Biology subjects

Pereda, A.

Publications and source records attributed to Pereda, A..

2 recordsLinked to original sources

bioRxiv2026

The structure of the Salmonella phage epsilon15 tailspike reveals multiple O-antigen binding sites and a protruding esterase domain

Many bacteriophages use tailspikes to degrade host bacterial polysaccharides, facilitating access to the outer membrane. The homotrimeric tailspikes of the Salmonella phage epsilon15 feature a slender phage-binding domain, a kink, and a barrel-shaped section with three petal-like protrusions. Here, we present the crystal structures of the monomeric protruding petal domain alone and of the trimeric barrel-shaped section with three petal domains. The barrel-shaped section includes a trimeric beta-helix, typical of phage tailspikes, alongside a trimeric beta-sandwich domain. The petal domain exhibits a fold characteristic of the serine-glycine-asparagine-histidine (SGNH) esterase family. Co-crystallisation with O-antigen fragments identified four binding sites on the tailspike: two adjacent sites on the surface of the triple beta-helix, one in the beta-sandwich domain and a fourth near the petal esterase site. These binding sites align with the expected orientation of the phage just before DNA transfer. Nuclear magnetic resonance spectroscopy and site-directed mutagenesis revealed an endorhamnosidase activity, showed that the reaction mechanism proceeds by inversion of the configuration and revealed that the active site is located at the junction of the two beta-helix binding sites. Analogous experiments also revealed an esterase site in the petal domain. Together, the structural and functional insights suggest a dual role for the phage epsilon15 tailspike: de-acetylation of the O-antigen, potentially affecting the local structure and lipopolysaccharide flexibility, plus cleavage of the O-antigen, enabling the phage to approach the bacterial membrane.

biochemistry↗

bioRxiv2024

Uncovering the electrical synapse proteome in retinal neurons via in vivo proximity labeling

AbstractElectrical synapses containing Connexin 36 (Cx36) represent the main means for direct electrical communication among neurons in the mammalian nervous system. However, little is known about the protein complexes that constitute these synapses. In the present study, we applied different BioID strategies to screen the interactomes of Connexin 36 and its zebrafish orthologue Cx35b in retinal neurons. For in vivo proximity labeling in mice, we took advantage of the Cx36-EGFP strain and expressed a GFP-nanobody-TurboID fusion construct selectively in AII amacrine cells. For in vivo BioID in zebrafish, we generated a transgenic line expressing a Cx35b-TurboID fusion under control of the Cx35b promoter. Both strategies allowed us to capture a plethora of molecules that were associated with electrical synapses and showed a high degree of evolutionary conservation in the proteomes of both species. Besides known interactors of Cx36 such as ZO-1 and ZO-2 we have identified more than 50 new proteins, such as scaffold proteins, adhesion molecules and regulators of the cytoskeleton. Moreover, we determined the subcellular localization of these proteins in mouse retina and tested potential binding interactions with Cx36. Amongst these new interactors, we identified signal induced proliferation associated 1 like 3 (Sipa1l3), a protein that has been implicated in cell junction formation and cell polarity, as a new scaffold of electrical synapses. Interestingly, Sipa1l3 was able to interact with ZO-1, ZO-2 and Cx36, suggesting a pivotal role in electrical synapse function. In summary, our study provides the first detailed view of the electrical synapse proteome in retinal neurons, which is likely to apply to electrical synapses elsewhere.

neuroscience↗