Hub proteins participate in cellular regulation by dynamic binding of multiple proteins within interaction networks. The hub protein LC8 reversibly interacts with more than 100 partners through a flexible pocket at its dimer interface. To explore the diversity of the LC8 partner pool, we screened for LC8 binding partners using a proteomic phage display library composed of peptides from the human proteome, which had no bias toward a known LC8 motif. Of the identified hits, we validated binding of 29 peptides using isothermal titration calorimetry. Of the 29 peptides, 19 were entirely novel, and all had the canonical TQT motif anchor. A striking observation is that numerous peptides containing the TQT anchor do not bind LC8, indicating that residues outside of the anchor facilitate LC8 interactions. Using both LC8-binding and nonbinding peptides containing the motif anchor, we developed the “LC8Pred” algorithm that identifies critical residues flanking the anchor and parses random sequences to predict LC8-binding motifs with ∼78% accuracy. Our findings significantly expand the scope of the LC8 hub interactome.

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.

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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. We examine the hub protein LC8, which binds to disordered regions of 100+ client proteins in a wide range of stoichiometries. Despite evidence that LC8 binds clients cooperatively, prior ITC thermodynamic analyses have relied on models that do not accommodate allostery, and furthermore do not account for critical uncertainties in analyte concentrations. To characterize allostery in a more rigorous fashion, we build on existing Bayesian approaches to ITC to quantify thermodynamic parameters for multi-step binding interactions impacted by significant uncertainty in protein concentration. Notably, we account for a previously unrecognized intrinsic ambiguity in concentrations in standard binding models and clarify how this ambiguity impacts the extent to which binding parameters can be determined in cases of highly uncertain analyte concentrations. Our approach is applicable to a host of multi-step binding interactions, and we use it to investigate two systems. First, we deeply examine 2:2 LC8 binding and find it to be significantly positively cooperative with high confidence for multiple clients. Building on observations in the LC8 system, we develop a system-agnostic "phase diagram" calculated from synthetic data demonstrating that certain binding parameters intrinsically inflate parameter uncertainty in ITC analysis, independent of experimental uncertainties. Second, we study 2:2 binding between the dynein intermediate chain and binding protein NudE, where in contrast, we find little evidence of allostery.

14‐3‐3 proteins are central hub regulators of hundreds of phosphorylated “client” proteins. They are subject to over 60 post‐translational modifications (PTMs), yet little is known how these PTMs alter 14‐3‐3 function and its ability to regulate downstream signaling pathways. An often neglected, but well‐documented 14‐3‐3 PTM found under physiological and immune‐stimulatory conditions is the conversion of tyrosine to 3‐nitro‐tyrosine at several Tyr sites, two of which are located at sites considered important for 14‐3‐3 function: Y130 (β‐isoform numbering) is located in the primary phospho‐client peptide‐binding groove, while Y213 is found on a secondary binding site that engages with clients for full 14‐3‐3/client complex formation and client regulation. By genetically encoding 3‐nitro‐tyrosine, we sought to understand if nitration at Y130 and Y213 effectively modulated 14‐3‐3 structure, function, and client complexation. The 1.5 Å resolution crystal structure of 14‐3‐3 nitrated at Y130 showed the nitro group altered the conformation of key residues in the primary binding site, while functional studies confirmed client proteins failed to bind this variant of 14‐3‐3. But, in contrast to other client‐binding deficient variants, it did not localize to the nucleus. The 1.9 Å resolution structure of 14‐3‐3 nitrated at Y213 revealed unusual flexibility of its C‐terminal α‐helix resulting in domain swapping, suggesting additional structural plasticity though its relevance is not clear as this nitrated form retained its ability to bind clients. Collectively, our data suggest that nitration of 14‐3‐3 will alter downstream signaling systems, and if uncontrolled could result in global dysregulation of the 14‐3‐3 interactome.