Limits...
Mutational analysis of the rotavirus NSP4 enterotoxic domain that binds to caveolin-1.

Ball JM, Schroeder ME, Williams CV, Schroeder F, Parr RD - Virol. J. (2013)

Bottom Line: Analyses of mutant synthetic peptides in which the hydrophobic residues in the enterotoxic domain of NSP4 were altered suggested a critical hydrophobic residue.Both NSP4HydroMut112-140, that contains three charged amino acids (aa113, 124, 131) changed from the original hydrophobic residues and NSP4AlaAcidic112-140 that contained three alanine residues substituted for negatively charged (aa114, 125, 132) amino acids failed to induce diarrhea.Whereas peptides NSP4wild type 112-140 and NSP4AlaBasic112-140 that contained three alanine substituted for positively charged (aa115, 119, 133) amino acids, induced diarrhea.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathobiology, Texas A&M University, TVMC, College Station, Texas 77843-4467, USA. rebeccadparr@gmail.com.

ABSTRACT

Background: Rotavirus (RV) nonstructural protein 4 (NSP4) is the first described viral enterotoxin, which induces early secretory diarrhea in neonatal rodents. Our previous data show a direct interaction between RV NSP4 and the structural protein of caveolae, caveolin-1 (cav-1), in yeast and mammalian cells. The binding site of cav-1 mapped to the NSP4 amphipathic helix, and led us to examine which helical face was responsible for the interaction.

Methods: A panel of NSP4 mutants were prepared and tested for binding to cav-1 by yeast two hybrid and direct binding assays. The charged residues of the NSP4 amphipathic helix were changed to alanine (NSP446-175-ala6); and three residues in the hydrophobic face were altered to charged amino acids (NSP4(46-175)-HydroMut). In total, twelve mutants of NSP4 were generated to define the cav-1 binding site. Synthetic peptides corresponding to the hydrophobic and charged faces of NSP4 were examined for structural changes by circular dichroism (CD) and diarrhea induction by a neonatal mouse study.

Results: Mutations of the hydrophilic face (NSP4(46-175)-Ala6) bound cav-1 akin to wild type NSP4. In contrast, disruption of the hydrophobic face (NSP4(46-175)-HydroMut) failed to bind cav-1. These data suggest NSP4 and cav-1 associate via a hydrophobic interaction. Analyses of mutant synthetic peptides in which the hydrophobic residues in the enterotoxic domain of NSP4 were altered suggested a critical hydrophobic residue. Both NSP4HydroMut112-140, that contains three charged amino acids (aa113, 124, 131) changed from the original hydrophobic residues and NSP4AlaAcidic112-140 that contained three alanine residues substituted for negatively charged (aa114, 125, 132) amino acids failed to induce diarrhea. Whereas peptides NSP4wild type 112-140 and NSP4AlaBasic112-140 that contained three alanine substituted for positively charged (aa115, 119, 133) amino acids, induced diarrhea.

Conclusions: These data show that the cav-1 binding domain is within the hydrophobic face of the NSP4 amphipathic helix. The integrity of the helical structure is important for both cav-1 binding and diarrhea induction implying a connection between NSP4 functional and binding activities.

Show MeSH

Related in: MedlinePlus

A and B Organization of the Polar and Hydrophobic faces of the NSP4 crystal structure of the enterotoxic AAH (A and B respectively). A shows the acidic amino acids, D114, E122, E125 and D132 depicted in a maroon color. The basic amino acids K115, R119, R129 and K133 are shown in blue. B shows the hydrophobic amino acids of the AAH of NSP4 I113, L116, V124, L127, I130, Y131 and L134 in purple. C PyMol representations of the hydrophobic face of NSP4-(residues 46-175) and mutants. Amino acids in purple (I113, V124 and Y131) are indicated by arrows represent the wild type NSP4. Eight mutant clones are viewed below the wild type NSP4. The mutations, I113R, V124D and Y131K are orange, and amino acids that are mutated to alanines are shown as blue. To the right of each represented clone, the results of the yeast two hybrid and the peptide binding assays are given.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3924327&req=5

Figure 1: A and B Organization of the Polar and Hydrophobic faces of the NSP4 crystal structure of the enterotoxic AAH (A and B respectively). A shows the acidic amino acids, D114, E122, E125 and D132 depicted in a maroon color. The basic amino acids K115, R119, R129 and K133 are shown in blue. B shows the hydrophobic amino acids of the AAH of NSP4 I113, L116, V124, L127, I130, Y131 and L134 in purple. C PyMol representations of the hydrophobic face of NSP4-(residues 46-175) and mutants. Amino acids in purple (I113, V124 and Y131) are indicated by arrows represent the wild type NSP4. Eight mutant clones are viewed below the wild type NSP4. The mutations, I113R, V124D and Y131K are orange, and amino acids that are mutated to alanines are shown as blue. To the right of each represented clone, the results of the yeast two hybrid and the peptide binding assays are given.

Mentions: To identify the face of the amphipathic helix that binds cav-1, a 3-D structural model was generated using the crystallographic determinants for NSP4 95-135 [42] and visualized using PyMol (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC). The six charged residues between 114 and 135 were mutated to alanine (D114A, K115A, R119A, E125A, D132A, and K133A) in FLNSP4-Ala6, and three hydrophobic residues were mutated to charged residues (I113R, V124K, and Y131D), in FLNSP4-HydroMut (Figure 1A and B, Table 1).


Mutational analysis of the rotavirus NSP4 enterotoxic domain that binds to caveolin-1.

Ball JM, Schroeder ME, Williams CV, Schroeder F, Parr RD - Virol. J. (2013)

A and B Organization of the Polar and Hydrophobic faces of the NSP4 crystal structure of the enterotoxic AAH (A and B respectively). A shows the acidic amino acids, D114, E122, E125 and D132 depicted in a maroon color. The basic amino acids K115, R119, R129 and K133 are shown in blue. B shows the hydrophobic amino acids of the AAH of NSP4 I113, L116, V124, L127, I130, Y131 and L134 in purple. C PyMol representations of the hydrophobic face of NSP4-(residues 46-175) and mutants. Amino acids in purple (I113, V124 and Y131) are indicated by arrows represent the wild type NSP4. Eight mutant clones are viewed below the wild type NSP4. The mutations, I113R, V124D and Y131K are orange, and amino acids that are mutated to alanines are shown as blue. To the right of each represented clone, the results of the yeast two hybrid and the peptide binding assays are given.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3924327&req=5

Figure 1: A and B Organization of the Polar and Hydrophobic faces of the NSP4 crystal structure of the enterotoxic AAH (A and B respectively). A shows the acidic amino acids, D114, E122, E125 and D132 depicted in a maroon color. The basic amino acids K115, R119, R129 and K133 are shown in blue. B shows the hydrophobic amino acids of the AAH of NSP4 I113, L116, V124, L127, I130, Y131 and L134 in purple. C PyMol representations of the hydrophobic face of NSP4-(residues 46-175) and mutants. Amino acids in purple (I113, V124 and Y131) are indicated by arrows represent the wild type NSP4. Eight mutant clones are viewed below the wild type NSP4. The mutations, I113R, V124D and Y131K are orange, and amino acids that are mutated to alanines are shown as blue. To the right of each represented clone, the results of the yeast two hybrid and the peptide binding assays are given.
Mentions: To identify the face of the amphipathic helix that binds cav-1, a 3-D structural model was generated using the crystallographic determinants for NSP4 95-135 [42] and visualized using PyMol (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC). The six charged residues between 114 and 135 were mutated to alanine (D114A, K115A, R119A, E125A, D132A, and K133A) in FLNSP4-Ala6, and three hydrophobic residues were mutated to charged residues (I113R, V124K, and Y131D), in FLNSP4-HydroMut (Figure 1A and B, Table 1).

Bottom Line: Analyses of mutant synthetic peptides in which the hydrophobic residues in the enterotoxic domain of NSP4 were altered suggested a critical hydrophobic residue.Both NSP4HydroMut112-140, that contains three charged amino acids (aa113, 124, 131) changed from the original hydrophobic residues and NSP4AlaAcidic112-140 that contained three alanine residues substituted for negatively charged (aa114, 125, 132) amino acids failed to induce diarrhea.Whereas peptides NSP4wild type 112-140 and NSP4AlaBasic112-140 that contained three alanine substituted for positively charged (aa115, 119, 133) amino acids, induced diarrhea.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathobiology, Texas A&M University, TVMC, College Station, Texas 77843-4467, USA. rebeccadparr@gmail.com.

ABSTRACT

Background: Rotavirus (RV) nonstructural protein 4 (NSP4) is the first described viral enterotoxin, which induces early secretory diarrhea in neonatal rodents. Our previous data show a direct interaction between RV NSP4 and the structural protein of caveolae, caveolin-1 (cav-1), in yeast and mammalian cells. The binding site of cav-1 mapped to the NSP4 amphipathic helix, and led us to examine which helical face was responsible for the interaction.

Methods: A panel of NSP4 mutants were prepared and tested for binding to cav-1 by yeast two hybrid and direct binding assays. The charged residues of the NSP4 amphipathic helix were changed to alanine (NSP446-175-ala6); and three residues in the hydrophobic face were altered to charged amino acids (NSP4(46-175)-HydroMut). In total, twelve mutants of NSP4 were generated to define the cav-1 binding site. Synthetic peptides corresponding to the hydrophobic and charged faces of NSP4 were examined for structural changes by circular dichroism (CD) and diarrhea induction by a neonatal mouse study.

Results: Mutations of the hydrophilic face (NSP4(46-175)-Ala6) bound cav-1 akin to wild type NSP4. In contrast, disruption of the hydrophobic face (NSP4(46-175)-HydroMut) failed to bind cav-1. These data suggest NSP4 and cav-1 associate via a hydrophobic interaction. Analyses of mutant synthetic peptides in which the hydrophobic residues in the enterotoxic domain of NSP4 were altered suggested a critical hydrophobic residue. Both NSP4HydroMut112-140, that contains three charged amino acids (aa113, 124, 131) changed from the original hydrophobic residues and NSP4AlaAcidic112-140 that contained three alanine residues substituted for negatively charged (aa114, 125, 132) amino acids failed to induce diarrhea. Whereas peptides NSP4wild type 112-140 and NSP4AlaBasic112-140 that contained three alanine substituted for positively charged (aa115, 119, 133) amino acids, induced diarrhea.

Conclusions: These data show that the cav-1 binding domain is within the hydrophobic face of the NSP4 amphipathic helix. The integrity of the helical structure is important for both cav-1 binding and diarrhea induction implying a connection between NSP4 functional and binding activities.

Show MeSH
Related in: MedlinePlus