Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Rapali P

PROVIDER: S-EPMC3078936 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Rapali Péter P Radnai László L Süveges Dániel D Harmat Veronika V Tölgyesi Ferenc F Wahlgren Weixiao Y WY Katona Gergely G Nyitray László L Pál Gábor G

PloS one 20110418 4

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 flanke ...[more]

PMID: 21533121

Similar Datasets

Project description:Multistep protein-protein interactions underlie most biological processes, but their characterization through methods such as isothermal titration calorimetry (ITC) is largely confined to simple models that provide little information on the intermediate, individual steps. In this study, we primarily examine the essential hub protein LC8, a small dimer that binds disordered regions of 100+ client proteins in two symmetrical grooves at the dimer interface. Mechanistic details of LC8 binding have remained elusive, hampered in part by ITC data analyses employing simple models that treat bivalent binding as a single event with a single binding affinity. We build on existing Bayesian ITC approaches to quantify thermodynamic parameters for multi-site binding interactions impacted by significant uncertainty in protein concentration. Using a two-site binding model, we identify positive cooperativity with high confidence for LC8 binding to multiple client peptides. In contrast, application of an identical model to the two-site binding between the coiled-coil NudE dimer and the intermediate chain of dynein reveals little evidence of cooperativity. We propose that cooperativity in the LC8 system drives the formation of saturated induced-dimer structures, the functional units of most LC8 complexes. In addition to these system-specific findings, our work advances general ITC analysis in two ways. First, we describe a previously unrecognized mathematical ambiguity in concentrations in standard binding models and clarify how it impacts the precision with which binding parameters are determinable in cases of high uncertainty in analyte concentrations. Second, building on observations in the LC8 system, we develop a system-agnostic heat map of practical parameter identifiability calculated from synthetic data which demonstrates that the ability to determine microscopic binding parameters is strongly dependent on both the parameters themselves and experimental conditions. The work serves as a foundation for determination of multi-step binding interactions, and we outline best practices for Bayesian analysis of ITC experiments.

Project description:Protein-protein interactions (PPIs) formed between short linear motifs and globular domains play important roles in many regulatory and signaling processes but are highly underrepresented in current protein-protein interaction databases. These types of interactions are usually characterized by a specific binding motif that captures the key amino acids shared among the interaction partners. However, the computational proteome-level identification of interaction partners based on the known motif is hindered by the huge number of randomly occurring matches from which biologically relevant motif hits need to be extracted. In this work, we established a novel bioinformatic filtering protocol to efficiently explore interaction network of a hub protein. We introduced a novel measure that enabled the optimization of the elements and parameter settings of the pipeline which was built from multiple sequence-based prediction methods. In addition, data collected from PPI databases and evolutionary analyses were also incorporated to further increase the biological relevance of the identified motif hits. The approach was applied to the dynein light chain LC8, a ubiquitous eukaryotic hub protein that has been suggested to be involved in motor-related functions as well as promoting the dimerization of various proteins by recognizing linear motifs in its partners. From the list of putative binding motifs collected by our protocol, several novel peptides were experimentally verified to bind LC8. Altogether 71 potential new motif instances were identified. The expanded list of LC8 binding partners revealed the evolutionary plasticity of binding partners despite the highly conserved binding interface. In addition, it also highlighted a novel, conserved function of LC8 in the upstream regulation of the Hippo signaling pathway. Beyond the LC8 system, our work also provides general guidelines that can be applied to explore the interaction network of other linear motif binding proteins or protein domains.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets