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.
Highlights
The Adenosine triphosphate (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 magnetic resonance approaches using EPR and solid-state NMR to obtain spectroscopic insights into the transition state of ATP hydrolysis which we trap for the oligomeric bacterial DnaB helicase from Helicobacter pylori (Hp, monomeric molecular weight 59 kDa) by using the transition-state analogue ADP:AlF4−
That we report a proton-detected 31P,1H correlation spectrum at fast magic-angle spinning (MAS) frequencies using a sub-milligram sample amount, which was so far, to the best of our knowledge, not possible with any of the previous equipment. This is an important step for proving hydrogen bonding in protein–nucleic acid complexes ranging from proteins involved in DNA replication or virus assemblies by solid-state NMR and to derive nucleotide-binding modes, even in quite large systems as the one we looked at
Summary
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. Bacterial DnaB helicases, which use the energy of ATP hydrolysis to unwind the DNA double helix, belong to the ASCE division of P-loop NTPases The members of this division are characterized by an additional β-strand in the P-loop and a catalytic glutamate (E) residue next to the attacking water molecule[14,15,16]. RecA-type ATPases generally differ from most other P-loop NTPases in that their stimulating residues, which operate in a tandem, interact upon activation only with the γ-phosphate, but not with the α-phosphate group This interaction triggers the ATP hydrolysis; the triggering mechanism, has yet to be determined[21]
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