Structural basis of substrate recognition and allosteric activation of the proapoptotic mitochondrial HtrA2 protease.

The mitochondrial serine protease HtrA2 is a human homolog of the Escherichia coli Deg-proteins exhibiting chaperone and proteolytic roles. HtrA2 is involved in both apoptotic regulation via its ability to degrade inhibitor-of-apoptosis proteins (IAPs), as well as in cellular maintenance as part of the cellular protein quality control machinery, by preventing the possible toxic accumulation of aggregated proteins. In this study, we use advanced solution NMR spectroscopy methods combined with biophysical characterization and biochemical assays to elucidate the crucial role of the substrate recognizing PDZ domain. This domain regulates the protease activity of HtrA2 by triggering an intricate allosteric network involving the regulatory loops of the protease domain. We further show that divalent metal ions can both positively and negatively modulate the activity of HtrA2, leading to a refined model of HtrA2 regulation within the apoptotic pathway.

Structure and dynamics of the mitochondrial DNA-compaction factor Abf2 from S. cerevisiae.

Mitochondria are essential organelles that produce most of the energy via the oxidative phosphorylation (OXPHOS) system in all eukaryotic cells. Several essential subunits of the OXPHOS system are encoded by the mitochondrial genome (mtDNA) despite its small size. Defects in mtDNA maintenance and expression can lead to severe OXPHOS deficiencies and are amongst the most common causes of mitochondrial disease. The mtDNA is packaged as nucleoprotein structures, referred to as nucleoids. The conserved mitochondrial proteins, ARS-binding factor 2 (Abf2) in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and mitochondrial transcription factor A (TFAM) in mammals, are nucleoid associated proteins (NAPs) acting as condensing factors needed for packaging and maintenance of the mtDNA. Interestingly, gene knockout of Abf2 leads, in yeast, to the loss of mtDNA and respiratory growth, providing evidence for its crucial role. On a structural level, the condensing factors usually contain two DNA binding domains called high-mobility group boxes (HMG boxes). The co-operating mechanical activities of these domains are crucial in establishing the nucleoid architecture by bending the DNA strands. Here we used advanced solution NMR spectroscopy methods to characterize the dynamical properties of Abf2 together with its interaction with DNA. We find that the two HMG-domains react notably different to external cues like temperature and salt, indicating distinct functional properties. Biophysical characterizations show the pronounced difference of these domains upon DNA-binding, suggesting a refined picture of the Abf2 functional cycle.

Thumb-domain dynamics modulate the functional repertoire of DNA-Polymerase IV (DinB).

In order to cope with the risk of stress-induced mutagenesis, cells in all kingdoms of life employ Y-family DNA polymerases to resolve resulting DNA lesions and thus maintaining the integrity of the genome. In Escherichia coli, the DNA polymerase IV, or DinB, plays this crucial role in coping with these type of mutations via the so-called translesion DNA synthesis. Despite the availability of several high-resolution crystal structures, important aspects of the functional repertoire of DinB remain elusive. In this study, we use advanced solution NMR spectroscopy methods in combination with biophysical characterization to elucidate the crucial role of the Thumb domain within DinB's functional cycle. We find that the inherent dynamics of this domain guide the recognition of double-stranded (ds) DNA buried within the interior of the DinB domain arrangement and trigger allosteric signals through the DinB protein. Subsequently, we characterized the RNA polymerase interaction with DinB, revealing an extended outside surface of DinB and thus not mutually excluding the DNA interaction. Altogether the obtained results lead to a refined model of the functional repertoire of DinB within the translesion DNA synthesis pathway.