Understanding structural features of biomolecular interactions : from classical simulations to ab initio calculations

The structures of biomolecules and their interactions dictate their functions. In this thesis, five papers are presented to illustrate how the dynamics of biomolecules can be investigated and derivation of desired thermodynamic quantities obtained by utilising a diverse range of computational techniques, from simulations utilising classical mechanical descriptions to calculations employing quantum mechanical descriptions. Classical simulation, referring to molecular dynamics simulation with atomistic force fields, has been used in every paper in this thesis. In Paper I, classical simulation and homology modelling are used to investigate the dynamics of a protein as well as that of its homologues, which have a missing region. Protein purification and production of these homologues was also attempted. When state transitions like protonation and tautomerisation equilibria are central to the query, we employed lambda-dynamics, an extension to conventional simulation that can describe transitions between states by including coupling parameter lambda in the dynamics. In Papers II and III, protonation and tautomerisation equilibria respectively are central to the query. In Paper II, lambda-dynamics and multiple pH regime are both used to calculate the pK shifts of cytidine in triplex nucleic acid environments. In some of the triplex nucleic acid systems, sugar modification LNA is present. The force field parameters of LNA have been updated to provider better descriptions for pK calculations. In Paper III, lambda-dynamics is used to describe tautomerisation equilibrium between two tautomers of pseudoisocytidine in singlestranded and triple-stranded nucleic acids in order to observe how the equilibrium shifts in different environments. In vitro binding assay is used to corroborate the computational results. When greater accuracy for certain properties like electrostatics or energetics is required, we employed quantum mechanical calculations as well as hybrid methods which combine classical and quantum mechanical descriptions. In Paper IV, QM and QM/MM calculations were performed to calculate the energetic difference between two tautomers in the ribosome. In Paper V, protein-specific polarised charge, a charge update scheme that updates the atomic charges with QM and Poisson-Boltzmann calculations during classical simulation, is used for better electrostatics description of a peptide

Cannabinoid biosynthesis using non-canonical enzymes

Please click Additional Files below to see the full abstract

Adsorption and folding dynamics of MPER of HIV-1 gp41 in the presence of dpc micelle

Membrane-proximal ectodomain region (MPER) of HIV-1 gp41 is known to have several epitopes of monoclonal antibodies. It also plays an important role in the membrane fusion process that is well-evidenced, though not well-elucidated. There are also disputes over the true structure of MPER. In this study, MPER NMR structure in the presence of dodecylphosphatidylcholine micelle is used in the molecular dynamic simulation to elucidate structural dynamics and adsorption to model MPER interaction in a membrane environment. Polarized protein-specific charge derived from its NMR structure is found to better preserve the helical structure found in the NMR structure compared to AMBER03 calculation. The preserved helical structure also adsorb to the micelle using the hydrophobic side-chains, consistent to the NMR structure. Ab initio folding of MPER predicts a structure quite in well agreement with the NMR structure (RMSd 3.9 Å) and shows that the micelle plays a role in the folding process

Computational study of bindings of HK20 Fab and D5 Fab to HIV-1 gp41

Antibodies HK20 and D5 have been shown to target HIV-1 gp41, thereby inhibiting membrane fusion that facilitates viral entry. The binding picture is static, based on the X-ray crystal structures of the Fab regions and gp41 mimetic five-helix bundle. In this study, we carried out molecular dynamics simulation to provide the dynamic binding picture. Calculated binding free energies are within reasonable range of and follow the trend of the experimental values: −15.28 kcal/mol for HK20 Fab (expt. −11.60 kcal/mol) and −17.90 kcal/mol for D5 Fab (expt. −11.70 kcal/mol). Alanine scanning at protein–protein interface reveals that the highest contributors to binding for HK20 Fab are F54 and I56, both of VH region, as well as R30′ of VL region; whereas for D5 Fab, F54 of VH region, as well as W32′ and Y94′ of VL region. HK20 F54 and I56, as well as D5 I52, F54, and T56, bind to the gp41 hydrophobic binding pocket, an important region targeted by many other fusion inhibitors. Hydrogen bonding analysis also identifies high-occupancy hydrogen bonds at the periphery of gp41 hydrophobic pocket. Considering that almost all interface residues are turn residues, further work may be directed to turn mimics. Pre-orientation by the hydrogen bonds to poise this particular turn towards the binding pocket may also be a point worth pursuing

Modeling pK shift in DNA triplexes containing locked nucleic acids

The protonation states for nucleic acid bases are difficult to assess experimentally. In the context of DNA triplex, the protonation state of cytidine in the third strand is particularly important, because it needs to be protonated in order to form Hoogsteen hydrogen bonds. A sugar modification, locked nucleic acid (LNA), is widely used in triplex forming oligonucleotides to target sites in the human genome. In this study, the parameters for LNA are developed in line with the CHARMM nucleic acid force field and validated toward the available structural experimental data. In conjunction, two computational methods were used to calculate the protonation state of the third strand cytidine in various DNA triplex environments: λ-dynamics and multiple pH regime. Both approaches predict pK of this cytidine shifted above physiological pH when cytidine is in the third strand in a triplex environment. Both methods show an upshift due to cytidine methylation, and a small downshift when the sugar configuration is locked. The predicted pK values for cytidine in DNA triplex environment can inform the design of better-binding oligonucleotides.Published versio

Computational Study of Uracil Tautomeric Forms in the Ribosome: The Case of Uracil and 5‑Oxyacetic Acid Uracil in the First Anticodon Position of tRNA

Tautomerism is important in many biomolecular interactions, not least in RNA biology. Crystallographic studies show the possible presence of minor tautomer forms of transfer-RNA (tRNA) anticodon bases in the ribosome. The hydrogen positions are not resolved in the X-ray studies, and we have used ab initio calculations and molecular dynamics simulations to understand if and how the minor enol form of uracil (U), or the modified uracil 5-oxyacetic acid (cmo⁵U), can be accommodated in the tRNA–messenger-RNA interactions in the ribosome decoding center. Ab initio calculations on isolated bases show that the modification affects the keto–enol equilibrium of the uracil base only slightly; the keto form is dominant (>99.99%) in both U and cmo⁵U. Other factors such as interactions with the surrounding nucleotides or ions would be required to shift the equilibrium toward the enol tautomer. Classical molecular simulations show a better agreement with the X-ray structures for the enol form, but free energy calculations indicate that the most stable form is the keto. In the ribosome, the enol tautomers of U and cmo⁵U pair with a guanine forming two hydrogen bonds, which do not involve the enol group. The oxyacetic acid modification has a minor effect on the keto–enol equilibrium

Recent Advances in Structure, Function, and Pharmacology of Class A Lipid GPCRs: Opportunities and Challenges for Drug Discovery

10.3390/ph15010012PHARMACEUTICALS15

Recent Advances in Structure, Function, and Pharmacology of Class A Lipid GPCRs: Opportunities and Challenges for Drug Discovery

Great progress has been made over the past decade in understanding the structural, functional, and pharmacological diversity of lipid GPCRs. From the first determination of the crystal structure of bovine rhodopsin in 2000, much progress has been made in the field of GPCR structural biology. The extraordinary progress in structural biology and pharmacology of GPCRs, coupled with rapid advances in computational approaches to study receptor dynamics and receptor-ligand interactions, has broadened our comprehension of the structural and functional facets of the receptor family members and has helped usher in a modern age of structure-based drug design and development. First, we provide a primer on lipid mediators and lipid GPCRs and their role in physiology and diseases as well as their value as drug targets. Second, we summarize the current advancements in the understanding of structural features of lipid GPCRs, such as the structural variation of their extracellular domains, diversity of their orthosteric and allosteric ligand binding sites, and molecular mechanisms of ligand binding. Third, we close by collating the emerging paradigms and opportunities in targeting lipid GPCRs, including a brief discussion on current strategies, challenges, and the future outlook

Structure-Guided Engineering of Prenyltransferase NphB for High-Yield and Regioselective Cannabinoid Production

10.1021/acscatal.2c00786ACS Catalysis4628-463

Modeling p<i>K</i> Shift in DNA Triplexes Containing Locked Nucleic Acids

The protonation states for nucleic acid bases are difficult to assess experimentally. In the context of DNA triplex, the protonation state of cytidine in the third strand is particularly important, because it needs to be protonated in order to form Hoogsteen hydrogen bonds. A sugar modification, locked nucleic acid (LNA), is widely used in triplex forming oligonucleotides to target sites in the human genome. In this study, the parameters for LNA are developed in line with the CHARMM nucleic acid force field and validated toward the available structural experimental data. In conjunction, two computational methods were used to calculate the protonation state of the third strand cytidine in various DNA triplex environments: λ-dynamics and multiple pH regime. Both approaches predict p<i>K</i> of this cytidine shifted above physiological pH when cytidine is in the third strand in a triplex environment. Both methods show an upshift due to cytidine methylation, and a small downshift when the sugar configuration is locked. The predicted p<i>K</i> values for cytidine in DNA triplex environment can inform the design of better-binding oligonucleotides