| 1. |
- Lebrette, Hugo, 1986-, et al.
(författare)
-
Structure of a ribonucleotide reductase R2 protein radical
- 2023
-
Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 382:6666, s. 109-113
-
Tidskriftsartikel (refereegranskat)abstract
- Aerobic ribonucleotide reductases (RNRs) initiate synthesis of DNA building blocks by generating a free radical within the R2 subunit; the radical is subsequently shuttled to the catalytic R1 subunit through proton-coupled electron transfer (PCET). We present a high-resolution room temperature structure of the class Ie R2 protein radical captured by x-ray free electron laser serial femtosecond crystallography. The structure reveals conformational reorganization to shield the radical and connect it to the translocation path, with structural changes propagating to the surface where the protein interacts with the catalytic R1 subunit. Restructuring of the hydrogen bond network, including a notably short O-O interaction of 2.41 angstroms, likely tunes and gates the radical during PCET. These structural results help explain radical handling and mobilization in RNR and have general implications for radical transfer in proteins.
|
|
| 2. |
- Pata, Jorgaq, et al.
(författare)
-
Purification and characterization of Cdr1, the drug-efflux pump conferring azole resistance in Candida species
- 2024
-
Ingår i: Biochimie. - 0300-9084 .- 1638-6183. ; 220, s. 167-178
-
Tidskriftsartikel (refereegranskat)abstract
- Candida albicans and C. glabrata express exporters of the ATP -binding cassette (ABC) superfamily and address them to their plasma membrane to expel azole antifungals, which cancels out their action and allows the yeast to become multidrug resistant (MDR). In a way to understand this mechanism of defense, we describe the purification and characterization of Cdr1, the membrane ABC exporter mainly responsible for such phenotype in both species. Cdr1 proteins were functionally expressed in the baker yeast, tagged at their C -terminal end with either a His -tag for the glabrata version, cgCdr1-His, or a green fluorescent protein (GFP) preceded by a proteolytic cleavage site for the albicans version, caCdr1-P-GFP. A membrane Cdr1-enriched fraction was then prepared to assay several detergents and stabilizers, probing their level of extraction and the ATPase activity of the proteins as a functional marker. Immobilized metal -affinity and size -exclusion chromatographies (IMAC, SEC) were then carried out to isolate homogenous samples. Overall, our data show that although topologically and phylogenetically close, both proteins display quite distinct behaviors during the extraction and purification steps, and qualify cgCdr1 as a good candidate to characterize this type of proteins for developing future inhibitors of their azole antifungal efflux activity.
|
|
| 3. |
- Riepl, Daniel, et al.
(författare)
-
Molecular principles of proton-coupled quinone reduction in the membrane-bound superoxide oxidase
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Reactive oxygen species (ROS) are physiologically harmful radicals generated as biproducts of aerobic respiration. To detoxify ROS, most cells employ superoxide scavenging enzymes that disproportionate superoxide (O2•-) to oxygen (O2) and hydrogen peroxide (H2O2). However, the recently discovered membrane-bound superoxide oxidase (SOO) (Nature Chemical Biol 2018) is a minimal 4-helical bundle protein that catalyzes the direct oxidation of O2•- to O2 and drives quinone reduction by mechanistic principles that remain unknown. Here we combine multiscale hybrid quantum/classical (QM/MM) free energy calculations and microsecond molecular dynamics simulations with functional assays and site-directed mutagenesis experiments to probe the energetics and dynamics underlying the charge transfer reactions of the superoxide (O2•-)-driven quinone reduction. We identify a cluster of charged residues at the periplasmic side of the membrane that functions as a O2•- collecting antenna, which shuttles the electrons to the active site for quinone reduction. Based on multidimensional QM/MM string simulations, we suggest that a proton-coupled electron transfer (PCET) reaction from the active site heme b and nearby histidine residues (H87, H158) catalyzes the quinol (QH2) formation, followed by proton uptake from the cytoplasmic side of the membrane. The functional relevance of the identified residues is supported by site-directed mutagenesis and activity assays, with mutations leading to inhibition of the O2•--driven quinone reduction activity. We suggest that the coupled electron and proton transfer reactions build up a proton motive force that support the bacterial energy transduction machinery, with the PCET reactions providing unique design principles of a minimal oxidoreductase.
|
|
| 4. |
- Wiseman, Benjamin, 1976-, et al.
(författare)
-
Alternating L4 loop architecture of the bacterial polysaccharide co-polymerase WzzE
- 2023
-
Ingår i: Communications Biology. - 2399-3642. ; 6:1
-
Tidskriftsartikel (refereegranskat)abstract
- Lipopolysaccharides such as the enterobacterial common antigen are important components of the enterobacterial cell envelope that act as a protective barrier against the environment and are often polymerized by the inner membrane bound Wzy-dependent pathway. By employing cryo-electron microscopy we show that WzzE, the co-polymerase component of this pathway that is responsible for the length modulation of the enterobacterial common antigen, is octameric with alternating up-down conformations of its L4 loops. The alternating up-down nature of these essential loops, located at the top of the periplasmic bell, are modulated by clashing helical faces between adjacent protomers that flank the L4 loops around the octameric periplasmic bell. This alternating arrangement and a highly negatively charged binding face create a dynamic environment in which the polysaccharide chain is extended, and suggest a ratchet-type mechanism for polysaccharide elongation. Cryo-EM structure of bacterial polysaccharide co-polymerase WzzE provides insight into possible mechanisms of lipopolysaccharide elongation and length regulation.
|
|
