Biology subjects

Garboczi, D. N.

Publications and source records attributed to Garboczi, D. N..

4 recordsLinked to original sources

bioRxiv2026

Structure of a secreted lancelet carotenoid cleavage dioxygenase (CCD), from an ancestral CCD clade

Carotenoid cleavage dioxygenases (CCDs) are a superfamily of non-heme iron-containing enzymes ubiquitous across all kingdoms of life. Previously, we identified a new subfamily of CCDs termed beta-carotene oxygenase-like (BCOL) proteins, a chordate expansion of an ancestral sister clade of metazoan CCDs, which are widely distributed in marine invertebrates, including mollusks and lancelets. Here we present the three-dimensional structure of a representative BCOL protein from the lancelet Branchiostoma floridae (BCOLg) at 2.4 [A] resolution and its functional properties. BCOLg symmetrically cleaves {beta}-carotene into all-trans retinal. RNAseq of wild-caught animals detected six of the seven annotated BCOL transcripts in Branchiostoma gut tissue. We expressed and crystallized recombinant BCOLg. BCOLg is heavily glycosylated, with four N-linked glycosylation sites, and adopts the seven-bladed propeller fold characteristic of the superfamily, with one disulfide bond contributing to structural stability. BCOLg and several other BCOL proteins contain Asp substitutions at two of three Glu positions found in other CCDs, stabilizing three of four His residues involved in Fe2+ coordination. Notably, BCOLg lacks the canonical -PDPCK- motif characteristic of other metazoan CCDs.

biochemistry↗

bioRxiv2025

Specificity and mechanism of the double-stranded RNA-specific J2 monoclonal antibody

Double-stranded (ds) RNAs are major structural components of the transcriptome, hallmarks of viral infection, and primary triggers of innate immune responses. The J2 monoclonal antibody is the gold-standard method to discover and map endogenous dsRNAs across subcellular locations and cell surfaces, detect exogenous RNAs in viral infection, and surveil mRNA prophylactics and therapeutics for inflammatory dsRNAs. To define its epitope, specificity, and mechanism, we determine a 2.85 [A] co-crystal structure of J2 antigen-binding fragment (Fab) bound to dsRNA. J2 uses its heavy and light chains in tandem to track the dsRNA minor groove, recognizing a staggered 8-bp duplex. J2 is exquisitely selective for dsRNAs, requires 14 bp for robust binding, and exhibits greatly diminished binding for GC-rich dsRNAs. J2 and R-loop-specific S9.6 antibody share a common recognition strategy distinct from intracellular dsRNA-binding proteins. This study provides mechanistic insights into dsRNA recognition and establishes a framework for reliable application and data interpretation of the J2 antibody in RNA discovery.

molecular biology↗

bioRxiv2025

The 2.3 A Structure of A21, a Protein Component of the Conserved Poxvirus Entry-Fusion Complex

Poxviruses are exceptional in having an entry-fusion complex (EFC) consisting of eleven conserved proteins embedded in the membrane of mature virions. With the goal of understanding the function of the EFC, extensive efforts have been made to determine the structures and roles of its components, and to date, structures have been determined for nine of the eleven proteins. Here, we report the crystal structure of A21, the 10th EFC protein, comprising two -helices clasping a twisted antiparallel {beta}-sheet stabilized by two conserved disulfide bonds. The stability of each of the three A21 loops is provided by hydrogen bonds between main-chain atoms and several highly conserved residues, making the overall fold of A21 and its orthologs resilient to evolutionary change. Based on AlphaFold modeling and phylogenetic analysis of A21, we suggest that its highly conserved N-terminal transmembrane domain and C-terminal -helix enable A21 integration into EFC, where it primarily interacts with the G3/L5 subcomplex and the smallest of EFC components, the O3 protein. HighlightsO_LIWith the structure of A21 determined by us, the structures of 10 out of the 11 proteins forming the entry-fusion complex (EFC) of the poxvirus are now known. C_LIO_LIA21 features a highly conserved transmembrane (TM) domain, which, along with the C--helix, facilitates the integration of A21 and its orthologs into the EFC. C_LIO_LIWithin the EFC, A21 primarily interacts with the G3/L5 subcomplex and the O3 peptide. C_LI

molecular biology↗

bioRxiv2024

Chemokines Kill Bacteria by Binding Anionic Phospholipids without Triggering Antimicrobial Resistance

Classically, chemokines coordinate leukocyte trafficking during immune responses; however, many chemokines have also been reported to possess direct antibacterial activity in vitro. Yet, the bacterial killing mechanism of chemokines and the biochemical properties that define which members of the chemokine superfamily are antimicrobial remain poorly understood. Here we report that the antimicrobial activity of chemokines is defined by their ability to bind phosphatidylglycerol and cardiolipin, two anionic phospholipids commonly found in the bacterial plasma membrane. We show that only chemokines able to bind these two phospholipids kill Escherichia coli and Staphylococcus aureus and that they exert rapid bacteriostatic and bactericidal effects against E. coli with a higher potency than the antimicrobial peptide beta-defensin 3. Furthermore, our data support that bacterial membrane cardiolipin facilitates the antimicrobial action of chemokines. Both biochemical and genetic interference with the chemokine-cardiolipin interaction impaired microbial growth arrest, bacterial killing, and membrane disruption by chemokines. Moreover, unlike conventional antibiotics, E. coli failed to develop resistance when placed under increasing antimicrobial chemokine pressure in vitro. Thus, we have identified cardiolipin and phosphatidylglycerol as novel binding partners for chemokines responsible for chemokine antimicrobial action. Our results provide proof of principle for developing chemokines as novel antibiotics resistant to bacterial antimicrobial resistance mechanisms.

microbiology↗