Polybivalency and Disordered Proteins in Ordering Macromolecular Assemblies

Intrinsically disordered proteins (IDPs) are prevalent in macromolecular assemblies and are thought to mediate protein recognition in complex regulatory processes and signaling pathways. The formation of a polybivalent scaffold is a key process by which IDPs drive early steps in macromolecular assemblies. Three intrinsically disordered proteins, IC, Swallow and Nup159, are core components, respectively, of cytoplasmic dynein, bicoid mRNA localization apparatus, and nuclear pore complexes. In all three systems, the hub protein LC8 recognizes on the IDP, short linear motifs that are fully disordered in the apo form, but adopt a β-strand when bound to LC8. The IDP/LC8 complex forms a bivalent scaffold primed to bind additional bivalent ligands. Scaffold formation also promotes self-association and/or higher order organization of the IDP components at a site distant from LC8 binding. Rigorous thermodynamic analyses imply that association of additional bivalent ligands is driven by entropic effects where the first binding event is weak but subsequent binding of additional ligands occurs with higher affinity. Here, we review specific examples of macromolecular assemblies in which polybivalency of aligned IDP duplexes not only enhances binding affinity and results in formation of a stable complex but also compensates unfavorable steric and enthalpic interactions. We propose that polybivalent scaffold assembly involving IDPs and LC8-like proteins is a general process in the cell biology of a class of multi-protein structures that are stable yet fine-tuned for diverse cellular requirements.Keywords: Poly-bivalent scaffold, Macromolecular assembly, LC8, Bivalency, Intrinsically disordered proteins, Enthalpy–entropy compensatio

Interactions of LC8 with N-Terminal Segments of the Intermediate Chain of Cytoplasmic Dynein

LC8, a highly conserved 10-kDa light chain, and IC74, a 74-kDa intermediate chain, are presumed to promote the assembly of the cytoplasmic dynein motor protein complex and to be engaged in the controlled binding and release of cargo. The interactions of LC8 from Drosophila melanogaster with constructs of IC74 were characterized in vitro by affinity methods, limited proteolysis, and circular dichroism spectroscopy. Previously, we have performed limited proteolysis on the N-terminal domain of IC74, IC(1-289), when free and when bound to dynein light chains LC8 and Tctex-1[1]. We have also shown that upon addition of LC8, IC(1-289) undergoes a significant conformational change from a largely unfolded to a more ordered structure. The purpose of the work presented here is to determine whether residues 1-30 in IC74, predicted to be in a coiled coil, are involved in the stabilization of the protein upon binding to LC8. Constructs of IC74, IC(1-143), and IC(30-143) that include the LC8 binding site but with and without the first 30 residues were prepared, and their binding and protection patterns were compared to our previous results for IC(1-289). The results suggest that coiled coil residues 1-30 are not responsible for the increase in structure we observe when IC(1-289) binds to LC8

Interactions of yeast dynein with dynein light chain and dynactin: General implications for intrinsically disordered duplex scaffolds in multi-protein assemblies

Intrinsically disordered protein (IDP) duplexes composed of two IDP chains cross-linked by bivalent partner proteins form scaffolds for assembly of multiprotein complexes. The N-terminal domain of dynein intermediate chain (N-IC) is one such IDP that forms a bivalent scaffold with multiple dynein light chains including LC8, a hub protein that promotes duplex formation of diverse IDP partners. N-IC also binds a subunit of the dynein regulator, dynactin. Here we characterize interactions of a yeast ortholog of N-IC (N-Pac11) with yeast LC8 (Dyn2) or with the intermediate chain-binding subunit of yeast dynactin (Nip100). Residue level changes in Pac11 structure are monitored by NMR spectroscopy, and binding energetics are monitored by isothermal titration calorimetry (ITC). N-Pac11 is monomeric and primarily disordered except for a single α-helix (SAH) at the N terminus and a short nascent helix, LH, flanked by the two Dyn2 recognition motifs. Upon binding Dyn2, the only Pac11 residues making direct protein-protein interactions are in and immediately flanking the recognition motifs. Dyn2 binding also orders LH residues of Pac11. Upon binding Nip100, only Pac11 SAH residues make direct protein-protein interactions, but LH residues at a distant sequence position and L1 residues in an adjacent linker are also ordered. The long distance, ligand-dependent ordering of residues reveals new elements of dynamic structure within IDP linker regions.Keywords: isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR), protein assembly, intrinsically disordered protein, dyneinKeywords: isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR), protein assembly, intrinsically disordered protein, dynei

The Interplay of Ligand Binding and Quaternary Structure in the Diverse Interactions of Dynein Light Chain LC8

Dynein light chain LC8 is a small, dimeric, and very highly conserved globular protein that is an integral part of the dynein and myosin molecular motors but appears to have a broader role in multiple protein complexes unrelated to molecular motors. LC8 binds to two families of targets: those having a KXTQT sequence fingerprint and those having a GIQVD fingerprint. All known LC8 binding partners containing these fingerprints share a common binding site on LC8 that raises the question of what determines binding specificity. Here, we present the crystal structure of apo-LC8 at 1.7-Å resolution, which, when compared with the crystal structures of several LC8 complexes, gives insight into the mechanism underlying the binding diversity of LC8. Peptide binding is associated with a shift in quaternary structure that expands the hydrophobic binding surface available to the ligand, in addition to changes in tertiary structure and ordering of LC8 around the binding groove. The observed quaternary shift suggests a mechanism by which binding at one of the two identical sites can influence binding at the other. NMR spectra of titrations with peptides from each fingerprint family show evidence of allosteric interaction between the two binding sites, to a differing degree in the two ligand families. Allosteric interaction between the binding sites may be a mechanism to promote simultaneous binding of ligands from the same family, providing a physiological role for the two fingerprints.Keywords: folding scaffold, protein crystallography, dynein light chai

Charting a new frontier of science by integrating mathematical modeling to understand and predict complex biological systems

Biological systems are staggeringly complex. To untangle this complexity and make predictions about biological systems is a continuous goal of biological research. One approach to achieve these goals is to emphasize the use of quantitative measures of biological processes. Advances in quantitative biology data collection and analysis across scales (molecular, cellular, organismal, ecological) has transformed how we understand, categorize, and predict complex biological systems. Simultaneously, thanks to increased computational power, mathematicians, engineers and physical scientists -- collectively termed theoreticians -- have developed sophisticated models of biological systems at different scales. But there is still a disconnect between the two fields. This surge of quantitative data creates an opportunity to apply, develop, and evaluate mathematical models of biological systems and explore novel methods of analysis. The novel modeling schemes can also offer deeper understanding of principles in biology. In the context of this paper, we use “models” to refer to mathematical representations of biological systems. This data revolution puts scientists in a unique position to leverage information-rich datasets to improve descriptive modeling. Moreover, advances in technology allow inclusion of heterogeneity and variability within these datasets and mathematical models. This inclusion may lead to identifying previously undetermined variables driving or maintaining heterogeneity and diversity. Improved inclusion of variation may even improve biologically meaningful predictions about how systems will respond to perturbations. Although some of these practices are mainstream in specific sub-fields of biology, such practices are not widespread across all fields of biological sciences. With resources dedicated to better integrating biology and mathematical modeling, we envision a transformational improvement in the ability to describe and predict complex biological systems

Structural basis for assembly and function of the Nup82 complex in the nuclear pore scaffold

Nuclear pore complexes (NPCs) are huge assemblies formed from ∼30 different nucleoporins, typically organized in subcomplexes. One module, the conserved Nup82 complex at the cytoplasmic face of NPCs, is crucial to terminate mRNA export. To gain insight into the structure, assembly, and function of the cytoplasmic pore filaments, we reconstituted in yeast the Nup82–Nup159–Nsp1–Dyn2 complex, which was suitable for biochemical, biophysical, and electron microscopy analyses. Our integrative approach revealed that the yeast Nup82 complex forms an unusual asymmetric structure with a dimeric array of subunits. Based on all these data, we developed a three-dimensional structural model of the Nup82 complex that depicts how this module might be anchored to the NPC scaffold and concomitantly can interact with the soluble nucleocytoplasmic transport machinery

Dimerisation of the PICTS complex via LC8/Cut-up drives co-transcriptional transposon silencing in Drosophila

In animal gonads, the PIWI-interacting RNA (piRNA) pathway guards genome integrity in part through the co-transcriptional gene silencing of transposon insertions. In Drosophila ovaries, piRNA-loaded Piwi detects nascent transposon transcripts and instructs heterochromatin formation through the Panoramix-induced co-transcriptional silencing (PICTS) complex, containing Panoramix, Nxf2 and Nxt1. Here, we report that the highly conserved dynein light chain LC8/Cut-up (Ctp) is an essential component of the PICTS complex. Loss of Ctp results in transposon de-repression and a reduction in repressive chromatin marks specifically at transposon loci. In turn, Ctp can enforce transcriptional silencing when artificially recruited to RNA and DNA reporters. We show that Ctp drives dimerisation of the PICTS complex through its interaction with conserved motifs within Panoramix. Artificial dimerisation of Panoramix bypasses the necessity for its interaction with Ctp, demonstrating that conscription of a protein from a ubiquitous cellular machinery has fulfilled a fundamental requirement for a transposon silencing complex

Structural basis for assembly and function of the Nup82 complex in the nuclear pore scaffold

Nuclear pore complexes (NPCs) are huge assemblies formed from ~30 different nucleoporins, typically organized in subcomplexes. One module, the conserved Nup82 complex at the cytoplasmic face of NPCs, is crucial to terminate mRNA export. To gain insight into the structure, assembly, and function of the cytoplasmic pore filaments, we reconstituted in yeast the Nup82–Nup159–Nsp1–Dyn2 complex, which was suitable for biochemical, biophysical, and electron microscopy analyses. Our integrative approach revealed that the yeast Nup82 complex forms an unusual asymmetric structure with a dimeric array of subunits. Based on all these data, we developed a three-dimensional structural model of the Nup82 complex that depicts how this module might be anchored to the NPC scaffold and concomitantly can interact with the soluble nucleocytoplasmic transport machinery

A network of assembly factors is involved in remodeling rRNA elements during preribosome maturation

The following corrections appear in the attached pdf labelled "Correction to Version of Record". The authors inadvertently omitted Woonghee Lee from the list of authors. The corrected author list and affiliations are noted. Revised acknowledgment paragraphs including Woonghee Lee’s funding source and contribution also appear in the correction. In addition, the authors noted the omission of details of NMR structure calculation from the Materials and Methods section. The relevant paragraph and the references associated with it are appended.Eukaryotic ribosome biogenesis involves ∼200 assembly factors, but how these contribute to ribosome maturation is poorly understood. Here, we identify a network of factors on the nascent 60S subunit that actively remodels preribosome structure. At its hub is Rsa4, a direct substrate of the force-generating ATPase Rea1. We show that Rsa4 is connected to the central protuberance by binding to Rpl5 and to ribosomal RNA (rRNA) helix 89 of the nascent peptidyl transferase center (PTC) through Nsa2. Importantly, Nsa2 binds to helix 89 before relocation of helix 89 to the PTC. Structure-based mutations of these factors reveal the functional importance of their interactions for ribosome assembly. Thus, Rsa4 is held tightly in the preribosome and can serve as a “distribution box,” transmitting remodeling energy from Rea1 into the developing ribosome. We suggest that a relay-like factor network coupled to a mechano-enzyme is strategically positioned to relocate rRNA elements during ribosome maturation