11 research outputs found
Ligand-Induced Proton Transfer and Low-Barrier Hydrogen Bond Revealed by X-ray Crystallography
Ligand binding can change the pKa of protein residues and influence enzyme catalysis. Herein, we report three sub-Angstrom resolution X-ray crystal structures of CTX-M \u3b2-lactamase, representing three stages of the enzymatic pathway, apo protein (0.79 \uc5), pre-covalent complex (0.89 \uc5), and acylation transition state analog (0.84 \uc5). The binding of a non-covalent ligand induces a proton transfer from the catalytic Ser70 to the general base Glu166, and the formation of a low-barrier hydrogen bond (LBHB) between Ser70 and Lys73. QM/MM reaction path calculations determined the proton transfer barrier between Ser70 and Lys73 to be 1.53 kcal/mol, further confirming the presence of a LBHB. This LBHB is absent in the other two structures. Our data represents the first evidence of a direct and transient LBHB stabilizing a nucleophilic serine, as hypothesized by Cleland and Kreevoy. These results have important implications for the study of enzyme mechanisms as well as protein-inhibitor interactions
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
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