Biology subjects

Hvorecny, K. L.

Publications and source records attributed to Hvorecny, K. L..

8 recordsLinked to original sources

bioRxiv2025

De Novo Design of Miniprotein Inhibitors of Bacterial Adhesins

The rise of multidrug-resistant bacterial infections necessitates the discovery of novel antimicrobial strategies. Here, we show that protein design provides a generalizable means of generating new antimicrobials by neutralizing the function of bacterial adhesins, which are virulence factors critical in host-pathogen interactions. We de novo designed high-affinity miniprotein binders to FimH and Abp chaperone usher pili adhesins from uropathogenic Escherichia coli and Acinetobacter baumannii, respectively, which are implicated in mediating both uncomplicated and catheter-associated urinary tract infections (UTI) responsible for significant morbidity worldwide. The designed antagonists have high specificity and stability, disrupt bacterial recognition of host receptors, block biofilm formation, and are effective in treating and preventing murine models of uncomplicated and catheter- associated UTIs in vivo.

microbiology↗

bioRxiv2024

Antibodies disrupt bacterial adhesion by ligand mimicry and allosteric interference

A critical step in infections is the attachment of many microorganisms to host cells using lectins that bind surface glycans, making lectins promising antimicrobial targets. Upon binding mannosylated glycans, FimH, the most studied lectin adhesin of type 1 fimbriae in E. coli, undergoes an allosteric transition from an inactive to an active conformation that can act as a catch-bond. Monoclonal antibodies that alter FimH glycan binding in various ways are available, but the mechanisms of these antibodies remain unclear. Here, we use cryoEM, mass spectrometry, binding assays, and molecular dynamics simulations to determine the structure-function relationships underlying antibody-FimH binding. Our study reveals four distinct antibody mechanisms of action: ligand mimicry by an N-linked, high-mannose glycan; stabilization of the ligand pocket in the inactive state; conformational trapping of the active and inactive states; and locking of the ligand pocket through long-range allosteric effects. These structures reveal multiple mechanisms of antibody responses to an allosteric protein and provide blueprints for new antimicrobial that target adhesins.

immunology↗

bioRxiv2024

Molecular basis for the transcriptional regulation of an epoxide-based virulence circuit in Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa infects cystic fibrosis (CF) patient airways and produces a virulence factor Cif that is associated with worse outcomes. Cif is an epoxide hydrolase that reduces cell-surface abundance of the cystic fibrosis transmembrane conductance regulator (CFTR) and sabotages pro-resolving signals. Its expression is regulated by a divergently transcribed TetR family transcriptional repressor. CifR represents the first reported epoxide-sensing bacterial transcriptional regulator, but neither its interaction with cognate operator sequences nor the mechanism of activation has been investigated. Using biochemical and structural approaches, we uncovered the molecular mechanisms controlling this complex virulence operon. We present here the first molecular structures of CifR alone and in complex with operator DNA, resolved in a single crystal lattice. Significant conformational changes between these two structures suggest how CifR regulates the expression of the virulence gene cif. Interactions between the N-terminal extension of CifR with the DNA minor groove of the operator play a significant role in the operator recognition of CifR. We also determined that cysteine residue Cys107 is critical for epoxide sensing and DNA release. These results offer new insights into the stereochemical regulation of an epoxide-based virulence circuit in a critically important clinical pathogen.

biochemistry↗

bioRxiv2023

Toxoplasma gondii actin filaments are tuned for rapid disassembly and turnover

The cytoskeletal protein actin plays a critical role in the pathogenicity of Toxoplasma gondii, mediating invasion and egress, cargo transport, and organelle inheritance. Advances in live cell imaging have revealed extensive filamentous actin networks in the Apicomplexan parasite, but there is conflicting data regarding the biochemical and biophysical properties of Toxoplasma actin. Here, we imaged the in vitro assembly of individual Toxoplasma actin filaments in real time, showing that native, unstabilized filaments grow tens of microns in length. Unlike skeletal muscle actin, Toxoplasma filaments intrinsically undergo rapid treadmilling due to a high critical concentration, fast monomer dissociation, and rapid nucleotide exchange. Cryo-EM structures of stabilized and unstabilized filaments show an architecture like skeletal actin, with differences in assembly contacts in the D-loop that explain the dynamic nature of the filament, likely a conserved feature of Apicomplexan actin. This work demonstrates that evolutionary changes at assembly interfaces can tune dynamic properties of actin filaments without disrupting their conserved structure.

biophysics↗

bioRxiv2022

Human PRPS1 filaments stabilize allosteric sites to regulate activity

The universally conserved enzyme phosphoribosyl pyrophosphate synthetase (PRPS) assembles filaments in evolutionarily diverse organisms. PRPS is a key regulator of nucleotide metabolism, and mutations in the human enzyme PRPS1 lead to a spectrum of diseases. Here, we determine structures of PRPS1 filaments in active and inhibited states, with fixed assembly contacts accommodating both conformations. The conserved assembly interface stabilizes the binding site for the essential activator phosphate, increasing activity in the filament. Some disease mutations alter assembly, supporting the link between filament stability and activity. Structures of active PRPS1 filaments turning over substrate also reveal coupling of catalysis in one active site with product release in an adjacent site. PRPS1 filaments therefore provide an additional layer of allosteric control, conserved throughout evolution, with likely impact on metabolic homeostasis. Stabilization of allosteric binding sites by polymerization adds to the growing diversity of assembly-based enzyme regulatory mechanisms.

biochemistry↗

bioRxiv2021

A second specificity-determining loop in Class A sortases: Biochemical characterization of natural sequence variation in chimeric SrtA enzymes

Gram-positive bacteria contain sortase enzymes on their cell surfaces that catalyze transpeptidation reactions critical for proper cellular function. In vitro, sortases are used in sortase-mediated ligation (SML) reactions for a variety of protein engineering applications. Historically, sortase A from Staphylococcus aureus (saSrtA) has been the enzyme of choice for SML reactions. However, the stringent specificity of saSrtA for the sequence motif LPXTG limits its uses. Here, we use principal component analysis to identify a structurally conserved loop with a high degree of variability in all classes of sortases. We investigate the contribution of this {beta}7-{beta}8 loop, located between the catalytic cysteine and arginine residues and immediately adjacent to the target binding cleft, by designing and testing chimeric sortase enzymes. Our chimeras utilize natural sequence variation of Class A sortases from 8 species engineered into the SrtA sequence from Streptococcus pneumoniae (spSrtA). While some of our chimeric enzymes mimic the activity and selectivity of the wild-type protein from which the loop sequence is derived (e.g., that of saSrtA), others result in chimeric spSrtA enzymes able to accommodate a range of residues in the final position of the substrate motif (LPXTX). Using mutagenesis, structural, and sequence analyses, we identify three interactions facilitated by {beta}7-{beta}8 loop residues that appear to be broadly characteristic of Class A sortase enzymes. These studies provide the foundation for a deeper understanding of sortase target selectivity and can expand the sortase toolbox for future SML applications.

biochemistry↗

bioRxiv2021

The Giardia lamellipodium-like ventrolateral flange supports attachment and rapid cytokinesis

Attachment to the intestinal epithelium is critical to the lifestyle of the ubiquitous parasite Giardia lamblia. The microtubule cytoskeleton plays a well characterized role in attachment via the ventral adhesive disc, whereas the role of the unconventional actin cytoskeleton is controversial. We identified a novel actin associated protein with putative WH2-like actin binding domains we named Flangin. Flangin complexes with Giardia actin and is enriched in the ventrolateral flange (VLF), a lamellipodium-like membrane protrusion at the interface between parasites and attached surfaces. Live imaging revealed that the VLF grows to ~1 m in width after cytokinesis, then remains size-uniform in interphase, grows during mitosis, and is resorbed during cytokinesis. A Flangin truncation mutant stabilizes the VLF and blocks cytokinesis, indicating that the VLF is a membrane reservoir supporting rapid myosin-independent cytokinesis in Giardia. Rho family GTPases are important regulators of membrane protrusions, GlRac, the sole Rho family GTPase in Giardia, was localized to the VLF. Knockdown of Flangin, actin, and GlRac result in VLF formation defects indicating a conserved role for GlRac and actin in forming membrane protrusions, despite the absence of canonical actin binding proteins that link Rho GTPase signaling to lamellipodia formation. Flangin-depleted parasites challenged with fluid shear force in flow chambers had a reduced ability to remain attached, indicating a role for the VLF in attachment. This secondary attachment mechanism complements the microtubule based adhesive ventral disc, a feature that is particularly important during mitosis when the parental ventral disc begins disassembly in preparation for cytokinesis. ImportanceThe ventrolateral flange (VLF) is a lamellipodium-like structure found at the host-parasite interface that has long been thought to be involved in parasite attachment. The proteins responsible for building the VLF have remained unidentified precluding manipulation of the VLF to determine its role in Giardia biology. We identified Flangin, a novel actin associated protein that localizes to the VLF, implicating Giardia actin in VLF formation. We demonstrate that: 1.) Flangin, actin, and GlRac are required for VLF formation, 2.) the VLF serves as a membrane reservoir to support Giardias incredibly fast cytokinesis, and 3) the VLF augments attachment, which is critical to parasitism. The microtubule-based adhesive ventral disc and the actin-based ventrolateral flange represent redundant means of maintaining attachment, the presence of redundant systems illustrate the importance of attachment to the lifestyle of this ubiquitous parasite.

cell biology↗

bioRxiv2021

Biochemical and Structural Characterization of Two Cif-Like Epoxide Hydrolases from Burkholderia cenocepacia

Epoxide hydrolases catalyze the conversion of epoxides to vicinal diols in a range of cellular processes such as signaling, detoxification, and virulence. These enzymes typically utilize a pair of tyrosine residues to orient the substrate epoxide ring in the active site and stabilize the hydrolysis intermediate. A new subclass of epoxide hydrolases that utilize a histidine in place of one of the tyrosines was established with the discovery of the CFTR Inhibitory Factor (Cif) from Pseudomonas aeruginosa. Although the presence of such Cif-like epoxide hydrolases was predicted in other opportunistic pathogens based on sequence analyses, only Cif and its homologue aCif from Acinetobacter nosocomialis have been characterized. Here we report the biochemical and structural characteristics of Cfl1 and Cfl2, two Cif-like epoxide hydrolases from Burkholderia cenocepacia. Cfl1 is able to hydrolyze xenobiotic as well as biological epoxides that might be encountered in the environment or during infection. In contrast, Cfl2 shows very low activity against a diverse set of epoxides. The crystal structures of the two proteins reveal quaternary structures that build on the well-known dimeric assembly of the /{beta} hydrolase domain, but broaden our understanding of the structural diversity encoded in novel oligomer interfaces. Analysis of the interfaces reveals both similarities and key differences in sequence conservation between the two assemblies, and between the canonical dimer and the novel oligomer interfaces of each assembly. Finally, we discuss the effects of these higher-order assemblies on the intra-monomer flexibility of Cfl1 and Cfl2 and their possible roles in regulating enzymatic activity.

biochemistry↗