NMR studies on the coiled-coil domain and RPA-ssDNA

Abstract

ATRIP, the regulatory protein of ATR, exits as one stable complex with ATR in human cells. Recent studies showed that ATR-ATRIP complex was localized to sites of DNA damage through interaction between N-terminal of ATRIP and RPA-ssDNA. And coiled-coil domain of ATRIP was predicated to be essential for stabilization of ATR binding and accumulation of ATR-ATRIP at DNA lesions. To characterize the structural feature of ATRIP, coiled-coil domain and N-terminal of ATRIP were investigated by NMR and biological approaches. CD spectra of N-terminal of ATRIP reveal that the second structure of N-terminal of ATRIP is dominated by a “random coil” contribution and is absent of tertiary structure. The unstructured N-terminal of ATRIP is further confirmed by 2-D 1H-15N HSQC characterized with narrow spreaded resonance frequency for amide protons both in 15N and 1H regions. However, NMR titration experiment shows that the folding structure of N-terminal of ATRIP can not be induced upon addition with RPA-ssDNA. Additionally, apparent binding between N-terminal of ATRIP and RPA-ssDNA can not be observed by pull-down assay using Ni-NTA agarose and chemical cross-linking with EDC in vitro. Regarding to coiled-coil domain of ATRIP, CD spectra reveals that coiled-coil domain of ATRIP is contributed by highly α-helix. And $2-D^1H-^{15}N$ HSQC of coiled-coil domain of ATRIP is characterized in highly order oligomerization. Furthermore, the highly order oligomerization of coiled-coil domain of ATRIP is determined to be likely induced by the common oligomeric interaction between coiled-coil domain $(ATRIP_{108-139aa})$ and coiled-coil domain $(ATRIP_{169-217aa})$.