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

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. P-31 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.

Automated summary

The study utilizes solid-state NMR and a structure-calculation protocol to determine the position of metal ions in the bacterial DnaB helicase from Helicobacter pylori complexed with ATP-analogue ADP:AlF4- and single-stranded DNA. The use of Mn2+ and Co2+ in localizing the ATP cofactor in large oligomeric protein assemblies is discussed and compared. P-31 PCSs induced in the Co2+-containing sample are then used to localize DNA phosphate groups and provide structural insights into DNA binding to the DnaB helicase.