding site at the C-terminus of RAD4， and through in vitro binding experiments we analyzed the interaction between a 17-mer peptide representing this site and CML19. We found that the peptide shows a high affinity for CML19 in the presence of Ca (stoichiometry 1:1) and the interaction primarily involves the C-terminal half of CML19.
