Biology subjects

Mulholland, A. J.

Publications and source records attributed to Mulholland, A. J..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Double crossed? Structural and computational studies of an unusual crosslinked heme in Methylococcus capsulatus cytochrome P460

Cytochromes P460 oxidise hydroxylamine within the nitrogen cycle and contain as their active site an unusual catalytic c-type heme where the porphyrin is cross-linked to the protein via a lysine residue in addition to the canonical cross links from cysteine residues. Understanding how enzymes containing P460 heme oxidise hydroxylamine into either nitrous oxide or nitric oxide has implications for climate change. Interestingly the P460 containing hydroxylamine oxidoreductase utilises a tyrosine cross link to heme and performs similar chemistry. Previous crystal structures of cytochrome P460 from Nitrosomonas europaea (NeP460) clearly show the existence of a single crosslink between the Nz atom of lysine and the heme porphyrin with mutagenesis studies indicating roles for the crosslink in positioning a proton transfer residue and/or influencing the distortion of the heme. Here we describe the evidence for a novel double cross link between lysine and heme in the cytochrome P460 from Methylococcus capsulatus (Bath). In order to understand the complexities of this enzyme system we applied high resolution structural biology approaches at synchrotron and XFEL sources paired with crystal spectroscopies. Linked to this we carried out QM/MM simulations that enabled the prediction of electronic absorption spectra providing a crucial validation to linking simulations and experimental structures. Our work demonstrates the feasibility of a double crosslink in McP460 and provides an opportunity to investigate how simulations can interact with experimental structures.

biochemistry↗

bioRxiv2024

Electrostatic interactions define nacubactam potency against OXA-48-like β-lactamases

Carbapenemases, {beta}-lactamases hydrolysing carbapenem antibiotics, severely challenge treatment of multi-drug resistant bacterial infections. OXA-48 is among the most widely disseminated carbapenemases in Enterobacterales, consequently new treatment options for OXA-48 producers are urgently required. Development of diazabicyclooctane (DBO) inhibitors to overcome {beta}-lactamase-mediated resistance is one attractive option, due to their efficacy against a wide range of {beta}-lactamases. The DBO avibactam is already licensed for clinical use, with the related compound nacubactam currently in phase III trials for carbapenem-resistant infections. Here we investigate the activities of avibactam and nacubactam towards OXA-48 and two variants, OXA-163 and OXA-405, that contain deletions in the {beta}5 - {beta}6 loop adjacent to the active site and show modified activity towards different {beta}-lactam classes. Compared to avibactam, nacubactam is c. 80-fold less potent towards OXA-48, but this difference is reduced in OXA-163 and OXA-405. Crystal structures of the respective avibactam and nacubactam complexes, and molecular dynamics simulations based upon these, reveal residue Arg214 on the OXA-48 {beta}5 - {beta}6 active-site loop to be electrostatically repelled by nacubactam, but not avibactam, binding. This increases flexibility of the OXA-48 {beta}5 - {beta}6 loop, as well as neighbouring active site loops, in simulations of the OXA-48:nacubactam, compared to the avibactam, complex. Such effects are not observed in simulations of the respective complexes of OXA-163 and OXA-405, which lack Arg214. These data indicate that interactions with Arg214 can determine DBO potency towards OXA-48-like enzymes, and suggest that sequence variation in this {beta}-lactamase family affects reactivity towards inhibitors as well as {beta}-lactam substrates.

biochemistry↗

bioRxiv2024

Dynamical Responses Predict a Distal Site that Modulates Activity in an Antibiotic Resistance Enzyme

{beta}-Lactamases, which hydrolyse {beta}-lactam antibiotics, are key determinants of antibiotic resistance. Predicting the sites and effects of distal mutations in enzymes is challenging. For {beta}-lactamases, the ability to make such predictions would contribute to understanding activity against, and development of, antibiotics and inhibitors to combat resistance. Here, using dynamical non-equilibrium molecular dynamics (D-NEMD) simulations combined with experiments, we demonstrate that intramolecular communication networks differ in three class A SulpHydryl Variant (SHV)-type {beta}-lactamases). Differences in network architecture and correlated motions link to catalytic efficiency and {beta}-lactam substrate spectrum. Further, the simulations identify a distal residue 89 in the clinically important Klebsiella pneumoniae carbapenemase 2 (KPC-2), as a participant in similar networks, suggesting that mutation at this position would modulate enzyme activity. Experimental kinetics, biophysical and structural characterisation of the naturally occurring, but previously biochemically uncharacterised, KPC-2G89D mutant with several antibiotics and inhibitors reveals significant changes in hydrolytic spectrum, specifically reducing activity towards carbapenems without effecting major structural or stability changes. These results show that D-NEMD simulations can predict distal sites where mutation affects enzyme activity. This approach could have broad application in understanding enzyme evolution, and in engineering of natural and de novo enzymes.

biochemistry↗

bioRxiv2023

Substrate recognition and selectivity in SARS-CoV-2 main protease: Unveiling the role of subsite interactions through dynamical nonequilibrium molecular dynamics simulations

The main protease (Mpro) of the SARS-CoV-2 coronavirus employs a cysteine-histidine dyad in its active site to catalyse hydrolysis of the viral polyproteins. It is well established that binding of the substrate P1-Gln in the S1 subsite of Mpro active site is crucial for catalysis and this interaction has been employed to inform inhibitor design; however, how Mpro dynamically recognises and responds to substrate binding remains difficult to probe by experimental methods. We thus employed the dynamical nonequilibrium molecular dynamics (D-NEMD) approach to probe the response of Mpro to systematic substrate variations. The results emphasise the importance of P1-Gln for initiating a productive enzymatic reaction. Specifically, substituting P1-Gln with alanine disrupts the conformations of the Cys145 and His41 dyad, causing Cys145 to transition from the productive gauche conformation to the non-productive trans conformation. Importantly, our findings indicate that Mpro exhibits dynamic responses to substrate binding and likely to substrate-mimicking inhibitors within each of the S4-S2' subsites. The results inform on the substrate selectivity requirements and shed light on the observed variations in hydrolytic efficiencies of Mpro towards different substrates. Some interactions between substrate residues and enzyme subsites involve more induced fit than others, implying that differences in functional group flexibility may optimise the binding of a substrate or inhibitor in a particular subsite.

biochemistry↗

bioRxiv2023

Allosteric modulation by the fatty acid site in the glycosylated SARS-CoV-2 spike

The trimeric spike protein plays an essential role in the SARS-CoV-2 virus lifecycle, facilitating virus entry through binding to the cellular receptor angiotensin-converting enzyme 2 (ACE2) and mediating viral-host membrane fusion. The SARS-CoV-2 spike contains a fatty acid (FA) binding site at the interface between two neighbouring receptor-binding domains. This site, also found in some other coronaviruses, binds free fatty acids such as linoleic acid. Binding at this site locks the spike in a non-infectious, closed conformation. This site is coupled to functionally important regions, but the effects of glycans on these allosteric effects have not been investigated. Understanding allostery and how this site modulates the behaviour of the spike protein could potentiate the development of promising alternative strategies for new coronavirus therapies. Here, we apply dynamical nonequilibrium molecular dynamics (D-NEMD) simulations to investigate allosteric effects of the FA site in the fully glycosylated spike of the original SARS-CoV-2 ancestral variant. The results show allosteric networks that connect the FA site to important functional regions of the protein, including some more than 40 [A] away, including the receptor binding motif, an antigenic supersite in the N-terminal domain, the furin cleavage site, regions surrounding the fusion peptide, and another allosteric site known to bind heme and biliverdin. The networks identified here highlight the complexity of the allosteric modulation in this protein and reveal a striking and unexpected connection between different allosteric sites. Notably, 65% of amino acid substitutions, deletions and insertions in the Alpha, Beta, Delta, Gamma and Omicron variants map onto or close to the identified allosteric pathways. Comparison of the FA site connections from D-NEMD in the glycosylated and non-glycosylated spikes revealed that the presence of glycans does not qualitatively change the internal allosteric pathways within the protein, with some glycans facilitating the transmission of the structural changes within and between subunits. Significance statementThe spike protein is crucial for the SARS-CoV-2 virus, enabling the fusion of the viral and host cell membranes. This protein contains several allosteric sites, including a fatty acid binding site at the interface between every two neighbouring receptor-binding domains. This site modulates the behaviour of the protein, with the binding of various free fatty acids and other small molecules influencing the spikes structure. In particular, the binding of linoleic acid, an essential fatty acid molecule, stabilizes the protein in a non-infectious locked conformation, thus making it inaccessible for binding to human receptors. Here, we investigate how the fatty acid site modulates the structural and dynamical behaviour of the fully glycosylated protein. Our work reveals complex patterns of communication between the fatty acid site and functionally important regions of the spike (including the receptor binding motif, the antigenic supersite in the N-terminal domain, the heme/biliverdin site, furin cleavage site and the fusion-peptide surrounding regions) and shed new light on the roles of glycans in this protein.

biochemistry↗

bioRxiv2023

Electric fields determine carbapenemase activity in class A β-lactamases

Antimicrobial resistance is a public health crisis. Limited understanding of the catalytic drivers in resistance-mediating enzymes such as {beta}-lactamases hinders our ability to combat this crisis. Here, we dissect the catalytic contributions of active-site electric fields in class A {beta}-lactamases. We studied the enzymatic hydrolysis of a carbapenem antibiotic by QM/MM molecular dynamics simulations and quantified active-site fields with a custom-made script. We discovered that the fields correlate well with activity and identified seven positions, some distal, that distinguish efficient carbapenemases. Electric-field analysis may help predict the activity of {beta}-lactamases and guide antibiotic and enzyme design. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/565607v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@15ee41aorg.highwire.dtl.DTLVardef@d1a11corg.highwire.dtl.DTLVardef@a83bd7org.highwire.dtl.DTLVardef@f0abdf_HPS_FORMAT_FIGEXP M_FIG C_FIG Electric field script: www.github.com/bunzela/FieldTools

biochemistry↗

bioRxiv2023

Cooperative conformational transitions and the temperature dependence of enzyme catalysis

Many enzymes display non-Arrhenius behaviour with curved Arrhenius plots in the absence of denaturation. There has been significant debate about the origin of this behaviour and recently the role of the activation heat capacity [Formula] has been widely discussed. If enzyme-catalysed reactions occur with appreciable negative values of [Formula] (arising from narrowing of the conformational space along the reaction coordinate), then curved Arrhenius plots are a consequence. To investigate these phenomena in detail, we have collected high precision temperature-rate data over a wide temperature interval for a model glycosidase enzyme MalL, and a series of mutants that change the temperature-dependence of the enzyme-catalysed rate. We use these data to test a range of models including macromolecular rate theory (MMRT) and an equilibrium model. In addition, we have performed extensive molecular dynamics (MD) simulations to characterise the conformational landscape traversed by MalL in the enzyme-substrate complex and an enzyme-transition state complex. We have crystallised the enzyme in a transition state-like conformation in the absence of a ligand and determined an X-ray crystal structure at very high resolution (1.10 [A]). We show (using simulation) that this enzyme-transition state conformation has a more restricted conformational landscape than the wildtype enzyme. We coin the term "transition state-like conformation (TLC)" to apply to this state of the enzyme. Together, these results imply a cooperative conformational transition between an enzyme-substrate conformation (ES) and a transition-state-like conformation (TLC) that precedes the chemical step. We present a two-state model as an extension of MMRT (MMRT-2S) that describes the data along with a convenient approximation with linear temperature dependence of the activation heat capacity (MMRT-1L) that can be used where fewer data points are available. Our model rationalises disparate behaviour seen for MalL and a thermophilic alcohol dehydrogenase and is consistent with a raft of data for other enzymes. Our model can be used to characterise the conformational changes required for enzyme catalysis and provides insights into the role of cooperative conformational changes in transition state stabilisation that are accompanied by changes in heat capacity for the system along the reaction coordinate. TLCs are likely to be of wide importance in understanding the temperature dependence of enzyme activity, and other aspects of enzyme catalysis.

biochemistry↗

bioRxiv2023

Epistasis Arises from Shifting the Rate-Limiting Step during Enzyme Evolution

The molecular mechanisms by which epistasis boosts enzyme activity remain elusive, undermining our ability to predict the evolution of pathogens and engineer novel biocatalysts. Here, we reveal how directed evolution of a {beta}-lactamase yielded highly epistatic activity enhancements. Evolution selected four mutations that increase antibiotic resistance 40-fold, despite their marginal individual effects ([≤] 2-fold). Synergistic improvements coincided with the introduction of super-stochiometric burst kinetics, indicating that epistasis is rooted in the enzymes conformational dynamics. Kinetic, structural, and dynamical analyses reveal that epistasis was driven by distinct effects of each mutation on the catalytic cycle. The first mutation acquired during evolution increases protein flexibility and accelerates substrate binding, which is rate-limiting in the wild-type enzyme. The ensuing mutations predominantly boosted the chemical steps by fine-tuning substrate interactions. Our work identifies an overlooked cause for epistasis: changing the rate-limiting step can result in substantial positive synergy boosting enzyme activity.

evolutionary biology↗

bioRxiv2023

Antibodies generated in vitro and in vivo elucidate design of a thermostable ADDomer COVID-19 nasal nanoparticle vaccine

COVID-19 continues to damage populations, communities and economies worldwide. Vaccines have reduced COVID-19-related hospitalisations and deaths, primarily in developed countries. Persisting infection rates, and highly transmissible SARS-CoV-2 Variants of Concern (VOCs) causing repeat and breakthrough infections, underscore the ongoing need for new treatments to achieve a global solution. Based on ADDomer, a self-assembling protein nanoparticle scaffold, we created ADDoCoV, a thermostable COVID-19 candidate vaccine displaying multiple copies of a SARS-CoV-2 receptor binding motif (RBM)-derived epitope. In vitro generated neutralising nanobodies combined with molecular dynamics (MD) simulations and electron cryo-microscopy (cryo-EM) established authenticity and accessibility of the epitopes displayed. A Gigabody comprising multimerized nanobodies prevented SARS-CoV-2 virion attachment with picomolar EC50. Antibodies generated by immunising mice cross-reacted with VOCs including Delta and Omicron. Our study elucidates nasal administration of ADDomer-based nanoparticles for active and passive immunisation against SARS-CoV-2 and provides a blueprint for designing nanoparticle reagents to combat respiratory viral infections.

biochemistry↗

bioRxiv2022

Photovoltaic enzymes by design and evolution

The global energy crisis challenges us to develop more efficient strategies for the sustainable production of energy. Given the excellent efficiency of the natural photosynthetic apparatus, biohybrid photovoltaic devices present an attractive solution for solar energy conversion. However, their composition, stability, and complexity can limit their inclusion into photovoltaic devices. Here, we combined computational design and directed evolution to overcome these limitations and create tailor-made photoenzymes. Photo-biocatalysts were designed by introducing photosensitizer binding sites into heme-containing helical bundle proteins. The designed binding sites were specific for the target photosensitizer and readily transplanted into other helical bundles. The best design was highly evolvable and reached nanomolar ligand affinity after mutagenesis and screening. The evolved enzyme generated 2.6 times higher photocurrents than the photosensitizer alone, primarily driven by increased photostability. Evolvability is a unique advantage of our protein-based approach over abiological photovoltaic and will be critical to developing efficient biohybrid systems. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/521207v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@2b6031org.highwire.dtl.DTLVardef@1b83f69org.highwire.dtl.DTLVardef@14b39a4org.highwire.dtl.DTLVardef@1b569bb_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

Dynamical nonequilibrium molecular dynamics simulations identify allosteric sites and positions associated with drug resistance in the SARS-CoV-2 main protease

The SARS-CoV-2 main protease (Mpro) plays an essential role in the coronavirus lifecycle by catalysing hydrolysis of the viral polyproteins at specific sites. Mpro is the target of drugs, such as nirmatrelvir, though resistant mutants have emerged that threaten drug efficacy. Despite its importance, questions remain on the mechanism of how Mpro binds its substrates. Here, we apply dynamical nonequilibrium molecular dynamics (D-NEMD) simulations to evaluate structural and dynamical responses of Mpro to the presence and absence of a substrate. The results highlight communication between the Mpro dimer subunits and identify networks, including some far from the active site, that link the active site with a known allosteric inhibition site, or which are associated with nirmatrelvir resistance. They imply that some mutations enable resistance by altering the allosteric behaviour of Mpro. More generally, the results show the utility of the D-NEMD technique for identifying functionally relevant allosteric sites and networks including those relevant to resistance.

biochemistry↗

bioRxiv2022

Tautomer-specific deacylation and {Omega}-loop flexibility explain carbapenem-hydrolyzing, broad-spectrum activity of the KPC-2 β-lactamase

KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-{beta}-lactamase (SBL) responsible for extensive {beta}-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate {beta}-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent {beta}-lactams, evade activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently catalyze deacylation of carbapenem-derived acyl-enzymes. We present high-resolution (1.25-1.4 [A]) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin) and carbapenems (imipenem, meropenem and ertapenem), obtained utilizing an isosteric deacylation-deficient mutant (E166Q). Mobility of the {Omega}-loop (residues 165-170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different {beta}-lactams. Carbapenem-derived acyl-enzyme structures reveal predominance of the {Delta}1-(2R) imine tautomer, except for the imipenem acyl-enzyme, which is present in dual occupancy in both {Delta}1-(2R) and (2S) configurations. Quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations of deacylation of the KPC-2:meropenem acyl-enzyme, using an adaptive string method (ASM), show that the {Delta}1-(2R) isomer has a 7 kcal/mol higher barrier for the (rate-determining) formation of the tetrahedral deacylation intermediate than the {Delta}2 tautomer. The simulations identify tautomer-specific differences in hydrogen bonding networks involving the carbapenem C-3 carboxylate and the deacylating water, that, together with stabilization by protonated N-4 of accumulating negative charge during oxyanion formation, accelerate deacylation of the {Delta}2-enamine acyl-enzyme compared to the {Delta}1-imine. Taken together, our data show how the flexible {Omega}-loop helps confer broad spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the {Delta}2-enamine acyl-enzyme tautomer. Differentiation of the barriers associated with deacylation of these subtly different {beta}-lactam isomers further identifies ASM as a sensitive method for calculation of reaction energetics that can accurately model turnover and, potentially, predict the impact of substrate modifications or point mutations upon activity.

biochemistry↗

bioRxiv2022

Molecular dynamics of spike variants in the locked conformation: RBD interfaces, fatty acid binding and furin cleavage sites.

Since December 2019 the SARS-CoV-2 virus has infected billions of people around the world and caused millions of deaths. The ability for this RNA virus to mutate has produced variants that have been responsible for waves of infections across the globe. The spike protein on the surface of the SARS-CoV-2 virion is responsible for cell entry in the infection process. Here we have studied the spike proteins from the Original, Alpha (B.1.1.7), Delta (B1.617.2), Delta-plus (B1.617.2-AY1), Omicron BA.1 and Omicron BA.2 variants. Using models built from cryo-EM structures with linoleate bound (6BZ5.pdb) and the N-terminal domain from 7JJI.pdb, each is built from the first residue, with missing loops modelled and 45 disulphides per trimer. Each spike variant was modified from the same Original model framework to maximise comparability. Three replicate, 200 ns atomistic molecular dynamics simulations were performed for each case. (These data also provide the basis for further, non-equilibrium molecular dynamics simulations, published elsewhere.) The analysis of our equilibrium molecular dynamics reveals that sequence variation at the closed receptor binding domain interface particularly for Omicron BA.2 has implications for the avidity of the locked conformation, with potential effects on Omicron BA.1 and Delta-plus. Linoleate binding has a mildly stabilizing effect on furin cleavage site motions in the Original and Alpha variants, but has no effect in Delta, Delta-plus and slightly increases motions at this site for Omicron BA.1, but not BA.2, under these simulation conditions.

biochemistry↗

bioRxiv2022

SARS-CoV-2 spike variants differ in their allosteric response to linoleic acid

The SARS-CoV-2 spike protein contains a fatty acid binding site, also found in some other coronaviruses (e.g. SARS-CoV), which binds linoleic acid and is functionally important. When occupied by linoleic acid, it reduces infectivity, by locking the spike in a less infectious conformation. Here, we use dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations to compare the response of spike variants to linoleic acid removal. These simulations show that the fatty acid site is coupled to functional regions of the protein, some of them far from the site (e.g. in the receptor-binding motif, N-terminal domain, the furin cleavage site located in position 679-685 and the fusion peptide-surrounding regions) and identify the allosteric networks involved in these connections. Comparison of the response of the original ( Wuhan) spike with four variants: Alpha, Delta, Delta plus and Omicron BA.1 show that the variants differ significantly in their response to linoleic acid removal. The allosteric connections to the fatty acid site on Alpha are generally similar to the original protein, except for the receptor-binding motif and S71-R78 region which show a weaker link to the FA site. In contrast, Omicron is the most affected variant exhibiting significant differences in the receptor-binding motif, N-terminal domain, V622-L629 and the furin cleavage site. These differences in allosteric modulation may be of functional relevance, e.g. in differences in transmissibility and virulence. Experimental comparison of the effects of linoleic acid on different variants is warranted.

biochemistry↗

bioRxiv2022

Structural and temporal basis for agonism in the α4β2 nicotinic acetylcholine receptor

Despite decades of study, the structural mechanisms underpinning agonist efficacy in pentameric ligand-gated ion channels remain poorly understood. Here, a combination of extensive equilibrium and dynamical-nonequilibrium molecular dynamics simulations was used to obtain a detailed description of the structural and dynamic changes induced within the human 4{beta}2 nicotinic acetylcholine receptor by a full and a partial agonist, namely acetylcholine and nicotine, and map how these rearrangements propagate within this receptor. These simulations reveal how the agonists modulate the patterns associated with intra and inter-domain communication and the evolution of the agonist-specific structural rearrangements. For the first time, we show that full and partial agonists, although generally using similar routes for through-receptor signal transmission, induce different amplitudes of conformational rearrangements in key functional motifs, thus impacting the rates of signal propagation within the protein. The largest agonist-induced conformational differences are located in the Cys loop, loops C and 1-{beta}1 in the 4 subunit, loops F and {beta}1-{beta}2 in the {beta}2 subunit and in the extracellular selectivity filter.

biochemistry↗

bioRxiv2021

Ligand-induced unfolding mechanism of an RNA G-quadruplex

The cationic porphyrin, TMPyP4, is a well-established DNA G-quadruplex (G4) binding ligand that can stabilize different topologies via multiple binding modes. However, TMPyP4 has completely opposite destabilizing and unwinding effect on RNA G4 structures. The structural mechanisms that mediate RNA G4 unfolding remains unknown. Here, we report on the TMPyP4-induced RNA G4 unfolding mechanism studied by well-tempered metadynamics (WT-MetaD) with supporting biophysical experiments. The simulations predict a two-state mechanism of TMPyP4 interaction via a groove-bound and a top-face bound conformation. The dynamics of TMPyP4 stacking on the top tetrad disrupts Hoogsteen H-bonds between guanine bases resulting in the consecutive TMPyP4 intercalation from top-to-bottom G-tetrads. The results reveal a striking correlation between computational and experimental approaches and validate WT-MetaD simulations as a powerful tool for studying RNA G4-ligand interactions.

biophysics↗

bioRxiv2021

Discovery of SARS-CoV-2 Mpro Peptide Inhibitors from Modelling Substrate and Ligand Binding

The main protease (Mpro) of SARS-CoV-2 is central to its viral lifecycle and is a promising drug target, but little is known concerning structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of classical molecular mechanics and quantum mechanical techniques, including automated docking, molecular dynamics (MD) simulations, linear-scaling DFT, QM/MM, and interactive MD in virtual reality, to investigate the molecular features underlying recognition of the natural Mpro substrates. Analyses of the subsite interactions of modelled 11-residue cleavage site peptides, ligands from high-throughput crystallography, and designed covalently binding inhibitors were performed. Modelling studies reveal remarkable conservation of hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular at the P2/S2 sites. The binding modes of the natural substrates, together with extensive interaction analyses of inhibitor and fragment binding to Mpro, reveal new opportunities for inhibition. Building on our initial Mpro-substrate models, computational mutagenesis scanning was employed to design peptides with improved affinity and which inhibit Mpro competitively. The combined results provide new insight useful for the development of Mpro inhibitors.

bioinformatics↗

bioRxiv2021

The fatty acid site is coupled to functional motifs in the SARS-CoV-2 spike protein and modulates spike allosteric behaviour

The SARS-CoV-2 spike protein is the first contact point between the SARS-CoV-2 virus and host cells and mediates membrane fusion. Recently, a fatty acid binding site was identified in the spike (Toelzer et al. Science 2020). The presence of linoleic acid at this site modulates binding of the spike to the human ACE2 receptor, stabilizing a locked conformation of the protein. Here, dynamical-nonequilibrium molecular dynamics simulations reveal that this fatty acid site is coupled to functionally relevant regions of the spike, some of them far from the fatty acid binding pocket. Removal of a ligand from the fatty acid binding site significantly affects the dynamics of distant, functionally important regions of the spike, including the receptor-binding motif, furin cleavage site and fusion-peptide-adjacent regions. The results also show significant differences in behaviour between clinical variants of the spike: e.g. the D614G mutation shows a significantly different conformational response for some structural motifs relevant for binding and fusion. The simulations identify structural networks through which changes at the fatty acid binding site are transmitted within the protein. These communication networks significantly involve positions that are prone to mutation, indicating that observed genetic variation in the spike may alter its response to linoleate binding and associated allosteric communication.

biochemistry↗