Prolyl Isomerization-Mediated Conformational Changes Define ATR Subcellular Compartment-Specific Functions

Abstract

ATR is a PI3K-like kinase protein, regulating checkpoint responses to DNA damage and replication stress. Apart from its checkpoint function in the nucleus, ATR actively engages in an antiapoptotic role at mitochondria following DNA damage. The different functions of ATR in the nucleus and cytoplasm are carried out by two prolyl isomeric forms of ATR: <i>trans</i>- and <i>cis</i>-ATR, respectively. The isomerization occurs at the Pin1 Ser428-Pro429 motif of ATR. Here, we investigated the structural basis of the subcellular location-specific functions of human ATR. Using a mass spectrometry-based footprinting approach, the surface accessibility of ATR lysine residues to sulfo-NHS-LC-biotin modification was monitored and compared between the <i>cis</i>- and the <i>trans</i>-isomers. We have identified two biotin-modified lysine residues, K459 and K469, within the BH3-like domain of <i>cis</i>-ATR that were not accessible in <i>trans</i>-ATR, indicating a conformational change around the BH3 domain between <i>cis-</i> and <i>trans</i>-ATR. The conformational alteration also involved the N-terminal domain and the middle HEAT domain. Moreover, experimental results from an array of complementary assays show that <i>cis</i>-ATR with the accessible BH3 domain was able to bind to tBid while <i>trans</i>-ATR could not. In addition, both <i>cis</i>- and <i>trans</i>-ATR can directly form homodimers <i>via</i> their C-terminal domains without ATRIP, while nuclear (<i>trans</i>-ATR) in the presence of ATRIP forms dimer-dimer complexes involving both N- and C-termini of ATR and ATRIP after UV. Structural characteristics around the Ser428-Pro429 motif and the BH3 domain region are also analyzed by molecular modeling and dynamics simulation. In support, <i>cis</i> conformation was found to be significantly more energetically favorable than <i>trans</i> at the Ser428-Pro429 bond in a 20-aa wild-type ATR peptide. Taken together, our results suggest that the isomerization-induced structural changes of ATR define both its subcellular location and compartment-specific functions and play an essential role in promoting cell survival and DNA damage responses.