The Mre11-Rad50-Nbs1 protein complex is one of the first responders to DNA double strand breaks. Studies have shown that the catalytic activities of the evolutionarily conserved Mre11-Rad50 (MR) core complex depend on an ATP-dependent global conformational change that takes the macromolecule from an open, extended structure in the absence of ATP to a closed, globular structure when ATP is bound. We have previously identified an additional ‘partially open’ conformation using Luminescence Resonance Energy Transfer (LRET) experiments. Here, a combination of LRET and the molecular docking program HADDOCK was used to further investigate this partially open state and identify three conformations of MR in solution: closed, partially open, and open, which are in addition to the extended, apo conformation. Mutants disrupting specific Mre11-Rad50 interactions within each conformation were used in nuclease activity assays on a variety of DNA substrates to help put the three states into a functional perspective. LRET data collected on MR bound to DNA demonstrate that the three conformations also exist when nuclease substrates are bound. These models were further supported with SAXS data which corroborate the presence of multiple states in solution. Together, the data suggest a mechanism for the nuclease activity of the MR complex along the DNA.
All data generated or analysed during this study are available from the DRYAD database under the doi: https://doi.org/10.5061/dryad.qfttdz0h6
Data from: LRET-derived HADDOCK structural models describe the conformational heterogeneity required for processivity of the Mre11-Rad50 DNA damage repair complexDryad Digital Repository, doi:10.5061/dryad.qfttdz0h6.
- Michael Latham
- Michael Latham
- Michael Latham
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Maria Spies, University of Iowa, United States
© 2022, Canny & Latham
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Akt is a Ser/Thr protein kinase that plays a central role in metabolism and cancer. Regulation of Akt's activity involves an autoinhibitory intramolecular interaction between its pleckstrin homology (PH) domain and its kinase domain that can be relieved by C-tail phosphorylation. PH domain mutant E17K Akt is a well-established oncogene. Previously, we reported that the conformation of autoinhibited Akt may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds (Chu, Viennet, et al, 2020). Here we discover unexpectedly that a single mutation R86A Akt exhibits intensified autoinhibitory features with enhanced PH domain-kinase domain affinity. Structural and biochemical analysis uncovers the importance of a key interaction network involving Arg86, Glu17, and Tyr18 that controls Akt conformation and activity. Our studies also shed light on the molecular basis for E17K Akt activation as an oncogenic driver.
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