24 research outputs found
Use of Nonequilibrium Work Methods to Compute Free Energy Differences Between Molecular Mechanical and Quantum Mechanical Representations of Molecular Systems
Carrying out free energy simulations
(FES) using quantum mechanical
(QM) Hamiltonians remains an attractive, albeit elusive goal. Renewed
efforts in this area have focused on using “indirect”
thermodynamic cycles to connect “low level” simulation
results to “high level” free energies. The main obstacle
to computing converged free energy results between molecular mechanical
(MM) and QM (Δ<i>A</i><sup>MM→QM</sup>), as
recently demonstrated by us and others, is differences in the so-called
“stiff” degrees of freedom (e.g., bond stretching) between
the respective energy surfaces. Herein, we demonstrate that this problem
can be efficiently circumvented using nonequilibrium work (NEW) techniques,
i.e., Jarzynski’s and Crooks’ equations. Initial applications
of computing Δ<i>A</i><sub>NEW</sub><sup>MM→QM</sup>, for blocked amino acids
alanine and serine as well as to generate butane’s potentials
of mean force via the indirect QM/MM FES method, showed marked improvement
over traditional FES approaches
Unlocking the Binding and Reaction Mechanism of Hydroxyurea Substrates as Biological Nitric Oxide Donors
Hydroxyurea is the only FDA approved treatment of sickle
cell disease. It is believed that the primary mechanism of action
is associated with the pharmacological elevation of nitric oxide in
the blood; however, the exact details of this are still unclear. In
the current work, we investigate the atomic level details of this
process using a combination of flexible-ligand/flexible-receptor virtual
screening coupled with energetic analysis that decomposes interaction
energies. Utilizing these methods, we were able to elucidate the previously
unknown substrate binding modes of a series of hydroxyurea analogs
to hemoglobin and the concomitant structural changes of the enzyme.
We identify a backbone carbonyl that forms a hydrogen bond with bound
substrates. Our results are consistent with kinetic and electron paramagnetic resonance (EPR) measurements of hydroxyurea–hemoglobin reactions, and a full
mechanism is proposed that offers new insights into possibly improving
substrate binding and/or reactivity
How Does Catalase Release Nitric Oxide? A Computational Structure–Activity Relationship Study
Hydroxyurea (HU) is the only FDA
approved medication for treating sickle cell disease in adults. The
primary mechanism of action is pharmacological elevation of nitric
oxide (NO) levels which induces propagation of fetal hemoglobin. HU
is known to undergo redox reactions with heme based enzymes like hemoglobin
and catalase to produce NO. However, specific details about the HU
based NO release remain unknown. Experimental studies indicate that
interaction of HU with human catalase compound I produces NO. Presently,
we combine flexible receptor–flexible substrate induced fit
docking (IFD) with energy decomposition analyses to examine the atomic
level details of a possible key step in the clinical conversion of
HU to NO. Substrate binding modes of nine HU analogs with catalase
compound I were investigated to determine the essential properties
necessary for effective NO release. Three major binding orientations
were found that provide insight into the possible reaction mechanisms
for producing NO. Further results show that anion/radical intermediates
produced as part of these mechanisms would be stabilized by hydrogen
bonding interactions from distal residues His75, Asn148, Gln168, and
oxoferryl-heme. These details will ideally contribute to both a clearer
mechanistic picture and provide insights for future structure based
drug design efforts
Can Molecular Dynamics and QM/MM Solve the Penicillin Binding Protein Protonation Puzzle?
Benzylpenicillin, a member of the
β-lactam antibiotic class, has been widely used to combat bacterial
infections since 1947. The general mechanism is well-known: a serine
protease enzyme (i.e., DD-peptidase) forms a long lasting intermediate
with the lactam ring of the antibiotic known as acylation, effectively
preventing biosynthesis of the bacterial cell wall. Despite this overall
mechanistic understanding, many details of binding and catalysis are
unclear. Specifically, there is ongoing debate about active site protonation
states and the role of general acids/bases in the reaction. Herein,
a unique combination of MD simulations, QM/MM minimizations, and QM/MM
orbital analyses is combined with systematic variation of active site
residue protonation states. Critical interactions that maximize the
stability of the bound inhibitor are examined and used as metrics.
This approach was validated by examining cefoxitin interactions in
the CTX-M β-lactamase from E. coli and compared to an ultra high-resolution (0.88 Å) crystal structure.
Upon confirming the approach used, an investigation of the preacylated Streptomyces R61 active site with bound benzylpenicillin
was performed, varying the protonation states of His298 and Lys65.
We concluded that protonated His298 and deprotonated Lys65 are most
likely to exist in the R61 active site
Identification and Characterization of Noncovalent Interactions That Drive Binding and Specificity in DD-Peptidases and β‑Lactamases
Bacterial
resistance to standard (i.e., β-lactam-based) antibiotics
has become a global pandemic. Simultaneously, research into the underlying
causes of resistance has slowed substantially, although its importance
is universally recognized. Key to unraveling critical details is characterization
of the noncovalent interactions that govern binding and specificity
(DD-peptidases, antibiotic targets, versus β-lactamases, the
evolutionarily
derived enzymes that play a major role in resistance) and ultimately
resistance as a whole. Herein, we describe a detailed investigation
that elicits new chemical insights into these underlying intermolecular
interactions. Benzylpenicillin and a novel β-lactam peptidomimetic
complexed to the Stremptomyces R61
peptidase are examined using an arsenal of computational techniques:
MD simulations, QM/MM calculations, charge perturbation analysis,
QM/MM orbital analysis, bioinformatics, flexible receptor/flexible
ligand docking, and computational ADME predictions. Several key molecular
level interactions are identified that not only shed light onto fundamental
resistance mechanisms, but also offer explanations for observed specificity.
Specifically, an extended π–π network is elucidated
that suggests antibacterial resistance has evolved, in part, due to
stabilizing aromatic interactions. Additionally, interactions between
the protein and peptidomimetic substrate are identified and characterized.
Of particular interest is a water-mediated salt bridge between Asp217
and the positively charged N-terminus of the peptidomimetic, revealing
an interaction that may significantly contribute to β-lactam
specificity. Finally, interaction information is used to suggest modifications
to current β-lactam compounds that should both improve binding
and specificity in DD-peptidases and their physiochemical properties
Disruption of an Active Site Network Leads to Activation of C2α-Lactylthiamin Diphosphate on the Antibacterial Target 1‑Deoxy‑d‑xylulose-5-phosphate Synthase
The bacterial metabolic enzyme 1-deoxy-d-xylulose-5-phosphate
synthase (DXPS) catalyzes the thiamin diphosphate (ThDP)-dependent
formation of DXP from pyruvate and d-glyceraldehyde-3-phosphate
(d-GAP). DXP is an essential bacteria-specific metabolite
that feeds into the biosynthesis of isoprenoids, pyridoxal phosphate
(PLP), and ThDP. DXPS catalyzes the activation of pyruvate to give
the C2α-lactylThDP (LThDP) adduct that is long-lived on DXPS
in a closed state in the absence of the cosubstrate. Binding of d-GAP shifts the DXPS-LThDP complex to an open state which coincides
with LThDP decarboxylation. This gated mechanism distinguishes DXPS
in ThDP enzymology. How LThDP persists on DXPS in the absence of cosubstrate,
while other pyruvate decarboxylases readily activate LThDP for decarboxylation,
is a long-standing question in the field. We propose that an active
site network functions to prevent LThDP activation on DXPS until the
cosubstrate binds. Binding of d-GAP coincides with a conformational
shift and disrupts the network causing changes in the active site
that promote LThDP activation. Here, we show that the substitution
of putative network residues, as well as nearby residues believed
to contribute to network charge distribution, predictably affects
LThDP reactivity. Substitutions predicted to disrupt the network have
the effect to activate LThDP for decarboxylation, resulting in CO2 and acetate production. In contrast, a substitution predicted
to strengthen the network fails to activate LThDP and has the effect
to shift DXPS toward the closed state. Network-disrupting substitutions
near the carboxylate of LThDP also have a pronounced effect to shift
DXPS to an open state. These results offer initial insights to explain
the long-lived LThDP intermediate and its activation through disruption
of an active site network, which is unique to DXPS. These findings
have important implications for DXPS function in bacteria and its
development as an antibacterial target
Disruption of an Active Site Network Leads to Activation of C2α-Lactylthiamin Diphosphate on the Antibacterial Target 1‑Deoxy‑d‑xylulose-5-phosphate Synthase
The bacterial metabolic enzyme 1-deoxy-d-xylulose-5-phosphate
synthase (DXPS) catalyzes the thiamin diphosphate (ThDP)-dependent
formation of DXP from pyruvate and d-glyceraldehyde-3-phosphate
(d-GAP). DXP is an essential bacteria-specific metabolite
that feeds into the biosynthesis of isoprenoids, pyridoxal phosphate
(PLP), and ThDP. DXPS catalyzes the activation of pyruvate to give
the C2α-lactylThDP (LThDP) adduct that is long-lived on DXPS
in a closed state in the absence of the cosubstrate. Binding of d-GAP shifts the DXPS-LThDP complex to an open state which coincides
with LThDP decarboxylation. This gated mechanism distinguishes DXPS
in ThDP enzymology. How LThDP persists on DXPS in the absence of cosubstrate,
while other pyruvate decarboxylases readily activate LThDP for decarboxylation,
is a long-standing question in the field. We propose that an active
site network functions to prevent LThDP activation on DXPS until the
cosubstrate binds. Binding of d-GAP coincides with a conformational
shift and disrupts the network causing changes in the active site
that promote LThDP activation. Here, we show that the substitution
of putative network residues, as well as nearby residues believed
to contribute to network charge distribution, predictably affects
LThDP reactivity. Substitutions predicted to disrupt the network have
the effect to activate LThDP for decarboxylation, resulting in CO2 and acetate production. In contrast, a substitution predicted
to strengthen the network fails to activate LThDP and has the effect
to shift DXPS toward the closed state. Network-disrupting substitutions
near the carboxylate of LThDP also have a pronounced effect to shift
DXPS to an open state. These results offer initial insights to explain
the long-lived LThDP intermediate and its activation through disruption
of an active site network, which is unique to DXPS. These findings
have important implications for DXPS function in bacteria and its
development as an antibacterial target
Example of Structure Editing module setup for iron-sulfur containing proteins.
<p>Example of Structure Editing module setup for iron-sulfur containing proteins.</p
Web-Based Computational Chemistry Education with CHARMMing III: Reduction Potentials of Electron Transfer Proteins
<div><p>A module for fast determination of reduction potentials, <i>E°</i>, of redox-active proteins has been implemented in the CHARMM INterface and Graphics (CHARMMing) web portal (<a href="http://www.charmming.org" target="_blank">www.charmming.org</a>). The free energy of reduction, which is proportional to <i>E°</i>, is composed of an intrinsic contribution due to the redox site and an environmental contribution due to the protein and solvent. Here, the intrinsic contribution is selected from a library of pre-calculated density functional theory values for each type of redox site and redox couple, while the environmental contribution is calculated from a crystal structure of the protein using Poisson-Boltzmann continuum electrostatics. An accompanying lesson demonstrates a calculation of <i>E°</i>. In this lesson, an ionizable residue in a [4Fe-4S]-protein that causes a pH-dependent <i>E°</i> is identified, and the <i>E°</i> of a mutant that would test the identification is predicted. This demonstration is valuable to both computational chemistry students and researchers interested in predicting sequence determinants of <i>E°</i> for mutagenesis.</p></div
Example of the graphic interface for making point mutations in CHARMMing.
<p>Example of the graphic interface for making point mutations in CHARMMing.</p