Evolution of cation binding in the active sites of P-loop nucleoside triphosphatases in relation to the basic catalytic mechanism
- University of Osnabrück, Germany
- Lomonosov Moscow State University, Russia
- Russian Academy of Sciences, Russia
- National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, United States
Figures
Figure 1
Mg-NTP complexes and their binding in the active sites of P-loop NTPases.
Phosphate chains of NTP molecules and their analogs are colored by atoms: oxygen atoms in red, phosphorus in orange. The K+ ion is shown as a purple sphere, Na+ ion is shown as a blue sphere, Mg2+ …
Figure 2 with 3 supplements
Binding of monovalent cations to the Mg-ATP in water.
The color scheme is as in Figure 1. (A) Superposition of the ATP phosphate chain conformations observed in the MD simulations in the presence of K+ ions (shown in purple); Na+ ions (shown in blue) …
Figure 2—figure supplement 1
Radial distribution of cations in the proximity of each oxygen atom.
Radial distributions are shown for all atoms of the ATP phosphate chain. (A) Atom names are in accordance with the CHARMM naming scheme (Vanommeslaeghe et al., 2010) and the recent IUPAC …
Figure 2—figure supplement 2
Properties of cation binding to the ATP as derived from MD simulations.
(A) Probability distribution functions for cations around the phosphate chain. We have plotted the number of atoms inside the area centered on phosphorus atoms of the ATP phosphate chain as a …
Figure 2—figure supplement 3
Binding of monovalent cations to the Mg-GTP in water.
Distances to the AG and BG binding sites (RAG and RBG) were calculated as averages of the distances to the two corresponding oxygen atoms (see Figure 2 in the main text). The distances to the oxygen …
Figure 3 with 1 supplement
Cation binding induces eclipsed conformation of the phosphate chain.
(A) Conformations of Mg-ATP complexes with one and two K+ ions bound as inferred from MD simulations; left structure, no K+ ion bound in the AG site; right structure, a K+ ion is bound in the AG …
Figure 3—figure supplement 1
Coupling between cation binding in the AG site and rotation of γ-phosphate relative to α- and β-phosphates.
Data from MD simulations with restraints on the positions of K+ ions (see the text and Supplementary file 1C). The top graph shows free energy calculated from normalized probabilities of ATP …
Figure 4 with 3 supplements
Dynamics of the phosphate chain of the Mg-ATP complex with and without monovalent cations.
Each left panel shows the PA-PG distance (upper trace) and the PB-O3B-PG angle (bottom trace) in the course of MD simulations. Thin gray lines show actual values measured from each frame of the MD …
Figure 4—figure supplement 1
Coordination of the Mg22+ion by the oxygen atoms of the ATP phosphate chain during MD simulations.
Black vertical lines indicate borders between independent simulations, thick colored lines show moving averages of distances measured during MD simulations. Oxygen atoms are labeled as in Figure 1D. …
Figure 4—figure supplement 2
Estimation of correlation times for the PA-PG distances.
A,B, Changes of the distance value upon MD simulations of βγ-coordinated Mg-ATP complexes with no additional monovalent cations (A) and with K+ ions (B) provided as examples. (C) Autocorrelation …
Figure 4—figure supplement 3
Estimation of correlation times for the PB-O-PG angles.
A, B, Changes of the angle value upon MD simulations of βγ-coordinated Mg-ATP complexes with no additional monovalent cations (A) and with K+ ions (B) provided as examples. (C) Autocorrelation …
Figure 5 with 2 supplements
Heat maps of the Mg-ATP phosphate chain conformations distribution characterized by the PA-PG distances (X-axis) and PB-O3B-PG angles (Y-axis).
Heat maps for systems with monovalent cations include only conformations of Mg-ATP complexes with at least one cation present within 4 Å radius. The color intensity is proportional to the …
Figure 5—figure supplement 1
Heat maps of the Mg-GTP phosphate chain conformations distribution characterized by the PA-PG distances (X-axis) and PB-O3B-PG angles (Y-axis).
Heat maps for systems with monovalent cations include only conformations of Mg-GTP complexes with at least one cation present within 4 Å area, and with Mg2+ in βγ coordination. The color intensity …
Figure 5—figure supplement 2
Phosphate chain shape of ATP and GTP analogs in the X-ray structures of P-loop NTPases.
PDB entries for structures of P-loop NTPases were extracted from InterPro database entry IPR027417 ‘P-loop containing nucleoside triphosphate hydrolase’ and filtered to contain only those X-ray …
Figure 6 with 4 supplements
Location of positive charges around the phosphate chain of Mg-NTP complexes in solution and in protein structures.
The color scheme is as in Figure 1; dark blue spheres indicate positions of positively charged side-chain nitrogen atoms of Lys and Arg residues, P-loop regions are shown as cartoons in grey. (A) …
Figure 6—figure supplement 1
Active sites of P-loop NTPases with established K+-dependent activity (see Supplementary file 1A for the full list and references).
Each of the proteins shown has both Asn residues that were shown to be associated with binding of monovalent cations in related proteins (Ash et al., 2012). Switch I, including the K-loop, and its …
Figure 6—figure supplement 2
Activation of the MnmE GTPase upon dimerization.
(A) Inactive dimer of the full-length MnmE in the GTP-bound form (the structure (PDB: 3GEI) was resolved with non-hydrolyzable GTP analogs). The P-loop domain is shown in grey, the K-loop is not …
Figure 6—figure supplement 3
Activation of the GTPase Era upon RNA binding.
(A) Inactive Era in the GDP-bound form [PDB: 3IEU] (Tu et al., 2009) in two projections. (B) Active Era in complex with nucleotides 1506–1542 of 16S rRNA and a non-hydrolyzable analog of GTP [PDB: …
Figure 6—figure supplement 4
Positively charged moieties in the active site of RecA-like recombinases.
(A) Cation-dependent RadA recombinase from Methanococcus voltae [PDB: 2F1H] (Qian et al., 2006). (B) Cation-independent RecA recombinase from E. coli [PDB: 3CMX] (Chen et al., 2008). The protein …
Figure 7 with 2 supplements
Molecular dynamics of MnmE GTPase.
(A) Superposition of the GTP-binding sites of the inactive, monomeric G-domain of MnmE (the 2GJ8W system, blue) and the active K+-bound dimer of G-domains (the 2GJ8K system, red); representative …
Figure 7—figure supplement 1
Hydrogen bonds lengths during MD simulations of the G-domain of MnmE.
Distances between phosphate chain oxygen atoms and surrounding amino acid residues were measured in the course of 100-ns MD simulations. (A) inactive monomer without a full-fledged K-loop (the 3GEI …
Figure 7—figure supplement 2
The distance between O2A and O3G in GTP bound to MnmE as inferred from MD simulations.
Plotted are data for the active dimer of G-domains with K+ ions bound (the 2GJ8K system, red and magenta for individual monomers) and inactive, monomeric G-domain of MnmE with the K+ ion replaced by …
Figure 8
Effects of Na+ binding on the shape of phosphate chain in solution and in Na+-adapted P-loop NTPases.
The color scheme is as in Figure 1, except that Al and F atoms in the GDP-AlF4- complexes are colored grey and cyan, respectively. (A) Superposition of the K+-bound (solid structure) and Na+-bound …
Tables
Table 1
Effects of monovalent cations on the shape of the triphosphate chain of the Mg-ATP complex in water, as inferred from the MD simulation data.
https://doi.org/10.7554/eLife.37373.013| Added cation | Conformation of the triphosphate chain of Mg-ATP* | |||||
|---|---|---|---|---|---|---|
| βγ-coordination | βγ-coordination, ‘curled’ phosphate chain | αβγ-coordination | ||||
| PA-PG distance, Å | PB-O3B-PG angle | PA-PG distance, Å | PB-O3B-PG angle | PA-PG distance, Å | PB-O3B-PG angle | |
| None | 5.46 ± 0.34 | 122.3 ± 3.5 | N/A | 4.76 ± 0.18 | 124.9 ± 3.3 | |
| K+ | 4.91 ± 0.24 | 122.0 ± 3.3 | N/A | 4.32 ± 0.24 | 128.0 ± 3.5 | |
| Na+ | 4.69 ± 0.22 | 122.9 ± 3.2 | 4.60 ± 0.22 | 124.0 ± 3.3 | 4.26 ± 0.37 | 127.7 ± 3.6 |
| NH4+ | 4.85 ± 0.22 | 122.3 ± 3.3 | 4.56 ± 0.21 | 124.6 ± 3.3 | 4.22 ± 0.16 | 127.8 ± 3. |
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*The conformations of the Mg-ATP complex were determined as described in the text. Mean values and standard deviations of PA-PG distance (in Å) and the PB-O3B-PG angle (in degrees) were measured over the respective parts of the simulations. Simulation periods corresponding to βγ and αβγ conformations were identified by tracking distances between Mg2+ and non-bridging oxygen atoms of the phosphate chain (Figure 4—figure supplement 1); simulation periods corresponding to the ‘curled’ conformation were identified from PA-PG distance tracks and visual inspection of the phosphate chain shape (Figure 4). Data for the αβγ coordination of the Mg-ATP complex and conformations with curled phosphate chain were calculated from simulations 1–4 in Supplementary file 1C; characterization of the βγ-coordination was based on simulations 5–8 in Supplementary file 1C, see Supplementary file 1E for further details.
Table 2
Activation mechanisms within the classes of P-loop NTPases that contain both cation-dependent and cation-independent enzymes.
https://doi.org/10.7554/eLife.37373.022| Superfamily | Family | Activating charge | Activation mechanism |
|---|---|---|---|
| Kinase-GTPase division, TRAFAC class | |||
| Classic translation factor GTPases | EF-G/EF-2 | K+ | Functional interaction with ribosomal RNA/other protein(s)/other domain(s) of the same protein (Hwang and Inouye, 2001;Moreau et al., 2008; Tomar et al., 2011; Achila et al., 2012;Fasano et al., 1982; Ebel et al., 1992; Dubnoff and Maitra, 1972;Kuhle and Ficner, 2014; Manikas et al., 2016; Daigle and Brown, 2004; Foucher et al., 2012;Rafay et al., 2012; Pérez-Arellano et al., 2013; Villarroya et al., 2008) |
| EF-Tu/EF-1A | K+ | ||
| EIF2G | K+ | ||
| ERF3 | K+ | ||
| IF-2 | K+ | ||
| LepA | K+ | ||
| OBG-HflX-like GTPases | HflX | K+ | |
| OBG | K+ | ||
| NOG | K+ | ||
| YchF/OLA1 | K+ | ||
| YlqF/YawG GTPases | NOG2 | K+ | |
| RsgA | K+ | ||
| TrmE-Era-EngA-EngB-Septin-like GTPases | EngA (Der) | K+ | |
| EngB | K+ | ||
| Era | K+ | ||
| FeoB | K+ | Dimerization (e.g. mRNA-associated in the case of MnmE) (Chappie et al., 2010; Koenig et al., 2008; Gasper et al., 2009) | |
| MnmE | K+ | ||
| Septin | Arg finger | ||
| Toc34-like | Arg finger | ||
| Dynamin-like GTPases | hGBP | Arg finger | |
| Dynamin | K+/Na+ | ||
| Extended Ras | Ras family | Arg finger | Interaction with a specialized activating protein or domain(Bos et al., 2007; Cherfils and Zeghouf, 2013) |
| Gα subunits | Arg finger | ||
| Myosin/kinesin | Myosin | Arg finger | |
| Kinesin | Arg finger | ||
| ASCE division, RecA/F1-like class | |||
| DNA-repair and recombination ATPases | RecA | Lys finger | DNA/RNA-dependent oligomerization(Chen et al., 2008) |
| RadA | K+ | ||
| Rho helicases | Rho | Arg finger | Interaction with the neighboring subunit within a conformationally coupled hexamer (Komoriya et al., 2012; Walker, 1998;Senior et al., 2002; Skordalakes and Berger, 2006) |
| T3SS ATPases | YscN | Arg finger | |
| Flil | Arg finger | ||
| F-/V-type ATPases | V-type A | Arg finger | |
| F-type β | |||
| V-type B | |||
| F-type α | |||
Table 3
Monovalent cation binding in crystal structures of P-loop NTPases.
https://doi.org/10.7554/eLife.37373.023| Protein | PDB entry | Bound NTP analog | Occupation of the AG site | Phosphate chain shape | |||
|---|---|---|---|---|---|---|---|
| Cation | Distance to the closest O atom of PA, Å* | Distance to the closest O atom of PG, Å*,† | PA-PG distance, Å* | PB-O3B-PG angle, degrees† | |||
| TRAFAC class NTPases | |||||||
| GTPase MnmE(TrmE) | 2gj8 | GDP AlF4- | K+ | 2.8 | 2.6 | 5.4 | 136.3 |
| 2gja | GDP AlF4- | NH4+ | 2.9 | 2.5 | 5.4 | 136.9 | |
| 2gj9 | GDP AlF4- | Rb+ | 2.9 | 2.8 | 5.5 | 131.6 | |
| GTPase FeoB | 3ss8 | GDP AlF4- | K+ | 2.8 | 2.6 | 5.4 | 144.9 |
| Dynamin-like proteins | 2x2e | GDP AlF4- | Na+ | 4.0 | 2.5 | 5.3 | 131.2 |
| 2x2f | GDP AlF4- | Na+ | 4.1 | 2.6 | 5.3 | 133.6 | |
| 3w6p | GDP AlF4- | Na+ | 4 | 2.4 | 5.5 | 135.3 | |
| 3t34 | GDP AlF4- | Na+ | 3.8 | 2.4 | 5.6 | 149.3 | |
| GTPase Era | 3r9w | GNP | H2O‡ | 3 | 3.4 | 5.1 | 129.2 |
| Eukaryotic translation initiation factor eIF5B | 4ncn | GTP | Na+ | 2.4 | 2.4 | 5.0 | 126.6 |
| 4tmv | GSP | Na+ | 2.4 | 2.8 (S)§ | 4.9 | 126.3 | |
| 4tmw | GTP | Na+ | 2.4 | 2.4 | 4.9 | 125.9 | |
| 4tmz | GSP | K+ | 2.7 | 3.3 (S)§ | 4.9 | 122.1 | |
| RecA/F1-like class NTPases | |||||||
| DNA recombinase RadA | 3ew9 | ANP | K+ | 6.2 | 3.3 | 5.1 | 124.5 |
| 2f1h | ANP | K+ | 6.6 | 3.5 | 5.3 | 125.3 | |
| 2fpm | ANP | K+ | 5.9 | 2.6 | 5.1 | 124.2 | |
| 1xu4 | ANP | K+ | 6.1 | 2.7 | 5.2 | 125.0 | |
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*The values were measured directly in the respective protein structures displayed in PyMOL.
† If the γ-phosphate was replaced by an AlF4- complex, the distance was measured to the closest F atom
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‡ While GTPase Era has been shown to be K+-dependent (Rafay et al., 2012; Meier et al., 2000), the crystallization solution contained no K+, only Na+, so that the likely cation-binding site is occupied by a water molecule, which forms hydrogen bonds with K+ ligands.
§ Non-hydrolyzable GTP analog GDP-monothiophosphate (GSP) contains a sulfur atom in the place of the O1G atom of γ-phosphate; this atom in involved in coordination of monovalent cations in respective structures.
Additional files
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Supplementary file 1
(A) Monovalent cation requirements of P-loop GTPases and ATPases. (B) Properties of monovalent cations and their interactions with the Mg2+-ATP complex. (C) Molecular dynamics simulations performed in this work. (D) Values of dihedral angles of the phosphate chains of Mg-ATP in the presence of K+ ions. (E) Lifetimes of the βγ-conformation of Mg-ATP complex during MD simulations. (F) Characteristics of the triphosphate chain for different interactions between the Mg2+ ion and ATP. (G) Comparison of the PA-PG distance measurements of the βγ-coordinated Mg-ATP complexes. (H) Comparison of the PA-PG distance measurements of the αβγ-coordinated Mg-ATP complexes. (I) Comparison of the PA-PG distance measurements for the αβγ-coordinated and ‘curled’ βγ-coordinated Mg-ATP complexes in different systems. (J) Comparison of the PB-O3B-PG angle measurements for the βγ-coordinated Mg-ATP complexes. (K) Comparison of the PB-O3B-PG angle measurements for the αβγ-coordinated Mg-ATP complexes. (L) Comparison of the PA-PG distance measurements for the αβγ-coordinated and ‘curled’ βγ-coordinated Mg-ATP complexes.
- https://doi.org/10.7554/eLife.37373.028
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Transparent reporting form
- https://doi.org/10.7554/eLife.37373.029
