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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

Display of the final Navβ1 3D model with the postulated interface with the α subunit. The location of the successful double mutant TANA becomes evident when comparing it to the template with the projected reversible and irreversible contact surface areas (Fig. 5). Each beta strand is labeled by its letter (from A to G). TANA (T109 → A N110 → A) lies on a prominent loop (flap) between strands E and F. The amino terminal side shows toward the extracellular space, while the carboxy terminal end of the ectodomain of Navβ1 is followed by the transmembrane and intracellular parts (Fig. 1). The protein backbone is displayed in rainbow colors from blue (N-term) over green, yellow and orange to red (C-term). Space-filling atoms mark the model endings. 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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f0045: Display of the final Navβ1 3D model with the postulated interface with the α subunit. The location of the successful double mutant TANA becomes evident when comparing it to the template with the projected reversible and irreversible contact surface areas (Fig. 5). Each beta strand is labeled by its letter (from A to G). TANA (T109 → A N110 → A) lies on a prominent loop (flap) between strands E and F. The amino terminal side shows toward the extracellular space, while the carboxy terminal end of the ectodomain of Navβ1 is followed by the transmembrane and intracellular parts (Fig. 1). The protein backbone is displayed in rainbow colors from blue (N-term) over green, yellow and orange to red (C-term). Space-filling atoms mark the model endings. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.

Mentions: In good keeping with the literature attesting a protein binding role to prominent functional loop residues, the template's amino acids aspartate 349 and asparagine 350 were identified (Table 4, Fig. 8) and the corresponding target segment documented (cf. label “Asp349Asn350” in Fig. 7). Then both adjacent residues were mutated into alanine (T109A, NA) which gave the double mutant its name: TANA (Fig. 9). The combined SDM/electrophysiological studies with wild and mutant type TANA led to the long-awaited general loss of function of Nav1.4 channels in biological tests. Hence, the 3D model of Navβ1 successfully predicted two residues to disturb the interaction between the α/β1 subunit of the Nav1.4 channel.


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)

Display of the final Navβ1 3D model with the postulated interface with the α subunit. The location of the successful double mutant TANA becomes evident when comparing it to the template with the projected reversible and irreversible contact surface areas (Fig. 5). Each beta strand is labeled by its letter (from A to G). TANA (T109 → A N110 → A) lies on a prominent loop (flap) between strands E and F. The amino terminal side shows toward the extracellular space, while the carboxy terminal end of the ectodomain of Navβ1 is followed by the transmembrane and intracellular parts (Fig. 1). The protein backbone is displayed in rainbow colors from blue (N-term) over green, yellow and orange to red (C-term). Space-filling atoms mark the model endings. 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

f0045: Display of the final Navβ1 3D model with the postulated interface with the α subunit. The location of the successful double mutant TANA becomes evident when comparing it to the template with the projected reversible and irreversible contact surface areas (Fig. 5). Each beta strand is labeled by its letter (from A to G). TANA (T109 → A N110 → A) lies on a prominent loop (flap) between strands E and F. The amino terminal side shows toward the extracellular space, while the carboxy terminal end of the ectodomain of Navβ1 is followed by the transmembrane and intracellular parts (Fig. 1). The protein backbone is displayed in rainbow colors from blue (N-term) over green, yellow and orange to red (C-term). Space-filling atoms mark the model endings. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
Mentions: In good keeping with the literature attesting a protein binding role to prominent functional loop residues, the template's amino acids aspartate 349 and asparagine 350 were identified (Table 4, Fig. 8) and the corresponding target segment documented (cf. label “Asp349Asn350” in Fig. 7). Then both adjacent residues were mutated into alanine (T109A, NA) which gave the double mutant its name: TANA (Fig. 9). The combined SDM/electrophysiological studies with wild and mutant type TANA led to the long-awaited general loss of function of Nav1.4 channels in biological tests. Hence, the 3D model of Navβ1 successfully predicted two residues to disturb the interaction between the α/β1 subunit of the Nav1.4 channel.

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