Hydrophobic residues in small ankyrin 1 participate in binding to obscurin.
Identifieur interne : 002251 ( Main/Exploration ); précédent : 002250; suivant : 002252Hydrophobic residues in small ankyrin 1 participate in binding to obscurin.
Auteurs : Chris D. Willis [États-Unis] ; Taiji Oashi ; Ben Busby ; Alexander D. Mackerell ; Robert J. BlochSource :
- Molecular membrane biology [ 1464-5203 ] ; 2012.
Descripteurs français
- KwdFr :
- Alanine (génétique), Alanine (métabolisme), Animaux, Ankyrines (), Ankyrines (génétique), Ankyrines (métabolisme), Données de séquences moléculaires, Facteurs d'échange de nucléotides guanyliques (), Facteurs d'échange de nucléotides guanyliques (métabolisme), Interactions hydrophobes et hydrophiles, Isoformes de protéines (), Isoformes de protéines (génétique), Isoformes de protéines (métabolisme), Leucine (génétique), Leucine (métabolisme), Liaison aux protéines, Mutagenèse dirigée, Protéines du muscle (), Protéines du muscle (métabolisme), Protéines recombinantes (), Protéines recombinantes (génétique), Protéines recombinantes (métabolisme), Rats, Résonance plasmonique de surface, Simulation de dynamique moléculaire, Sites de fixation, Séquence d'acides aminés.
- MESH :
- génétique : Alanine, Ankyrines, Isoformes de protéines, Leucine, Protéines recombinantes.
- métabolisme : Alanine, Ankyrines, Facteurs d'échange de nucléotides guanyliques, Isoformes de protéines, Leucine, Protéines du muscle, Protéines recombinantes.
- Animaux, Ankyrines, Données de séquences moléculaires, Facteurs d'échange de nucléotides guanyliques, Interactions hydrophobes et hydrophiles, Isoformes de protéines, Liaison aux protéines, Mutagenèse dirigée, Protéines du muscle, Protéines recombinantes, Rats, Résonance plasmonique de surface, Simulation de dynamique moléculaire, Sites de fixation, Séquence d'acides aminés.
English descriptors
- KwdEn :
- Alanine (genetics), Alanine (metabolism), Amino Acid Sequence, Animals, Ankyrins (chemistry), Ankyrins (genetics), Ankyrins (metabolism), Binding Sites, Guanine Nucleotide Exchange Factors (chemistry), Guanine Nucleotide Exchange Factors (metabolism), Hydrophobic and Hydrophilic Interactions, Leucine (genetics), Leucine (metabolism), Molecular Dynamics Simulation, Molecular Sequence Data, Muscle Proteins (chemistry), Muscle Proteins (metabolism), Mutagenesis, Site-Directed, Protein Binding, Protein Isoforms (chemistry), Protein Isoforms (genetics), Protein Isoforms (metabolism), Rats, Recombinant Proteins (chemistry), Recombinant Proteins (genetics), Recombinant Proteins (metabolism), Surface Plasmon Resonance.
- MESH :
- chemical , chemistry : Ankyrins, Guanine Nucleotide Exchange Factors, Muscle Proteins, Protein Isoforms, Recombinant Proteins.
- chemical , genetics : Alanine, Ankyrins, Leucine, Protein Isoforms, Recombinant Proteins.
- chemical , metabolism : Alanine, Ankyrins, Guanine Nucleotide Exchange Factors, Leucine, Muscle Proteins, Protein Isoforms, Recombinant Proteins.
- Amino Acid Sequence, Animals, Binding Sites, Hydrophobic and Hydrophilic Interactions, Molecular Dynamics Simulation, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Binding, Rats, Surface Plasmon Resonance.
Abstract
Abstract Small ankyrin-1 is a splice variant of the ANK1 gene that binds to obscurin A. Previous studies have identified electrostatic interactions that contribute to this interaction. In addition, molecular dynamics (MD) simulations predict four hydrophobic residues in a 'hot spot' on the surface of the ankyrin-like repeats of sAnk1, near the charged residues involved in binding. We used site-directed mutagenesis, blot overlays and surface plasmon resonance assays to study the contribution of the hydrophobic residues, V70, F71, I102 and I103, to two different 30-mers of obscurin that bind sAnk1, Obsc₆₃₁₆₋₆₃₄₅ and Obsc₆₂₃₁₋₆₂₆₀. Alanine mutations of each of the hydrophobic residues disrupted binding to the high affinity binding site, Obsc₆₃₁₆₋₆₃₄₅. In contrast, V70A and I102A mutations had no effect on binding to the lower affinity site, Obsc₆₂₃₁₋₆₂₆₀. Alanine mutagenesis of the five hydrophobic residues present in Obsc₆₃₁₆₋₆₃₄₅ showed that V6328, I6332, and V6334 were critical to sAnk1 binding. Individual alanine mutants of the six hydrophobic residues of Obsc₆₂₃₁₋₆₂₆₀ had no effect on binding to sAnk1, although a triple alanine mutant of residues V6233/I6234/I6235 decreased binding. We also examined a model of the Obsc₆₃₁₆₋₆₃₄₅-sAnk1 complex in MD simulations and found I102 of sAnk1 to be within 2.2Å of V6334 of Obsc₆₃₁₆₋₆₃₄₅. In contrast to the I102A mutation, mutating I102 of sAnk1 to other hydrophobic amino acids such as phenylalanine or leucine did not disrupt binding to obscurin. Our results suggest that hydrophobic interactions contribute to the higher affinity of Obsc₆₃₁₆₋₆₃₄₅ for sAnk1 and to the dominant role exhibited by this sequence in binding.
DOI: 10.3109/09687688.2012.660709
PubMed: 22416964
Affiliations:
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Willis</name><affiliation wicri:level="4"><nlm:affiliation>Program in Biochemistry and Molecular Biology, University of Maryland, Baltimore, Baltimore, MD, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Program in Biochemistry and Molecular Biology, University of Maryland, Baltimore, Baltimore, MD</wicri:regionArea><placeName><region type="state">Maryland</region><settlement type="city">College Park (Maryland)</settlement></placeName><orgName type="university">Université du Maryland</orgName></affiliation></author><author><name sortKey="Oashi, Taiji" sort="Oashi, Taiji" uniqKey="Oashi T" first="Taiji" last="Oashi">Taiji Oashi</name></author><author><name sortKey="Busby, Ben" sort="Busby, Ben" uniqKey="Busby B" first="Ben" last="Busby">Ben Busby</name></author><author><name sortKey="Mackerell, Alexander D" sort="Mackerell, Alexander D" uniqKey="Mackerell A" first="Alexander D" last="Mackerell">Alexander D. Mackerell</name></author><author><name sortKey="Bloch, Robert J" sort="Bloch, Robert J" uniqKey="Bloch R" first="Robert J" last="Bloch">Robert J. 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Willis</name><affiliation wicri:level="4"><nlm:affiliation>Program in Biochemistry and Molecular Biology, University of Maryland, Baltimore, Baltimore, MD, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Program in Biochemistry and Molecular Biology, University of Maryland, Baltimore, Baltimore, MD</wicri:regionArea><placeName><region type="state">Maryland</region><settlement type="city">College Park (Maryland)</settlement></placeName><orgName type="university">Université du Maryland</orgName></affiliation></author><author><name sortKey="Oashi, Taiji" sort="Oashi, Taiji" uniqKey="Oashi T" first="Taiji" last="Oashi">Taiji Oashi</name></author><author><name sortKey="Busby, Ben" sort="Busby, Ben" uniqKey="Busby B" first="Ben" last="Busby">Ben Busby</name></author><author><name sortKey="Mackerell, Alexander D" sort="Mackerell, Alexander D" uniqKey="Mackerell A" first="Alexander D" last="Mackerell">Alexander D. Mackerell</name></author><author><name sortKey="Bloch, Robert J" sort="Bloch, Robert J" uniqKey="Bloch R" first="Robert J" last="Bloch">Robert J. Bloch</name></author></analytic><series><title level="j">Molecular membrane biology</title><idno type="eISSN">1464-5203</idno><imprint><date when="2012" type="published">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Alanine (genetics)</term><term>Alanine (metabolism)</term><term>Amino Acid Sequence</term><term>Animals</term><term>Ankyrins (chemistry)</term><term>Ankyrins (genetics)</term><term>Ankyrins (metabolism)</term><term>Binding Sites</term><term>Guanine Nucleotide Exchange Factors (chemistry)</term><term>Guanine Nucleotide Exchange Factors (metabolism)</term><term>Hydrophobic and Hydrophilic Interactions</term><term>Leucine (genetics)</term><term>Leucine (metabolism)</term><term>Molecular Dynamics Simulation</term><term>Molecular Sequence Data</term><term>Muscle Proteins (chemistry)</term><term>Muscle Proteins (metabolism)</term><term>Mutagenesis, Site-Directed</term><term>Protein Binding</term><term>Protein Isoforms (chemistry)</term><term>Protein Isoforms (genetics)</term><term>Protein Isoforms (metabolism)</term><term>Rats</term><term>Recombinant Proteins (chemistry)</term><term>Recombinant Proteins (genetics)</term><term>Recombinant Proteins (metabolism)</term><term>Surface Plasmon Resonance</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Alanine (génétique)</term><term>Alanine (métabolisme)</term><term>Animaux</term><term>Ankyrines ()</term><term>Ankyrines (génétique)</term><term>Ankyrines (métabolisme)</term><term>Données de séquences moléculaires</term><term>Facteurs d'échange de nucléotides guanyliques ()</term><term>Facteurs d'échange de nucléotides guanyliques (métabolisme)</term><term>Interactions hydrophobes et hydrophiles</term><term>Isoformes de protéines ()</term><term>Isoformes de protéines (génétique)</term><term>Isoformes de protéines (métabolisme)</term><term>Leucine (génétique)</term><term>Leucine (métabolisme)</term><term>Liaison aux protéines</term><term>Mutagenèse dirigée</term><term>Protéines du muscle ()</term><term>Protéines du muscle (métabolisme)</term><term>Protéines recombinantes ()</term><term>Protéines recombinantes (génétique)</term><term>Protéines recombinantes (métabolisme)</term><term>Rats</term><term>Résonance plasmonique de surface</term><term>Simulation de dynamique moléculaire</term><term>Sites de fixation</term><term>Séquence d'acides aminés</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Ankyrins</term><term>Guanine Nucleotide Exchange Factors</term><term>Muscle Proteins</term><term>Protein Isoforms</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Alanine</term><term>Ankyrins</term><term>Leucine</term><term>Protein Isoforms</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Alanine</term><term>Ankyrins</term><term>Guanine Nucleotide Exchange Factors</term><term>Leucine</term><term>Muscle Proteins</term><term>Protein Isoforms</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Alanine</term><term>Ankyrines</term><term>Isoformes de protéines</term><term>Leucine</term><term>Protéines recombinantes</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Alanine</term><term>Ankyrines</term><term>Facteurs d'échange de nucléotides guanyliques</term><term>Isoformes de protéines</term><term>Leucine</term><term>Protéines du muscle</term><term>Protéines recombinantes</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term><term>Animals</term><term>Binding Sites</term><term>Hydrophobic and Hydrophilic Interactions</term><term>Molecular Dynamics Simulation</term><term>Molecular Sequence Data</term><term>Mutagenesis, Site-Directed</term><term>Protein Binding</term><term>Rats</term><term>Surface Plasmon Resonance</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Ankyrines</term><term>Données de séquences moléculaires</term><term>Facteurs d'échange de nucléotides guanyliques</term><term>Interactions hydrophobes et hydrophiles</term><term>Isoformes de protéines</term><term>Liaison aux protéines</term><term>Mutagenèse dirigée</term><term>Protéines du muscle</term><term>Protéines recombinantes</term><term>Rats</term><term>Résonance plasmonique de surface</term><term>Simulation de dynamique moléculaire</term><term>Sites de fixation</term><term>Séquence d'acides aminés</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract Small ankyrin-1 is a splice variant of the ANK1 gene that binds to obscurin A. Previous studies have identified electrostatic interactions that contribute to this interaction. In addition, molecular dynamics (MD) simulations predict four hydrophobic residues in a 'hot spot' on the surface of the ankyrin-like repeats of sAnk1, near the charged residues involved in binding. We used site-directed mutagenesis, blot overlays and surface plasmon resonance assays to study the contribution of the hydrophobic residues, V70, F71, I102 and I103, to two different 30-mers of obscurin that bind sAnk1, Obsc₆₃₁₆₋₆₃₄₅ and Obsc₆₂₃₁₋₆₂₆₀. Alanine mutations of each of the hydrophobic residues disrupted binding to the high affinity binding site, Obsc₆₃₁₆₋₆₃₄₅. In contrast, V70A and I102A mutations had no effect on binding to the lower affinity site, Obsc₆₂₃₁₋₆₂₆₀. Alanine mutagenesis of the five hydrophobic residues present in Obsc₆₃₁₆₋₆₃₄₅ showed that V6328, I6332, and V6334 were critical to sAnk1 binding. Individual alanine mutants of the six hydrophobic residues of Obsc₆₂₃₁₋₆₂₆₀ had no effect on binding to sAnk1, although a triple alanine mutant of residues V6233/I6234/I6235 decreased binding. We also examined a model of the Obsc₆₃₁₆₋₆₃₄₅-sAnk1 complex in MD simulations and found I102 of sAnk1 to be within 2.2Å of V6334 of Obsc₆₃₁₆₋₆₃₄₅. In contrast to the I102A mutation, mutating I102 of sAnk1 to other hydrophobic amino acids such as phenylalanine or leucine did not disrupt binding to obscurin. Our results suggest that hydrophobic interactions contribute to the higher affinity of Obsc₆₃₁₆₋₆₃₄₅ for sAnk1 and to the dominant role exhibited by this sequence in binding.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Maryland</li></region><settlement><li>College Park (Maryland)</li></settlement><orgName><li>Université du Maryland</li></orgName></list><tree><noCountry><name sortKey="Bloch, Robert J" sort="Bloch, Robert J" uniqKey="Bloch R" first="Robert J" last="Bloch">Robert J. Bloch</name><name sortKey="Busby, Ben" sort="Busby, Ben" uniqKey="Busby B" first="Ben" last="Busby">Ben Busby</name><name sortKey="Mackerell, Alexander D" sort="Mackerell, Alexander D" uniqKey="Mackerell A" first="Alexander D" last="Mackerell">Alexander D. Mackerell</name><name sortKey="Oashi, Taiji" sort="Oashi, Taiji" uniqKey="Oashi T" first="Taiji" last="Oashi">Taiji Oashi</name></noCountry><country name="États-Unis"><region name="Maryland"><name sortKey="Willis, Chris D" sort="Willis, Chris D" uniqKey="Willis C" first="Chris D" last="Willis">Chris D. Willis</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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