Microsecond Timescale Simulations at the Transition State of PmHMGR Predict Remote Allosteric Residues

Understanding the mechanisms of enzymatic catalysis requires a detailed understanding of the complex interplay of structure and dynamics of large systems that is a challenge for both experimental and computational approaches. QM/MM methods have been extensively used to study these reactions, but the difficulties arising from the hybrid treatment of the system are well documented. More importantly, the computational demands of QM/MM simulations mean that the dynamics of the reaction can only be considered on a timescale of nanoseconds even though the conformational changes needed to react the catalytically active state happen on a much slower timescale. Here we demonstrate an alternative approach that uses transition state force fields (TSFFs) derived by the quantum-guided molecular mechanics (Q2MM) method that provides a consistent treatment of the entire system at the classical molecular mechanics level and allows simulations at the microsecond timescale. Application of this approach the second hydride transfer transition state of HMG-CoA reductase from Pseudomonas mevalonii (PmHMGR) identified three remote residues, R396 E399 and L407, (15-27 Å away from the active site) that have a remote dynamic effect on enzyme activity. The predictions were subsequently validated experimentally via site-directed mutagenesis. These results show that microsecond timescale MD simulations of transition states are possible and can predict rather than just rationalize remote allosteric residues.

Download Full-text

pH dependent inhibition from ammonium ions in the Pseudomonas mevalonii HMG-CoA Reductase crystallization environment

10.1101/2020.06.03.132290 ◽

2020 ◽

Author(s):

Vatsal Purohit ◽

C. Nicklaus Steussy ◽

Anthony R. Rosales ◽

Chandra J. Critchelow ◽

Tim Schmidt ◽

...

Keyword(s):

Conformational Changes ◽

Pathogenic Bacteria ◽

Coenzyme A ◽

Enzymatic Reaction ◽

Hydride Transfer ◽

Reverse Direction ◽

Ammonium Ions ◽

Hmg Coa Reductase ◽

Pseudomonas Mevalonii ◽

Coa Reductase

ABSTRACTHMG-CoA reductase (Pseudomonas mevalonii) utilizes mevalonate, coenzyme A (CoA) and the cofactor NAD in a complex mechanism involving two hydride transfers with cofactor exchange, accompanied by large conformational changes by a 50 residue subdomain, to generate HMG-CoA. Details about this mechanism such as the conformational changes that allow intermediate formation, cofactor exchange and product release remain unknown. The formation of the proposed intermediates has also not been observed in structural studies with natural substrates. Having been shown to be an essential enzyme for the survival of gram-positive antibiotic resistant pathogenic bacteria, studying its mechanism in detail will be beneficial in developing novel antibacterials. The enzyme has been shown to be catalytically active inside the crystal with dithio-HMG-CoA and NADH but curiously is found to be inactive in the reverse direction in the structure bound to mevalonate, CoA and NAD.To understand the factors limiting activity in the HMGR crystal with mevalonate, CoA and NAD, we studied the effect of crystallization components and pH on enzymatic activity. We observed a strong inhibition in the crystallization buffer and an increase in activity with increasing pH. We attribute this inhibitive effect to the presence of ammonium ions present in the crystal since inhibition is also observed with several other ammonium salt buffers. Additionally, the lack of inhibition was observed in the absence of ammonium. The effect of each ligand (mevalonate, CoA and NAD) on the rate of the enzymatic reaction in the crystallization environment was further investigated by measuring their Km in the crystallization buffer. The Km measurements indicate that the hydride transfer step between NAD and mevalonate is inhibited in the crystallization environment. To test this further, we solved a crystal structure of pmHMGR bound to the post-hydride transfer intermediate (mevaldehyde) and cofactor Coenzyme A. The resulting turnover with the formation of a thiohemiacetal indicated that the crystallization environment inhibited the oxidative acylation of mevalonate and the reaction intermediate mevaldyl-CoA.

Download Full-text

Automated Fitting of Transition State Force Fields for Biomolecular Simulations

10.26434/chemrxiv.13206038 ◽

2020 ◽

Author(s):

Olaf Wiest ◽

Taylor R. Quinn ◽

Himani Patel ◽

Paul Helquist ◽

Per-Ola Norrby ◽

...

Keyword(s):

Transition State ◽

Force Field ◽

Molecular Mechanics ◽

Small Molecule ◽

Functional Form ◽

Enzymatic Reactions ◽

Hmgcoa Reductase ◽

Biomolecular Simulations ◽

Pseudomonas Mevalonii ◽

State Force

The application of the Quantum Guided Molecular Mechanics (Q2MM) method to transition states of enzymatic reactions to generate a transition state force field (TSFF) with the functional form of AMBER. The differences to fitting of small-molecule TSFFs and the similarities of the approach to transfer learning are discussed. The application to the transition state of the second hydride transfer in HMGCoA Reductase from Pseudomonas mevalonii is discussed.

Download Full-text

Microsecond Timescale MD Simulations at the Transition State of PmHMGR Predict Remote Allosteric Residues

Chemical Science ◽

10.1039/d1sc00102g ◽

2021 ◽

Author(s):

Taylor R. Quinn ◽

Olaf Wiest ◽

Per-Ola Norrby ◽

Xuhui Huang ◽

Cynthia V. Stauffacher ◽

...

Keyword(s):

Transition State ◽

Enzymatic Catalysis ◽

Md Simulations ◽

Complex Interplay ◽

Detailed Understanding ◽

Structure And Dynamics ◽

Large Systems

Understanding the mechanisms of enzymatic catalysis requires a detailed understanding of the complex interplay of structure and dynamics of large systems that is a challenge for both experimental and computational...

Download Full-text

Automated Fitting of Transition State Force Fields for Biomolecular Simulations

10.26434/chemrxiv.13206038.v1 ◽

2020 ◽

Author(s):

Olaf Wiest ◽

Taylor R. Quinn ◽

Himani Patel ◽

Paul Helquist ◽

Per-Ola Norrby ◽

...

Keyword(s):

Transition State ◽

Force Field ◽

Molecular Mechanics ◽

Small Molecule ◽

Functional Form ◽

Enzymatic Reactions ◽

Hmgcoa Reductase ◽

Biomolecular Simulations ◽

Pseudomonas Mevalonii ◽

State Force

The application of the Quantum Guided Molecular Mechanics (Q2MM) method to transition states of enzymatic reactions to generate a transition state force field (TSFF) with the functional form of AMBER. The differences to fitting of small-molecule TSFFs and the similarities of the approach to transfer learning are discussed. The application to the transition state of the second hydride transfer in HMGCoA Reductase from Pseudomonas mevalonii is discussed.

Download Full-text

Molecular Basis of Glucose Transport Mechanism in Plants

10.26434/chemrxiv.8049848.v1 ◽

2019 ◽

Cited By ~ 2

Author(s):

Balaji Selvam ◽

Ya-Chi Yu ◽

Liqing Chen ◽

Diwakar Shukla

Keyword(s):

Molecular Dynamics ◽

Free Energy ◽

Molecular Dynamics Simulations ◽

Conformational Changes ◽

Transport Mechanism ◽

Conformational Dynamics ◽

Site Directed Mutagenesis ◽

Free Energy Barrier ◽

Transport Cycle ◽

Dynamics Simulations

The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane. However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as 1 for the glucose the transport mechanism. SWEETs undergoes structural transition to outward-facing (OF), Occluded (OC) and inward-facing (IF) and strongly support alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly same for OF, OC and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and act as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.

Download Full-text

Closing the gap: an atomistic structural and functional perspective of S. mansoni universal stress G4LZI3 protein in complex with phenolic compounds

Current Drug Discovery Technologies ◽

10.2174/1570163817666201001122436 ◽

2020 ◽

Vol 17 ◽

Author(s):

Priscilla Masamba ◽

Geraldene Munsamy ◽

Abidemi Paul Kappo

Keyword(s):

Phenolic Compounds ◽

Conformational Changes ◽

Stress Protein ◽

Md Simulations ◽

Binding Energies ◽

Developmental Cycle ◽

Structure Based Drug Design ◽

Bioinformatic Tools ◽

Drug Of Choice ◽

Functional Perspective

Background: For decades, Praziquantel has been the undisputed drug of choice for all schistosome infections, but rising concerns due to the unelucidated mechanism of action of the drug and unavoidable reports of emerging drug resistant strains has necessitated the need for alternative treatment drug. Moreover, current apprehension has been reinforced by total dependence on the drug for treatment hence, the search for novel and effective anti-schistosomal drugs. Uses: This study made use of bioinformatic tools to determine the structural binding of the Universal G4LZI3 stress protein (USP) in complex with ten polyphenol compounds, thereby highlighting the effectiveness of these recently identified ‘lead’ molecules in the design of novel therapeutics targeted against schistosomiasis. Upregulation of the G4LZI3 USP throughout the schistosome multifaceted developmental cycle sparks interest in its potential role as a druggable target. The integration of in silico tools provides an atomistic perspective into the binding of potential inhibitors to target proteins. Conclusion: This study therefore, implemented the use of molecular dynamic (MD) simulations to provide functional and structural insight into key conformational changes upon binding of G4ZLI3 to these key phenolic compounds. Post-MD analyses revealed unique structural and conformational changes in the G4LZI3 protein in complex with curcumin and catechin respectively. These systems exhibited the highest binding energies, while the major interacting residues conserved in all the complexes provides a route map for structure-based drug design of novel compounds with enhanced inhibitory potency against the G4LZI3 protein. This study suggests an alternative approach for the development of anti-schistosomal drugs using natural compounds.

Download Full-text

Correlation of membrane protein conformational and functional dynamics

Nature Communications ◽

10.1038/s41467-021-24660-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Raghavendar Reddy Sanganna Gari ◽

Joel José Montalvo‐Acosta ◽

George R. Heath ◽

Yining Jiang ◽

Xiaolong Gao ◽

...

Keyword(s):

Membrane Protein ◽

Conformational Changes ◽

Single Channel ◽

Conformational Dynamics ◽

Md Simulations ◽

High Temporal Resolution ◽

Dependent Manner ◽

Functional Dynamics ◽

Ph Dependent ◽

Open States

AbstractConformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. However, correlation between functional and conformational dynamics remained difficult, lacking experimental techniques to monitor sub-millisecond conformational changes. Here, we use the outer membrane protein G (OmpG) as a model system where loop-6 opens and closes the β-barrel pore like a lid in a pH-dependent manner. Functionally, single-channel electrophysiology shows that while closed states are favored at acidic pH and open states are favored at physiological pH, both states coexist and rapidly interchange in all conditions. Using HS-AFM height spectroscopy (HS-AFM-HS), we monitor sub-millisecond loop-6 conformational dynamics, and compare them to the functional dynamics from single-channel recordings, while MD simulations provide atomistic details and energy landscapes of the pH-dependent loop-6 fluctuations. HS-AFM-HS offers new opportunities to analyze conformational dynamics at timescales of domain and loop fluctuations.

Download Full-text