ATP Analogues for Structural Investigations: Case Studies of a DnaB Helicase and an ABC Transporter.

Nucleoside triphosphates (NTPs) are used as chemical energy source in a variety of cell systems. Structural snapshots along the NTP hydrolysis reaction coordinate are typically obtained by adding stable, nonhydrolyzable adenosine triphosphate (ATP) -analogues to the proteins, with the goal to arrest a state that mimics as closely as possible a physiologically relevant state, e.g., the pre-hydrolytic, transition and post-hydrolytic states. We here present the lessons learned on two distinct ATPases on the best use and unexpected pitfalls observed for different analogues. The proteins investigated are the bacterial DnaB helicase from Helicobacter pylori and the multidrug ATP binding cassette (ABC) transporter BmrA from Bacillus subtilis, both belonging to the same division of P-loop fold NTPases. We review the magnetic-resonance strategies which can be of use to probe the binding of the ATP-mimics, and present carbon-13, phosphorus-31, and vanadium-51 solid-state nuclear magnetic resonance (NMR) spectra of the proteins or the bound molecules to unravel conformational and dynamic changes upon binding of the ATP-mimics. Electron paramagnetic resonance (EPR), and in particular W-band electron-electron double resonance (ELDOR)-detected NMR, is of complementary use to assess binding of vanadate. We discuss which analogues best mimic the different hydrolysis states for the DnaB helicase and the ABC transporter BmrA. These might be relevant also to structural and functional studies of other NTPases.

Open questions on the origin of life at anoxic geothermal fields.

We have recently reconstructed the 'hatcheries' of the first cells by combining geochemical analysis with phylogenomic scrutiny of the inorganic ion requirements of universal components of modern cells (Mulkidjanian et al. Proc Natl Acad Sci U S A 109:E821-830, 2012). These ubiquitous, and by inference primordial, proteins and functional systems show affinity to and functional requirement for K⁺, Zn²âº, Mn²âº, and phosphate. Thus, protocells must have evolved in habitats with a high K⁺/Na⁺ ratio and relatively high concentrations of Zn, Mn and phosphorous compounds. Geochemical reconstruction shows that the ionic composition conducive to the origin of cells could not have existed in marine settings but is compatible with emissions of vapor-dominated zones of inland geothermal systems. Under an anoxic, CO2-dominated atmosphere, the ionic composition of pools of cool, condensed vapor at anoxic geothermal fields would resemble the internal milieu of modern cells. Such pools would be lined with porous silicate minerals mixed with metal sulfides and enriched in K⁺ ions and phosphorous compounds. Here we address some questions that have appeared in print after the publication of our anoxic geothermal field scenario. We argue that anoxic geothermal fields, which were identified as likely cradles of life by using a top-down approach and phylogenomics analysis, could provide geochemical conditions similar to those which were suggested as most conducive for the emergence of life by the chemists who pursuit the complementary bottom-up strategy.