Steered Molecular Dynamics Simulations of NAD Unbinding from GAPDH and LDH

Protein-ligand interactions play an important role in understanding biophysical processes including the glycolytic pathway. Calculation of the energy profile of ligand unbinding is essential for understanding possible substrate channeling of nicotinamide adenine dinucleotide (NAD) between lactate dehydrogenase (LDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Herein, steered molecular dynamics (SMD) simulations elucidate the process of NAD unbinding from LDH and GAPDH. Brownian dynamics (BD) simulate, using the energy reaction criterion, NAD diffusion towards the binding site of GAPDH or LDH to identify potential residues where strong protein-ligand coulombic interactions exist. These residues are used to design several dissociation pathways for the SMD simulations. Simulations either apply a harmonic guiding potential or a constant force SMD to perform center of mass (COM) pulling of the NAD. The two ligands in the tetrameric GAPDH protein are successfully released from the binding pocket using a force constant k ≥ 5000 kJ/mol/nm2 or a constant force F ≥ 600 pN, within the first 4.2 ns of simulation time. A constant force of 600 pN is enough to pull out three of the four ligands from their corresponding LDH binding sites within the first 0.5 to 1.2 ns of simulation time. Upon releasing the ligand from the binding site, NAD conformational changes are traced, starting with a stretched, open conformation in the binding site and ending with a bent structure in solution. The bent structure is consistent with previous experimental and simulation data of NAD free in solution. The unbinding free energies associated with the NAD release along the proposed pathways are calculated using the Jarzynski equality, in the stiff-spring approximation of pulling

Steered Molecular Dynamics Simulations of NAD Unbinding from GAPDH and LDH

Protein-ligand interactions play an important role in understanding biophysical processes including the glycolytic pathway. Calculation of the energy profile of ligand unbinding is essential for understanding possible substrate channeling of nicotinamide adenine dinucleotide (NAD) between lactate dehydrogenase (LDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Herein, steered molecular dynamics (SMD) simulations elucidate the process of NAD unbinding from LDH and GAPDH. Brownian dynamics (BD) simulate, using the energy reaction criterion, NAD diffusion towards the binding site of GAPDH or LDH to identify potential residues where strong protein-ligand coulombic interactions exist. These residues are used to design several dissociation pathways for the SMD simulations. Simulations either apply a harmonic guiding potential or a constant force SMD to perform center of mass (COM) pulling of the NAD. The two ligands in the tetrameric GAPDH protein are successfully released from the binding pocket using a force constant k ≥ 5000 kJ/mol/nm2 or a constant force F ≥ 600 pN, within the first 4.2 ns of simulation time. A constant force of 600 pN is enough to pull out three of the four ligands from their corresponding LDH binding sites within the first 0.5 to 1.2 ns of simulation time. Upon releasing the ligand from the binding site, NAD conformational changes are traced, starting with a stretched, open conformation in the binding site and ending with a bent structure in solution. The bent structure is consistent with previous experimental and simulation data of NAD free in solution. The unbinding free energies associated with the NAD release along the proposed pathways are calculated using the Jarzynski equality, in the stiff-spring approximation of pulling

Correction: Identification of specific calcitonin-like receptor residues important for calcitonin gene-related peptide high affinity binding

This is a correction article. After publication of this work [1], we became aware of the fact that Robert C. Speth was not included as an author. Dr. Speth put a considerable amount of time and effort into developing and preparing the radiopeptide used to carry out the radioligand binding studies reported in this manuscript and therefore should have originally been included as an author. We apologize to Dr. Speth for any inconvenience that this oversight might have caused and thank him for his invaluable contribution to this project

Introducing DInaMo: A Package for Calculating Protein Circular Dichroism Using Classical Electromagnetic Theory

The dipole interaction model is a classical electromagnetic theory for calculating circular dichroism (CD) resulting from the π-π* transitions of amides. The theoretical model, pioneered by J. Applequist, is assembled into a package, DInaMo, written in Fortran allowing for treatment of proteins. DInaMo reads Protein Data Bank formatted files of structures generated by molecular mechanics or reconstructed secondary structures. Crystal structures cannot be used directly with DInaMo; they either need to be rebuilt with idealized bond angles and lengths, or they need to be energy minimized to adjust bond lengths and bond angles because it is common for crystal structure geometries to have slightly short bond lengths, and DInaMo is sensitive to this. DInaMo reduces all the amide chromophores to points with anisotropic polarizability and all nonchromophoric aliphatic atoms including hydrogens to points with isotropic polarizability; all other atoms are ignored. By determining the interactions among the chromophoric and nonchromophoric parts of the molecule using empirically derived polarizabilities, the rotational and dipole strengths are determined leading to the calculation of CD. Furthermore, ignoring hydrogens bound to methyl groups is initially explored and proves to be a good approximation. Theoretical calculations on 24 proteins agree with experiment showing bands with similar morphology and maxima

Theoretical Circular Dichroism Spectra of the α-Helical Protein Calexitin with the Dipole Interaction Model Including the n-π* Transition

Circular dichroism (CD) is an important structural biology technique used to study protein dynamics, and most especially the secondary structure of peptides and proteins. Although CD is a technique that is relatively easy to introduce to undergraduate students, the high cost of obtaining a conventional CD instrument and the time required for sample preparation prevents a good number of students from having hands-on experiments demonstrating the principle of CD. Herein, theoretical circular dichroism with the dipole interaction model, DInaMo, is proposed as a tool for introducing students to CD. Using the dipole interaction model, the CD spectra of an α-helical protein, calexcitin, is predicted with a good morphology, and peak intensity and location of the π– π* transition. The n–π* transition is well approximated with normal modes obtained in the correct location and sign

Computer Simulations of NAD Channeling between GAPDH and LDH

Functional protein-protein interactions are essential for many physiological processes and may play important roles in substrate channeling, coenzyme transfer, and compartmentation in glycolysis. Herein, Brownian dynamics (BD) elucidates the interactions between the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) and the transfer of the cofactor nicotinamide adenine dinucleotide (NAD) between LDH and GAPDH. BD channeling simulation results strongly depend on choice of reactive atom set. When the reactive atoms set comprise of atoms in the vicinity of the NAD binding site of either enzyme, short NAD trajectories between enzyme subunits occur. If the reactive atoms set were chosen from atoms belonging to NAD binding sites, the efficiency of reaching LDH decreased significantly, and even the shortest trajectories spent time equilibrating with solvent before binding the next active site. Transfer of NAD from GAPDH to LDH is sensitive to overall structure of enzyme-enzyme complex. Small variations in orientation of one enzyme relative to the other cause changes in channeling efficiency. The process of NAD release from GAPDH and LDH binding sites was studied with steered molecular dynamics. The GAPDH/LDH complex with two LDH subunits facing two GAPDH subunits was chosen as initial structure. Six NAD molecules were included - two molecules in GAPDH active subunits and 4 NAD molecules bound to each LDH subunit. External forces were applied to O3 NAD atom of each NAD molecule. MD trajectories (2 ns) with external force (≤ 1000 pN) were able to pull the NAD out of GAPDH, but not out of LDH. Such strong binding of NAD by LDH is because the nicotinic moiety is buried deep inside LDH subunit globule. Additional studies are needed to confirm the hypothesis that intermolecular contacts soften GAPDH subunit and its affinity to NAD reduces