Inhibitory Proteins Block Substrate Access to the Active Site of Bacillus subtilis Intramembrane Metalloprotease SpoIVFB

Intramembrane proteases of diverse signaling pathways use membrane-embedded active sites to cleave membrane-associated substrates. Interactions of intramembrane metalloproteases with modulators are poorly understood. Inhibition of Bacillus subtilis intramembrane metalloprotease SpoIVFB requires BofA and SpoIVFA, which transiently prevent cleavage of Pro-σK during endosporulation. Three conserved BofA residues (N48, N61, T64) in or near predicted transmembrane segment (TMS) 2 were found to be required for SpoIVFB inhibition. Disulfide cross-linking indicated that BofA TMS2 occupies the SpoIVFB active site region. BofA and SpoIVFA neither prevented SpoIVFB from interacting with Pro-σK in co-purification assays nor interfered with cross-linking between the C-terminal regions of Pro-σK and SpoIVFB. However, BofA and SpoIVFA did interfere with cross-linking between the N-terminal Proregion of Pro-σK and the SpoIVFB active site region and interdomain linker. A BofA variant lacking predicted TMS1, in combination with SpoIVFA, was less effective at interfering with some of the cross-links and slightly less effective at inhibiting cleavage of Pro-σK by SpoIVFB. A structural model was built of SpoIVFB in complex with BofA and parts of SpoIVFA and Pro-σK, using partial homology and constraints from cross-linking and co-evolutionary analyses. The model predicts that N48 in BofA TMS2 interacts with T64 (and possibly N61) of BofA to stabilize a membrane-embedded C-terminal region. SpoIVFA is predicted to bridge the BofA C-terminal region and SpoIVFB. Thus, the two inhibitory proteins block access of the Pro-σK N-terminal region to the SpoIVFB active site region. Our findings may inform efforts to develop selective inhibitors of intramembrane metalloproteases.

Accurately positioning functional residues with robotics-inspired computational protein design

Accurate positioning of functional residues is critical for the design of new protein functions, but has remained difficult because of the prevalence of irregular local geometries in active sites. Here we introduce two computational methods that build local protein geometries from sequence with atomic accuracy: fragment kinematic closure (FKIC) and loophash kinematic closure (LHKIC). FKIC and LHKIC integrate two approaches: robotics-inspired kinematics of protein backbones and insertion of peptide fragments, and show up to 140-fold improvements in native-like predictions over either approach alone. We then integrate these methods into a new design protocol, pull-into-place (PIP), to position functionally important sidechains via design of new structured loop conformations. We validate PIP by remodeling a sizeable active site region in an enzyme and confirming the engineered new conformations of two designs with crystal structures. The described methods can be applied broadly to the design of many new protein geometries and functions.

in silico Study Reveals Potential Docking Sites of δ 2-isoxazolines derivates for Inhibiting Russell’s Viper PLA2 Toxin

Snake venom phospholipase A2s (svPLA2s) has been known as the most abundant component and predominant cause of Russell’s viper envenomation. Limitation to serum therapy and considerable pharmacological interest led the researcher to synthesized multi-toxic PLA2 inhibitors, δ2-isoxazolines derivate. Although δ2- isoxazolines derivate already proved inhibitor activity in Group II svPLA2 with known IC50, their mechanism of action remains unveiled. Our recent study investigated their inhibitory activity via molecular docking. The virtual screening revealed that the ligand with diverse structures tied to the relatively same active site region. The result sheds light on the significance of His48 and Asp49 as part of the pro-inflammatory eliciting region. ADME analysis was also performed to filter and identify the best potential inhibitor acceptable for human use. This moiety leads to finding a better therapeutic strategy of svPLA2 inhibitors both as an alternative to serum anti-venom treatment.

Molecular dynamics simulations of the interactions between triose phosphate isomerase and sulfonamides

Malaria is a disease with debilitating health and negative economic impacts in regions at high risk of infection. Parasitic resistance and side effects of current antimalarial drugs are major setbacks to the successful campaigns that have reduced malaria incidence by 40% in the last decade. The parasite’s dependence on glycolysis for energy requirements makes pathway enzymes suitable targets for drug development. Specifically, triose phosphate isomerase (TPI) from Plasmodium falciparum (pTPI) and human (hTPI) cells show striking structural features that can be used in development of new antimalarial agents. In this study MD simulations were used to characterize binding sites on hTPI and pTPI interactions with sulfonamides. The molecular mechanics Poisson–Boltzmann surface area (MM–PBSA) method was used to estimate the interaction energies of four sulfonamide-TPI docked complexes. A unique combination of key residues at the dimer interface of pTPI is responsible for the observed selective affinity to pTPI compared to hTPI. The representative sulfonamide; 4-amino-N-(3,5-dimethylphenyl)-3-fluorbenzenesulfonamide (sulfaE) shows a strong affinity with pTPI (dimer interface, −42.91 kJ/mol and active site region, −71.62 kJ/mol), hTPI (dimer interface, −41.32 kJ/mol and active site region, −84.40 kJ/mol). Strong and favorable Van der Waals interactions and increases in non-polar solvation energies explain the difference in affinity between pTPI with sulfaE compared to hTPI at the dimer interface. This is an indication that the dimer interface of TPI glycolytic enzyme is vital for development of sulfonamide based antimalarial drugs.

Copper(II) Inhibition of the SARS-CoV-2 Main Protease

In an analysis of the structural stability of the coronavirus main protease (Mpro), we identified regions of the protein that could be disabled by cobalt(III)-cation binding to histidines and cysteines [1]. Here we have extended our work to include copper(II) chelates, which we have docked to HIS 41 and CYS 145 in the Mpro active-site region. We have found stable docked structures where Cu(II) could readily bond to the CYS 145 thiolate, which would be lethal to the enzyme. We also started studying the Spike Protein, PDB ID: 6VXX and the region around the D614G mutant.

Copper(II) Inhibition of the SARS-CoV-2 Main Protease

In an analysis of the structural stability of the coronavirus main protease (Mpro), we identified regions of the protein that could be disabled by cobalt(III)-cation binding to histidines and cysteines [1]. Here we have extended our work to include copper(II) chelates, which we have docked to HIS 41 and CYS 145 in the Mpro active-site region. We have found stable docked structures where Cu(II) could readily bond to the CYS 145 thiolate, which would be lethal to the enzyme. We also started studying the Spike Protein, PDB ID: 6VXX and the region around the D614G mutant.

Lanthanide-dependent alcohol dehydrogenases require an essential aspartate residue for metal coordination and enzymatic function

The lanthanide elements (Ln3+), those with atomic numbers 57–63 (excluding promethium, Pm3+), form a cofactor complex with pyrroloquinoline quinone (PQQ) in bacterial XoxF methanol dehydrogenases (MDHs) and ExaF ethanol dehydrogenases (EDHs), expanding the range of biological elements and opening novel areas of metabolism and ecology. Other MDHs, known as MxaFIs, are related in sequence and structure to these proteins, yet they instead possess a Ca2+-PQQ cofactor. An important missing piece of the Ln3+ puzzle is defining what features distinguish enzymes that use Ln3+-PQQ cofactors from those that do not. Here, using XoxF1 MDH from the model methylotrophic bacterium Methylorubrum extorquens AM1, we investigated the functional importance of a proposed lanthanide-coordinating aspartate residue. We report two crystal structures of XoxF1, one with and another without PQQ, both with La3+ bound in the active-site region and coordinated by Asp320. Using constructs to produce either recombinant XoxF1 or its D320A variant, we show that Asp320 is needed for in vivo catalytic function, in vitro activity, and La3+ coordination. XoxF1 and XoxF1 D320A, when produced in the absence of La3+, coordinated Ca2+ but exhibited little or no catalytic activity. We also generated the parallel substitution in ExaF to produce ExaF D319S and found that this variant loses the capacity for efficient ethanol oxidation with La3+. These results provide evidence that a Ln3+-coordinating aspartate is essential for the enzymatic functions of XoxF MDHs and ExaF EDHs, supporting the notion that sequences of these enzymes, and the genes that encode them, are markers for Ln3+ metabolism.

Lanthanide-dependent alcohol dehydrogenases require an essential aspartate residue for metal coordination and function

ABSTRACTThe presence of lanthanide elements (Ln3+) and pyrroloquinoline quinone (PQQ) containing cofactors in XoxF methanol dehydrogenases (MDHs) and ExaF ethanol dehydrogenases (EDHs) has expanded the list of biological elements and opened novel areas of metabolism and ecology. Other MDHs known as MxaFIs are related in sequence and structure to these proteins, yet they instead possess a Ca2+-PQQ cofactor. An important missing piece of the Ln3+ puzzle is defining what protein features distinguish enzymes using Ln3+-PQQ cofactors from those that do not. In this study, we investigated the functional importance of a proposed lanthanide-coordinating aspartate using XoxF1 MDH from the model methylotrophic bacterium Methylorubrum extorquens AM1. We report two crystal structures of XoxF1, one containing PQQ and the other free of this organic molecule, both with La3+ bound in the active site region and coordinated by Asp320. Using constructs to produce either recombinant XoxF1 or its D320A variant, we show Asp320 is needed for in vivo catalytic function, in vitro activity of purified enzyme, and coordination of La3+. XoxF1 and XoxF1 D320A, when produced in the absence of La3+, coordinate Ca2+, but exhibit little or no catalytic activity. In addition, we generated the parallel substitution to produce ExaF D319S, and showed the enzyme loses the capacity for efficient ethanol oxidation with La3+. These results provide empirical evidence of an essential Ln3+-coordinating aspartate for the function of XoxF MDHs and ExaF EDHs; thus, supporting the suggestion that sequences of these enzymes, and the genes that encode them, are markers for Ln3+ metabolism.

Molecular Docking Studies on Gingerol Analogues toward Mushroom Tyrosinase

The gingerol presents the starting point of our work which aims to discover new inhibitors of the tyrosinase enzyme. Therefore, we have studied the activity of gingerol derivatives as inhibitors against mushroom tyrosinase based on the molecular docking. Molecular docking studies were performed on a series of gingerol analogues retrieved from Zinc database (with 70% as similarity threshold). The gingerol analogues were docked within the active site region of mushroom tyrosinase (PDB: 2Y9X) using Molegro Virtual Docker V.5.0. The results of molecular docking studies revealed that some analogues of gingerol have higher Moldock score (in terms of negative energy) than gingerol and the experimentally known inhibitors of tyrosinase, and showed favourable molecular interactions exhibiting common molecular interaction with Ala323, Met280 and Asn260 residues of tyrosinase. Furthermore, the top docked compounds used in this work do not violate the Lipinsky rule of five.