The crystal structure of human Navβ3-Ig domain and its implications

The mammalian Voltage-gated sodium (Na_v) channel is composed of a single α subunit (~ 260 kDa), a multi-pass membrane protein that renders ion selectivity and two or more Na_vβ subunits (25‒40 kDa), that are Type I single-pass membrane proteins and regulate Na_vα subunit function. These subunits are assembled on the plasma membrane of electrically-excitable cells as an intrinsic membrane protein complex and help to initiate and propagate the action potential. The four major mammalian Na_vβ-subunit isoforms, Na_vβ1‒4 proteins possess an N-terminal extracellular Immunoglobulin (Ig) domain (ECD), a single transmembrane α-helix, and an intracellular C-terminal region (ICD). This thesis is mainly focused on the structural biology aspects of the human Na_vβ3 subunit. It reports the atomic structure of the Na_vβ3-Ig domain as determined by X-ray crystallography. Interestingly, the Na_vβ3-Ig domain is observed as a trimer in the crystal structure. The homo-trimer assembly interface lies at the N-terminus and is constrained by a disulphide bond not normally present in Ig domains. The Na_vβ3 subunit Ig domain is known to be glycosylated and contains four potential N-linked glycosylation sites. However, the X-ray crystallography was conducted on deglycosylated protein. Using computational modelling, it is shown that glycan addition would not interfere with Na_vβ3-Ig domain trimerization. Independent evidence gathered using Analytical Ultracentrifugation (crosslinked, glycosylated Na_vβ3-Ig domain, *in vitro*), Proximity Ligation Assay (full-length Na_vβ3, *in vivo*), Atomic Force Microscopy (isolated full-length Na_vβ3, *in vitro*) and Photo-activated Localisation Microscopic experiments (full-length Na_vβ3, *in situ*) support the view that the Na_vβ3 subunit can form trimers when expressed in cells. The biological significance of Na_vβ3 subunit trimerization is discussed. Strategies to express and purify the Na_vβ1/β2/β4-Ig domains were made. Wild type Na_vβ2- and Na_vβ4-Ig domains exist as monomers and dimers, simultaneously in solution, although crystals that diffracted to the necessary resolution were not produced.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Namadurai, Sivakumar

Advisors dc:contributor.advisor

• Jackson, Antony
• Chirgadze, Dimitri

• crystal structure of Navβ3-Ig domain

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f57616d3-c0c4-4500-9442-893ca9b49d4d/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.107561

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/366790