Paramagnetic Solid‐State NMR to Localize the Metal‐Ion Cofactor in an Oligomeric DnaB Helicase

Abstract

Paramagnetic metal ions can be inserted into ATP‐fueled motor proteins by exchanging the diamagnetic Mg2+ cofactor with Mn2+ or Co2+. Then, paramagnetic relaxation enhancement (PRE) or pseudo‐contact shifts (PCSs) can be measured to report on the localization of the metal ion within the protein. We determine the metal position in the oligomeric bacterial DnaB helicase from Helicobacter pylori complexed with the transition‐state ATP‐analogue ADP:AlF4− and single‐stranded DNA using solid‐state NMR and a structure‐calculation protocol employing CYANA. We discuss and compare the use of Mn2+ and Co2+ in localizing the ATP cofactor in large oligomeric protein assemblies. 31P PCSs induced in the Co2+‐containing sample are then used to localize the DNA phosphate groups on the Co2+ PCS tensor surface enabling structural insights into DNA binding to the DnaB helicase.

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

“… a Sketch of hydrogen-bond formation and spatial proximities as revealed by the hPH and chemical-shift temperature-dependence experiments for ADP and DNA coordination to Hp DnaB. The Mg 2+ co-factor has been placed in spatial proximity to the AlF 4 − unit supported by the results from the EDNMR spectra (the coordinating aspartate located in the Walker B motif is shown additionally 113 ). b Zoom into the nucleotide-binding domain for Bst DnaB:GDP:AlF 4 − :DNA (PDB accession code 4ESV).…”

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

“…For a detailed description of paramagnetic NMR in solution and solid-state, readers are referred to a comprehensive review by Pell and coworkers ( Pell et al, 2019 ). In ssNMR, PRE measurements were first employed to identify residues of metalloproteins in proximity to the paramagnetic metal ( Balayssac et al, 2007b , Balayssac et al, 2007a , Pintacuda et al, 2007 ); recently, PRE experiments have also been used to study nucleic acid–protein complexes, ( Ahmed et al, 2020 , Wiegand et al, 2017a , Zehnder et al, 2021 ). Notably, Ahmed et al report the first structure of an RNP complex obtained solely from ssNMR data.…”

“…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 ).…”

Paramagnetic spin labeling of a bacterial DnaB helicase for solid-state NMR

“…We created ensemble conformation models [57] for PROXYL-M and Gd 3+ -DOTA-M and determined effective distances between the spin labels and every amino-acid residue in DnaB. First, we generated While the determination of PREs in 2D spectra of DnaB is difficult due to the large protein size, we have recently described for DnaB that this problem can, in principle, be mitigated by increasing the number of spectral dimensions [20]. Thus, we also recorded 3D NCACB spectra of PROXYL-M-tagged DnaB and apo DnaB, respectively.…”

“…In case of metalloproteins, naturally occurring metal binding sites can be used and loaded with transition metals or lanthanide ions [19]. For instance, the diamagnetic Mg 2+ cofactor in ATPdriven proteins can be exchanged with paramagnetic metal ions [20,21]. Additionally, diamagnetic proteins and nucleic acids [22] can be modified at specific positions with covalently bound paramagnetic tags [12,23], e.g.…”