Elisar Barbar

What's in a name? Why these proteins are intrinsically disordered: Why these proteins are intrinsically disordered.

“What’s in a name? That which we call a rose By any other name would smell as sweet.” From “Romeo and Juliet”, William Shakespeare (1594) This article opens a series of publications on disambiguation of the basic terms used in the field of intrinsically disordered proteins. We start from the beginning, namely from the explanation of what the expression “intrinsically disordered protein” actually means and why this particular term has been chosen as the common denominator for this class of proteins characterized by broad structural, dynamic and functional characteristics.

Multivalency in the assembly of intrinsically disordered dynein intermediate chain

Dynein light chains are thought to increase binding efficiency of dynein intermediate chain to both dynein heavy chain and dynactin, but their exact role is not clear. Isothermal titration calorimetry and x-ray crystallography reported herein indicate that multivalency effects underlie efficient dynein assembly and regulation. For a ternary complex of a 60-amino acid segment of dynein intermediate chain (IC) bound to two homodimeric dynein light chains Tctex1 and LC8, there is a 50-fold affinity enhancement for the second light chain binding. For a designed IC construct containing two LC8 sites, observed the 1000-fold enhancement reflects a remarkably pure entropic chelate effect of a magnitude commensurate with theoretical predictions. The lower enhancement in wild-type IC is attributed to unfavorable free energy changes associated with incremental interactions of IC with Tctex1. Our results show assembled dynein IC as an elongated, flexible polybivalent duplex, and suggest that polybivalency is an important general mechanism for constructing stable yet reversible and functionally versatile complexes.

The Intermediate Chain of Cytoplasmic Dynein Is Partially Disordered and Gains Structure upon Binding to Light-Chain LC8 †

The N-terminal domain of dynein intermediate chain, IC(1-289), is highly disordered, but upon binding to dynein light-chain LC8, it undergoes a significant conformational change to a more ordered structure. Using circular dichroism and fluorescence spectroscopy, we demonstrate that the change in conformation is due to an increase in the helical structure and to enhanced compactness in the environment of tryptophan 161. An increase in helical structure and compactness is also observed with trimethylamine-N-oxide (TMAO), a naturally occurring osmolyte used here as a probe to identify regions with a propensity for induced folding. Global protection of IC(1-289) from protease digestion upon LC8 binding was localized to a segment that includes residues downstream of the LC8-binding site. Several smaller constructs of IC(1-289) containing the LC8-binding site and one of the predicted helix or coiled-coil segments were made. IC(1-143) shows no increase in helical structure upon binding, while IC(114-260) shows an increase in helical structure similar to what is observed with IC(1-289). Binding of IC(114-260) to LC8 was monitored by fluorescence and native gel electrophoresis and shows saturation of binding, a stoichiometry of 1:1, and moderate binding affinity. The induced folding of IC(1-289) upon LC8 binding suggests that LC8 could act through the intermediate chain to facilitate dynein assembly or regulate cargo-binding interactions.

Potential role for phosphorylation in differential regulation of the assembly of dynein light chains

The homodimeric light chains LC8 and Tctex-1 are integral parts of the microtubule motor cytoplasmic dynein, as they directly associate with dynein intermediate chain IC and various cellular cargoes. These light chains appear to regulate assembly of the dynein complex by binding to and promoting dimerization of IC. In addition, both LC8 and Tctex-1 play roles in signaling, apoptosis, and neuronal development that are independent of their function in dynein, but it is unclear how these various activities are modulated. Both light chains undergo specific phosphorylation, and here we present biochemical and NMR analyses of phosphomimetic mutants that indicate how phosphorylation may regulate light chain function. For both LC8 and Tctex-1, phosphorylation promotes dissociation from IC while retaining their binding activity with other non-dynein proteins. Although LC8 and Tctex-1 are homologs having a common fold, their reduced affinity for IC upon phosphorylation arises by different mechanisms. In the case of Tctex-1, phosphorylation directly masks the IC binding site at the dimer interface, whereas for LC8, phosphorylation dissociates the dimer and indirectly eliminates the binding site. This modulation of the monomer-dimer equilibrium by phosphorylation provides a novel mechanism for discrimination among LC8 binding partners.

Heme catalyzes tyrosine 385 nitration and inactivation of prostaglandin H2 synthase-1 by peroxynitrite

The mechanism by which the inflammatory enzyme prostaglandin H2 synthase-1 (PGHS-1) deactivates remains undefined. This study aimed to determine the stabilizing parameters of PGHS-1 and identify factors leading to deactivation by nitric oxide species (NOx). Purified PGHS-1 was stabilized when solubilized in β-octylglucoside (rather than Tween-20 or CHAPS) and when reconstituted with hemin chloride (rather than hematin). Peroxynitrite (ONOO−) activated the peroxidase site of PGHS-1 independently of the cyclooxygenase site. After ONOO− exposure, holoPGHS-1 could not metabolize arachidonic acid and was structurally compromised, whereas apoPGHS-1 retained full activity once reconstituted with heme. After incubation of holoPGHS-1 with ONOO−, heme absorbance was diminished but to a lesser extent than the loss in enzymatic function, suggesting the contribution of more than one process to enzyme inactivation. Hydroperoxide scavengers improved enzyme activity, whereas hydroxyl radical scavengers provided no protection from the effects of ONOO−. Mass spectral analyses revealed that tyrosine 385 (Tyr 385) is a target for nitration by ONOO− only when heme is present. Multimer formation was also observed and required heme but could be attenuated by arachidonic acid substrate. We conclude that the heme plays a role in catalyzing Tyr 385 nitration by ONOO− and the demise of PGHS-1.

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-A 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.

The solution structure of the pH-induced monomer of dynein light-chain LC8 from Drosophila

The structure of Drosophila LC8 pH‐induced monomer has been determined by NMR spectroscopy using the program AutoStructure. The structure at pH 3 and 30°C is similar to the individual subunits of mammalian LC8 dimer with the exception that a β strand, which crosses between monomers to form an intersubunit β‐sheet in the dimer, is a flexible loop with turnlike conformations in the monomer. Increased flexibility in the interface region relative to the rest of the protein is confirmed by dynamic measurements based on 15N relaxation. Comparison of the monomer and dimer structures indicates that LC8 is not a domain swapped dimer.

The Crystal Structure of Dynein Intermediate Chain-Light Chain Roadblock Complex Gives New Insights into Dynein Assembly

The roadblock/LC7 dynein light chain is a ubiquitous component of all dyneins and is essential for many diverse processes including proper axonal transport and dendrite growth. In addition, LC7 functions in non-dynein transcriptional activation of the transforming growth factor-β complex. Crystal structures of Drosophila melanogaster LC7 in the apo form and in complex with a segment of the disordered N-terminal domain of dynein intermediate chain (IC) provide the first definitive identification of the IC sequence recognized by LC7. The site, confirmed by isothermal titration calorimetry studies, overlaps the IC sequence considered in the literature to be an IC self-association domain. The IC peptide binds as two amphipathic helices that lie along an extensive hydrophobic cleft on LC7 and ends with a polar side-chain interaction network that includes conserved residues from both proteins. The LC7 recognition sequence on IC and its interface with LC7 are well conserved and are, thus, likely representative of all IC·LC7 structures. Interestingly, the position of bound IC in the IC·LC7 complex mimics a helix that is integrated into the primary structure in distantly related LC7 homologs. The IC·LC7 structure further shows that the naturally occurring roblZ deletion mutation contains the majority of the IC binding site and suggests that promotion of IC binding by phosphorylation of LC7 is an indirect effect.

NMR Characterization of Partially Folded and Unfolded Conformational Ensembles of Proteins

Studies of unfolded and partially folded proteins provide important insight into the initiation and process of protein folding. This review focuses on the use of nmr in characterization of ensembles of proteins that model the early stages of folding. Analysis of an ensemble includes description of the number of conformations, their structure, relative populations, interconversion rates, and dynamics of subconformations. A chemically synthesized analogue of bovine pancreatic trypsin inhibitor (BPTI), [14-38](Abu), has provided a rare system for characterization of multiple partially folded conformations in slow exchange at near physiological conditions. Multidimensional nmr techniques coupled with selective labeling were used to probe different segments of the polypeptide chain. At each labeled site, there is evidence of slow interconversion between two families of partially folded conformations that in themselves are ensembles of rapidly interconverting conformers. All these conformers display significantly more order in the core relative to the rest of the molecule. For other variants of BPTI that are unfolded at equilibrium, the most ordered structure is also favored in the hydrophobic core residues of the native protein. This is consistent with the hypothesis that the residues that are the first to fold go on to form the most stable, structure-determining part of the protein.

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

The assembly factor heterodimer Rsa4–Nsa2 binds to the preribosome and transmits remodeling energy from the force-generating ATPase Rea1 to facilitate relocation of ribosomal RNA elements during ribosome maturation.