14‐3‐3 protein binding blocks the dimerization interface of caspase‐2

Among all species, caspase 2 C2 is the most evolutionarily conserved caspase required for effective initiation of apoptosis following death stimuli. C2 is activated through dimerization and autoproteolytic cleavage and inhibited through phosphorylation at Ser139 and Ser164, within the linker between the caspase recruitment and p19 domains of the zymogen, followed by association with the adaptor protein 14 3 3, which maintains C2 in its immature form procaspase proC2 . However, the mechanism of 14 3 3 dependent inhibition of C2 activation remains unclear. Here, we report the structural characterization of the complex between proC2 and 14 3 3 by hydrogen deuterium mass spectrometry and protein crystallography to determine the molecular basis for 14 3 3 mediated inhibition of C2 activation. Our data reveal that the 14 3 3 dimer interacts with proC2 not only through ligand binding grooves but also through other regions outside the central channel, thus explaining the isoform dependent specificity of 14 3 3 protein binding to proC2 and the substantially higher binding affinity of 14 3 3 protein to proC2 than to the doubly phosphorylated peptide. The formation of the complex between 14 3 3 protein and proC2 does not induce any large conformational change in proC2. Furthermore, 14 3 3 protein interacts with and masks both the nuclear localization sequence and the C terminal region of the p12 domain of proC2 through transient interactions in which both the p19 and p12 domains of proC2 are not firmly docked onto the surface of 14 3 3. This masked region of p12 domain is involved in C2 dimerization. Therefore, 14 3 3 protein likely inhibits proC2 activation by blocking its dimerization surfac

Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition methodology

Abstract We present a hydrogen/deuterium exchange workflow coupled to tandem mass spectrometry (HX-MS2) that supports the acquisition of peptide fragment ions alongside their peptide precursors. The approach enables true auto-curation of HX data by mining a rich set of deuterated fragments, generated by collisional-induced dissociation (CID), to simultaneously confirm the peptide ID and authenticate MS1-based deuteration calculations. The high redundancy provided by the fragments supports a confidence assessment of deuterium calculations using a combinatorial strategy. The approach requires data-independent acquisition (DIA) methods that are available on most MS platforms, making the switch to HX-MS2 straightforward. Importantly, we find that HX-DIA enables a proteomics-grade approach and wide-spread applications. Considerable time is saved through auto-curation and complex samples can now be characterized and at higher throughput. We illustrate these advantages in a drug binding analysis of the ultra-large protein kinase DNA-PKcs, isolated directly from mammalian cells

Novobiocin blocks nucleic acid binding to Polθ and inhibits stimulation of its ATPase activity.

Polymerase theta (Polθ) acts in DNA replication and repair, and its inhibition is synthetic lethal in BRCA1 and BRCA2-deficient tumor cells. Novobiocin (NVB) is a first-in-class inhibitor of the Polθ ATPase activity, and it is currently being tested in clinical trials as an anti-cancer drug. Here, we investigated the molecular mechanism of NVB-mediated Polθ inhibition. Using hydrogen deuterium exchange-mass spectrometry (HX-MS), biophysical, biochemical, computational and cellular assays, we found NVB is a non-competitive inhibitor of ATP hydrolysis. NVB sugar group deletion resulted in decreased potency and reduced HX-MS interactions, supporting a specific NVB binding orientation. Collective results revealed that NVB binds to an allosteric site to block DNA binding, both in vitro and in cells. Comparisons of The Cancer Genome Atlas (TCGA) tumors and matched controls implied that POLQ upregulation in tumors stems from its role in replication stress responses to increased cell proliferation: this can now be tested in fifteen tumor types by NVB blocking ssDNA-stimulation of ATPase activity, required for Polθ function at replication forks and DNA damage sites. Structural and functional insights provided in this study suggest a path for developing NVB derivatives with improved potency for Polθ inhibition by targeting ssDNA binding with entropically constrained small molecules