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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

Consensus estimation for the secondary structure of the target Navβ1 amino acid sequence (third line) by NPSA (first line) and JPRED (second line) web tools [40,41]. Symbols: one letter coded amino acids, in red (helical, H), in brown (beta strands/sheet, E), in blue (loop/coiled C). The signal peptide (in positions1 to 18) was typed in lower case letters. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
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f0020: Consensus estimation for the secondary structure of the target Navβ1 amino acid sequence (third line) by NPSA (first line) and JPRED (second line) web tools [40,41]. Symbols: one letter coded amino acids, in red (helical, H), in brown (beta strands/sheet, E), in blue (loop/coiled C). The signal peptide (in positions1 to 18) was typed in lower case letters. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.

Mentions: After secondary structure prediction (Fig. 4) the primary sequence of Navβ1 was threaded through the aforementioned PDB templates, resulting in the empirical selection of our 3D template where the strands, loops and turns matched the predicted secondary structure of the target subunit.


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)

Consensus estimation for the secondary structure of the target Navβ1 amino acid sequence (third line) by NPSA (first line) and JPRED (second line) web tools [40,41]. Symbols: one letter coded amino acids, in red (helical, H), in brown (beta strands/sheet, E), in blue (loop/coiled C). The signal peptide (in positions1 to 18) was typed in lower case letters. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0020: Consensus estimation for the secondary structure of the target Navβ1 amino acid sequence (third line) by NPSA (first line) and JPRED (second line) web tools [40,41]. Symbols: one letter coded amino acids, in red (helical, H), in brown (beta strands/sheet, E), in blue (loop/coiled C). The signal peptide (in positions1 to 18) was typed in lower case letters. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
Mentions: After secondary structure prediction (Fig. 4) the primary sequence of Navβ1 was threaded through the aforementioned PDB templates, resulting in the empirical selection of our 3D template where the strands, loops and turns matched the predicted secondary structure of the target subunit.

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