Active site geometry stabilization of a presenilin homolog by the lipid bilayer promotes intramembrane proteolysis

Cleavage of membrane proteins in the lipid bilayer by intramembrane proteases is crucial for health and disease. Although different lipid environments can potently modulate their activity, how this is linked to their structural dynamics is unclear. Here we show that the carboxy-peptidase-like activity of the archaeal intramembrane protease PSH, a homolog of the Alzheimer's disease-associated presenilin/γ-secretase is impaired in micelles and promoted in a lipid bilayer. Comparative molecular dynamics simulations revealed that important elements for substrate binding such as transmembrane domain 6a of PSH are more labile in micelles and stabilized in the lipid bilayer. Moreover, consistent with an enhanced interaction of PSH with a transition-state analog inhibitor, the bilayer promoted the formation of the enzyme's catalytic active site geometry. Our data indicate that the lipid environment of an intramembrane protease plays a critical role in structural stabilization and active site arrangement of the enzyme-substrate complex thereby promoting intramembrane proteolysis.

N-glycosylation modulates enzymatic activity of Trypanosoma congolense trans-sialidase

Trypanosomes cause the devastating disease trypanosomiasis, in which the action of trans-sialidase (TS) enzymes harbored on their surface is a key virulence factor. TS are highly N-glycosylated, but the biological functions of the glycans remain elusive. In this study, we investigated the influence of N-glycans on the enzymatic activity and structure stability of TconTS1, a recombinant TS from the African parasite Trypanosoma congolense. MALDI-TOF MS revealed that eight asparagine sites were glycosylated with high-mannose type N-glycans. Deglycosylation of TconTS1 led to a 5-fold decrease in substrate affinity but to the same conversion rate relative to the untreated enzyme. After deglycosylation, no changes in secondary structure elements were observed in circular dichroism experiments. Molecular dynamics simulations revealed interactions between the highly flexible N-glycans and some conserved amino acids belonging to the catalytic site. These interactions led to conformational changes, possibly enhancing substrate accessibility and promoting enzyme/substrate complex stability. The here-observed modulation of catalytic activity via the N-glycan shield may be a structure-function relationship intrinsic of several members of the TS family.

Room-temperature dynamic nuclear polarization enhanced NMR spectroscopy of small biological molecules in water

Nuclear magnetic resonance (NMR) spectroscopy is a powerful and popular technique for probing the molecular structures, dynamics and chemical properties. However the conventional NMR spectroscopy is bottlenecked by its low sensitivity. Dynamic nuclear polarization (DNP) boosts NMR sensitivity by orders of magnitude and resolves this limitation. In liquid-state this revolutionizing technique has been restricted to a few specific non-biological model molecules in organic solvents. Here we show that the carbon polarization in small biological molecules, including carbohydrates and amino acids, can be enhanced sizably by in situ Overhauser DNP (ODNP) in water at room temperature and at high magnetic field. An observed connection between ODNP 13C enhancement factor and paramagnetic 13C NMR shift has led to the exploration of biologically relevant heterocyclic compound indole. The QM/MM MD simulation underscores the dynamics of intermolecular hydrogen bonds as the driving force for the scalar ODNP in a long-living radical-substrate complex. Our work reconciles results obtained by DNP spectroscopy, paramagnetic NMR and computational chemistry and provides new mechanistic insights into the high-field scalar ODNP.

Design, synthesis, and biological evaluation of symmetrical azine derivatives as novel tyrosinase inhibitors

A series of symmetrical azine derivatives containing different substituted benzyl moieties were designed, synthesized, and evaluated for their inhibitory activity against tyrosinase. The results showed that compounds 3e, 3f, 3h, 3i, 3j, and 3k possess effective tyrosinase inhibition with IC50 values ranging from 7.30 μM to 62.60 μM. Particularly, compounds 3f displayed around three-fold improvement in the potency (IC50 = 7.30 ± 1.15 μM) compared to that of kojic acid (IC50 = 20.24 ± 2.28 μM) as the positive control. Kinetic study of compound 3f confirmed uncompetitive inhibitory activity towards tyrosinase indicating that it can bind to enzyme–substrate complex. Next, molecular docking analysis was performed to study the interactions and binding mode of the most potent compound 3f in the tyrosinase active site. Besides, the cytotoxicity of 3f, as well as its potency to reduce the melanin content were also measured on invasive melanoma B16F10 cell line. Also, 3f exhibited above 82% cell viability in the A375 cell line at 10 µM. Consequently, compounds 3f could be introduced as a potent tyrosinase inhibitor that might be a promising candidate in the cosmetics, medicine, and food industry.

Apixaban, an orally available anticoagulant, inhibits SARS-CoV-2 replication by targeting its major protease in a non-competitive way

Anticoagulants are associated with clinical benefit against the 2019 coronavirus disease (COVID-19), preventing COVID-19 associated coagulopathy. Blood coagulation factor Xa (FXa) and SARS-CoV-2 major protease (Mpro) share over 80% homology at the three-dimensional protein level. Thus, it is worth interrogating whether there is crosstalk between inhibitors and substrates between these enzymes. Here, we found that the clinically-approved FXa inhibitor apixaban targets SARS-CoV-2 Mpro with a 21-fold higher potency than boceprevir (GC376). Apixaban displayed a non-competitive mechanism of inhibition towards Mpro, since it targets the enzyme/substrate complex and the allosteric site onto the viral protease. Enzymatic assays were further validated in infected Calu-3 cells, which reveal that apixaban decreases the production of infectious viral particles in a dose-dependent manner, with an inhibitory potency in the micromolar range. Our results are in line with the proposed early use of anticoagulants, including FXa inhibitors, to improve clinical outcome of COVID-19 patients. In this context, apixaban may display a dual mechanism of action by targeting FXa to prevent coagulopathy and, at some level, SARS-CoV-2 Mpro.

Specificity of AMPylation of the human chaperone BiP is mediated by TPR motifs of FICD

To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis.

N 6-Methyladenosines in mRNAs reduce the accuracy of codon reading by transfer RNAs and peptide release factors

We used quench flow to study how N6-methylated adenosines (m6A) affect the accuracy ratio between kcat/Km (i.e. association rate constant (ka) times probability (Pp) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated kcat/Km for Glu-tRNAGlu, EF-Tu and GTP forming ternary complex (T3) reading cognate (GAA and Gm6AA) or near-cognate (GAU and Gm6AU) codons. ka decreased 10-fold by m6A introduction in cognate and near-cognate cases alike, while Pp for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated kcat/Km for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um6AA) and near-cognate (UAG and Um6AG) stop codons to decrease 6-fold or 3-fold by m6A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m6A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate kcat/Km, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate kcat/Km.

Spontaneous Cleavages of a Heterologous Protein, the CenA Endoglucanase of Cellulomonas fimi, in Escherichia coli

CenA is an endoglucanase secreted by the Gram-positive cellulolytic bacterium, Cellulomonas fimi, to the environment as a glycosylated protein. The role of glycosylation in CenA is unclear. However, it seems not crucial for functional activity and secretion since the unglycosylated counterpart, recombinant CenA (rCenA), is both bioactive and secretable in Escherichia coli. Using a systematic screening approach, we have demonstrated that rCenA is subjected to spontaneous cleavages (SC) in both the cytoplasm and culture medium of E. coli, under the influence of different environmental factors. The cleavages were found to occur in both the cellulose-binding (CellBD) and catalytic domains, with a notably higher occurring rate detected in the former than the latter. In CellBD, the cleavages were shown to occur close to potential N-linked glycosylation sites, suggesting that these sites might serve as ‘attributive tags’ for differentiating rCenA from endogenous proteins and the points of initiation of SC. It is hypothesized that glycosylation plays a crucial role in protecting CenA from SC when interacting with cellulose in the environment. Subsequent to hydrolysis, SC would ensure the dissociation of CenA from the enzyme-substrate complex. Thus, our findings may help elucidate the mechanisms of protein turnover and enzymatic cellulolysis.

Molecular mechanism of chromogenic substrate hydrolysis in the active site of human carboxylesterase-1

Human carboxylesterases are involved in the protective processes of detoxification during the hydrolytic metabolism of xenobiotics. Knowledge of the molecular mechanisms of substrates hydrolysis in the enzymes active site is necessary for the rational drug design. In this work, the molecular mechanism of the hydrolysis reaction of para-nitrophenyl acetate in the active site of human carboxylesterase was determined using modern methods of molecular modeling. According to the combined method of quantum mechanics/molecular mechanics calculations, the chemical reaction occurs within four elementary steps, including two steps of the acylation stage, and two steps of the deacylation stage. All elementary steps have low energy barriers, with the gradual lowering of the intermediate energies that stimulates reaction in the forward direction. The molecular docking was used to estimate the binding constants of the enzyme-substrate complex and the dissociation constant of enzyme-product complexes. The effective kinetic parameters of the enzymatic hydrolysis in the active site of carboxylesterase are determined by numerical solution of the differential kinetic equations.

Attempt on Magnification of the Mechanism of Enzyme Catalyzed Reaction through Bio-geometric Model for the Five Points Circle in the Triangular Form of Lineweaver-Burk Plot

The bio-geometrical model is dealing with correlation between the “five events for enzyme catalyzed reaction” and “triple point event serving groups on the circle” in triangle obtained for the graphical presentation of the double reciprocal for magnification of the mechanism of enzyme catalyzed reaction. This model is based on the nine point circle in triangle of the double reciprocal plot. The five significant points (B, D, E, F and G) resulted for the circle with x – and y – coordinates. The present attempt is considering interactions among enzymes and substrates for the successful release of product through each and every point on the circle in triangle. The controlling role of the point, “O”, center of circle in each and every event of the biochemical reaction is obligatory. The model is allotting specific role for the significant events in the biochemical reaction catalyzed by the enzymes. The enzymatic catalysis is supposed to be completed through five events, which may be named as, “Bio-geometrical events of enzyme catalyzed reaction”. These five events for enzyme catalyzed reaction include: (1) Initial event of enzymatic interaction with the substrates; (2) Event of the first transition state for the formation of “enzyme-substrate” complex; (3) Event of the second transition state for the formation of “enzyme-product” complex; (4) Event of release of the product and relieve enzyme and (5) The event of directing the enzyme to continue the reaction. The model utilizes the “triple point serving group on the circle” for the success of each and every event in the biochemical reaction. Thus, there is involvement of the three points including the point “O” for each event in the enzyme catalyzed reaction. The group of points serving for carrying out the event may be classified into five conic sections like: B-O-E; E-O-G; G-O-D; D-O-F and F-O-B. The bio-geometrical model is correlation between the “five events for enzyme catalyzed reaction” and “triple point event serving groups on the circle” in a triangle of the double reciprocal plot.