The LarB carboxylase/hydrolase forms a transient cysteinyl-pyridine intermediate during nickel-pincer nucleotide cofactor biosynthesis

Enzymes possessing the nickel-pincer nucleotide (NPN) cofactor catalyze C2 racemization or epimerization reactions of α-hydroxyacid substrates. LarB initiates synthesis of the NPN cofactor from nicotinic acid adenine dinucleotide (NaAD) by performing dual reactions: pyridinium ring C5 carboxylation and phosphoanhydride hydrolysis. Here, we show that LarB uses carbon dioxide, not bicarbonate, as the substrate for carboxylation and activates water for hydrolytic attack on the AMP-associated phosphate of C5-carboxylated-NaAD. Structural investigations show that LarB has an N-terminal domain of unique fold and a C-terminal domain homologous to aminoimidazole ribonucleotide carboxylase/mutase (PurE). Like PurE, LarB is octameric with four active sites located at subunit interfaces. The complex of LarB with NAD+, an analog of NaAD, reveals the formation of a covalent adduct between the active site Cys221 and C4 of NAD+, resulting in a boat-shaped dearomatized pyridine ring. The formation of such an intermediate with NaAD would enhance the reactivity of C5 to facilitate carboxylation. Glu180 is well positioned to abstract the C5 proton, restoring aromaticity as Cys221 is expelled. The structure of as-isolated LarB and its complexes with NAD+ and the product AMP identify additional residues potentially important for substrate binding and catalysis. In combination with these findings, the results from structure-guided mutagenesis studies lead us to propose enzymatic mechanisms for both the carboxylation and hydrolysis reactions of LarB that are distinct from that of PurE.

Computational Identification of a Putative Allosteric Binding Pocket in TMPRSS2

Camostat, nafamostat, and bromhexine are inhibitors of the transmembrane serine protease TMPRSS2. The inhibition of TMPRSS2 has been shown to prevent the viral infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viruses. However, while camostat and nafamostat inhibit TMPRSS2 by forming a covalent adduct, the mode of action of bromhexine remains unclear. TMPRSS2 is autocatalytically activated from its inactive form, zymogen, through a proteolytic cleavage that promotes the binding of Ile256 to a putative allosteric pocket (A-pocket). Computer simulations, reported here, indicate that Ile256 binding induces a conformational change in the catalytic site, thus providing the atomistic rationale to the activation process of the enzyme. Furthermore, computational docking and molecular dynamics simulations indicate that bromhexine competes with the N-terminal Ile256 for the same binding site, making it a potential allosteric inhibitor. Taken together, these findings provide the atomistic basis for the development of more selective and potent TMPRSS2 inhibitors.

The BU72-μ opioid receptor crystal structure is a covalent adduct

<div>In the crystal structure of BU72 bound to the μ opioid receptor (μOR), the opioid clashes with an adjacent residue in the N-terminus; strong and unexplained electron density connects the two, centered on a point ~1.6 Å from each. This is too short for non-covalent interactions, implying covalent bonds to an unmodeled non-hydrogen atom. A magnesium ion has recently been proposed as a candidate. However, this would require unrealistically short bonds and an incomplete coordination shell. Moreover, the crystals were prepared without magnesium salts, but with components that can generate reactive oxygen species (ROS): HEPES buffer, nickel ions, and an N-terminus that forms redox-active nickel complexes. Here I show that an oxygen atom fits the unexplained density well, giving a type of covalent adduct known to form in the presence of ROS, with reasonable geometry and no clashes. While the precise structure is tentative, the observed density firmly establishes covalent bonds linking ligand and residue. Severe strain is evident in the ligand, the tethered N-terminus, and the connecting bonds. This strain, along with interactions between the N-terminus and surrounding residues, is likely to distort the receptor conformation. The subsequent μOR-Gi structure, which differs in several features associated with activation, is therefore likely to be a more accurate model of the active receptor. The possibility of reactions like this should be considered in the choice of protein truncation sites and purification conditions.</div>

Mapping the role of aromatic amino acids within a blue-light sensing LOV domain

Photosensing LOV (Light, Oxygen, Voltage) domains detect and respond to UVA/Blue (BL) light by forming a covalent adduct between the flavin chromophore and a nearby cysteine, via the decay of...

Parameters for Irreversible Inactivation of Monoamine Oxidase

The irreversible inhibitors of monoamine oxidases (MAO) slow neurotransmitter metabolism in depression and neurodegenerative diseases. After oxidation by MAO, hydrazines, cyclopropylamines and propargylamines form a covalent adduct with the flavin cofactor. To assist the design of new compounds to combat neurodegeneration, we have updated the kinetic parameters defining the interaction of these established drugs with human MAO-A and MAO-B and analyzed the required features. The Ki values for binding to MAO-A and molecular models show that selectivity is determined by the initial reversible binding. Common to all the irreversible inhibitor classes, the non-covalent 3D-chemical interactions depend on a H-bond donor and hydrophobic-aromatic features within 5.7 angstroms apart and an ionizable amine. Increasing hydrophobic interactions with the aromatic cage through aryl halogenation is important for stabilizing ligands in the binding site for transformation. Good and poor inactivators were investigated using visible spectroscopy and molecular dynamics. The initial binding, close and correctly oriented to the FAD, is important for the oxidation, specifically at the carbon adjacent to the propargyl group. The molecular dynamics study also provides evidence that retention of the allenyl imine product oriented towards FADH− influences the formation of the covalent adduct essential for effective inactivation of MAO.

The BU72-μ Opioid Receptor Crystal Structure Is a Covalent Adduct

<div>In the crystal structure of BU72 bound to the μ opioid receptor, the opioid clashes with an adjacent residue, and unexplained electron density connects the two. It has been reported that this density can be filled by a magnesium ion. However, this proposal requires unrealistically short bonds and an incomplete coordination shell. Moreover, the crystals were prepared without magnesium salts, but with components that can generate reactive oxygen species: HEPES buffer, nickel ions, and an N-terminus that forms redox-active nickel complexes. Here I show that an oxygen atom fills the unexplained density, giving a known type of covalent adduct with reasonable geometry and no clashes. Strain is evident, but is consistent with tension from the tethered N-terminus.</div>

The BU72-μ Opioid Receptor Crystal Structure Is a Covalent Adduct

<div>In the crystal structure of BU72 bound to the μ opioid receptor, the opioid clashes with an adjacent residue, and unexplained electron density connects the two. It has been reported that this density can be filled by a magnesium ion. However, this proposal requires unrealistically short bonds and an incomplete coordination shell. Moreover, the crystals were prepared without magnesium salts, but with components that can generate reactive oxygen species: HEPES buffer, nickel ions, and an N-terminus that forms redox-active nickel complexes. Here I show that an oxygen atom fills the unexplained density, giving a known type of covalent adduct with reasonable geometry and no clashes. Strain is evident, but is consistent with tension from the tethered N-terminus.</div>

Covalent and Non-Covalent Binding Free Energy Calculations for Peptidomimetic Inhibitors of SARS-CoV-2 Main Protease

COVID-19, the disease caused by the newly discovered coronavirus — SARS-CoV-2, has created global health, social, and economic crisis. At the time of writing (November 12, 2020), there are over 50 million confirmed cases and more than 1 million reported deaths due to COVID-19. Currently, there are no approved vaccines, and recently Veklury (remdesivir) was approved for the treatment of COVID-19 requiring hospitalization. The main protease (M^pro) of the virus is an attractive target for the development of effective antiviral therapeutics because it is required for proteolytic cleavage of viral polyproteins. Furthermore, the M^pro has no human homologues, so drugs designed to bind to this target directly have less risk for off-target reactivity. Recently, several high-resolution crystallographic structures of the M^pro in complex with inhibitors have been determined — to guide drug development and to spur efforts in structure-based drug design. One of the primary objectives of modern structure-based drug design is the accurate prediction of receptor­-ligand binding affinities for rational drug design and discovery. Here, we perform rigorous alchemical absolute binding free energy calculations and QM/MM calculations to give insight into the total binding energy of two recently crystallized inhibitors of SARS-CoV-2 M^pro, namely, N3 and α-ketoamide 13b. The total binding energy consists of both covalent and non-covalent binding components since both compounds are covalent inhibitors of the M^pro. Our results indicate that the covalent and non-covalent binding free energy contributions of both inhibitors to the M^pro target differ significantly. The N3 inhibitor has more favourable non-covalent interactions, particularly hydrogen bonding, in the binding site of the M^pro than the α-ketoamide inhibitor. But the Gibbs energy of reaction for the M^pro–α-ketoamide covalent adduct is greater than the Gibbs reaction energy for the M^pro–N3 covalent adduct. These differences in the covalent and non-covalent binding free energy contributions for both inhibitors could be a plausible explanation for their in vitro differences in antiviral activity. Our findings highlight the importance of both covalent and non-covalent binding free energy contributions to the absolute binding affinity of a covalent inhibitor towards its target.