Biology subjects

Lovell, S.

Publications and source records attributed to Lovell, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Expanding Macrocyclic Topology through Cysteine-to-N-Terminal Cyclisation Enables Covalent Peptide Inhibitor Discovery

Macrocyclic peptides are an attractive therapeutic modality capable of engaging challenging protein targets while retaining many favourable drug-like properties. Their high-affinity binding also provides an ideal framework for proximity-driven covalent inhibition through incorporation of latent electrophiles. Phage display enables the high-throughput screening of billion-member macrocyclic peptide libraries; however, existing libraries rely predominantly on cysteine-mediated cyclisation, restricting the range of macrocyclic topologies available for ligand discovery. Here, we report a mild and efficient cyclisation strategy based on a bromomethyl picolinaldehyde (BMP) linker that reacts with a cysteine side chain and the peptide N-terminus to generate a previously unexplored macrocyclic topology incorporating neighbouring pyridine and imidazolidinone rings. The chemistry is compatible with phage display and enabled screening of BMP-cyclised peptide libraries against plasma kallikrein, yielding a potent macrocyclic inhibitor. The BMP-cyclised peptide displayed substantially greater potency than analogous peptides cyclised through either a disulfide bond or the widely used linker 1,4-bis(bromomethyl)benzene (DBMB). Furthermore, comparison with an equivalent DBMB-cyclised library demonstrated that BMP-mediated cyclisation enabled access to binding motifs not identified by conventional cysteine-to-cysteine cyclisation. Finally, positional sulfur(VI) fluoride exchange (SuFEx) electrophile scanning converted the BMP-derived hit into a selective covalent macrocyclic activity-based probe capable of labelling plasma kallikrein in human plasma. Together, these findings establish BMP-mediated cyclisation as a versatile strategy for expanding the topological diversity of phage-displayed macrocycles and accelerating the discovery of both reversible and covalent macrocyclic peptide ligands.

biochemistry↗

bioRxiv2026

Beyond the Catalytic Serine: Selective Protease Engagement with Covalent Macrocyclic Activity-based Probes

Activity-based probes (ABPs) are widely used to profile serine protease activity - enzymes central to diverse physiological and pathological processes - but most rely on covalent modification of the conserved catalytic serine residue, often resulting in poor selectivity across related proteases. Here, we introduce covalent macrocyclic activity-based probes (cmABPs) that selectively target non-catalytic residues within serine protease active sites. By combining phage display with systematic electrophile scanning, we identify macrocyclic scaffolds that position sulfur(VI) fluoride (SuFEx) electrophiles to covalently engage alternative nucleophiles such as lysine and tyrosine. Applied to plasma kallikrein, this approach yielded a macrocyclic scaffold that was converted into covalent probes via fluorosulfate scanning. Remarkably, small changes in electrophile structure produced large, tuneable differences in covalent kinetics, with benzenesulfonyl fluoride derivative 23 achieving rapid and complete protein modification. Biochemical and mass spectrometry analyses confirmed selective modification of an active-site lysine by 23, along with robust performance in complex biological samples. Extension to urokinase plasminogen activator further demonstrates the generality of this strategy. More broadly, this work establishes electrophile scanning within macrocyclic scaffolds as a general approach for tuning covalent reactivity and provides a blueprint for designing selective probes that move beyond catalytic-residue targeting.

biochemistry↗

bioRxiv2026

A truncated soil phage catechol 1,2-dioxygenase illustrates how viruses preserve and disseminate auxiliary catalytic functions in the soil microbiome

Bacteriophages can rewire host chemistry via auxiliary viral genes (AVGs). Using metagenomic and metatranscriptomic data from the native soil microbiome, we identified transcriptionally active AVGs, including a viral catechol 1,2-dioxygenase (V-C12DO). V-C12DO shares [~]40% sequence identity with its nearest bacterial homologs and lacks the helical dimerization domain. Despite truncation, V-C12DO retains more than [~]25% of the global consensus residues compared to C12DOs across domains of life, including the two tyrosines and two histidines that coordinate the non-heme Fe(III) active site. A 1.7 [A] crystal structure also showed the conservation of the canonical {beta}-sandwich scaffold for the iron. We next confirmed that V-C12DO cleaves catechol and remains highly active across a broad range of temperatures (30-60 {degrees}C), pH (5.5-9), and salinity (up to 2 M), exceeding those of known bacterial CD12Os. This work shows that truncated phage enzymes preserve the core catalytic chemistry and potentially further expand host metabolic versatility across dynamic environmental conditions.

microbiology↗

bioRxiv2025

An orally available PfPKG inhibitor blocks sporozoite infection of the liver

Malaria remains a global health threat exacerbated by emerging resistance to antimalarial therapies and insecticides, climate-driven outbreaks, and limited chemoprotective options. Here, we report the characterization of RUPB-61, the first orally bioavailable inhibitor of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG). RUPB-61 prevents infection by P. falciparum and P. cynomolgi sporozoites, including the formation of hypnozoites by the latter. A single oral dose blocks liver infection by P. berghei sporozoites in vivo, demonstrating efficacy consistent with further development as a once-weekly prophylaxis based on pharmacokinetic modeling. The compound retains activity against field isolates resistant to chloroquine, mefloquine, cycloguanil, sulfadoxine and pyrimethamine, suggesting low likelihood of cross-resistance to existing antimalarials. Structural studies and free energy-based modeling guided-compound design prospectively validated the predictive accuracy of an in silico model of PfPKG interactions with this chemotype. While selectivity profiling identified off-target activity against human kinases, structural modeling provides a clear path for optimization. These results establish PfPKG inhibitors as promising candidates for chemoprevention and support further preclinical development of the RUPB-61 chemotype. Author SummaryMalaria remains a serious global health threat, made worse by growing resistance to existing drugs and the spread of insecticide-resistant mosquitoes. A critical gap in our prevention toolkit is the lack of safe, long-acting oral drugs that stop malaria parasites from infecting the liver - the first and obligatory step of every new infection. Here, we report the characterization of RUPB-61, a compound that blocks an enzyme that the malaria parasite needs to invade and survive in the liver. We show that a single oral dose completely prevents liver infection in mice, and that the compound remains active against parasites that are resistant to several currently used antimalarial drugs. Using X-ray crystallography and computer-based modeling, we determined how RUPB-61 binds to its target in atomic detail, providing a roadmap for designing improved versions. While the compound shows some unwanted interactions with human proteins, our structural data offer clear strategies to solve this challenge. Together, these findings provide a solid foundation for developing a new class of once-weekly oral malaria prevention drugs.

microbiology↗

bioRxiv2025

Macrocyclic phage display for identification of selective protease substrates

Traditional methods for identifying selective protease substrates have primarily relied on synthetic libraries of linear peptides, which offer limited sequence and structural diversity. Here, we present an approach that leverages phage display technology to screen large libraries of chemically modified cyclic peptides, enabling the identification of highly selective substrates for a protease of interest. Our method uses a reactive chemical linker to cyclize peptides on the phage surface, while simultaneously incorporating an affinity tag and a fluorescent reporter. The affinity tag enables capture of the phage library and subsequent release of phages expressing optimal substrates upon incubation with a protease of interest. The addition of a turn-on fluorescent reporter allows direct quantification of cleavage efficiency throughout each selection round. The resulting identified substrates can then be chemically synthesized, optimized and validated using recombinant enzymes and cells. We demonstrate the utility of this approach using Fibroblast Activation Protein alpha (FAP) and the related proline-specific protease, dipeptidyl peptidase-4 (DPP4), as targets. Phage selection and subsequent optimization identified substrates with selectivity for each target that have the potential to serve as valuable tools for applications in basic biology and fluorescence image-guided surgery (FIGS). Overall, our strategy provides a rapid and unbiased platform for effectively discovering highly selective, non-natural protease substrates, overcoming key limitations of existing methods.

biochemistry↗

bioRxiv2024

Identification of Covalent Cyclic Peptide Inhibitors Targeting Protein-Protein Interactions Using Phage Display

Peptide macrocycles are promising therapeutics for a variety of disease indications due to their overall metabolic stability and potential to make highly selective binding interactions with targets. Recent advances in covalent macrocycle peptide discovery, driven by phage and mRNA display methods, have enabled the rapid identification of highly potent and selective molecules from large libraires of diverse macrocycles. However, there are currently limited examples of macrocycles that can be used to disrupt protein-protein interactions and even fewer examples that function by formation of a covalent bond to a target protein. In this work, we describe a directed counter-selection method that enables identification of covalent macrocyclic ligands targeting a protein-protein interaction using a phage display screening platform. This method utilizes binary and ternary screenings of a chemically modified phage display library, employing the stable and weakly reactive aryl fluorosulfate electrophile. We demonstrate the utility of this approach using the SARS-CoV-2 Spike-ACE2 protein-protein interaction and identify multiple covalent macrocyclic inhibitors that disrupt this interaction. The resulting compounds displayed antiviral activity against live virus that was irreversible after washout due to the covalent binding mechanism. These results highlight the potential of this screening platform for developing covalent macrocyclic drugs that disrupt protein-protein interactions with long lasting effects.

microbiology↗

bioRxiv2024

Mechanistic Studies of Small Molecule Ligands Selective to RNA Single G Bulges

Small-molecule RNA binders have emerged as an important pharmacological modality. A profound understanding of the ligand selectivity, binding mode, and influential factors governing ligand engagement with RNA targets is the foundation for rational ligand design. Here, we report a novel class of coumarin derivatives exhibiting selective binding affinity towards single G RNA bulges. Harnessing the computational power of all-atom Gaussian accelerated Molecular Dynamics (GaMD) simulations, we unveiled a rare minor groove binding mode of the ligand with a key interaction between the coumarin moiety and the G bulge. This predicted binding mode is consistent with results obtained from structure-activity-relationship (SAR) studies and transverse relaxation measurements by NMR spectroscopy. We further generated 444 molecular descriptors from 69 coumarin derivatives and identified key contributors to the binding events, such as charge state and planarity, by lasso (least absolute shrinkage and selection operator) regression. Strikingly, small structure perturbations on these key contributors, such as the addition of a methyl group that disrupts the planarity of the ligand resulted in > 100-fold reduction in the binding affinity. Our work deepened the understanding of RNA-small molecule interactions and integrated a new generalizable platform for the rational design of selective small-molecule RNA binders.

biochemistry↗

bioRxiv2024

Borrelia burgdorferi BB0346 is an Essential, Structurally Variant LolA Homolog that is Primarily Required for Homeostatic Localization of Periplasmic Lipoproteins

In diderm bacteria, the Lol pathway canonically mediates the periplasmic transport of lipoproteins from the inner membrane (IM) to the outer membrane (OM) and therefore plays an essential role in bacterial envelope homeostasis. After extrusion of modified lipoproteins from the IM via the LolCDE complex, the periplasmic chaperone LolA carries lipoproteins through the periplasm and transfers them to the OM lipoprotein insertase LolB, itself a lipoprotein with a LolA-like fold. Yet, LolB homologs appear restricted to {psi}-proteobacteria and are missing from spirochetes like the tick-borne Lyme disease pathogen Borrelia burgdorferi, suggesting a different hand-off mechanism at the OM. Here, we solved the crystal structure of the B. burgdorferi LolA homolog BB0346 (LolABb) at 1.9 [A] resolution. We identified multiple structural deviations in comparative analyses to other solved LolA structures, particularly a unique LolB-like protruding loop domain. LolABb failed to complement an Escherichia coli lolA knockout, even after codon optimization, signal I peptide adaptation, and a C-terminal chimerization which had allowed for complementation with an -proteobacterial LolA. Analysis of a conditional B. burgdorferi lolA knockout strain indicated that LolABb was essential for growth. Intriguingly, protein localization assays indicated that initial depletion of LolABb led to an emerging mislocalization of both IM and periplasmic OM lipoproteins, but not surface lipoproteins. Together, these findings further support the presence of two separate primary secretion pathways for periplasmic and surface OM lipoproteins in B. burgdorferi and suggest that the distinct structural features of LolABb allow it to function in a unique LolB-deficient lipoprotein sorting system. SIGNIFICANCEBorrelia spirochetes causing Lyme disease and relapsing fever have unusual double-membrane envelopes that instead of lipopolysaccharide (LPS) display abundant surface lipoproteins. We recently showed that secretion of these surface lipoproteins in Borrelia burgdorferi depends on a distant homolog of the canonical LPS outer membrane translocase LptD. Here, we probed the role of the B. burgdorferi Lol pathway in lipoprotein sorting and secretion. We show that the periplasmic chaperone LolA is essential, functionally different from E. coli LolA, with structural features of a bifunctional lipoprotein carrier protein operating without a downstream LolB outer membrane lipoprotein insertase. Depletion of LolA did not impact surface lipoprotein localization but led to a marked mislocalization of inner membrane lipoproteins to the outer membrane. This further supports two parallel, yet potentially interacting Borrelia lipoprotein transport pathways that are responsible for either secreting surface lipoprotein virulence factors or maintaining proper distribution of lipoproteins within the periplasmic space.

microbiology↗

bioRxiv2024

Molecular basis for the activation of the Fatty Acid Kinase complex of Staphylococcus aureus

Gram-positive bacteria utilize a Fatty Acid Kinase (FAK) complex to harvest fatty acids from the environment. The complex, consisting of the fatty acid kinase, FakA, and an acyl carrier protein, FakB, is known to impact virulence and disease outcomes. However, FAKs structure and enzymatic mechanism remain poorly understood. Here, we used a combination of modeling, biochemical, and cell-based approaches to establish critical details of FAK activity. Solved structures of the apo and ligand-bound FakA kinase domain captured the protein state through ATP hydrolysis. Additionally, targeted mutagenesis of an understudied FakA Middle domain identified critical residues within a metal-binding pocket that contribute to FakA dimer stability and protein function. Regarding the complex, we demonstrated nanomolar affinity between FakA and FakB and generated computational models of the complexs quaternary structure. Together, these data provide critical insight into the structure and function of the FAK complex which is essential for understanding its mechanism.

microbiology↗

bioRxiv2024

HSV-1 ICP0 Dimer Domain Adopts a Novel β-barrel Fold

Infected cell protein 0 (ICP0) is an immediate-early regulatory protein of herpes simplex virus 1 (HSV-1) that possesses E3 ubiquitin ligase activity. ICP0 transactivates viral genes, in part, through its C-terminal dimer domain (residues 555-767). Deletion of this dimer domain results in reduced viral gene expression, lytic infection, and reactivation from latency. Since ICP0s dimer domain is associated with its transactivation activity and efficient viral replication, we wanted to determine the structure of this specific domain. The C-terminus of ICP0 was purified from bacteria and analyzed by X-ray crystallography to solve its structure. Each subunit or monomer in the ICP0 dimer is composed of nine {beta}-strands and two -helices. Interestingly, two adjacent {beta}-strands from one monomer "reach" into the adjacent subunit during dimer formation, generating two {beta}-barrel-like structures. Additionally, crystallographic analyses indicate a tetramer structure is formed from two {beta}-strands of each dimer, creating a "stacking" of the {beta}-barrels. The structural protein database searches indicate the fold or structure adopted by the ICP0 dimer is novel. The dimer is held together by an extensive network of hydrogen bonds. Computational analyses reveal that ICP0 can either form a dimer or bind to SUMO1 via its C-terminal SUMO-interacting motifs but not both. Understanding the structure of the dimer domain will provide insights into the activities of ICP0 and, ultimately, the HSV-1 life cycle.

microbiology↗

bioRxiv2024

Mixed Alkyl/Aryl Phosphonates Identify Metabolic Serine Hydrolases as Antimalarial Targets

Malaria, caused by Plasmodium falciparum, remains a significant health burden. A barrier for developing anti-malarial drugs is the ability of the parasite to rapidly generate resistance. We demonstrated that Salinipostin A (SalA), a natural product, kills parasites by inhibiting multiple lipid metabolizing serine hydrolases, a mechanism with a low propensity for resistance. Given the difficulty of employing natural products as therapeutic agents, we synthesized a library of lipidic mixed alkyl/aryl phosphonates as bioisosteres of SalA. Two constitutional isomers exhibited divergent anti-parasitic potencies which enabled identification of therapeutically relevant targets. We also confirm that this compound kills parasites through a mechanism that is distinct from both SalA and the pan-lipase inhibitor, Orlistat. Like SalA, our compound induces only weak resistance, attributable to mutations in a single protein involved in multidrug resistance. These data suggest that mixed alkyl/aryl phosphonates are a promising, synthetically tractable anti-malarials with a low-propensity to induce resistance.

microbiology↗

bioRxiv2023

Intra-prostatic tumour evolution, steps in metastatic spread and histogenomic associations revealed by integration of multi-region whole genome sequencing with histopathological features

Extension of prostate cancer beyond the primary site into the surrounding organs by local invasion or nodal metastasis is associated with poor prognosis. The emergence and evolution of cancer clones at this early stage of expansion and spread has not been studied in detail. We performed whole genome sequencing on 42 prostate cancer samples from the prostate, seminal vesicles and regional lymph nodes of five treatment-naive patients with locally advanced disease who underwent radical prostatectomy. Using cancer cell fractions computed from single nucleotide variants and copy number alterations, we reconstructed the tumour phylogenies, which in turn allowed us to infer key molecular steps in the progression of prostate cancer in these individuals. We mapped the clonal composition of cancer sampled across the prostate in each individual and inferred the routes of spread of cancer cells within the prostate and to seminal vesicles and lymph nodes. Based on these data, we delineated the route of tumour progression and metastasis following the transformation of adenocarcinoma to amphicrine morphology, the molecular events leading to whole genome duplication associated with a single clonal expansion and identified putative driver events associated with local invasion and lymph node metastasis. We also correlated genomic changes associated with differences in morphology and identified putative driver events associated with spread to seminal vesicle invasion and lymph node metastasis. Taken together, these findings have implications for diagnosis and risk stratification, in addition to providing a rationale for further studies to characterise the genetic changes associated with morphological transformation. Our results demonstrate the value of integrating multi-region sequencing with histopathological data to study tumour evolution and identify mechanisms of prostate cancer spread.

cancer biology↗

bioRxiv2023

Solid Phase Synthesis of Fluorosulfate Containing Macrocycles for Chemoproteomic Workflows

Macrocyclic peptides are attractive for chemoproteomic applications due to their modular synthesis and potential for high target selectivity. We describe a solid phase synthesis method for the efficient generation of libraries of small macrocycles that contain an electrophile and alkyne handle. The modular synthesis produces libraries that can be directly screened using simple SDS-PAGE readouts and then optimal lead molecules applied to proteomic analysis. We generated a library of 480 macrocyclic peptides containing the weakly reactive fluorosulfate (OSF) electrophile. Initial screening of a subset of the library containing each of the various diversity elements identified initial molecules of interest. The corresponding positional and confirmational isomers were then screened to select molecules that showed specific protein labeling patterns that were dependent on the probe structure. The most promising hits were applied to standard chemoproteomic workflows to identify protein targets. Our results demonstrate the feasibility of rapid, on-resin synthesis of diverse macrocyclic electrophiles to generate new classes of covalent ligands.

biochemistry↗

bioRxiv2022

Potent protease inhibitors of deadly lagoviruses: rabbit hemorrhagic disease virus and European brown hare syndrome virus

Rabbit hemorrhagic disease (RHD) and European brown hare syndrome (EBHS) are highly contagious diseases caused by lagoviruses in the Caliciviridae family and mainly affect rabbits and hares, respectively. These infectious diseases are associated with high mortality and a serious threat to domesticated and wild rabbits and hares, including endangered species such as Riparian brush rabbits. In the US, only isolated cases of RHD had been reported until Spring 2020. However, RHD caused by RHD type 2 virus (RHDV2) was unexpectedly reported in April 2020 in New Mexico and has subsequently spread to several US states infecting wild rabbits and hares, making it highly likely that RHD will become endemic in the US. Vaccines are available for RHD, however, there is no specific treatment for these diseases. RHDV and EBHSV encode a 3C-like protease (3CLpro), which is essential for virus replication and a promising target for antiviral drug development. We have previously generated focused small molecule libraries of 3CLpro inhibitors and demonstrated the in vitro potency and in vivo efficacy of some protease inhibitors against viruses that encode 3CLpro including caliciviruses and coronaviruses. Here we established the enzyme and cell-based assays for these uncultivable viruses to determine the in vitro activity of 3CLpro inhibitors, including GC376, a protease inhibitor being developed for feline infectious peritonitis, and identified potent inhibitors of RHDV1 and 2 and EBHSV. In addition, structure-activity relationship study and homology modelling of the 3CLpros and inhibitors revealed that lagoviruses share similar structural requirements for 3CLpro inhibition with other caliciviruses.

microbiology↗

bioRxiv2021

A suite of activity-based probes to dissect the KLK activome in drug-resistant prostate cancer

Kallikrein-related peptidases (KLKs) are a family of secreted serine proteases, which form a network - the KLK activome - with an important role in proteolysis and signaling. In prostate cancer (PCa), increased KLK activity promotes tumor growth and metastasis through multiple biochemical pathways, and specific quantification and tracking of changes in the KLK activome could contribute to validation of KLKs as potential drug targets. Herein we report a technology platform based on novel activity-based probes (ABPs) and inhibitors with unprecedented potency and selectivity enabling simultaneous orthogonal analysis of KLK2, KLK3 and KLK14 activity in hormone-responsive PCa cell lines and tumor homogenates. Using selective inhibitors and multiplexed fluorescent activity-based protein profiling (ABPP) we dissect the KLK activome in PCa cells and show that increased KLK14 activity leads to a migratory phenotype. Furthermore, using biotinylated ABPs we show that active KLK molecules are secreted into the bone microenvironment by PCa cells following stimulation by osteoblasts suggesting KLK-mediated signaling mechanisms could contribute to PCa metastasis to bone. Together our findings show that ABPP is a powerful approach to dissect dysregulation of the KLK activome as a promising and previously underappreciated therapeutic target in advanced PCa.

biochemistry↗

bioRxiv2021

Post-infection treatment with a protease inhibitor increases survival of mice with a fatal SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a serious global public health threat. The 3C-like protease (3CLpro) is a virus protease encoded by SARS-CoV-2, which is essential for virus replication. We have previously reported a series of small molecule 3CLpro inhibitors effective for inhibiting replication of human coronaviruses including SARS-CoV-2 in cell culture and in animal models. Here we generated a series of deuterated variants of a 3CLpro inhibitor, GC376, and evaluated the antiviral effect against SARS-CoV-2. The deuterated GC376 displayed potent inhibitory activity against SARS-CoV-2 in the enzyme and the cell-based assays. The K18-hACE2 mice develop mild to lethal infection commensurate with SARS-CoV-2 challenge doses and was proposed as a model for efficacy testing of antiviral agents. We treated lethally infected mice with a deuterated derivative of GC376. Treatment of K18-hACE2 mice at 24 hr post infection with a derivative (compound 2) resulted in increased survival of mice compared to vehicle-treated mice. Lung virus titers were decreased, and histopathological changes were ameliorated in compound 2-treated mice compared to vehicle-treated mice. Structural investigation using high-resolution crystallography illuminated binding interactions of 3CLpro of SARS-CoV-2 and SARS-CoV with deuterated variants of GC376. Taken together, deuterated GC376 variants have excellent potential as antiviral agents against SARS-CoV-2.

microbiology↗

bioRxiv2020

Challenges for targeting SARS-CoV-2 proteases as a therapeutic strategy for COVID-19

Two proteases produced by the SARS-CoV-2 virus, Mpro and PLpro, are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19. We screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both Mpro and PLpro proteases. These efforts identified a small number of hits for the Mpro protease and no viable hits for the PLpro protease. Of the Mpro hits identified as inhibitors of the purified recombinant protease, only two compounds inhibited viral infectivity in cellular infection assays. However, we observed a substantial drop in antiviral potency upon expression of TMPRSS2, a transmembrane serine protease that acts in an alternative viral entry pathway to the lysosomal cathepsins. This loss of potency is explained by the fact that our lead Mpro inhibitors are also potent inhibitors of host cell cysteine cathepsins. To determine if this is a general property of Mpro inhibitors, we evaluated several recently reported compounds and found that they are also effective inhibitors of purified human cathepsin L and B and showed similar loss in activity in cells expressing TMPRSS2. Our results highlight the challenges of targeting Mpro and PLpro proteases and demonstrate the need to carefully assess selectivity of SARS-CoV-2 protease inhibitors to prevent clinical advancement of compounds that function through inhibition of a redundant viral entry pathway.

microbiology↗

bioRxiv2020

The Structures of SctK and SctD from Pseudomonas aeruginosa Reveal the Interface of the Type III Secretion System Basal Body and Sorting Platform

Many Gram-negative bacterial pathogens use type III secretion systems (T3SS) to inject effector proteins into eukaryotic cells to subvert normal cellular functions. The T3SS apparatus (injectisome) shares a common overall architecture in all systems studied thus far, comprising three major components - the cytoplasmic sorting platform, the envelope-spanning basal body and the external needle with controlling tip complex. The sorting platform consists of an ATPase (SctN) connected to "pods" (SctQ) having six-fold symmetry via radial spokes (SctL). These pods interface with the 24-fold symmetric SctD inner membrane ring (IR) via an adaptor protein (SctK). Here we report the first high-resolution structure of a SctK protein family member, PscK from Pseudomonas aeruginosa, as well as the structure of its interacting partner, the cytoplasmic domain of PscD (SctD). The cytoplasmic domain of PscD forms a forkhead-associated (FHA) fold, like that of its homologues from other T3SS. PscK, on the other hand, forms a helix-rich structure that does not resemble any known protein fold. Based on these structural findings, we present the first model for an interaction between proteins from the sorting platform and the IR. We also test the importance of the PscD residues predicted to mediate this electrostatic interaction using a two-hybrid analysis. The functional need for Arg96 in vivo was then confirmed by monitoring secretion of the effector ExoU. These structures will contribute to the development of atomic-resolution models of the entire sorting platform and to our understanding of the mechanistic interface between the sorting platform and the basal body of the injectisome. HighlightsO_LIThe structures of Pseudomonas aeruginosa PscD (SctD) and PscK (SctK) were solved C_LIO_LIThe interface between the T3SS basal body and sorting platform was modeled C_LIO_LIThe crystal structure of PscK is the first for any SctK family member C_LIO_LIPscK represents a novel protein fold C_LIO_LISite-directed mutagenesis supports a computational model of the PscD-PscK interface C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/308627v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1efcf14org.highwire.dtl.DTLVardef@1043d81org.highwire.dtl.DTLVardef@161f21corg.highwire.dtl.DTLVardef@15e4357_HPS_FORMAT_FIGEXP M_FIG Graphical abstract. The first reported structure for a T3SS SctK protein family member was solved for PscK from Pseudomonas aeruginosa and this allowed for modeling of the interface between this sorting platform protein and the cytoplasmic domain of PscD (a SctD protein family member). This allowed for identification of amino acid residues that may play a role in the interaction between these proteins. The interface appears to be dominated by electrostatic interactions and mutagenesis confirmed the importance of key residues in driving their interaction based on two-hybrid analysis. C_FIG

biophysics↗