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.

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.

Microbial rhodopsins and G-protein coupled receptors (GPCRs, which include animal rhodopsins) are two distinct (super) families of heptahelical (7TM) membrane proteins that share obvious structural similarities but no significant sequence similarity. Comparison of the recently solved high-resolution structures of the sodium-translocating bacterial rhodopsin and various Na+-binding GPCRs revealed striking similarity of their sodium-binding sites. This similarity allowed us to construct a structure-guided sequence alignment for the two (super)families, which highlighted their evolutionary relatedness. Our analysis supports a common underlying molecular mechanism for both families that involves a highly conserved aromatic residue playing a pivotal role in rotation of the 6th transmembrane helix.ReviewersThis article was reviewed by Oded Beja, G. P. S. Raghava and L. Aravind.Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-015-0091-4) contains supplementary material, which is available to authorized users.

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).

The assembly of respiratory complexes into macromolecular supercomplexes is currently a hot topic, especially in the context of newly available structural details. However, most work to date has been done with purified detergent-solubilized material and in situ confirmation is absent. We here set out to enable the recording of respiratory supercomplex formation in living cells. Fluorescent sensor proteins were placed at specific positions at cytochrome c oxidase suspected to either be at the surface of a CI1CIII2CIV1 supercomplex or buried within this supercomplex. In contrast to other loci, sensors at subunits CoxVIIIa and CoxVIIc reported a dense protein environment, as detected by significantly shortened fluorescence lifetimes. According to 3D modelling CoxVIIIa and CoxVIIc are buried in the CI1CIII2CIV1 supercomplex. Suppression of supercomplex scaffold proteins HIGD2A and CoxVIIa2l was accompanied by an increase in the lifetime of the CoxVIIIa-sensor in line with release of CIV from supercomplexes. Strikingly, our data provide strong evidence for defined stable supercomplex configuration in situ.

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.

Proteins within a single family usually share a common function but differ in more specific features and can be divided into subfamilies with different properties. Availability of genomic, structural, and functional information implemented into numerous databases provides new opportunities for bioinformatic analysis of homologous proteins. In this work, new method of bioinformatic analysis has been developed to identify subfamily-specific positions (SSPs)--conserved only within protein subfamilies, but different between subfamilies--that seem to play important role in functional diversity. A novel scoring function is suggested to consider structural information as well as physicochemical and residue conservation in protein subfamilies. Random shuffling is performed to rank results by significance, and Bernoulli statistics is applied to calculate p-values. Algorithm does not require predefined subfamily classification and can propose it automatically by graph-based clustering. This method can be used as a tool to explore SSPs with different structural localization in order to understand their implication to structure-function relationship and protein function. Web interface to the program is available at http://biokinet.belozersky.msu.ru/zebra.

BackgroundBinding of cytochrome c, released from the damaged mitochondria, to the apoptotic protease activating factor 1 (Apaf-1) is a key event in the apoptotic signaling cascade. The binding triggers a major domain rearrangement in Apaf-1, which leads to oligomerization of Apaf-1/cytochrome c complexes into an apoptosome. Despite the availability of crystal structures of cytochrome c and Apaf-1 and cryo-electron microscopy models of the entire apoptosome, the binding mode of cytochrome c to Apaf-1, as well as the nature of the amino acid residues of Apaf-1 involved remain obscure.ResultsWe investigated the interaction between cytochrome c and Apaf-1 by combining several modeling approaches. We have applied protein-protein docking and energy minimization, evaluated the resulting models of the Apaf-1/cytochrome c complex, and carried out a further analysis by means of molecular dynamics simulations. We ended up with a single model structure where all the lysine residues of cytochrome c that are known as functionally-relevant were involved in forming salt bridges with acidic residues of Apaf-1. This model has revealed three distinctive bifurcated salt bridges, each involving a single lysine residue of cytochrome c and two neighboring acidic resides of Apaf-1. Salt bridge-forming amino acids of Apaf-1 showed a clear evolutionary pattern within Metazoa, with pairs of acidic residues of Apaf-1, involved in bifurcated salt bridges, reaching their highest numbers in the sequences of vertebrates, in which the cytochrome c-mediated mechanism of apoptosome formation seems to be typical.ConclusionsThe reported model of an Apaf-1/cytochrome c complex provides insights in the nature of protein-protein interactions which are hard to observe in crystallographic or electron microscopy studies. Bifurcated salt bridges can be expected to be stronger than simple salt bridges, and their formation might promote the conformational change of Apaf-1, leading to the formation of an apoptosome. Combination of structural and sequence analyses provides hints on the evolution of the cytochrome c-mediated apoptosis.ReviewersThis article was reviewed by Andrei L. Osterman, Narayanaswamy Srinivasan, Igor N. Berezovsky, and Gerrit Vriend (nominated by Martijn Huynen).Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-015-0059-4) contains supplementary material, which is available to authorized users.