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

Nat Commun. 2021 Sep 6;12(1):5293. doi: 10.1038/s41467-021-25599-z.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Diphosphate / chemistry*
  • Adenosine Diphosphate / metabolism
  • Adenosine Triphosphate / chemistry*
  • Adenosine Triphosphate / metabolism
  • Aluminum Compounds / chemistry
  • Aluminum Compounds / metabolism
  • Arginine / chemistry
  • Arginine / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Catalytic Domain
  • Cloning, Molecular
  • DNA, Bacterial / chemistry*
  • DNA, Bacterial / genetics
  • DNA, Bacterial / metabolism
  • DnaB Helicases / chemistry*
  • DnaB Helicases / genetics
  • DnaB Helicases / metabolism
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Fluorides / chemistry
  • Fluorides / metabolism
  • Gene Expression
  • Helicobacter pylori / enzymology*
  • Helicobacter pylori / genetics
  • Hydrolysis
  • Lysine / chemistry
  • Lysine / metabolism
  • Models, Molecular
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • Thermodynamics

Substances

  • Aluminum Compounds
  • Bacterial Proteins
  • DNA, Bacterial
  • Recombinant Proteins
  • Adenosine Diphosphate
  • Adenosine Triphosphate
  • Arginine
  • DnaB Helicases
  • Lysine
  • Fluorides
  • aluminum fluoride