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

Abstract

The ATP hydrolysis transition state of motor proteins is a weakly populated protein state that can be stabilized and investigated by replacing ATP with chemical mimics. We present atomic-level structural and dynamic insights on a state created by ADP aluminum fluoride binding to the bacterial DnaB helicase from Helicobacter pylori. We determined the positioning of the metal ion cofactor within the active site using electron paramagnetic resonance, and identified the protein protons coordinating to the phosphate groups of ADP and DNA using proton-detected 31P,1H solid-state nuclear magnetic resonance spectroscopy at fast magic-angle spinning > 100 kHz, as well as temperature-dependent proton chemical-shift values to prove their engagements in hydrogen bonds. 19F and 27Al MAS NMR spectra reveal a highly mobile, fast-rotating aluminum fluoride unit pointing to the capture of a late ATP hydrolysis transition state in which the phosphoryl unit is already detached from the arginine and lysine fingers.

Proton-phosphorous connectivities revealed by high-resolution proton-detected solid-state NMR

“…We have recently presented the first hPH correlation spectrum recorded at 100 kHz MAS in the context of a large motor protein coordinating to DNA and ADP to probe hydrogen bonding. 11 To establish and setup such experiments, we have chosen a small model compound as an external standard, namely the phosphorylated amino acid tyrosine ( ortho -phospho-tyrosine) which has already been subject to solid-state NMR studies. 96,97 Figure S8 summarizes the 1 H MAS spectra, the proton line broadening contributions measured at 100 kHz MAS as well as the hPH spectra (for more details see the Section S2, ESI†).…”

“…6,7 We herein introduce phosphorus-31 as an additional radiofrequency channel in proton-detected fast MAS experiments (≥100 kHz) 6–8 allowing the investigation of a variety of functionalized phosphorus-containing materials (for instance, heterogeneous catalysts, modified zeolites 9 or aluminophosphates 10 ), as well as the characterization of biomolecular protein–nucleotide complexes. 11 Compared to previously reported 31 P-detected 1 H– 31 P heteronuclear correlation experiments (HETCOR) combined with rather high-power 1 H homonuclear decoupling performed at relatively slow MAS frequencies, 9,12–15 the herein proposed 1 H-detected 31 P– 1 H correlation experiment at fast MAS benefits in particular from requiring small sample amounts (<0.5 mg) and low-power decoupling. In addition, inverse detection schemes as used in solution (where one starts and ends with proton polarization) is turning into the method-of-choice at fast MAS frequencies due to the high sensitivity in 1 H-detection of the NMR probes for small rotor diameters.…”

Nucleic acid–protein interfaces studied by MAS solid-state NMR spectroscopy

“…The approaches used so far to characterize intermolecular contacts in nucleic acid–protein complexes can be divided in three classes, whereby many of the published studies use a combination of these approaches. Similar to solution-state NMR, the involvement of a molecular surface in interactions with a binding partner can be detected by either chemical shift perturbations (CSPs) or intensity changes of the ssNMR peaks of the surface atoms, when comparing the free and the bound-state of the molecule ( Ahmed et al, 2020 , Boudet et al, 2019 , Lacabanne et al, 2020 , Malär et al, 2021b , Wiegand et al, 2020b , Wiegand et al, 2019 , Wiegand et al, 2016 , Williamson, 2013 ). For example, the formation of an hydrogen bond at a nucleic acid–protein interface causes a downfield shift of the involved 1 H atom ( Wagner et al, 1983 ).…”

“…The chemical shifts of residues in (D) have a larger dependence on temperature and are thus not involved in hydrogen bonds. Figures (C–D) are reproduced from ( Malär et al, 2021b ) ©2021 with permission from Springer Nature. …”

“…Despite these limitations, ssNMR has been applied to large viral assemblies ( Andreas et al, 2016 , Goldbourt et al, 2007 , Lusky et al, 2021 , Morag et al, 2015 , Morag et al, 2014 , Sergeyev et al, 2011 , Yu and Schaefer, 2008 ) and site specific structural information was obtained for the 46-residue-long major coat protein subunit of the filamentous bacteriophage Pf1, as part of the 36 MDa virion ( Goldbourt et al, 2007 ), thanks to the fact that the 7300 subunits of the virion all adopt the same conformation. Over the years, the ssNMR toolbox has been extended for the application to RNA in isolation ( Leppert et al, 2004 , Lusky et al, 2021 , Riedel et al, 2006 , Riedel et al, 2005a , Riedel et al, 2005b , Yang et al, 2017 ), RNA bound to short peptides ( Huang et al, 2010 , Huang et al, 2011 , Huang et al, 2017 , Olsen et al, 2005 , Olsen et al, 2010 ), RNA as part of RNP complexes ( Aguion et al, 2021 , Ahmed et al, 2020 , Marchanka et al, 2013 , Marchanka et al, 2015 , Marchanka et al, 2018b ) and DNA–protein complexes ( Boudet et al, 2019 , Lacabanne et al, 2020 , Malär et al, 2021b , Wiegand et al, 2020b , Wiegand et al, 2019 , Wiegand et al, 2017b , Wiegand et al, 2016 , Zehnder et al, 2021 ).…”