In ubiquitous P-loop fold nucleoside triphosphatases (also known as Walker NTPases), hydrolysis of ATP or GTP is triggered by interaction with an activating partner (usually another protein domain), which is accompanied by insertion of stimulatory moieties (usually arginine or lysine residues) into the catalytic sites. After inspecting over 3600 Mg-NTP-containing structures of P-loop NTPases, we identified those with stimulator(s) inserted into catalytic sites and analysed the patterns of stimulatory interactions. In most cases, at least one stimulator twists gamma-phosphate counter-clockwise by linking the oxygen atoms of alpha- and gamma-phosphates; the twisted gamma-phosphate is stabilized by a hydrogen bond with the backbone amino group of the fourth residue of the Walker A motif. In the remaining cases, the stimulators only interact with gamma-phosphate. The all-pervasive mechanistic interactions of diverse stimulators with the gamma phosphate group suggests its twisting/turning as the trigger for NTP hydrolysis.

Nucleoside triphosphates (NTPs) are used as chemical energy source in a variety of cell systems. Structural snapshots along the NTP hydrolysis reaction coordinate are typically obtained by adding stable, nonhydrolyzable adenosine triphosphate (ATP) -analogues to the proteins, with the goal to arrest a state that mimics as closely as possible a physiologically relevant state, e.g., the pre-hydrolytic, transition and post-hydrolytic states. We here present the lessons learned on two distinct ATPases on the best use and unexpected pitfalls observed for different analogues. The proteins investigated are the bacterial DnaB helicase from Helicobacter pylori and the multidrug ATP binding cassette (ABC) transporter BmrA from Bacillus subtilis, both belonging to the same division of P-loop fold NTPases. We review the magnetic-resonance strategies which can be of use to probe the binding of the ATP-mimics, and present carbon-13, phosphorus-31, and vanadium-51 solid-state nuclear magnetic resonance (NMR) spectra of the proteins or the bound molecules to unravel conformational and dynamic changes upon binding of the ATP-mimics. Electron paramagnetic resonance (EPR), and in particular W-band electron-electron double resonance (ELDOR)-detected NMR, is of complementary use to assess binding of vanadate. We discuss which analogues best mimic the different hydrolysis states for the DnaB helicase and the ABC transporter BmrA. These might be relevant also to structural and functional studies of other NTPases.

The P-loop fold nucleoside triphosphate (NTP) hydrolases (also known as Walker NTPases) function as ATPases, GTPases, and ATP synthases, are often of medical importance, and represent one of the largest and evolutionarily oldest families of enzymes. There is still no consensus on their catalytic mechanism. To clarify this, we performed the first comparative structural analysis of more than 3,100 structures of P-loop NTPases that contain bound substrate Mg-NTPs or their analogues. We proceeded on the assumption that structural features common to these P-loop NTPases may be essential for catalysis. Our results are presented in two articles. Here, in the first, we consider the structural elements that stimulate hydrolysis. Upon interaction of P-loop NTPases with their cognate activating partners (RNA/DNA/protein domains), specific stimulatory moieties, usually Arg or Lys residues, are inserted into the catalytic site and initiate the cleavage of gamma phosphate. By analyzing a plethora of structures, we found that the only shared feature was the mechanistic interaction of stimulators with the oxygen atoms of gamma-phosphate group, capable of causing its rotation. One of the oxygen atoms of gamma phosphate coordinates the cofactor Mg ion. The rotation must pull this oxygen atom away from the Mg ion. This rearrangement should affect the properties of the other Mg ligands and may initiate hydrolysis according to the mechanism elaborated in the second article (reference, Biomolecules 1832871).

Although P-loop fold nucleoside triphosphatases (also known as Walker NTPases) are ubiquitous, their catalytic mechanism remains obscure. Based on a comparative structural analysis of 3136 Mg-NTP-containing catalytic sites, we propose a common scheme of activated catalysis for P-loop NTPases where a hydrogen bond (H-bond) between the strictly conserved, Mg-coordinating Ser/Thr of the Walker A motif ([Ser/Thr]WA) and the conserved aspartate of the Walker B motif (AspWB) plays the key role. We found that this H-bond is very short in the structures with bound transition state (TS) analogs. We suggest that the proton affinities of these two residues reverse in the TS so that the proton relocates from [Ser/Thr]WA to AspWB. The anionic [Ser/Thr]WA withdraws then a proton from the (catalytic) water molecule, and the nascent hydroxyl anion attacks gamma-phosphate. When the gamma-phosphate group breaks away, the trapped proton relays from AspWB, via [Ser/Thr]WA, to beta-phosphate and compensates for its developing negative charge.

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.