The Basic Cleft of RPA70N Binds Multiple Checkpoint Proteins, Including RAD9, To Regulate ATR Signaling▿

ATR kinase activation requires the recruitment of the ATR-ATRIP and RAD9-HUS1-RAD1 (9-1-1) checkpoint complexes to sites of DNA damage or replication stress. Replication protein A (RPA) bound to single-stranded DNA is at least part of the molecular recognition element that recruits these checkpoint complexes. We have found that the basic cleft of the RPA70 N-terminal oligonucleotide-oligosaccharide fold (OB-fold) domain is a key determinant of checkpoint activation. This protein-protein interaction surface is able to bind several checkpoint proteins, including ATRIP, RAD9, and MRE11. RAD9 binding to RPA is mediated by an acidic peptide within the C-terminal RAD9 tail that has sequence similarity to the primary RPA-binding surface in the checkpoint recruitment domain (CRD) of ATRIP. Mutation of the RAD9 CRD impairs its localization to sites of DNA damage or replication stress without perturbing its ability to form the 9-1-1 complex or bind the ATR activator TopBP1. Disruption of the RAD9-RPA interaction also impairs ATR signaling to CHK1 and causes hypersensitivity to both DNA damage and replication stress. Thus, the basic cleft of the RPA70 N-terminal OB-fold domain binds multiple checkpoint proteins, including RAD9, to promote ATR signaling

Copper Binding Affinity of S100A13, a Key Component of the FGF-1 Nonclassical Copper-Dependent Release Complex

S100A13 is a member of the S100 protein family that is involved in the copper-dependent nonclassical secretion of signal peptideless proteins fibroblast growth factor 1 and interleukin 1α. In this study, we investigate the effects of interplay of Cu(2+) and Ca(2+) on the structure of S100A13 using a variety of biophysical techniques, including multi-dimensional NMR spectroscopy. Results of the isothermal titration calorimetry experiments show that S100A13 can bind independently to both Ca(2+) and Cu(2+) with almost equal affinity (K(d) in the micromolar range). Terbium binding and isothermal titration calorimetry data reveal that two atoms of Cu(2+)/Ca(2+) bind per subunit of S100A13. Results of the thermal denaturation experiments monitored by far-ultraviolet circular dichroism, limited trypsin digestion, and hydrogen-deuterium exchange (using (1)H-(15)N heteronuclear single quantum coherence spectra) reveal that Ca(2+) and Cu(2+) have opposite effects on the stability of S100A13. Binding of Ca(2+) stabilizes the protein, but the stability of the protein is observed to decrease upon binding to Cu(2+). (1)H-(15)N chemical shift perturbation experiments indicate that S100A13 can bind simultaneously to both Ca(2+) and Cu(2+) and the binding of the metal ions is not mutually exclusive. The results of this study suggest that the Cu(2+)-binding affinity of S100A13 is important for the formation of the FGF-1 homodimer and the subsequent secretion of the signal peptideless growth factor through the nonclassical release pathway

Chemical scaffold recycling: Structure-guided conversion of an HIV integrase inhibitor into a potent influenza virus RNA-dependent RNA polymerase inhibitor designed to minimize resistance potential

Influenza is one of the leading causes of disease-related mortalities worldwide. Several strategies have been implemented during the past decades to hinder the replication cycle of influenza viruses, all of which have resulted in the emergence of resistant virus strains. The most recent example is baloxavir marboxil, where a single mutation in the active site of the target endonuclease domain of the RNA-dependent-RNA polymerase renders the recent FDA approved compound ∼1000-fold less effective. Raltegravir is a first-in-class HIV inhibitor that shows modest activity to the endonuclease. Here, we have used structure-guided approaches to create rationally designed derivative molecules that efficiently engage the endonuclease active site. The design strategy was driven by our previously published structures of endonuclease-substrate complexes, which allowed us to target functionally conserved residues and reduce the likelihood of resistance mutations. We succeeded in developing low nanomolar equipotent inhibitors of both wild-type and baloxavir-resistant endonuclease. We also developed macrocyclic versions of these inhibitors that engage the active site in the same manner as their 'open' counterparts but with reduced affinity. Structural analyses provide clear avenues for how to increase the affinity of these cyclic compounds