An extensive ion-pair/hydrogen-bond network contributes to the thermostability of the MutL ATPase domain from Aquifex aeolicus.

Proteins from hyperthermophiles often contain a large number of ionic interactions. Close examination of the previously determined crystal structure of the ATPase domain of MutL from a hyperthermophile, Aquifex aeolicus, revealed that the domain contains a continuous ion-pair/hydrogen-bond network consisting of 11 charged amino acid residues on a ß-sheet. Mutations were introduced to disrupt the network, showing that the more extensively the network was disrupted, the greater the thermostability of the protein was decreased. Based on urea denaturation analysis, a thermodynamic parameter, energy for the conformational stability, was evaluated, which indicated that amino acid residues in the network contributed additively to the protein stability. A continuous network rather than a cluster of isolated interactions would pay less entropic penalty upon fixing the side chains to make the same number of ion pairs/hydrogen bonds, which might contribute more favorably to the structural formation of thermostable proteins.

Adenylate kinases of thermophiles Aquifex aeolicus and Geobacillus stearothermophilus: biochemical and kinetic studies.

Adenylate kinases (AK) play a pivotal role in the regulation of cellular energy. The aim of our work was to achieve the overproduction and purification of AKs from two groups of bacteria and to determine, for the first time, the comprehensive biochemical and kinetic properties of adenylate kinase from Gram-negative Aquifex aeolicus (AKaq) and Gram-positive Geobacillus stearothermophilus (AKst). Therefore we determined KM and Vmax values, and the effects of temperature, pH, metal ions, donors of the phosphate groups and inhibitor Ap5A for both thermophilic AKs. The kinetic studies indicate that both AKs exhibit significantly higher affinity for substrates with the pyrophosphate group than for adenosine monophosphate. AK activation by Mg2+ and Mn2+ revealed that both ions are efficient in the synthesis of adenosine diphosphate and adenosine triphosphate; however, Mn2+ ions at 0.2-2.0 mmol/L concentration were more efficient in the activation of the ATP synthesis than Mg2+ ions. Our research demonstrates that zinc ions inhibit the activity of enzymes in both directions, while Ap5A at a concentration of 10 µmol/L and 50 µmol/L inhibited both enzymes with a different efficiency. Sigmoid-like kinetics were detected at high ATP concentrations not balanced by Mg2+, suggesting the allosteric effect of ATP for both bacterial AKs.

Fluorescent iron­sulfur centers: Photochemistry of the PetA Rieske protein from Aquifex aeolicus.

Cytochrome bc1 complexes are energy-transducing enzymes and key components of respiratory electron chains. They contain Rieske 2Fe2S proteins that absorb very weakly in the visible absorption region compared to the heme cofactors of the cytochromes, but are known to yield photoproducts. Here, the photoreactions of isolated Rieske proteins from the hyperthermophilic bacterium Aquifex aeolicus are studied in two redox states using ultrafast transient fluorescence and absorption spectroscopy. We provide evidence, for the first time in iron­sulfur proteins, of very weak fluorescence of the excited state, in the oxidized as well as the reduced state. The excited states of the oxidized and reduced forms decay in 1.5 ps and 30 ps, respectively. In both cases they give rise to product states with lifetimes beyond 1 ns, reflecting photo-reduction of oxidized centers as well as photo-oxidation of reduced centers. Potential reaction partners are discussed and studied using site-directed mutagenesis. For the reduced state, a nearby disulfide bridge is suggested as an electron acceptor. The resulting photoproducts in either state may play a role in photoactivation processes.

Reconstitution and functional characterization of the FtsH protease in lipid nanodiscs.

FtsH is a membrane-bound protease that plays a crucial role in proteolytic regulation of many cellular functions. It is universally conserved in bacteria and responsible for the degradation of misfolded or misassembled proteins. A recent study has determined the structure of bacterial FtsH in detergent micelles. To properly study the function of FtsH in a native-like environment, we reconstituted the FtsH complex into lipid nanodiscs. We found that FtsH in membrane scaffold protein (MSP) nanodiscs maintains its native hexameric conformation and is functionally active. We further investigated the effect of the lipid bilayer composition (acyl chain length, saturation, head group charge and size) on FtsH proteolytic activity. We found that the lipid acyl chain length influences AaFtsH activity in nanodiscs, with the greatest activity in a bilayer of di-C18:1 PC. We conclude that MSP nanodiscs are suitable model membranes for further in vitro studies of the FtsH protease complex.

The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry.

AAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore toward a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. How substrates reach the FtsH central pore is an open key question that is not resolved by the available atomic structures of cytoplasmic and periplasmic domains. In this work, we used both negative stain TEM and cryo-EM to determine 3D maps of the full-length Aquifex aeolicus FtsH protease. Unexpectedly, we observed that detergent solubilization induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. The striking tilted conformation of the cytosolic domain in the FtsH dodecamer visualized by negative stain TEM suggests a lateral substrate entrance between the membrane and cytosolic domain. Such a substrate path was then resolved in the cryo-EM structure of the FtsH hexamer. By mapping the available structural information and structure predictions for the transmembrane helices to the amino acid sequence we identified a linker of ∼20 residues between the second transmembrane helix and the cytosolic domain. This unique polypeptide appears to be highly flexible and turned out to be essential for proper functioning of FtsH as its deletion fully eliminated the proteolytic activity of FtsH.

A Lynch syndrome-associated mutation at a Bergerat ATP-binding fold destabilizes the structure of the DNA mismatch repair endonuclease MutL.

In humans, mutations in genes encoding homologs of the DNA mismatch repair endonuclease MutL cause a hereditary cancer that is known as Lynch syndrome. Here, we determined the crystal structures of the N-terminal domain (NTD) of MutL from the thermophilic eubacterium Aquifex aeolicus (aqMutL) complexed with ATP analogs at 1.69-1.73 Å. The structures revealed significant structural similarities to those of a human MutL homolog, postmeiotic segregation increased 2 (PMS2). We introduced five Lynch syndrome-associated mutations clinically found in human PMS2 into the aqMutL NTD and investigated the protein stability, ATPase activity, and DNA-binding ability of these protein variants. Among the mutations studied, the most unexpected results were obtained for the residue Ser34. Ser34 (Ser46 in PMS2) is located at a previously identified Bergerat ATP-binding fold. We found that the S34I aqMutL NTD retains ATPase and DNA-binding activities. Interestingly, CD spectrometry and trypsin-limited proteolysis indicated the disruption of a secondary structure element of the S34I NTD, destabilizing the overall structure of the aqMutL NTD. In agreement with this, the recombinant human PMS2 S46I NTD was easily digested in the host Escherichia coli cells. Moreover, other mutations resulted in reduced DNA-binding or ATPase activity. In summary, using the thermostable aqMutL protein as a model molecule, we have experimentally determined the effects of the mutations on MutL endonuclease; we discuss the pathological effects of the corresponding mutations in human PMS2.

A nucleotide-dependent oligomerization of the Escherichia coli replication initiator DnaA requires residue His136 for remodeling of the chromosomal origin.

Escherichia coli replication initiator protein DnaA binds ATP with high affinity but the amount of ATP required to initiate replication greatly exceeds the amount required for binding. Previously, we showed that ATP-DnaA, not ADP-DnaA, undergoes a conformational change at the higher nucleotide concentration, which allows DnaA oligomerization at the replication origin but the association state remains unclear. Here, we used Small Angle X-ray Scattering (SAXS) to investigate oligomerization of DnaA in solution. Whereas ADP-DnaA was predominantly monomeric, AMP-PNP-DnaA (a non-hydrolysable ATP-analog bound-DnaA) was oligomeric, primarily dimeric. Functional studies using DnaA mutants revealed that DnaA(H136Q) is defective in initiating replication in vivo. The mutant retains high-affinity ATP binding, but was defective in producing replication-competent initiation complexes. Docking of ATP on a structure of E. coli DnaA, modeled upon the crystallographic structure of Aquifex aeolicus DnaA, predicts a hydrogen bond between ATP and imidazole ring of His136, which is disrupted when Gln is present at position 136. SAXS performed on AMP-PNP-DnaA (H136Q) indicates that the protein has lost its ability to form oligomers. These results show the importance of high ATP in DnaA oligomerization and its dependence on the His136 residue.

Homologs of aquifex aeolicus protein-only RNase P are not the major RNase P activities in the archaea haloferax volcanii and methanosarcina mazei.

The mature 5'-ends of tRNAs are generated by RNase P in all domains of life. The ancient form of the enzyme is a ribonucleoprotein consisting of a catalytic RNA and one or more protein subunits. However, in the hyperthermophilic bacterium Aquifex aeolicus and close relatives, RNase P is a protein-only enzyme consisting of a single type of polypeptide (Aq_880, ~23 kDa). In many archaea, homologs of Aq_880 were identified (termed HARPs for Homologs of Aquifex RNase P) in addition to the RNA-based RNase P, raising the question about the functions of HARP and the classical RNase P in these archaea. Here we investigated HARPs from two euryarchaeotes, Haloferax volcanii and Methanosarcina mazei. Archaeal strains with HARP gene knockouts showed no growth phenotypes under standard conditions, temperature and salt stress (H. volcanii) or nitrogen deficiency (M. mazei). Recombinant H. volcanii and M. mazei HARPs were basically able to catalyse specific tRNA 5'-end maturation in vitro. Furthermore, M. mazei HARP was able to rescue growth of an Escherichia coli RNase P depletion strain with comparable efficiency as Aq_880, while H. volcanii HARP was unable to do so. In conclusion, both archaeal HARPs showed the capacity (in at least one functional assay) to act as RNases P. However, the ease to obtain knockouts of the singular HARP genes and the lack of growth phenotypes upon HARP gene deletion contrasts with the findings that the canonical RNase P RNA gene cannot be deleted in H. volcanii, and a knockdown of RNase P RNA in H. volcanii results in severe tRNA processing defects. We conclude that archaeal HARPs do not make a major contribution to global tRNA 5'-end maturation in archaea, but may well exert a specialised, yet unknown function in (t)RNA metabolism. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1109-1116, 2019.