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

Abstract

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.

Spectroscopic glimpses of the transition state of ATP hydrolysis trapped in a bacterial DnaB helicase

“…Binding of ADP:AlF 4 − to the protein is revealed in EDNMR experiments, which employ the hyperfine couplings between a paramagnetic center and nearby nuclei to detect the latter. EDNMR has been used to characterize transition states of ATP hydrolysis, often in the context of ABC transporters for which such a state is successfully mimicked by ADP-vanadate 63 , 64 . Figure 2a shows the Mn 2+ EDNMR spectrum of DnaB complexed with ADP:AlF 4 − (red) compared to the reference spectrum of DnaB complexed only with ADP (cyan), here using a non- 13 C/ 15 N labeled protein (see below for 13 C/ 15 N labeling).…”

“…The cyan spectrum is reproduced from ref. 64 . Asterisks mark a broad, currently unassigned feature that is possibly due to Mn double quantum or combination lines 23 .…”

Spectroscopic glimpses of the transition state of ATP hydrolysis trapped in a bacterial DnaB helicase

“…EDNMR has been used to characterize transition states of ATP hydrolysis, often in the context of ABC transporters for which such a state is successfully mimicked by ADP-vanadate 59,60 .…”

Special Issue—The Conformational Universe of Proteins and Peptides: Tales of Order and Disorder

“…Beat H. Meier, Anja Böckmann and collaborators describe within their review the lessons learned from studies on two ATPases (the bacterial DnaB helicase from Helicobacter pylori and the multidrug ATP binding cassette (ABC) transporter BmrA from Bacillus subtilis ) [ 10 ]. The authors report on NMR approaches that can be employed to examine proteins binding to ATP-mimics.…”

“…The reported information can surely be relevant to researchers conducting studies on other NTPases (Nucleoside TriPhosphatases) [ 10 ].…”