Common Patterns of Hydrolysis Initiation in P-loop Fold Nucleoside Triphosphatases

Abstract

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).

Common Mechanism of Activated Catalysis in P-loop Fold Nucleoside Triphosphatases—United in Diversity

“…As for the analysis presented in in the companion paper [ 30 ], the structures were selected among those PDB entries that matched the following criteria: (1) assigned to InterPro record IPR027417; (2) contained an ATP/GTP molecule, non-hydrolyzable analogue of NTP, or a transition-state analogue; (3) contained at least one Mg 2+ , Mn 2+ , or Ca 2+ ion; and (4) resolution of 5 Å or higher, if applicable. Proteins were assigned to major classes of P-loop NTPases according to their membership in Pfam families [ 9 , 10 ]; see the details in the companion paper [ 30 ]. This search yielded 1474 PDB structures with 3666 catalytic sites in them.…”

“…We investigated the reliability of each catalytic site by assessing the integrity of the NTP analogue/presence of γ-phosphate mimic, presence of a P-loop motif Lys residue within 5 Å of the β-phosphate. This analysis yielded 3136 complexes in 1383 structures with various substrates: ATP and GTP, their nonhydrolyzable analogues, and ADP/GDP molecules associated with γ-phosphate-mimicking moieties; see Figure 2 in the companion paper [ 30 ].…”

“…For quality control, we measured the distances from [Ser/Thr] K+1 to Mg 2+ to ensure the correct binding of the Mg 2+ and general reliability of the structure resolution at the binding site (i.e., a long distance would indicate a disturbed catalytic site or resolution at the site that is insufficient for our purposes of comparative analysis), and from [Asp/Glu] WB to Mg 2+ , to identify cases of direct coordination of Mg 2+ by the acidic residue (short distances) or disassembled binding sites (long distances). In addition, in those sites that passed quality control, we examined the presence of interactions of phosphate chain with the NH K−3 group and positively charged moieties (the details of analysis are described in the companion paper [ 30 ] and depicted in its Figure 2).…”

“…Hydrolysis, however, takes place only when the P-loop domain interacts with its cognate activating partner, which could be a domain of the same protein, a separate protein, and/or a DNA/RNA molecule. Upon this interaction, specific stimulatory moieties, usually Arg or Lys residues (“Arg/Lys fingers”, Figure 1 E,F), are inserted into the catalytic sites and promote the cleavage of γ-phosphate; see the companion article [ 30 ] and [ 4 , 15 , 16 , 29 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 ].…”

“…Our results are presented in two articles. In the first article (see the companion paper [ 30 ]), we inspected 3136 structures of Mg-NTP-containing catalytic sites of P-loop NTPases, identified the structures with inserted stimulator(s), and analysed the patterns of stimulatory interactions.…”