scholarly journals Comparative analysis of active sites in P-loop nucleoside triphosphatases suggests an ancestral activation mechanism

2018 ◽  
Author(s):  
Daria N. Shalaeva ◽  
Dmitry A. Cherepanov ◽  
Michael Y. Galperin ◽  
Armen Y. Mulkidjanian
Keyword(s):  
Active Sites ◽  
Catalytic Mechanism ◽  
Md Simulations ◽  
Activation Mechanism ◽  
Sequence Motifs ◽  
Backbone Amide ◽  
Comparative Structure ◽  
Combined Structure ◽  
Phosphate Groups ◽  
Positively Charged

AbstractP-loop nucleoside triphosphatases (NTPases) share common Walker A (P-loop) and Walker B sequence motifs and depend on activating moieties (Arg or Lys fingers or a K+ ion). In search for a common catalytic mechanism, we combined structure comparisons of active sites in major classes of P-loop NTPases with molecular dynamics (MD) simulations of the Ras GTPase, a well-studied oncoprotein. Comparative structure analysis showed that positively charged activating moieties interact with gamma-phosphate groups of NTP substrates in all major classes of P-loop NTPases. In MD simulations, interaction of the activating Arg finger with the Mg-GTP-Ras complex led to the rotation of the gamma-phosphate group by 40 degrees enabling its interaction with the backbone amide group of Gly13. In all analyzed structures, the residue that corresponds to Gly13 of Ras was in a position to stabilize gamma-phosphate after its rotation, suggesting a common ancestral activation mechanism within the entire superfamily.

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

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Daria N Shalaeva ◽  
Dmitry A Cherepanov ◽  
Michael Y Galperin ◽  
Andrey V Golovin ◽  
Armen Y Mulkidjanian
Keyword(s):  
Active Sites ◽  
Catalytic Mechanism ◽  
Md Simulations ◽  
Basic Mechanism ◽  
Cation Binding ◽  
Activation Mechanism ◽  
Potassium Ions ◽  
Ammonium Ions ◽  
Mechanism Of Hydrolysis ◽  
Comparative Structure

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.

scholarly journals Evolution of cation binding in the active sites of P-loop nucleoside triphosphatases

2018 ◽  
Author(s):  
Daria N. Shalaeva ◽  
Dmitry A. Cherepanov ◽  
Michael Y. Galperin ◽  
Armen Y. Mulkidjanian
Keyword(s):  
Active Sites ◽  
Atp Hydrolysis ◽  
Md Simulations ◽  
Cation Binding ◽  
Potassium Ions ◽  
Sodium Ions ◽  
Ammonium Ions ◽  
Phosphate Chain ◽  
Comparative Structure ◽  
Phosphate Groups

AbstractThe activity of cellular nucleoside triphosphatases (NTPases) must be tightly controlled to prevent spontaneous ATP hydrolysis leading to cell death. While most P-loop NTPases require activation by arginine or lysine fingers, some of the apparently ancestral ones are, instead, activated by potassium ions, but not by sodium ions. We combined comparative structure analysis of P-loop NTPases of various classes with molecular dynamics (MD) simulations of Mg-ATP complexes in water and in the presence of potassium, sodium, or ammonium ions. In all analyzed structures, the conserved P-loop motif keeps the triphosphate chains of enzyme-bound NTPs in an extended, catalytically prone conformation, similar to that attained by ATP in water in the presence of potassium or ammonium ions bound between alpha- and gamma-phosphate groups. The smaller sodium ions could not reach both alpha- and gamma-phosphates of a protein-bound extended phosphate chain and therefore are unable to activate most potassium-dependent P-loop NTPases.

scholarly journals Exploration of inositol 1,4,5-trisphosphate (IP3) regulated dynamics of N-terminal domain of IP3 receptor reveals early phase molecular events during receptor activation

2018 ◽  
Author(s):  
Aneesh Chandran ◽  
Xavier Chee ◽  
David L. Prole ◽  
Taufiq Rahman
Keyword(s):  
Conformational Dynamics ◽  
Md Simulations ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Ip3 Receptor ◽  
Dynamic Correlation ◽  
Molecular Events ◽  
Binding Core ◽  
Domain Dynamics ◽  
Phosphate Groups

Inositol 1, 4, 5-trisphosphate (IP3) binding at the N-terminus (NT) of IP3 receptor (IP3R) allosterically triggers the opening of a Ca2+-conducting pore located ~ 100 Å away from the IP3-binding core (IBC). However, the precise mechanism of IP3 binding and correlated domain dynamics in the NT that are central to the IP3R activation, remains unknown. Our all-atom molecular dynamics (MD) simulations recapitulate the characteristic twist motion of the suppresser domain (SD) and reveal correlated ‘clam closure’ dynamics of IBC with IP3-binding, complementing existing suggestions on IP3R activation mechanism. Our study further reveals the existence of inter-domain dynamic correlation in the NT and establishes the SD to be critical for the conformational dynamics of IBC. Also, a tripartite interaction involving Glu283-Arg54-Asp444 at the SD – IBC interface seemed critical for IP3R activation. Intriguingly, during the sub-microsecond long simulation, we observed Arg269 undergoing an SD-dependent flipping of hydrogen bonding between the first and fifth phosphate groups of IP3. This seems to play a major role in determining the IP3 binding affinity of IBC in the presence/absence of the SD. Our study thus provides atomistic details of early molecular events occurring within the NT during and following IP3 binding that lead to channel gating.

scholarly journals Computational Approach Choice in Modeling Flexible Enzyme Active Sites

2019 ◽  
Author(s):  
Hisham Dokainish ◽  
James Gauld
Keyword(s):  
Conformational Changes ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Experimental Studies ◽  
Md Simulations ◽  
Acid Formation ◽  
Nucleophilic Attack ◽  
Sulfenic Acid ◽  
Proper Position

<div><div><div><p>The last step in the reductase step of the catalytic mechanism of MsrB was re-investigated using several computational approaches. Our previous QM-cluster paper showed that two possible mechanisms could occur, however the direct formation of disulfide from sulfonium cation was favored over sulfenic acid formation. In contrary, experimental studies suggest sulfenic acid formation. Therefore, first, we investigated the effect of level of theory, which confirmed previous conclusion. In addition, the effect of model choice was also investigated using ONIOM including a large QM layer around Cys440. Interestingly, deprotonating Cys440 leads to direct nucleophilic attack on Cys495 forming disulfide. Second, to eliminate the possibility that all previous results are an artifact of the used crystal structure in which the S...S distance is 3.29 Å, we ran a 5 ns MD simulation on the sulfonium cation intermediate. Surprisingly, our results show that the distance between the two sulfur is significantly increased to 4.88 Å. More importantly a water molecule is located in a proper position for nucleophilic attack. QM/MM calculations shows that sulfenic acid is formed via low barrier of 16.7 kJ mol-1. Finally, the effect of substrate binding on the two Cys's distance were investigated via running several MD simulations of possible intermediates, showing that substrate binding induces conformational changes increasing the sulfur's distance which is decreased upon substrate removal upon sulfenic acid formation. These results question the applicability of QM cluster approach in systems including flexible turns. It also emphasizes the importance of proper preparation of the starting structure.</p></div></div></div>

scholarly journals Computational Approach Choice in Modeling Flexible Enzyme Active Sites

2019 ◽  
Author(s):  
Hisham Dokainish ◽  
James Gauld
Keyword(s):  
Conformational Changes ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Experimental Studies ◽  
Md Simulations ◽  
Acid Formation ◽  
Nucleophilic Attack ◽  
Sulfenic Acid ◽  
Proper Position

<div><div><div><p>The last step in the reductase step of the catalytic mechanism of MsrB was re-investigated using several computational approaches. Our previous QM-cluster paper showed that two possible mechanisms could occur, however the direct formation of disulfide from sulfonium cation was favored over sulfenic acid formation. In contrary, experimental studies suggest sulfenic acid formation. Therefore, first, we investigated the effect of level of theory, which confirmed previous conclusion. In addition, the effect of model choice was also investigated using ONIOM including a large QM layer around Cys440. Interestingly, deprotonating Cys440 leads to direct nucleophilic attack on Cys495 forming disulfide. Second, to eliminate the possibility that all previous results are an artifact of the used crystal structure in which the S...S distance is 3.29 Å, we ran a 5 ns MD simulation on the sulfonium cation intermediate. Surprisingly, our results show that the distance between the two sulfur is significantly increased to 4.88 Å. More importantly a water molecule is located in a proper position for nucleophilic attack. QM/MM calculations shows that sulfenic acid is formed via low barrier of 16.7 kJ mol-1. Finally, the effect of substrate binding on the two Cys's distance were investigated via running several MD simulations of possible intermediates, showing that substrate binding induces conformational changes increasing the sulfur's distance which is decreased upon substrate removal upon sulfenic acid formation. These results question the applicability of QM cluster approach in systems including flexible turns. It also emphasizes the importance of proper preparation of the starting structure.</p></div></div></div>

The first crystal structure of the peptidase domain of the U32 peptidase family

2015 ◽  
Vol 71 (12) ◽  
pp. 2505-2512 ◽  
Author(s):  
Magdalena Schacherl ◽  
Angelika A. M. Montada ◽  
Elena Brunstein ◽  
Ulrich Baumann
Keyword(s):  
Crystal Structure ◽  
Catalytic Mechanism ◽  
Quaternary Structure ◽  
Catalytic Domain ◽  
Three Dimensional ◽  
Zinc Ion ◽  
Crystal Structure Analysis ◽  
Dimensional Structure ◽  
Sequence Motifs ◽  
Conserved Sequence

The U32 family is a collection of over 2500 annotated peptidases in the MEROPS database with unknown catalytic mechanism. They mainly occur in bacteria and archaea, but a few representatives have also been identified in eukarya. Many of the U32 members have been linked to pathogenicity, such as proteins fromHelicobacterandSalmonella. The first crystal structure analysis of a U32 catalytic domain fromMethanopyrus kandleri(genemk0906) reveals a modified (βα)8TIM-barrel fold with some unique features. The connecting segment between strands β7 and β8 is extended and helix α7 is located on top of the C-terminal end of the barrel body. The protein exhibits a dimeric quaternary structure in which a zinc ion is symmetrically bound by histidine and cysteine side chains from both monomers. These residues reside in conserved sequence motifs. No typical proteolytic motifs are discernible in the three-dimensional structure, and biochemical assays failed to demonstrate proteolytic activity. A tunnel in which an acetate ion is bound is located in the C-terminal part of the β-barrel. Two hydrophobic grooves lead to a tunnel at the C-terminal end of the barrel in which an acetate ion is bound. One of the grooves binds to aStrep-Tag II of another dimer in the crystal lattice. Thus, these grooves may be binding sites for hydrophobic peptides or other ligands.

scholarly journals Computational Insights into Substrate and Site Specificities, Catalytic Mechanism, and Protonation States of the Catalytic Asp Dyad ofβ-Secretase

Scientifica ◽  
2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Arghya Barman ◽  
Rajeev Prabhakar
Keyword(s):  
Catalytic Mechanism ◽  
Md Simulations ◽  
Peptide Bond ◽  
Docking Studies ◽  
In Silico Screening ◽  
Rate Determining Step ◽  
Rate Limiting Step ◽  
Beta Peptide ◽  
Structural Differences ◽  
Rate Limiting

In this review, information regarding substrate and site specificities, catalytic mechanism, and protonation states of the catalytic Asp dyad ofβ-secretase (BACE1) derived from computational studies has been discussed. BACE1 catalyzes the rate-limiting step in the generation of Alzheimer amyloid beta peptide through the proteolytic cleavage of the amyloid precursor protein. Due to its biological functioning, this enzyme has been considered as one of the most important targets for finding the cure for Alzheimer’s disease. Molecular dynamics (MD) simulations suggested that structural differences in the key regions (inserts A, D, and F and the 10s loop) of the enzyme are responsible for the observed difference in its activities towards the WT- and SW-substrates. The modifications in the flap, third strand, and insert F regions were found to be involved in the alteration in the site specificity of the glycosylphosphatidylinositol bound form of BACE1. Our QM and QM/MM calculations suggested that BACE1 hydrolyzed the SW-substrate more efficiently than the WT-substrate and that cleavage of the peptide bond occurred in the rate-determining step. The results from molecular docking studies showed that the information concerning a single protonation state of the Asp dyad is not enough to run an in silico screening campaign.

scholarly journals Insight into Inhibitor Binding in the Eukaryotic Proteasome: Computations of the 20S CP

2018 ◽  
Vol 19 (12) ◽  
pp. 3858
Author(s):  
Milan Hodošček ◽  
Nadia Elghobashi-Meinhardt
Keyword(s):  
Electrostatic Interactions ◽  
Active Sites ◽  
Sampling Period ◽  
Md Simulations ◽  
Structural Features ◽  
Relaxation Dynamics ◽  
Inhibitor Binding ◽  
Computational Analyses ◽  
Insight Into ◽  
Proteolytic Chamber

A combination of molecular dynamics (MD) simulations and computational analyses uncovers structural features that may influence substrate passage and exposure to the active sites within the proteolytic chamber of the 20S proteasome core particle (CP). MD simulations of the CP reveal relaxation dynamics in which the CP slowly contracts over the 54 ns sampling period. MD simulations of the SyringolinA (SylA) inhibitor within the proteolytic B 1 ring chamber of the CP indicate that favorable van der Waals and electrostatic interactions account for the predominant association of the inhibitor with the walls of the proteolytic chamber. The time scale required for the inhibitor to travel from the center of the proteolytic chamber to the chamber wall is on the order of 4 ns, accompanied by an average energetic stabilization of approximately −20 kcal/mol.

scholarly journals The Role of Electrostatics in Enzymes: Do Biomolecular Force Fields Reflect Protein Electric Fields?

2020 ◽  
Author(s):  
Richard T Bradshaw ◽  
Jacek Dziedzic ◽  
Chris-Kriton Skylaris ◽  
Jonathan W. Essex
Keyword(s):  
Electric Fields ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Force Fields ◽  
Prolyl Isomerase ◽  
Peptidyl Prolyl Isomerase ◽  
Polarizable Force Field ◽  
Polarizable Force Fields ◽  
Protein Active Sites ◽  
Dynamics Simulations

<div><div><div><p>Preorganization of large, directionally oriented, electric fields inside protein active sites has been proposed as a crucial contributor to catalytic mechanism in many enzymes, and may be efficiently investigated at the atomistic level with molecular dynamics simulations. Here we evaluate the ability of the AMOEBA polarizable force field, as well as the additive Amber ff14SB and Charmm C36m models, to describe the electric fields present inside the active site of the peptidyl-prolyl isomerase cyclophilin A. We compare the molecular mechanical electric fields to those calculated with a fully first principles quantum mechanical (QM) representation of the protein, solvent, and ions, and find that AMOEBA consistently shows far greater correlation with the QM electric fields than either of the additive force fields tested. Catalytically-relevant fields calculated with AMOEBA were typically smaller than those observed with additive potentials, but were generally consistent with an electrostatically-driven mechanism for catalysis. Our results highlight the accuracy and the potential advantages of using polarizable force fields in systems where accurate electrostatics may be crucial for providing mechanistic insights.</p></div></div></div>

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