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Predicting a double mutant in the twilight zone of low homology modeling for the skeletal muscle voltage-gated sodium channel subunit beta-1 (Nav1.4 β1).

Scior T, Paiz-Candia B, Islas ÁA, Sánchez-Solano A, Millan-Perez Peña L, Mancilla-Simbro C, Salinas-Stefanon EM - Comput Struct Biotechnol J (2015)

Bottom Line: Despite the distant phylogenic relationships, we found a 3D-template to identify two adjacent amino acids leading to the long-awaited loss of function (inactivation) of Nav1.4 channels.Exhaustive and unbiased sampling of "all β proteins" (Ig-like, Ig) resulted in a plethora of 3D templates which were compared to the target secondary structure prediction.The location of TANA was made possible thanks to another "all β protein" structure in complex with an irreversible bound protein as well as a reversible protein-protein interface (our "Rosetta Stone" effect).

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias Químicas, Universidad Autónoma de Puebla, Puebla, Mexico.

ABSTRACT
The molecular structure modeling of the β1 subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4) was carried out in the twilight zone of very low homology. Structural significance can per se be confounded with random sequence similarities. Hence, we combined (i) not automated computational modeling of weakly homologous 3D templates, some with interfaces to analogous structures to the pore-bearing Nav1.4 α subunit with (ii) site-directed mutagenesis (SDM), as well as (iii) electrophysiological experiments to study the structure and function of the β1 subunit. Despite the distant phylogenic relationships, we found a 3D-template to identify two adjacent amino acids leading to the long-awaited loss of function (inactivation) of Nav1.4 channels. This mutant type (T109A, N110A, herein called TANA) was expressed and tested on cells of hamster ovary (CHO). The present electrophysiological results showed that the double alanine substitution TANA disrupted channel inactivation as if the β1 subunit would not be in complex with the α subunit. Exhaustive and unbiased sampling of "all β proteins" (Ig-like, Ig) resulted in a plethora of 3D templates which were compared to the target secondary structure prediction. The location of TANA was made possible thanks to another "all β protein" structure in complex with an irreversible bound protein as well as a reversible protein-protein interface (our "Rosetta Stone" effect). This finding coincides with our electrophysiological data (disrupted β1-like voltage dependence) and it is safe to utter that the Nav1.4 α/β1 interface is likely to be of reversible nature.

No MeSH data available.


Related in: MedlinePlus

Schematic display of the main chain with its principal domains for the Navβ1 subunit. It documents the location of the major structural domains of Scn1b sodium channel voltage-gated subunit β type 1 Rattus norvegicus (accession code: Q00954) [4]. Shown are the sequence (A) and topology (B) which embraces the signal peptide (pentagons, length: 1–18), extracellular immunoglobulin domain (circles), transmembrane domain (hexagons), and intracellular domain (diamonds). The triangles symbolize the linker domain. Every second symbol is filled black or left white to mark the alternative neighbors.
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f0005: Schematic display of the main chain with its principal domains for the Navβ1 subunit. It documents the location of the major structural domains of Scn1b sodium channel voltage-gated subunit β type 1 Rattus norvegicus (accession code: Q00954) [4]. Shown are the sequence (A) and topology (B) which embraces the signal peptide (pentagons, length: 1–18), extracellular immunoglobulin domain (circles), transmembrane domain (hexagons), and intracellular domain (diamonds). The triangles symbolize the linker domain. Every second symbol is filled black or left white to mark the alternative neighbors.

Mentions: The primary sequence of the rat Na+ channel subunit β1 (rNavβ1) was retrieved from the UniProt web service (accession code: Q00954 [4,32]). The extracellular domain was limited to 142 residues excluding the signal peptide (Fig. 1).


Predicting a double mutant in the twilight zone of low homology modeling for the skeletal muscle voltage-gated sodium channel subunit beta-1 (Nav1.4 β1).

Scior T, Paiz-Candia B, Islas ÁA, Sánchez-Solano A, Millan-Perez Peña L, Mancilla-Simbro C, Salinas-Stefanon EM - Comput Struct Biotechnol J (2015)

Schematic display of the main chain with its principal domains for the Navβ1 subunit. It documents the location of the major structural domains of Scn1b sodium channel voltage-gated subunit β type 1 Rattus norvegicus (accession code: Q00954) [4]. Shown are the sequence (A) and topology (B) which embraces the signal peptide (pentagons, length: 1–18), extracellular immunoglobulin domain (circles), transmembrane domain (hexagons), and intracellular domain (diamonds). The triangles symbolize the linker domain. Every second symbol is filled black or left white to mark the alternative neighbors.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402383&req=5

f0005: Schematic display of the main chain with its principal domains for the Navβ1 subunit. It documents the location of the major structural domains of Scn1b sodium channel voltage-gated subunit β type 1 Rattus norvegicus (accession code: Q00954) [4]. Shown are the sequence (A) and topology (B) which embraces the signal peptide (pentagons, length: 1–18), extracellular immunoglobulin domain (circles), transmembrane domain (hexagons), and intracellular domain (diamonds). The triangles symbolize the linker domain. Every second symbol is filled black or left white to mark the alternative neighbors.
Mentions: The primary sequence of the rat Na+ channel subunit β1 (rNavβ1) was retrieved from the UniProt web service (accession code: Q00954 [4,32]). The extracellular domain was limited to 142 residues excluding the signal peptide (Fig. 1).

Bottom Line: Despite the distant phylogenic relationships, we found a 3D-template to identify two adjacent amino acids leading to the long-awaited loss of function (inactivation) of Nav1.4 channels.Exhaustive and unbiased sampling of "all β proteins" (Ig-like, Ig) resulted in a plethora of 3D templates which were compared to the target secondary structure prediction.The location of TANA was made possible thanks to another "all β protein" structure in complex with an irreversible bound protein as well as a reversible protein-protein interface (our "Rosetta Stone" effect).

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias Químicas, Universidad Autónoma de Puebla, Puebla, Mexico.

ABSTRACT
The molecular structure modeling of the β1 subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4) was carried out in the twilight zone of very low homology. Structural significance can per se be confounded with random sequence similarities. Hence, we combined (i) not automated computational modeling of weakly homologous 3D templates, some with interfaces to analogous structures to the pore-bearing Nav1.4 α subunit with (ii) site-directed mutagenesis (SDM), as well as (iii) electrophysiological experiments to study the structure and function of the β1 subunit. Despite the distant phylogenic relationships, we found a 3D-template to identify two adjacent amino acids leading to the long-awaited loss of function (inactivation) of Nav1.4 channels. This mutant type (T109A, N110A, herein called TANA) was expressed and tested on cells of hamster ovary (CHO). The present electrophysiological results showed that the double alanine substitution TANA disrupted channel inactivation as if the β1 subunit would not be in complex with the α subunit. Exhaustive and unbiased sampling of "all β proteins" (Ig-like, Ig) resulted in a plethora of 3D templates which were compared to the target secondary structure prediction. The location of TANA was made possible thanks to another "all β protein" structure in complex with an irreversible bound protein as well as a reversible protein-protein interface (our "Rosetta Stone" effect). This finding coincides with our electrophysiological data (disrupted β1-like voltage dependence) and it is safe to utter that the Nav1.4 α/β1 interface is likely to be of reversible nature.

No MeSH data available.


Related in: MedlinePlus