Biology subjects

Gade, P.

Publications and source records attributed to Gade, P..

4 recordsLinked to original sources

bioRxiv2026

Phylogenetic Analysis and Structural Evaluation of Staphylococcus aureus Serine-Aspartate Repeat-Containing Protein D with a Focus on Periprosthetic Joint Infection

Serine-aspartate repeat-containing protein D (SdrD) is a Staphylococcus aureus cell wall-anchored, calcium-binding adhesin member of the MSCRAMM Sdr subfamily that may contribute to bacterial adhesion and virulence. S. aureus is the most common cause of periprosthetic joint infection (PJI). Population-level distribution and sequence diversity of SdrD among clinical PJI isolates have not been systematically characterized, and the SdrD binding mechanism is still not well understood. To address these gaps, sdrD alleles were queried across 156 newly sequenced PJI isolates and compared to publicly available S. aureus genomes, and nucleotide- and protein-level phylogenies of the sdrCDE locus constructed. The SdrD crystal structure from S. aureus JH1 was determined, with solution small-angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, and assessment of conformational changes with calcium depletion. Three dominant sdrD subtypes were defined, associating with USA300, JH1, and TCH60; the JH1 sdrD subtype was predominant among PJI isolates. Structural studies showed that the conformation of individual domains and interdomain organization of the multidomain SdrD have limited flexibility in solution, and that the calcium-binding B domain retains its core fold under conditions of calcium depletion. Together, the findings presented support functional diversification among Sdr family members in mediating host attachment and inform a re-evaluation of the ligand-binding mechanism previously proposed for SdrD. AUTHOR SUMMARYStaphylococcus aureus is the leading cause of infections that develop around joint implants (periprosthetic joint infection, PJI). This bacterium has a large arsenal of surface proteins that allow it to stick to human tissues and implanted devices. This work focused on one such protein, SdrD, which has been linked to implant-associated infections but the structure and diversity of which among patients with PJI had not been well characterized. The genetic sequences of SdrD were analyzed across thousands of bacterial genomes, including those from patients with PJI. Distinct genetic variants of the protein were found, one of which was particularly common with PJI. The three-dimensional structure of SdrD was determined at atomic resolution and solution small-angle X-ray scattering (SAXS) and molecular dynamics used to study how it moves and responds to changes in its environment. Contrary to what was previously described, SdrD was shown to be relatively rigid. These findings change how SdrDs mechanism of action should be considered, potentially informing design strategies to block bacterial attachment before infection takes hold.

biophysics↗

bioRxiv2026

Chromatin remodelling enables enhancer resetting to facilitate the ERK transcriptional response

During development, cellular identity is ultimately determined by transcriptional output: lineage-specific genes must be activated, while genes associated with alternative fates must be repressed. This process depends on the activity of chromatin remodelling complexes, which regulate the accessibility of transcription factors to chromatin regulatory elements. In addition, cellular identity is shaped by exposure to intercellular signals. Understanding the mechanisms by which extracellular signals are translated into changes in the transcriptional program is essential for understanding cell fate decisions during development, as well as in disease conditions such as cancer. Here we describe a rapid and widespread enhancer resetting event in response to ERK signalling in mouse ES cells. This process occurs in two distinct phases: an immediate, genome-wide alteration in transcription factor binding dynamics at regulatory regions which is dependent on the release of paused RNA Polymerase II, followed by the re-establishment of a context appropriate, stable chromatin state. We demonstrate that the chromatin remodelling complex NuRD is required for this reestablishment phase and for the appropriate transcriptional response to ERK signalling. We propose that enhancer resetting places genomic regulatory regions in a state which is permissive to the exchange of transcription factors in order to establish a new, stable enhancer topology enabling rapid yet precise transcriptional response to extracellular signals.

molecular biology↗

bioRxiv2025

PANCS-spec-Binders: A system for rapidly discovering isoform- or epitope-specific binders

Proteins that bind to a target protein of interest, termed "binders," are essential components of biological research reagents and therapeutics. Target proteins present multiple binding surfaces with varying interaction potential. High-potential surfaces, or "hot spots," are experimentally identified as the most probable binding sites in de novo discovery campaigns. However, hot spots and their default binding modes do not always confer the desired specificity. Related proteins or isoforms often share similar hot spots, resulting in promiscuous binding. Interaction with a hot spot may also fail to elicit the intended biological outcome. Consequently, methods that direct de novo binder discovery toward targets with defined specificity are critically needed. We recently developed phage-assisted non-continuous selection of binders (PANCS-Binders), a selection platform with unparalleled speed and sequence-function fidelity that enables routine de novo binder discovery within days. However, because PANCS-Binder selections enrich variants based primarily on affinity, secondary screening is unlikely to identify binders to lesser hot spots because of the high likelihood of convergence. These alternative binding surfaces with weaker inherent interactions may possess desirable specificity profiles. Here, we develop PANCS-spec-Binders, which incorporates simultaneous selection and counterselection to control the specificity of enriched binders. We demonstrate PANCS-spec-Binders in two proof- of-concept applications: (1) discovery of isoform-selective binders that bind HRAS with >100-fold higher affinity than the highly related KRAS isoform, and (2) discovery of epitope-specific binders that either target or avoid the LIR interaction region of LC3B. PANCS-spec-Binders enables rapid identification of binders with defined specificity within days. SIGNIFICANCEAffinity reagents, termed "binders", are essential tools in research and therapeutic development. Binders generally require high specificity either at the selectivity level (binding only the target protein, but not related proteins) or at the epitope level (binding only at a specific surface on the target rather than another). While methods to discover binders in general have progressed, identifying binders with defined specificity features often requires extensive secondary screening and frequently results in failure. Here, we adapt our recently developed binder discovery platform to solve these two selectivity problems. By rapidly screening billions of variants, we can direct specificity between highly related proteins, direct binding to a specific epitope, and, because of the fidelity of our selections, identify binders that specifically avoid a defined epitope.

synthetic biology↗

bioRxiv2022

Class II UvrA protein Ecm16 requires ATPase activity to render resistance against echinomycin

II.Bacteria use various strategies to become antibiotic resistant. The molecular details of these strategies are not fully understood. We can increase our understanding by investigating the same strategies found in antibiotic-producing bacteria. In this work, we characterize the self-resistance protein Ecm16 encoded by echinomycin-producing bacteria. Ecm16 is a structural homolog of the Nucleotide Excision Repair (NER) protein UvrA. Expression of ecm16 in the heterologous system Escherichia coli was sufficient to render resistance against echinomycin. Ecm16 preferentially binds double-stranded DNA over single-stranded DNA and is likely to primarily interact with the backbone of DNA using a nucleotide-independent binding mode. Ecm16s binding affinity for DNA increased significantly when the DNA is intercalated with echinomycin. Ecm16 can repair echinomycin-induced DNA damage independently of NER. Like UvrA, Ecm16 has ATPase activity and this activity is essential for Ecm16s ability to render echinomycin resistance. Notably, UvrA and Ecm16 were unable to complement each others function. Increasing the cellular levels of UvrA in E. coli was insufficient to render echinomycin resistance. Similarly, Ecm16 was unable to repair DNA damage that is specific to UvrA. Together, our findings identify new mechanistic details of how a refurbished DNA repair protein Ecm16 can specifically render resistance to the DNA intercalator echinomycin. Our results, together with past observations, suggest a model where Ecm16 recognizes double helix distortions caused by echinomycin and repairs the problem independently of NER.

microbiology↗