6 research outputs found

    Molecular Modelling of Monovalent Cations in Energy-Converting Proteins

    No full text
    In this work, the evolutionary biophysics approach is applied to the two of the largest protein superfamilies present in human genomes, namely P-loop fold nucleoside triphosphatases (P-loop NTPases) and G-protein coupled receptors (GPCRs). This approach combines comparative analysis of protein structures and sequences with molecular modeling techniques in order to reveal not only the conservation of particular residues among proteins within each superfamily but also their role in the fundamental mechanisms underlying common functions. The study of the hydrolysis activation mechanism in P-loop NTPases started with the molecular dynamics simulations of Mg-NTP complexes (Mg-ATP and Mg-GTP) in the presence of K+, NH4+, and Na+ ions. These simulations showed that in the presence of large cations (K+ and NH4+), the conformation of the phosphate chain of ATP and GTP is extended, with large distances between alpha- and gamma-phosphates. This conformation is similar to the shape of ATP and GTP molecules (or their analogs) in the crystal structures of various P-loop NTPases. To clarify the role of monovalent cations in P-loop NTPases, MD simulations were conducted for two cation-dependent GTPases: tRNA modification GTPase MnmE and translation factor EF-Tu. MD simulations of Mg-GTP/EF-Tu complex bound to the tRNA and ribosome fragment in the presence of K+ ions have shown consistent binding of a potassium ion from the solution between alpha- and gamma-phosphates (AG site), similar to the cation binding in MnmE and other cation-dependent P-loop GTPases. In both proteins, binding of K+ ion in the AG site led to the rotation of gamma-phosphate, making this group more eclipsed with alpha-phosphate. The new rotated position of gamma-phosphate was stabilized by a novel H-bond with the backbone nitrogen of the K-3 residue (relative to the ubiquitously conserved Lys) of the P-loop motif. The activation mechanism observed in MD simulations of MnmE and EF-Tu could be envisioned as basic for P-loop NTPases, as these cation-dependent proteins are among the most ancient members of the P-loop superfamily. This mechanism was used as a basis for extensive comparative analysis of representative proteins from all major classes of P-loop NTPases. Based on the established conservation and presence of the key features in active sites of P-loop NTPases, the chain of events where rotation of gamma-phosphate triggers the nucleophilic attack and gamma-phosphate cleavage has been proposed as the basic universal activation mechanism of NTP hydrolysis in P-loop NTPases. The second part of this work explores the activation of GPCRs as sodium-translocating receptors. Crystal structures of the novel Na-pumping microbial rhodopsin along with the recent avalanche of GPCR structures provided the basis for comparative structure analysis, focused on investigating the similarities in the Na-binding sites of the two superfamilies. Structure superposition of GPCRs and microbial rhodopsins (MRs) based on comparison of their Na-binding sites was used to produce structure-guided sequence alignments of the two superfamilies. The only residue universally conserved between the two superfamilies was Trp in the helix 6/F (Trp6.48 in GPCRs). In both families, the signaling mechanism directly involves this residue, which is likely to be an ancient feature inherited from the common ancestor of MRs and GPCRs – the Na-pumping light-activated rhodopsin. The similarity of GPCRs with light-activated sodium pumps endorses the suggestion that GPCRs may also function as Na+ ion translocators. A model of GPCR activation accompanied by translocation of Na+ was constructed to demonstrate how this mechanism can explain the voltage sensitivity of certain Class A GPCRs. Two modes of activation were modeled – one where Na+ ion is transported into the cytoplasm and the one where Na+ ion is expelled to the intracellular space. The two modes quantitatively describe the behavior of voltage-activated and voltage-suppressed GPCRs, respectively. Finally, further structure scrutiny and rotamer analysis provided a plausible pathway of Na+ transmembrane translocation through the helical bundle of GPCRs

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

    No full text
    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 3100 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

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

    No full text
    To clarify the obscure hydrolysis mechanism of ubiquitous P-loop-fold nucleoside triphosphatases (Walker NTPases), we analysed the structures of 3136 catalytic sites with bound Mg-NTP complexes or their analogues. Our results are presented in two articles; here, in the second of them, we elucidated whether the Walker A and Walker B sequence motifs—common to all P-loop NTPases—could be directly involved in catalysis. We found that the hydrogen bonds (H-bonds) between the strictly conserved, Mg-coordinating Ser/Thr of the Walker A motif ([Ser/Thr]WA) and aspartate of the Walker B motif (AspWB) are particularly short (even as short as 2.4 ångströms) in the structures with bound transition state (TS) analogues. Given that a short H-bond implies parity in the pKa values of the H-bond partners, we suggest that, in response to the interactions of a P-loop NTPase with its cognate activating partner, a proton relocates from [Ser/Thr]WA to AspWB. The resulting anionic [Ser/Thr]WA alkoxide withdraws a proton from the catalytic water molecule, and the nascent hydroxyl attacks the gamma phosphate of NTP. When the gamma-phosphate breaks away, the trapped proton at AspWB passes by the Grotthuss relay via [Ser/Thr]WA to beta-phosphate and compensates for its developing negative charge that is thought to be responsible for the activation barrier of hydrolysis

    Evolution of cation binding in the active sites of P-loop nucleoside triphosphatases in relation to the basic catalytic mechanism

    No full text
    The ubiquitous P-loop fold nucleoside triphosphatases (NTPases) are typically activated by an arginine or lysine ‘finger’. Some of the apparently ancestral NTPases are, instead, activated by potassium ions. To clarify the activation mechanism, we combined comparative structure analysis with molecular dynamics (MD) simulations of Mg-ATP and Mg-GTP complexes in water and in the presence of potassium, sodium, or ammonium ions. In all analyzed structures of diverse P-loop NTPases, the conserved P-loop motif keeps the triphosphate chain of bound NTPs (or their analogs) in an extended, catalytically prone conformation, similar to that imposed on NTPs in water by potassium or ammonium ions. MD simulations of potassium-dependent GTPase MnmE showed that linking of alpha- and gamma phosphates by the activating potassium ion led to the rotation of the gamma-phosphate group yielding an almost eclipsed, catalytically productive conformation of the triphosphate chain, which could represent the basic mechanism of hydrolysis by P-loop NTPases
    corecore