Directed evolution reveals the binding motif preference of the LC8/DYNLL hub protein and predicts large numbers of novel binders in the human proteome.

LC8 dynein light chain (DYNLL) is a eukaryotic hub protein that is thought to function as a dimerization engine. Its interacting partners are involved in a wide range of cellular functions. In its dozens of hitherto identified binding partners DYNLL binds to a linear peptide segment. The known segme...

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Autores principales: Péter Rapali, László Radnai, Dániel Süveges, Veronika Harmat, Ferenc Tölgyesi, Weixiao Y Wahlgren, Gergely Katona, László Nyitray, Gábor Pál
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Publicado: Public Library of Science (PLoS) 2011
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spelling oai:doaj.org-article:b7cb1bc7ab3c47f7b216f39213f9e3402021-11-18T06:55:38ZDirected evolution reveals the binding motif preference of the LC8/DYNLL hub protein and predicts large numbers of novel binders in the human proteome.1932-620310.1371/journal.pone.0018818https://doaj.org/article/b7cb1bc7ab3c47f7b216f39213f9e3402011-04-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21533121/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203LC8 dynein light chain (DYNLL) is a eukaryotic hub protein that is thought to function as a dimerization engine. Its interacting partners are involved in a wide range of cellular functions. In its dozens of hitherto identified binding partners DYNLL binds to a linear peptide segment. The known segments define a loosely characterized binding motif: [D/S](-4)K(-3)X(-2)[T/V/I](-1)Q(0)[T/V](1)[D/E](2). The motifs are localized in disordered segments of the DYNLL-binding proteins and are often flanked by coiled coil or other potential dimerization domains. Based on a directed evolution approach, here we provide the first quantitative characterization of the binding preference of the DYNLL binding site. We displayed on M13 phage a naïve peptide library with seven fully randomized positions around a fixed, naturally conserved glutamine. The peptides were presented in a bivalent manner fused to a leucine zipper mimicking the natural dimer to dimer binding stoichiometry of DYNLL-partner complexes. The phage-selected consensus sequence V(-5)S(-4)R(-3)G(-2)T(-1)Q(0)T(1)E(2) resembles the natural one, but is extended by an additional N-terminal valine, which increases the affinity of the monomeric peptide twentyfold. Leu-zipper dimerization increases the affinity into the subnanomolar range. By comparing crystal structures of an SRGTQTE-DYNLL and a dimeric VSRGTQTE-DYNLL complex we find that the affinity enhancing valine is accommodated in a binding pocket on DYNLL. Based on the in vitro evolved sequence pattern we predict a large number of novel DYNLL binding partners in the human proteome. Among these EML3, a microtubule-binding protein involved in mitosis contains an exact match of the phage-evolved consensus and binds to DYNLL with nanomolar affinity. These results significantly widen the scope of the human interactome around DYNLL and will certainly shed more light on the biological functions and organizing role of DYNLL in the human and other eukaryotic interactomes.Péter RapaliLászló RadnaiDániel SüvegesVeronika HarmatFerenc TölgyesiWeixiao Y WahlgrenGergely KatonaLászló NyitrayGábor PálPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 4, p e18818 (2011)
institution DOAJ
collection DOAJ
language EN
topic Medicine
R
Science
Q
spellingShingle Medicine
R
Science
Q
Péter Rapali
László Radnai
Dániel Süveges
Veronika Harmat
Ferenc Tölgyesi
Weixiao Y Wahlgren
Gergely Katona
László Nyitray
Gábor Pál
Directed evolution reveals the binding motif preference of the LC8/DYNLL hub protein and predicts large numbers of novel binders in the human proteome.
description LC8 dynein light chain (DYNLL) is a eukaryotic hub protein that is thought to function as a dimerization engine. Its interacting partners are involved in a wide range of cellular functions. In its dozens of hitherto identified binding partners DYNLL binds to a linear peptide segment. The known segments define a loosely characterized binding motif: [D/S](-4)K(-3)X(-2)[T/V/I](-1)Q(0)[T/V](1)[D/E](2). The motifs are localized in disordered segments of the DYNLL-binding proteins and are often flanked by coiled coil or other potential dimerization domains. Based on a directed evolution approach, here we provide the first quantitative characterization of the binding preference of the DYNLL binding site. We displayed on M13 phage a naïve peptide library with seven fully randomized positions around a fixed, naturally conserved glutamine. The peptides were presented in a bivalent manner fused to a leucine zipper mimicking the natural dimer to dimer binding stoichiometry of DYNLL-partner complexes. The phage-selected consensus sequence V(-5)S(-4)R(-3)G(-2)T(-1)Q(0)T(1)E(2) resembles the natural one, but is extended by an additional N-terminal valine, which increases the affinity of the monomeric peptide twentyfold. Leu-zipper dimerization increases the affinity into the subnanomolar range. By comparing crystal structures of an SRGTQTE-DYNLL and a dimeric VSRGTQTE-DYNLL complex we find that the affinity enhancing valine is accommodated in a binding pocket on DYNLL. Based on the in vitro evolved sequence pattern we predict a large number of novel DYNLL binding partners in the human proteome. Among these EML3, a microtubule-binding protein involved in mitosis contains an exact match of the phage-evolved consensus and binds to DYNLL with nanomolar affinity. These results significantly widen the scope of the human interactome around DYNLL and will certainly shed more light on the biological functions and organizing role of DYNLL in the human and other eukaryotic interactomes.
format article
author Péter Rapali
László Radnai
Dániel Süveges
Veronika Harmat
Ferenc Tölgyesi
Weixiao Y Wahlgren
Gergely Katona
László Nyitray
Gábor Pál
author_facet Péter Rapali
László Radnai
Dániel Süveges
Veronika Harmat
Ferenc Tölgyesi
Weixiao Y Wahlgren
Gergely Katona
László Nyitray
Gábor Pál
author_sort Péter Rapali
title Directed evolution reveals the binding motif preference of the LC8/DYNLL hub protein and predicts large numbers of novel binders in the human proteome.
title_short Directed evolution reveals the binding motif preference of the LC8/DYNLL hub protein and predicts large numbers of novel binders in the human proteome.
title_full Directed evolution reveals the binding motif preference of the LC8/DYNLL hub protein and predicts large numbers of novel binders in the human proteome.
title_fullStr Directed evolution reveals the binding motif preference of the LC8/DYNLL hub protein and predicts large numbers of novel binders in the human proteome.
title_full_unstemmed Directed evolution reveals the binding motif preference of the LC8/DYNLL hub protein and predicts large numbers of novel binders in the human proteome.
title_sort directed evolution reveals the binding motif preference of the lc8/dynll hub protein and predicts large numbers of novel binders in the human proteome.
publisher Public Library of Science (PLoS)
publishDate 2011
url https://doaj.org/article/b7cb1bc7ab3c47f7b216f39213f9e340
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