The structural integrity of the membrane-embedded bacterial division complex FtsQBL studied with molecular dynamics simulations.

The FtsQBL is an essential molecular complex sitting midway through bacterial divisome assembly. To visualize and understand its structure, and the consequences of its membrane anchorage, we produced a model of the E. coli complex using the deep-learning prediction utility, AlphaFold 2. The heterotrimeric model was inserted into a 3-lipid model membrane and subjected to a 500-ns atomistic molecular dynamics simulation. The model is superb in quality and captures most experimentally derived structural features, at both the secondary structure and the side-chain levels. The model consists of a uniquely interlocking module contributed by the C-terminal regions of all three proteins. The functionally important constriction control domain residues of FtsB and FtsL are located at a fixed vertical position of ∼43-49 Å from the membrane surface. While the periplasmic domains of all three proteins are well-defined and rigid, the single transmembrane helices of each are flexible and their collective twisting and bending contribute to most structural variations, according to principal component analysis. Considering FtsQ only, the protein is more flexible in its free state relative to its complexed state-with the biggest structural changes located at the elbow between the transmembrane helix and the α-domain. The disordered N-terminal domains of FtsQ and FtsL associate with the cytoplasmic surface of the inner membrane instead of freely venturing into the solvent. Contact network analysis highlighted the formation of the interlocking trimeric module in FtsQBL as playing a central role in mediating the overall structure of the complex.

The crystal structure of the H116Q mutant of NDM-1: An enzyme devoid of zinc ions.

New Delhi metallo-ß-lactamase 1 (NDM-1) is an important causative factor of antimicrobial resistance due to its efficient hydrolysis of a broad range of ß-lactam compounds. The two zinc ions at the active site play essential roles in the NDM-1 catalytic activities. In a previous work, H116, one of the three ligands at the Zn1 site, was mutated in order to investigate the nature of zinc ion chelation. We report here the crystal structure of the NDM-1 H116Q mutant, that was designed to convert a B1 di-zinc enzyme into a B3 type, which either still binds two zinc ions or binds only one at the Zn2 site. The effect of mutation on the overall structure is minimal. Unexpectedly, no zinc ion was observed in the crystal structure. The Zn2-site ligating residue C221 forms a covalent bond with the nearby K121, a residue important in maintaining the active-site structure. The largest conformational changes were found at main-chain and side-chain atoms at residues 232-236 (loop 10), the proper configuration of which is known to be essential for substrate binding. The catalytic-site mutation caused little local changes, yet the effects were amplified and propagated to the substrate binding residues. There were big changes in the ψ angles of residues G232 and L234, which resulted in the side chain of N233 being displaced away from the substrate-binding site. In summary, we failed in turning a B1 enzyme into a B3 enzyme, yet we produced a zinc-less NDM-1 with residual activities.

NDM-1 Zn1-binding residue His116 plays critical roles in antibiotic hydrolysis.

Bacteria expressing NDM-1 have been labeled as superbugs because it confers upon them resistance to a broad range of ß-lactam antibiotics. The enzyme has a di­zinc active centre, with the Zn2 site extensively studied. The roles of active-site Zn1 ligand residues are, however, still not fully understood. We carried out structure-function studies using the mutants, H116A, H116N, and H116Q. Zinc content analysis showed that Zn1 binding was weakened by 40 to 60% in the H116 mutants. The enzymatic-activity studies showed that the lower hydrolysis rates were mainly caused by their weaker substrate binding. The catalytic efficiency (kcat/Km) of the mutants followed the order: WT > > H116Q (decreased by 4-20 fold) > H116A (decreased by 20-700 fold) ≥ H116N (decreased by 6-800 fold). The maximum effect was observed on H116N against penicillin G, whereas ampicillin was not hydrolyzed at all. The fold-increase of Km values, which informs the weakening of substrate binding, were: H116A by 5-45 fold; H116N by 6-100 fold; H116Q by 2-10 fold. Molecular dynamics simulations suggested that the Zn1 site mutations affected the positions of Zn2 and the bridging hydroxide, by 0.8 to 1.2 Å, with the largest changes of ~1.5 Å observed on Zn2 ligand C221. A native hydrogen bond between H118 and D236 was disrupted in the H116N and H116Q mutants, which led to increased flexibility of loop 10. Consequently, residue N233 was no longer maintained at an optimal position for substrate binding. H116 connected loop 7 across Zn1 to loop 10, thereby contributed to the overall integrity. This work revealed that the H116-Zn1 interaction plays a critical role in defining the substrate-binding site. From these results, it can be inferred that inhibition strategies targeting the zinc ions may be a new direction for drug development.

The structural dynamics of full-length divisome transmembrane proteins FtsQ, FtsB, and FtsL in FtsQBL complex formation.

FtsQBL is a transmembrane protein complex in the divisome of Escherichia coli that plays a critical role in regulating cell division. Although extensive efforts have been made to investigate the interactions between the three involved proteins, FtsQ, FtsB, and FtsL, the detailed interaction mechanism is still poorly understood. In this study, we used hydrogen-deuterium exchange mass spectrometry to investigate these full-length proteins and their complexes. We also dissected the structural dynamic changes and the related binding interfaces within the complexes. Our data revealed that FtsB and FtsL interact at both the periplasmic and transmembrane regions to form a stable complex. Furthermore, the periplasmic region of FtsB underwent significant conformational changes. With the help of computational modeling, our results suggest that FtsBL complexation may bring the respective constriction control domains (CCDs) in close proximity. We show that when FtsBL adopts a coiled-coil structure, the CCDs are fixed at a vertical position relative to the membrane surface; thus, this conformational change may be essential for FtsBL's interaction with other divisome proteins. In the FtsQBL complex, intriguingly, we show only FtsB interacts with FtsQ at its C-terminal region, which stiffens a large area of the ß-domain of FtsQ. Consistent with this, we found the connection between the α- and ß-domains in FtsQ is also strengthened in the complex. Overall, the present study provides important experimental evidence detailing the local interactions between the full-length FtsB, FtsL, and FtsQ protein, as well as valuable insights into the roles of FtsQBL complexation in regulating divisome activity.

Interdomain flexibility and interfacial integrity of ß-lactamase inhibitory protein (BLIP) modulate its binding to class A ß-lactamases.

ß-Lactamase inhibitory protein (BLIP) consists of a tandem repeat of αß domains conjugated by an interdomain loop and can effectively bind and inactivate class A ß-lactamases, which are responsible for resistance of bacteria to ß-lactam antibiotics. The varied ability of BLIP to bind different ß-lactamases and the structural determinants for significant enhancement of BLIP variants with a point mutation are poorly understood. Here, we investigated the conformational dynamics of BLIP upon binding to three clinically prevalent class A ß-lactamases (TEM1, SHV1, and PC1) with dissociation constants between subnanomolar and micromolar. Hydrogen deuterium exchange mass spectrometry revealed that the flexibility of the interdomain region was significantly suppressed upon strong binding to TEM1, but was not significantly changed upon weak binding to SHV1 or PC1. E73M and K74G mutations in the interdomain region improved binding affinity toward SHV1 and PC1, respectively, showing significantly increased flexibility of the interdomain region compared to the wild-type and favorable conformational changes upon binding. In contrast, more rigidity of the interfacial loop 135-145 was observed in these BLIP mutants in both free and bound states. Consistently, molecular dynamics simulations of BLIP exhibited drastic changes in the flexibility of the loop 135-145 in all complexes. Our results indicated for the first time that higher flexibility of the interdomain linker, as well as more rigidity of the interfacial loop 135-145, could be desirable determinants for enhancing inhibition of BLIP to class A ß-lactamases. Together, these findings provide unique insights into the design of enhanced inhibitors.

Virtual screening on the web for drug repurposing: a primer.

We describe a procedure of performing in silico (virtual) screening using a web-based service, the MTiOpenScreen, which is freely accessible to non-commercial users. We shall use the SARS-CoV-2 main protease as an example. Starting from a structure downloaded from the Protein Data Bank, we discuss how to prepare the coordinates file, taking into account the known biochemical background information of the target protein. The reader will find that this preparation step takes up most of the effort before the target is ready for screening. The steps for uploading the target structure and defining the search volume by critical residues, and the main parameters to use, are outlined. When this protocol is followed, the user will expect to obtain a ranked list of small approved drug compounds docked into the target structure. The results can be readily examined graphically on the web site or downloaded for studying in a local molecular graphics program such as PyMOL.

Simeprevir Potently Suppresses SARS-CoV-2 Replication and Synergizes with Remdesivir.

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir not only inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp) but also modulates host immune responses. Our results thus reveal the possible anti-SARS-CoV-2 mechanism of simeprevir and highlight the translational potential of optimizing simeprevir as a therapeutic agent for managing COVID-19 and future outbreaks of CoV.

Crystal structures of influenza nucleoprotein complexed with nucleic acid provide insights into the mechanism of RNA interaction.

The nucleoprotein (NP) of influenza virus is the core component of the ribonucleoprotein (RNP) and performs multiple structural and functional roles. Structures of the influenza A, B and D NP molecules have been solved previously, but structural information on how NP interacts with RNA remains elusive. Here we present the crystal structure of an obligate monomer of H5N1 NP in complex with RNA nucleotides to 2.3 Å, and a C-terminal truncation of this mutant, also in complex with RNA nucleotides, to 3 Å. In both structures, three nucleotides were identified near two positive grooves of NP suggested to be important for RNA binding. Structural evidence supports that conformational changes of flexible loops and the C-terminal tail both play important roles in the binding of RNA. Based on the structure, we propose a mechanism by which NP captures RNA by flexible loops and transfers it onto the positive binding grooves. Binding of RNA by NP is a crucial step for template re-encapsidation during transcription and replication and cRNP formation. Our structures thus provide insights into the molecular virology of the influenza virus.

Fatal Attraction: The Case of Toxic Soluble Dimers of Truncated PQBP-1 Mutants in X-Linked Intellectual Disability.

The frameshift mutants K192Sfs*7 and R153Sfs*41, of the polyglutamine tract-binding protein 1 (PQBP-1), are stable intrinsically disordered proteins (IDPs). They are each associated with the severe cognitive disorder known as the Renpenning syndrome, a form of X-linked intellectual disability (XLID). Relative to the monomeric wild-type protein, these mutants are dimeric, contain more folded contents, and have higher thermal stabilities. Comparisons can be drawn to the toxic oligomerisation in the "conformational diseases", which collectively describe medical conditions involving a substantial protein structural transition in the pathogenic mechanism. At the molecular level, the end state of these diseases is often cytotoxic protein aggregation. The conformational disease proteins contain varying extents of intrinsic disorder, and the consensus pathogenesis includes an early oligomer formation. We reviewed the experimental characterisation of the toxic oligomers in representative cases. PQBP-1 mutant dimerisation was then compared to the oligomerisation of the conformational disease proteins. The PQBP-1 mutants are unique in behaving as stable soluble dimers, which do not further develop into higher oligomers or aggregates. The toxicity of the PQBP-1 mutant dimers lies in the native functions (in transcription regulation and possibly, RNA splicing) being compromised, rather than proceeding to aggregation. Other examples of stable IDP dimers were discussed and we speculated on the roles of IDP dimerisation in protein evolution.

Parameterization of Large Ligands for Gromacs Molecular Dynamics Simulation with LigParGen.

Molecular dynamics (MD) simulation is a powerful method of investigating the interaction between molecular species. Defining the mechanical properties and topologies for all components involved is critical. While parameters for proteins are well established, those for the wide range of ligands and substrates are not. Here we introduce a very useful service which is designed for small organic molecules. We describe a protocol to extend this tool to beyond its current size (200 atoms) and formal charge (2+ to 2-) limits.

Molecular Data Visualization on Mobile Devices: A Quick Starter's Guide.

With the rise of tablets, truly portable molecular graphics are now available for wide use by scientists to share structural information in real time. We have surveyed the existing software available on Apple iPads and on Android tablets in order to make a recommendation to potential users, primarily based on the product features. Among the three apps for high-quality 3-D display, iMolview (available on both platforms) stands out to be our choice, with PyMOL app (iOS) a close alternative and NDKmol (Android) offering some uniquely useful functions. Hence we include a tutorial on how to get started using iMolview to do some simple visualization in 10 min.

Molecular Data Visualization with Augmented Reality (AR) on Mobile Devices.

Augmented reality (AR) allows a computer-generated 3D model to be superimposed onto a real-world environment in real time. The model can then be manipulated or probed interactively as if it is part of the real world. The application of AR in visualizing macromolecular structures is growing, primarily in showing preset collections of scenes for education purpose. Here, our emphasis is, however, on exploiting AR as a tool to facilitate scientific communication on the go. We have searched for freely available mobile software and custom-built tools which allow the display of user-specified protein structures. We provide step-by-step guides on a standalone app Ollomol (iOS and Android), as well as an in-browser web app, WebAR-PDB. Both of them allow users to specify entries from the Protein Data Bank (PDB) for an elementary AR experience. The application of AR enhances interactivity and imaginativity in macromolecular visualization.

BADAN-conjugated ß-lactamases as biosensors for ß-lactam antibiotic detection.

ß-Lactam antibiotic detection has significant implications in food safety control, environmental monitoring and pharmacokinetics study. Here, we report the development of two BADAN-conjugated ß-lactamases, E166Cb and E166Cb/N170Q, as sensitive biosensors for ß-lactam antibiotic detection. These biosensors were constructed by coupling an environment-sensitive BADAN probe onto location 166 at the active site of the PenP ß-lactamase E166C and E166C/N170Q mutants. They gave fluorescence turn-on signals in response to ß-lactam antibiotics. Molecular dynamics simulation of E166Cb suggested that the turn-on signal might be attributed to a polarity change of the microenvironment of BADAN and the removal of the fluorescence quenching effect on BADAN exerted by a nearby Tyr-105 upon the antibiotic binding. In the detection of four ß-lactams (penicillin G, penicillin V, cefotaxime and moxalactam), both E166Cb and E166Cb/N170Q delivered signal outputs in an antibiotic-concentration dependent manner with a dynamic range spanning from 10 nM to 1 µM. Compared to E166Cb, E166Cb/N170Q generally exhibited more stable signals owing to its higher deficiency in hydrolyzing the antibiotic analyte. The overall biosensor performance of E166Cb and E166Cb/N170Q was comparable to that of their respective fluorescein-modified counterparts, E166Cf and E166Cf/N170Q. But comparatively, the BADAN-conjugated enzymes showed a higher sensitivity, displayed a faster response in detecting moxalactam and a more stable fluorescence signals towards penicillin G. This study illustrates the potential of BADAN-conjugated ß-lactamases as biosensing devices for ß-lactam antibiotics.

Design of a structure-based fluorescent biosensor from bioengineered arginine deiminase for rapid determination of L-arginine.

Rationally designed mutations on recombinant arginine deiminase (ADI) could act as a 'turn-off' L-arginine (L-Arg) fluorescent biosensor and provide an alternative method for rapid determination of L-Arg. Double mutations were introduced on the Cys251➔Ser251 and Thr265➔Cys265 of recombinant ADI, rendering a single cysteine present on the protein surface for the site-specific attachment of a fluorophore, fluorescein-5-maleimide. The double mutations on ADI (265C) and its fluorescein-labelled form (265Cf) conserved the catalytic efficiency of wild-type ADI. Upon binding to L-Arg, 265Cf induced structural conformational changes and rendered the fluorescein moiety to move closer to Trp264, resulting in fluorescence quenching. The duration of fluorescence quenching was dependant on the L-Arg concentration. A linear relationship between the time at the maximum rate of fluorescence change and L-Arg concentrations, which ranged from 2.5 to 100 µM, was found with R2 = 0.9988. The measurement time was within 0.15-4 min. Determination of L-Arg concentration in fetal bovine serum could be achieved by the standard addition method and without sample pre-treatment. The result showed a good agreement with the one determined by mass spectrometry, suggesting our biosensor as a promising tool for the detection of L-Arg in biological samples.

Conformational Dynamics of the Helix 10 Region as an Allosteric Site in Class A ß-Lactamase Inhibitory Binding.

ß-Lactamase inhibitory protein (BLIP) can effectively inactivate class A ß-lactamases, but with very different degrees of potency. Understanding the different roles of BLIP in class A ß-lactamases inhibition can provide insights for inhibitor design. However, this problem was poorly solved on the basis of the static structures obtained by X-ray crystallography. In this work, ion mobility mass spectrometry, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics simulation revealed the conformational dynamics of three class A ß-lactamases with varying inhibition efficiencies by BLIP. A more extended conformation of PC1 was shown compared to those of TEM1 and SHV1. Localized dynamics differed in several important loop regions, that is, the protruding loop, H10 loop, Ω loop, and SDN loop. Upon binding with BLIP, these loops cooperatively rearranged to enhance the binding interface and to inactivate the catalytic sites. In particular, unfavorable changes in conformational dynamics were found in the protruding loop of SHV1 and PC1, showing less effective binding. Intriguingly, the single mutation on BLIP could compensate for the unfavored changes in this region, and thus exhibit enhanced inhibition toward SHV1 and PC1. Additionally, the H10 region was revealed as an important allosteric site that could modulate the inhibition of class A ß-lactamases. It was suggested that the rigid protruding loop and flexible H10 region might be determinants for the effective inhibition of TEM1. Our findings provided unique and explicit insights into the conformational dynamics of ß-lactamases and their bindings with BLIP. This work can be extended to other ß-lactamases of interest and inspire the design of novel inhibitors.

Mono-PEGylation of a Thermostable Arginine-Depleting Enzyme for the Treatment of Lung Cancer.

L-arginine (L-Arg) depletion induced by randomly PEGylated arginine deiminase (ADI-PEG20) can treat arginosuccinate synthase (ASS)-negative cancers, and ADI-PEG20 is undergoing phase III clinical trials. Unfortunately, ASS-positive cancers are resistant to ADI-PEG20. Moreover, the yield of ADI production is low because of the formation of inclusion bodies. Here, we report a thermostable arginine-depleting enzyme, Bacillus caldovelox arginase mutant (BCA-M: Ser161->Cys161). An abundant amount of BCA-M was easily obtained via high cell-density fermentation and heat treatment purification. Subsequently, we prepared BCA-M-PEG20, by conjugating a single 20 kDa PEG monomer onto the Cys161 residue via thio-chemistry. Unlike ADI-PEG20, BCA-M-PEG20 significantly inhibited ASS-positive lung cancer cell growth. Pharmacodynamic studies showed that a single intraperitoneal injection (i.p). administration of 250 U/mouse of BCA-M-PEG20 induced low L-Arg level over 168 h. The mono-PEGylation of BCA-M prolonged its elimination half-life from 6.4 to 91.4 h (a 14-fold increase). In an A549 lung cancer xenograft model, a weekly administration of 250 U/mouse of BCA-M-PEG20 suppressed tumor growth significantly. We also observed that BCA-M-PEG20 did not cause any significant safety issue in mouse models. Overall, BCA-M-PEG20 showed excellent results in drug production, potency, and stability. Thereby, it has great potential to become a promising candidate for lung cancer therapy.

Prediction of the SARS-CoV-2 (2019-nCoV) 3C-like protease (3CL pro) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates.

We prepared the three-dimensional model of the SARS-CoV-2 (aka 2019-nCoV) 3C-like protease (3CL pro) using the crystal structure of the highly similar (96% identity) ortholog from the SARS-CoV. All residues involved in the catalysis, substrate binding and dimerisation are 100% conserved. Comparison of the polyprotein PP1AB sequences showed 86% identity. The 3C-like cleavage sites on the coronaviral polyproteins are highly conserved. Based on the near-identical substrate specificities and high sequence identities, we are of the opinion that some of the previous progress of specific inhibitors development for the SARS-CoV enzyme can be conferred on its SARS-CoV-2 counterpart.  With the 3CL pro molecular model, we performed virtual screening for purchasable drugs and proposed 16 candidates for consideration. Among these, the antivirals ledipasvir or velpatasvir are particularly attractive as therapeutics to combat the new coronavirus with minimal side effects, commonly fatigue and headache.  The drugs Epclusa (velpatasvir/sofosbuvir) and Harvoni (ledipasvir/sofosbuvir) could be very effective owing to their dual inhibitory actions on two viral enzymes.

Hydrophobic substituents on isatin derivatives enhance their inhibition against bacterial peptidoglycan glycosyltransferase activity.

Moenomycin A, the well-known natural product inhibitor of peptidoglycan glycosyltransferase (PGT), is a large amphiphilic molecule of molecular mass of 1583 g/mol and its bioavailablity as a drug is relatively poor. In searching for small-molecule ligands with high inhibition ability targeting the enzyme, we found that the addition of hydrophobic groups to an isatin-based inhibitor of bacterial PGT significantly improves its inhibition against the enzyme, as well as its antibacterial activity. The improvement in enzymatic inhibition can be attributed to a better binding of the small molecule inhibitor to the hydrophobic region of the membrane-bound bacterial cell wall synthesis enzyme and the plasma membrane. In the present study, a total of 20 new amphiphilic compounds were systematically designed and the relationship between molecular hydrophobicity and the antibacterial activity by targeting at PGT was demonstrated. The in vitro lipid II transglycosylation inhibitory effects (IC50) against E. coli PBP1b and MICs of the compounds were investigated. Optimized results including MIC values of 6 µg/mL for MSSA, MRSA, B. subtilis and 12 µg/mL for E. coli were obtained with an isatin derivative 5m which has a molecular mass of 335 g/mol.

Frameshift PQBP-1 mutants K192Sfs*7 and R153Sfs*41 implicated in X-linked intellectual disability form stable dimers.

Polyglutamine tract-binding protein-1 (PQBP-1) is a nuclear intrinsically disordered protein playing important roles in transcriptional regulation and RNA splicing during embryonic and postembryonic development. In human, its mutations lead to severe cognitive impairment known as the Renpenning syndrome, a form of X-linked intellectual disability (XLID). Here, we report a combined biophysical study of two PQBP-1 frameshift mutants, K192Sfs*7 and R153Sfs*41. Both mutants are dimeric in solution, in contrast to the monomeric wild-type protein. These mutants contain more folded contents and have increased thermal stabilities. Using small-angle X-ray scattering data, we generated three-dimensional envelopes which revealed their overall flat shapes. We also described each mutant using an ensemble model based on a native-like initial pool with a dimeric structural core. PQBP-1 is known to repress transcription by way of interacting with the C-terminal domain of RNA polymerase II, which consists of 52 repeats of a consensus heptapeptide sequence YSPTSPS. We studied the binding of PQBP-1 variants to the labelled peptide which is phosphorylated at positions 2 and 5 (YpSPTpSPS) and found that this interaction is significantly weakened in the two mutants.