Biology subjects

Hamaia, S. W.

Publications and source records attributed to Hamaia, S. W..

3 recordsLinked to original sources

bioRxiv2025

Structural implications of glycosylation on the voltage-gated sodium channel β3-subunit

Voltage-gated sodium (NaV) channel -subunits are modulated by associated {beta}-subunits that affect their localization, trafficking and gating behaviour. The {beta}-subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules and the interactions between their extracellular Ig-domains may modify channel clustering. The full-length {beta}3-subunit can form cis trimers on the plasma membrane. The atomic resolution structure of a deglycosylated trimeric {beta}3-subunit Ig-domain has been solved by X-ray crystallography. However, it is not clear whether this particular trimeric Ig-domain structure is plausible for cell-expressed, glycosylated {beta}3-subunits. Here we use glycan profiling to confirm an extensive and heterogeneous pattern of {beta}3-subunit glycosylation, with the majority of glycans being bi- and tri-antennary structures with one or two terminal sialic acids. Two tryptic peptides of the {beta}3 Ig-domain are predicted to contain potential N-linked glycosylation sites. When the isolated, glycosylated full-length {beta}3-subunit was trypsin-digested and analysed by LC-MS/MS, only one of these peptides - containing an N-linked glycosylation site at residue N95 and located close to the trimer interface - was identified in its unmodified form, suggesting that residue N95 is under-glycosylated. All-atom molecular dynamics simulations of the glycosylated, membrane-bound full-length {beta}3 trimer confirmed that glycans can be accommodated with the Ig-domain trimer and indeed, may contribute to protein-membrane and inter-protomer interactions within the full-length, membrane-embedded trimer. Further biochemical studies are warranted to explore the interactions between oligomeric {beta}-subunits with corresponding -subunit sodium channels.

biochemistry↗

bioRxiv2024

Structural basis for collagen recognition by the Streptococcus pyogenes M3 protein and its involvement in biofilm

The M protein is an essential virulence factor of Streptococcus pyogenes, or group A streptococcus (GAS), one of the most common and dangerous human pathogens. Molecular and functional characterization of M protein variants and their interactions with host components is crucial for understanding streptococcal pathogenesis and vaccine development. The M3 protein is produced by the prevalent emm3 GAS serotype, which is frequently associated with severe invasive diseases. Here we characterize the interaction of M3 with human collagens through detailed structural and biochemical binding analysis. High-resolution structures of the N-terminal M3 domain in the free state as well as bound to a collagen peptide derived from the Collagen Ligands Collection reveal a novel T-shaped protein fold that presents binding sites complementing the characteristic topology of collagen triple helices. The structure of the M3/collagen peptide complex explains how emm3 GAS and related streptococci, such as the emerging human pathogen Streptococcus dysgalactiae subsp. equisimilis, can target collagens to enable colonization of various tissues. In line with this, we demonstrate that the M3/collagen interaction promotes enhanced biofilm formation of emm3 GAS in an emm type specific manner, which can be inhibited with the recombinant M3 N-terminal domain fragment. Further, emm3 GAS are shown to colocalize with collagen in tissue biopsies from patients with necrotizing soft tissue infections, where GAS biofilms are common. This observation is reproduced in infected organotypic skin models. Together, these data provide detailed molecular insights into an important streptococcal virulence mechanism with implications for the understanding of invasive infections, strategies for treating biofilm and M-protein based vaccine design.

microbiology↗

bioRxiv2024

Isoform-specific N-linked glycosylation of voltage-gated sodium channel alpha-subunits alters beta-subunit binding sites

HighlightsO_LIThree N-linked glycosylation sites conserved among all Nav channels C_LIO_LIGlycan modelling and molecular dynamics simulations highlight 3D landscape C_LIO_LIUnique Nav1.5 N-linked glycans may prevent binding to Ig-domains of {beta}1 and {beta}3 C_LIO_LIUnique Nav1.8 N-linked glycan may prevent binding to Ig-domains of {beta}2 and {beta}4 C_LIO_LIN-linked glycans likely contribute to supra-molecular clustering of Nav channels C_LI Voltage-gated sodium channel -subunits (Nav1.1-1.9) initiate and propagate action potentials in neurons and myocytes. The Nav {beta}-subunits ({beta}1-4) have been shown to modulate -subunit properties. Homo-oligomerization of {beta}-subunits on neighboring or opposing plasma membranes has been suggested to facilitate cis or trans interactions, respectively. The interactions between several Nav channel isoforms and {beta}-subunits have been determined using cryogenic electron microscopy (cryo-EM). Interestingly, the Nav cryo-EM structures reveal the presence of N-linked glycosylation sites. However, only the first glycan moieties are typically resolved at each site due to the flexibility of mature glycan trees. Thus, existing cryo-EM structures may risk de-emphasizing the structural implications of glycans on the Nav channels. Herein, molecular modelling and all-atom molecular dynamics simulations were applied to investigate the conformational landscape of N-linked glycans on Nav channel surfaces. The simulations revealed that negatively-charged sialic acid residues of two glycan sites may interact with voltage-sensing domains. Notably, two Nav1.5 isoform-specific glycans extensively cover the -subunit region that, in other Nav channel -subunit isoforms, corresponds to the binding site for the {beta}1-(and likely {beta}3-) subunit immunoglobulin (Ig) domain. Nav1.8 contains a unique N-linked glycosylation site that likely prevents its interaction with the {beta}2 and {beta}4-subunit Ig domain. These isoform-specific glycans may have evolved to facilitate specific functional interactions, for example by redirecting {beta}-subunit Ig-domains outwards to permit cis or trans supra-clustering within specialized cellular compartments such as the cardiomyocyte perinexal space. Further experimental work is necessary to validate these predictions.

biochemistry↗