Structural analysis of the Candida albicans mitochondrial DNA maintenance factor Gcf1p reveals a dynamic DNA-bridging mechanism.

The compaction of mitochondrial DNA (mtDNA) is regulated by architectural HMG-box proteins whose limited cross-species similarity suggests diverse underlying mechanisms. Viability of Candida albicans, a human antibiotic-resistant mucosal pathogen, is compromised by altering mtDNA regulators. Among them, there is the mtDNA maintenance factor Gcf1p, which differs in sequence and structure from its human and Saccharomyces cerevisiae counterparts, TFAM and Abf2p. Our crystallographic, biophysical, biochemical and computational analysis showed that Gcf1p forms dynamic protein/DNA multimers by a combined action of an N-terminal unstructured tail and a long helix. Furthermore, an HMG-box domain canonically binds the minor groove and dramatically bends the DNA while, unprecedentedly, a second HMG-box binds the major groove without imposing distortions. This architectural protein thus uses its multiple domains to bridge co-aligned DNA segments without altering the DNA topology, revealing a new mechanism of mtDNA condensation.

DNA specificities modulate the binding of human transcription factor A to mitochondrial DNA control region.

Human mitochondrial DNA (h-mtDNA) codes for 13 subunits of the oxidative phosphorylation pathway, the essential route that produces ATP. H-mtDNA transcription and replication depends on the transcription factor TFAM, which also maintains and compacts this genome. It is well-established that TFAM activates the mtDNA promoters LSP and HSP1 at the mtDNA control region where DNA regulatory elements cluster. Previous studies identified still uncharacterized, additional binding sites at the control region downstream from and slightly similar to LSP, namely sequences X and Y (Site-X and Site-Y) (Fisher et al., Cell 50, pp 247-258, 1987). Here, we explore TFAM binding at these two sites and compare them to LSP by multiple experimental and in silico methods. Our results show that TFAM binding is strongly modulated by the sequence-dependent properties of Site-X, Site-Y and LSP. The high binding versatility of Site-Y or the considerable stiffness of Site-X tune TFAM interactions. In addition, we show that increase in TFAM/DNA complex concentration induces multimerization, which at a very high concentration triggers disruption of preformed complexes. Therefore, our results suggest that mtDNA sequences induce non-uniform TFAM binding and, consequently, direct an uneven distribution of TFAM aggregation sites during the essential process of mtDNA compaction.

Plastic degradation by insect hexamerins: Near-atomic resolution structures of the polyethylene-degrading proteins from the wax worm saliva.

Plastic waste management is a pressing ecological, social, and economic challenge. The saliva of the lepidopteran Galleria mellonella larvae is capable of oxidizing and depolymerizing polyethylene in hours at room temperature. Here, we analyze by cryo-electron microscopy (cryo-EM) G. mellonella's saliva directly from the native source. The three-dimensional reconstructions reveal that the buccal secretion is mainly composed of four hexamerins belonging to the hemocyanin/phenoloxidase family, renamed Demetra, Cibeles, Ceres, and a previously unidentified factor termed Cora. Functional assays show that this factor, as its counterparts Demetra and Ceres, is also able to oxidize and degrade polyethylene. The cryo-EM data and the x-ray analysis from purified fractions show that they self-assemble primarily into three macromolecular complexes with striking structural differences that likely modulate their activity. Overall, these results establish the ground to further explore the hexamerins' functionalities, their role in vivo, and their eventual biotechnological application.

Characterization of the interaction between hepatitis C virus NS5B and the human oestrogen receptor alpha.

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is part of the viral replicative complex and plays a crucial role in HCV replication. It has been described that NS5B interacts with cellular proteins, and that interactions between NS5B and host proteins are crucial for viral replication. Some of the host factors involved in the HCV replication cycle include the oestrogen receptor alpha (ESR1), protein kinases (c-Src) and chaperones (Hsp70). In this report, we determine the requirements for the interplay between NS5B and the domain C of ESR1 (ESR1C) by using Förster Resonance Energy Transfer. NS5B-ESR1C and ESR1C-ESR1C interactions are dependent on ionic strength, indicating that contacts are mainly electrostatic. Additionally, NS5B residues involved in NS5B oligomerization were also essential for NS5B-ESR1C interaction. The study of the interactions among viral and host factors will provide data to establish innovative therapeutic strategies and the development of new antiviral drugs.