Biology subjects

Warwicker, J.

Publications and source records attributed to Warwicker, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Prediction of pH-sensing histidine residues in human cells

Changes in pH play an important role in human physiology and cell behaviours, evidenced by studies of tissues and gene expression, and the biophysics of individual systems. Bridging between these scales to establish pH-sensing networks has only been partially explored. Here, amino acid conservation and structural context, and benchmarking with known pH-sensors, were used to predict histidine residues within the human proteome that could modulate pH-dependence. Proteins containing these histidines are enriched in many categories, most notably nucleic acid-binding and transcriptional regulation. DNA phosphate backbone interactions are proposed to play a major role in modulating some pH-sensors. Transcription factors with potential pH-sensors and the largest number of documented target genes are highlighted, including those involved in circadian and developmental processes. Certain CDK-cyclin pairs also feature, along with components of SWI/SNF and cohesin complexes. Focussing on histidines, including those that are solvent accessible in AlphaFold protomer models, this study generates benchmarked predictions for possible pH-sensors in the human proteome.

bioinformatics↗

bioRxiv2026

Charged amino acid propensities and solubility: lysine is elevated at the termini of helices in E. coli proteins.

An emerging result in the relationship between amino acid sequence and protein solubility is a preference, on average, for lysine over arginine in more soluble proteins. The termini of helices are known to be prone to partial unfolding, often employing N- and C-cap amino acids to maintain stability. Hypothesising that lysine/arginine differences in relation to solubility may be evident at helical termini, their propensities and predicted charge interactions in helices were examined. There is enrichment of lysine over arginine at helical termini in AlphaFold models of Escherichia coli proteins, more so (on average) for the most soluble proteins. Similar effects are seen for the sum of charged amino acids at helical termini. Regions other than helical termini also show correlation of lysine composition, and overall charged amino acid composition, with solubility. These results suggest that protein design protocols could improve solubility through targeting lysine enrichment in regions such as helical termini, in addition to the more conventional consideration of helix capping interactions.

bioinformatics↗

bioRxiv2025

Outward-facing P-glycoprotein bound to drug displays nucleotide-dependent drug egress mechanism

ABCB1/P-glycoprotein requires coordination between ligand movement in the trans-membrane cavity and ATP binding and hydrolysis at the nucleotide binding domains, yet the sequence of these events remains incompletely resolved. Here, atomistic simulations with paclitaxel and verapamil across outward facing P-glycoprotein ensembles in various nucleotide states were integrated with analysis of transmembrane domain contacts, geometric solvent/ligand pathways, extracellular gating distances, and nucleotide coordi-nation characterization. Collectively, our simulations delineate a mechanistic framework for P-glycoprotein in which ATP binding organizes an outward-facing ensemble and sequential hydrolysis at the two nucleotide-binding sites drives substrate progression toward egress. Asymmetry in the nucleotide state is correlated to distinct motions within the transmembrane domains, which suggests a possible transient extracellular gating mechanism that enables staged release and limits re-entry of the ligand. The resulting model connects nucleotide chemistry to transmembrane domain motions that regulate gate dynamics and may guide experiments and development of modulators for ABC exporter activity in physiological and pharmacological contexts.

molecular biology↗

bioRxiv2025

Drug-bound P-glycoprotein reveals nucleotide-dependent ligand pockets and ingress channels

P-glycoprotein, an ATP-binding cassette exporter, coordinates drug recognition in a central cavity with nucleotide handling at nucleotide binding domains, yet the sequence and coupling of these events remain incompletely defined. Here, we report an integrated computational analysis of inward facing P-glycoprotein ensembles across varying drug locations, nucleotide states, and starting conformations. The sampled inward-facing ensembles of P-glycoprotein display continuous ligand cavity, reshaped by nucleotide occupancy, which enables polyspecific ligand recognition while transmitting allosteric signals to the nucleotide binding domains through intracellular coupling loops. Asymmetry between the two nucleotide binding sites follows from this communication, which alters the nucleotide coordination to potentially affect the catalytic progression. Our simulations reveal dynamic cavity access routes from cytosol and inner membrane, together with transient secondary structure within the interdomain flexible linker. Overall, our study gives insights into how ligand redistribution, asymmetric nucleotide coordination, and linker flexibility may correlate to form a unified view of early transport steps, yielding hypotheses for mutational and kinetic evaluation and informing strategies for small-molecule modulation of transporter function in cellular contexts where multidrug transport affects therapeutic response.

molecular biology↗

bioRxiv2025

Computational investigation suggests that the cell adhesion sub-proteome is enriched for sites of pH-dependence and charge burial, whilst some key intracellular pathways may be shielded from pH fluctuations.

Prediction of protein pH-dependence is generally made for individual proteins or pathways, and is also increasingly being used to aid protein functional design. Combining high-throughput pKa prediction methods with AlphaFold models allows pH-dependence to be studied across a proteome. Here, two methods and a variety of features for detecting pH-dependence and charge burial that is physiologically relevant, have been applied to human proteins. Predictions are effective for a small benchmark subset of well-characterised proteins and, more broadly, identify an overlap between features associated with pH-dependence and enzymes and transporters. The most informative filters are those describing relatively buried ionisable groups, with pKas close to neutral pH and/or involvement in highly-coupled charge networks. The question is addressed of which human proteins not annotated as enzymes or transporters, are predicted to have these features of predicted pH-dependence and/or charge networking. A striking feature from gene ontology analysis of those proteins is a predicted enrichment at the cell periphery, in particular an association with cell adhesion, including protein families not currently known to exhibit pH-dependence. Gene ontology classifications that are depleted for proteins with buried charge networks and/or predicted functional pH-dependence, include some associated with ribosomal and nuclear structure. This overall result suggests a possible general resilience of some key processes to pH fluctuations, whilst not precluding specific instances where signalling pathways have evolved responses to pH changes. A drawback of the study is restriction to protomer models, thus omitting groups that mediate pH-dependence through burial at an interface. However predictions are already notable, with their details provided for experimental design.

bioinformatics↗

bioRxiv2025

Nucleotide Asymmetry and Flexible Linker Dynamics Modulate Drug Efflux Cycle of P-glycoprotein, a Computational Study

Despite advancements in oncology, multidrug resistance (MDR) mediated by P-glycoprotein (P-gp/ABCB1) remains a major barrier to chemotherapy. P-gp is an ATP-binding cassette transporter that undergoes nucleotide-driven structural rearrangements to efflux chemotherapeutics, but the mechanistic details of the substrate transport remain poorly resolved. Here, we performed high-throughput molecular dynamics to simulate P-gp in a lipid bilayer (totaling [~]110 {micro}s) to dissect nucleotide-dependent conformational changes across the transport cycle. Our adaptive sampling strategy reveals asymmetric nucleotide coordination at nucleotide-binding sites (NBS), which correlates with transmembrane domain (TMD) restructuring for substrate efflux. The experimentally unresolved flexible linker transiently forms up to five turn -helix that affects nucleotide binding domain (NBD) dimerization process. We identified conformation-dependent substrate/allocrite pathways including nucleotide-specific access routes, while TMD-linker interaction facilitates substrate access tunnel formation. Together, these pathways reveal that the concerted interplay of nucleotide occupancy, linker dynamics, and overall protein conformation governs the structural plasticity and broad substrate promiscuity of the substrate binding cavity in P-gp. By integrating these findings, this work bridges static structural data with dynamic functional insights to further our understanding of the P-gp substrate translocation cycle.

biochemistry↗

bioRxiv2024

Computational investigation of missense somatic mutations in cancer and potential links to pH-dependence and proteostasis

Metabolic changes during tumour development lead to acidification of the extracellular environment and a smaller increase of intracellular pH. Searches for somatic missense mutations that could reveal adaptation to altered pH have focussed on arginine to histidine changes, part of a general arginine depletion that originates from DNA mutational mechanisms. Analysis of mutations to histidine, potentially a simple route to the introduction of pH-sensing, shows no clear biophysical separation overall of subsets that are more and less frequently mutated in cancer genomes. Within the more frequently mutated subset, individual sites predicted to mediate pH-dependence upon mutation include NDST1 (a Golgi-resident heparan sulphate modifying enzyme), the HLA-C chain of MHCI complex, and the water channel AQP-7. Arginine depletion is a general feature that persists in the more frequently mutated subset, and is complemented by over-representation of mutations to lysine. Arginine to lysine balance is a known factor in determining protein solubility, with higher lysine content being more favourable. Proteins with greater change in arginine to lysine balance are enriched for cell periphery location, where proteostasis is likely to be challenged in tumour cells. Somatic missense mutations in a cancer genome number only in the 10s typically, although can be much higher. Whether the altered arginine to lysine balance is of sufficient scale to play a role in tumour development is unknown.

bioinformatics↗

bioRxiv2024

G6b-B antibody-based cis-acting platelet receptor inhibitors (CAPRIs) as a new family of anti-thrombotic therapeutics

Key PointsO_LIBispecific cis-acting platelet receptor inhibitors (CAPRIs) mediate hetero-clustering of the ITIM receptor G6B with ITAM receptors. C_LIO_LIG6B-GPVI and G6B-CD32A CAPRIs specifically inhibit collagen- and immune complex-induced thrombus formation, respectively. C_LI Platelets are highly reactive fragments of megakaryocytes that play a fundamental role in thrombosis and hemostasis. Predictably, all conventional anti-platelet therapies elicit bleeding, raising the question whether the thrombotic activity of platelets can be targeted separately. In this study, we describe a novel approach of inhibiting platelet activation through the use of bispecific single-chain variable fragments (bi-scFvs), termed cis-acting platelet receptor inhibitors (CAPRIs) that harness the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing co-inhibitory receptor G6b-B (G6B) to suppress immunoreceptor tyrosine-based (ITAM)-containing receptor-mediated platelet activation. CAPRI-mediated hetero-clustering of G6B with either the ITAM-containing GPVI-FcR {gamma}-chain complex or Fc{gamma}RIIA (CD32A) inhibited collagen- or immune complex-induced platelet aggregation. G6B-GPVI CAPRIs strongly and specifically inhibited thrombus formation on collagen under arterial shear, whereas G6B-CD32A CAPRI strongly and specifically inhibited thrombus formation to heparin-induced thrombocytopenia, vaccine-induced thrombotic thrombocytopenia and antiphospholipid syndrome complexes on Von Willebrand Factor-coated surfaces and photochemical-injured endothelial cells under arterial shear. Our findings provide proof-of-concept that CAPRIs are highly effective at inhibiting ITAM receptor-mediated platelet activation, laying the foundation for a novel family of anti-thrombotic therapeutics with potentially improved efficacy and fewer bleeding outcomes compared with current anti-platelet therapies.

cell biology↗

bioRxiv2022

Enrichment of charge-absent regions in phase separated proteins

Many studies focus on the relationship between protein charge and liquid-liquid phase separation (LLPS), generally finding that a large degree of charge neutralisation is involved for condensate formation. Here, sequences within human proteins that lack the charge-bearing residues Asp, Glu, Lys, and Arg (termed charge-absent) are analysed alongside annotation for involvement in LLPS. Scaffold proteins, central to condensate formation, on average possess longer charge-absent regions than those not key for LLPS. Charge-absent regions tend to have relatively high hydropathy scores. Overall, they are enriched in Ala, Gly, Pro, and Ser with more specific groupings evident when the subset is clustered by amino acid composition. For several proteins, segments with charge-absent regions have been identified as modulators of LLPS. It is hypothesised that for at least some of the charge-absent regions, a lack of charged group desolvation energy, together with a relatively hydrophobic sequence composition, may facilitate condensation through homomeric interactions. If this is the case, it should be relatively easy to modulate through incorporation of charge through engineering, potentially including pH-sensing.

bioinformatics↗

bioRxiv2022

Computational investigation of mechanisms for pH modulation of human chloride channels

Many transmembrane proteins are modulated by intracellular or extracellular pH. Investigation of pH-dependence generally proceeds by mutagenesis of a wide set of amino acids, guided by properties such as amino acid conservation and structure. This study shows how web-based calculation of pKas allows rapid and effective identification of amino acids of interest with respect to pH-dependence. Commencing with the calcium-activated chloride channel bestrophin 1, the carboxylate ligand structure around calcium sites relaxes in the absence of calcium, consistent with a measured lack of pH-dependence. By contrast, less relaxation in the absence of calcium in TMEM16A, and maintenance of elevated carboxylate sidechain pKas, is suggested to give rise to pH-dependent chloride channel activity. This hypothesis, modulation of calcium/proton coupling and pH-dependent activity through extent of structural relaxation, is shown to apply to the well-characterised cytosolic proteins calmodulin (pH-independent) and calbindin D9k (pH-dependent). Further application of destabilised ionisable charge sites, or electrostatic frustration, is made to other human chloride channels (that are not calcium-activated), ClC-2, GABAA, and GlyR. Experimentally-determined sites of pH modulation are readily identified. The structure-based tool is freely available, allowing users to focus mutagenesis studies, construct hypothetical proton pathways, and derive hypotheses such as the model for control of pH-dependent calcium activation through structural flexibility. Predicting altered pH-dependence for mutations in ion channel disorders can support experimentation and, ultimately, clinical intervention.

biophysics↗

bioRxiv2022

A pH-dependent cluster of charges in a conserved cryptic pocket on flaviviral envelopes

Flaviviruses are enveloped viruses which include numerous human pathogens of escalating global health concern that are predominantly transmitted by mosquitoes and ticks. Some, such as dengue virus, exhibit the phenomenon of antibody-dependent enhancement (ADE) of disease, making traditional vaccine-based routes of fighting viral infections problematic. The pH-dependent conformational change of the envelope (E) protein required for fusion between the viral and endosomal membranes is an attractive point of inhibition by antivirals as it also has the potential to diminish the effects of ADE. Here, we systematically examined six flaviviruses by employing large-scale molecular dynamics (MD) simulations of a [~]400,000 atom raft system that represents a substantial portion of the curved flaviviral envelope. We utilised a benzene-mapping approach over a total of 14 s of sampling time, leading to discovery of shared hotspots and elucidation of the dynamic behaviour of conserved cryptic sites. A cryptic pocket previously shown to bind a detergent molecule exhibited significant strain-specific characteristics. An alternative conserved cryptic site located at the E protein domain interfaces showed a more consistent dynamic behaviour across flaviviruses and contains a buried, conserved cluster of ionisable residues that includes His144, previously implicated in the pH-dependent conformational switch required for fusion. The dynamics of the cluster were further explored in extensive constant-pH simulations and revealed cluster and domain-interface disruption under low pH conditions. Based on this, we propose a cluster-dependent mechanism that addresses inconsistencies in the histidine-switch hypothesis and highlights the role of cluster protonation in orchestrating the domain dissociation pivotal for the formation of the fusogenic trimer. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/499710v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@2a90feorg.highwire.dtl.DTLVardef@9535a1org.highwire.dtl.DTLVardef@1f41ae0org.highwire.dtl.DTLVardef@1106e41_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Relationship between monomer packing and receptor binding domain pocket status in the spike trimer of SARS-CoV-2 variants

Existence of a SARS-CoV-2 spike protein trimer form with closer packing between monomers when receptor binding domains (RBDs) are all down, locked as opposed to closed, has been associated with linoleic acid (LA) binding at neutral pH, or can occur at acidic pH in the absence of LA binding. The relationship between degree of closure of the LA binding pocket of the RBD, and monomer burial in the trimer, is examined for a range of spike protein structures, including those with D614G mutation, and that of the Delta variant (which also carries D614G). Some spike protein structures with this aspartic acid mutation show monomer packing approaching that of the locked form (at neutral pH, without LA binding) for two segments, a third (around the RBD) remains less closely packed. Mutations in the RBD are a focus for the Omicron variant spike protein. Structure reports suggest that these mutations are involved in increased RBD-RBD interactions, and also that they could lead to a closing of the LA pocket, both of which could impact on pH-dependence. One potential outcome is that the extent of pH-dependent conformational transitions of the pre-fusion SARS-CoV-2 spike trimer are reduced in the Omicron variant.

bioinformatics↗

bioRxiv2021

Predicted pH-dependent stability of SARS-CoV-2 spike protein trimer from interfacial acidic groups

Transition between receptor binding domain (RBD) up and down forms of the SARS-CoV-2 spike protein trimer is coupled to receptor binding and is one route by which variants can alter viral properties. It is becoming apparent that key roles in the transition are played by pH and a more compact closed form, termed locked. Calculations of pH-dependence are made for a large set of spike trimers, including locked form trimer structures that have recently become available. Several acidic sidechains become sufficiently buried in the locked form to give a predicted pH-dependence in the mild acidic range, with stabilisation of the locked form as pH reduces from 7.5 to 5, consistent with emerging characterisation by cryo-electron microscopy. The calculated pH effects in pre-fusion spike trimers are modulated mainly by aspartic acid residues, rather than the more familiar histidine role at mild acidic pH. These acidic sidechains are generally surface located and weakly interacting when not in a locked conformation. In this model, their replacement (perhaps with asparagine) would remove the pH-dependent destabilisation of locked spike trimer conformations, and increase their recovery at neutral pH. This would provide an alternative or supplement to the insertion of disulphide linkages for stabilising spike protein trimers, with potential relevance for vaccine design.

bioinformatics↗

bioRxiv2021

Uncovering cryptic pockets in the SARS-CoV-2 spike glycoprotein

The recent global COVID-19 pandemic has prompted a rapid response in terms of vaccine and drug development targeting the viral pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this work, we modelled a complete membrane-embedded SARS-CoV-2 spike (S) protein, the primary target of vaccine and therapeutics development, based on available structural data and known glycan content. We then used molecular dynamics (MD) simulations to study the system in the presence of benzene probes designed to enhance discovery of cryptic, potentially druggable pockets on the S protein surface. We uncovered a novel cryptic pocket with promising druggable properties located underneath the 617-628 loop, which was shown to be involved in the formation of S protein multimers on the viral surface. A marked multi-conformational behaviour of this loop in simulations was validated using hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments, supportive of opening and closing dynamics. Interestingly, the pocket is also the site of the D614G mutation, known to be important for SARS-CoV-2 fitness, and within close proximity to mutations in the novel SARS-CoV-2 strains B.1.1.7 and B.1.1.28, both of which are associated with increased transmissibility and severity of infection. The pocket was present in systems emulating both immature and mature glycosylation states, suggesting its druggability may not be dependent upon the stage of virus maturation. Overall, the predominantly hydrophobic nature of the cryptic pocket, its well conserved surface, and proximity to regions of functional relevance in viral assembly and fitness are all promising indicators of its potential for therapeutic targeting. Our method also successfully recapitulated hydrophobic pockets in the receptor binding domain and N-terminal domain associated with detergent or lipid binding in prior cryo-electron microscopy (cryo-EM) studies. Collectively, this work highlights the utility of the benzene mapping approach in uncovering potential druggable sites on the surface of SARS-CoV-2 targets.

biophysics↗

bioRxiv2021

PhosIDP: a web tool to visualize the location of phosphorylation sites in disordered regions

Charge is a key determinant of intrinsically disordered protein (IDP) and intrinsically disordered region (IDR) properties. IDPs and IDRs are enriched in sites of phosphorylation, which alters charge. Visualizing the degree to which phosphorylation modulates the charge profile of a sequence would assist in the functional interpretation of IDPs and IDRs. PhosIDP is a web tool that shows variation of charge and fold propensity upon phosphorylation. In combination with the displayed location of protein domains, the information provided by the web tool can lead to functional inferences for the consequences of phosphorylation. IDRs are components of many proteins that form biological condensates. It is shown that IDR charge, and its modulation by phosphorylation, is more tightly controlled for proteins that are essential for condensate formation than for those present in condensates but inessential.

bioinformatics↗

bioRxiv2020

A model for pH coupling of the SARS-CoV-2 spike protein open/closed equilibrium.

SARS-CoV-2, causative agent of the COVID-19 pandemic, is thought to release its RNA genome at either the cell surface or within endosomes, the balance being dependent on spike protein stability, and the complement of receptors, co-receptors and proteases. To investigate possible mediators of pH-dependence, pKa calculations have been made on a set of structures for spike protein ectodomain and fragments from SARS-CoV-2 and other coronaviruses. Dominating a heat map of the aggregated predictions, 3 histidine residues in S2 are consistently predicted as destabilising in pre-fusion (all 3) and post-fusion (2 of 3) structures. Other predicted features include the more moderate energetics of surface salt-bridge interactions, and sidechain-mainchain interactions. Two aspartic acid residues in partially buried salt-bridges (D290 - R273 and R355 - D398) have pKas that are calculated to be elevated and destabilising in more open forms of the spike trimer. These aspartic acids are most stabilised in a tightly closed conformation that has been observed when linoleic acid is bound, and which also affects the interactions of D614. The D614G mutation is known to modulate the balance of closed to open trimer. It is suggested that D398 in particular contributes to a pH-dependence of the open/closed equilibrium, potentially coupled to the effects of linoleic acid binding and D614G mutation, and possibly also A570D mutation. These observations are discussed in the context of SARS-CoV-2 infection, mutagenesis studies, and other human coronaviruses.

bioinformatics↗

bioRxiv2020

protein-sol pKa: prediction of electrostatic frustration, with application to coronaviruses

Evolution couples differences in ambient pH to biological function through protonatable groups, in particular those that switch from buried to exposed and alter protonation state in doing so. We present a tool focusing on structure-based discovery and display of these groups. Since prediction of buried group pKas is computationally intensive, solvent accessibility of ionisable groups is displayed, from which the user can iteratively select pKa calculation centers. Results are color-coded, with emphasis on buried groups. Utility is demonstrated with coronaviruses, which exhibit variable dependence on the acidic pH of the endocytotic pathway. After benchmarking with variants of murine hepatitis virus, a pair of conserved histidine residues are identified that are predicted to be electrostatically frustrated at acidic pH in a common structural core of pre- and post-fusion coronavirus spike proteins. We suggest that an intermediate expanded conformation at endosomal pH could relax the frustration, allowing histidine protonation, and facilitating conformational conversion. This tool is available at http://www.protein-sol.manchester.ac.uk/pka/.

bioinformatics↗

bioRxiv2020

A benzene-mapping approach for uncovering cryptic pockets in membrane-bound proteins

Molecular dynamics (MD) simulations in combination with small organic probes present in the solvent have previously been used as a method to reveal cryptic pockets that may not have been identified in experimental structures. We report such a method implemented within the CHARMM forcefield to effectively explore cryptic pockets on the surfaces of membrane-embedded proteins using benzene as a probe molecule. This relies on modified non-bonded parameters in addition to repulsive potentials between membrane lipids and benzene molecules. The method was tested on part of the outer shell of the dengue virus (DENV), for which research into a safe and effective neutralizing antibody or drug molecule is still ongoing. In particular, the envelope (E) protein, associated with the membrane (M) protein, is a lipid membrane-embedded complex which forms a dimer in the mature viral envelope. Solvent mapping was performed for the full, membrane-embedded EM protein complex and compared with similar calculations performed for the isolated, soluble E protein ectodomain dimer in solvent. Ectodomain-only simulations with benzene exhibited unfolding effects not observed in the more physiologically relevant membrane-associated systems. A cryptic pocket which has been experimentally shown to bind n-octyl-{beta}-D-glucoside detergent was consistently revealed in all benzene-containing simulations. The addition of benzene also enhanced the flexibility and hydrophobic exposure of cryptic pockets at a key, functional interface in the E protein, and revealed a novel, potentially druggable pocket that may be targeted to prevent conformational changes associated with viral entry into the cell. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/025163v1_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@abe537org.highwire.dtl.DTLVardef@155a741org.highwire.dtl.DTLVardef@57bff6org.highwire.dtl.DTLVardef@fbee3a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗