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.

Functional and LC-MS/MS analysis of in vitro transcribed mRNAs carrying phosphorothioate or boranophosphate moieties reveal polyA tail modifications that prevent deadenylation without compromising protein expression

ABSTRACTChemical modifications enable preparation of mRNAs with augmented stability and translational activity. In this study, we explored how chemical modifications of 5’,3’-phosphodiester bonds in the mRNA body and polyA tail influence the biological properties of eukaryotic mRNA. To obtain modified and unmodified in vitro transcribed mRNAs, we used ATP and ATP analogues modified at the α-phosphate (containing either O-to-S or O-to-BH3 substitutions) and three different RNA polymerases—SP6, T7 and polyA polymerase. To verify the efficiency of incorporation of ATP analogues in the presence of ATP, we developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for quantitative assessment of modification frequency based on exhaustive degradation of the transcripts to 5’-mononucleotides. The method also estimated the average polyA tail lengths, thereby providing a versatile tool for establishing a structure-biological property relationship for mRNA. We found that mRNAs containing phosphorothioate groups within the polyA tail were substantially less susceptible to degradation by 3’-deadenylase than unmodified mRNA and were efficiently expressed in cultured cells, which makes them useful research tools and potential candidates for future development of mRNA-based therapeutics.

Molecular determinants for agonist recognition and discrimination in P2X2 receptors

P2X receptors (P2XRs) are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding. P2XRs contribute to synaptic transmission and are involved in pain and inflammation, thus representing valuable drug targets. Recent crystal structures have confirmed the findings of previous studies with regards to the amino acid chains involved in ligand recognition, but they have also suggested that backbone carbonyl atoms contribute to ATP recognition and discrimination. Here we use a combination of site-directed mutagenesis, amide-to-ester substitutions, and a range of ATP analogues with subtle alterations to either base or sugar component to investigate the contributions of backbone carbonyl atoms toward ligand recognition and discrimination in rat P2X2Rs. Our findings demonstrate that while the Lys69 backbone carbonyl makes an important contribution to ligand recognition, the discrimination between different ligands is mediated by both the side chain and the backbone carbonyl oxygen of Thr184. Together, our data demonstrate how conserved elements in P2X2Rs recognize and discriminate agonists.