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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 protein models (first row) and topology schemes (bottom row) of the used template (panel A, 1HCF [59]) in comparison to Navβ4 (panel B, 4MZ2 [30]) and myelin ectodomain (panel C, 1NEU [74]). The β strands (arrow symbols) are labeled. Between β strands C and D (leftmost strand) a larger loop segment allows the formation of two extra β strands, labeled C′ and C″ (panels B and C) [42]. They were analyzed in the following step.
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f0015: Display of protein models (first row) and topology schemes (bottom row) of the used template (panel A, 1HCF [59]) in comparison to Navβ4 (panel B, 4MZ2 [30]) and myelin ectodomain (panel C, 1NEU [74]). The β strands (arrow symbols) are labeled. Between β strands C and D (leftmost strand) a larger loop segment allows the formation of two extra β strands, labeled C′ and C″ (panels B and C) [42]. They were analyzed in the following step.

Mentions: At the end of the present modeling study the crystal structures of Navβ3 and β4 were published (PDB codes: 4L1D [29]; 4MZ2 [30]). Now – during Spring 2015 – we carried out fully automated homology modeling of the target protein and compared the results to our manually generated model (Fig. 3) [59]. It was found that aiming at a higher overall id score was not necessary. A lower id score could include a better structural conservation in just the local hot spot(s). Despite its poor id score (19% in Table 3) our 3D template (PDB code: 1HCF) [59]) yet outperformed the higher scoring templates (PDB codes: 4L1D [29]; 4MZ2 [30]) for four good reasons: (i) allowing to pinpoint unexplored target surface areas in search of residues to be mutated (hot spots). (ii) Even templates with lower id score still conserve the Ig-like fold unit (β-sandwich core). (iii) Even with a higher id score above the twilight zone of homology, templates like 1NEU or Navβ3 and β4 do not present reliable loop coordinates. (iv) Albeit the overall structure of our 3D template had large variation to show in the loop parts – and therein it was not worse than any other template – the lengths, distances or twists of its fold geometry closely resembled that of the two crystal structures of β3 and β4 subunits: strand A–turn–strand B, strand C–(initial part of longer) loop–strand D, strand E–turn–strand F, or strand F–turn–strand G.


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 protein models (first row) and topology schemes (bottom row) of the used template (panel A, 1HCF [59]) in comparison to Navβ4 (panel B, 4MZ2 [30]) and myelin ectodomain (panel C, 1NEU [74]). The β strands (arrow symbols) are labeled. Between β strands C and D (leftmost strand) a larger loop segment allows the formation of two extra β strands, labeled C′ and C″ (panels B and C) [42]. They were analyzed in the following step.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0015: Display of protein models (first row) and topology schemes (bottom row) of the used template (panel A, 1HCF [59]) in comparison to Navβ4 (panel B, 4MZ2 [30]) and myelin ectodomain (panel C, 1NEU [74]). The β strands (arrow symbols) are labeled. Between β strands C and D (leftmost strand) a larger loop segment allows the formation of two extra β strands, labeled C′ and C″ (panels B and C) [42]. They were analyzed in the following step.
Mentions: At the end of the present modeling study the crystal structures of Navβ3 and β4 were published (PDB codes: 4L1D [29]; 4MZ2 [30]). Now – during Spring 2015 – we carried out fully automated homology modeling of the target protein and compared the results to our manually generated model (Fig. 3) [59]. It was found that aiming at a higher overall id score was not necessary. A lower id score could include a better structural conservation in just the local hot spot(s). Despite its poor id score (19% in Table 3) our 3D template (PDB code: 1HCF) [59]) yet outperformed the higher scoring templates (PDB codes: 4L1D [29]; 4MZ2 [30]) for four good reasons: (i) allowing to pinpoint unexplored target surface areas in search of residues to be mutated (hot spots). (ii) Even templates with lower id score still conserve the Ig-like fold unit (β-sandwich core). (iii) Even with a higher id score above the twilight zone of homology, templates like 1NEU or Navβ3 and β4 do not present reliable loop coordinates. (iv) Albeit the overall structure of our 3D template had large variation to show in the loop parts – and therein it was not worse than any other template – the lengths, distances or twists of its fold geometry closely resembled that of the two crystal structures of β3 and β4 subunits: strand A–turn–strand B, strand C–(initial part of longer) loop–strand D, strand E–turn–strand F, or strand F–turn–strand G.

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