Phosphorylation in the Ser/Arg-rich region of the nucleocapsid of SARS-CoV-2 regulates phase separation by inhibiting self-association of a distant helix.

The nucleocapsid protein (N) of SARS-CoV-2 is essential for virus replication, genome packaging, evading host immunity, and virus maturation. N is a multidomain protein composed of an independently folded monomeric N-terminal domain that is the primary site for RNA binding and a dimeric C-terminal domain that is essential for efficient phase separation and condensate formation with RNA. The domains are separated by a disordered Ser/Arg-rich region preceding a self-associating Leu-rich helix. Phosphorylation in the Ser/Arg region in infected cells decreases the viscosity of N:RNA condensates promoting viral replication and host immune evasion. The molecular level effect of phosphorylation, however, is missing from our current understanding. Using NMR spectroscopy and analytical ultracentrifugation, we show that phosphorylation destabilizes the self-associating Leu-rich helix 30 amino-acids distant from the phosphorylation site. NMR and gel shift assays demonstrate that RNA binding by the linker is dampened by phosphorylation, whereas RNA binding to the full-length protein is not significantly affected presumably due to retained strong interactions with the primary RNA-binding domain. Introducing a switchable self-associating domain to replace the Leu-rich helix confirms the importance of linker self-association to droplet formation and suggests that phosphorylation not only increases solubility of the positively charged elongated Ser/Arg region as observed in other RNA-binding proteins but can also inhibit self-association of the Leu-rich helix. These data highlight the effect of phosphorylation both at local sites and at a distant self-associating hydrophobic helix in regulating liquid-liquid phase separation of the entire protein.

Quantifying cooperative multisite binding in the hub protein LC8 through Bayesian inference.

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.

The dynein light chain 8 (LC8) binds predominantly "in-register" to a multivalent intrinsically disordered partner.

Dynein light chain 8 (LC8) interacts with intrinsically disordered proteins (IDPs) and influences a wide range of biological processes. It is becoming apparent that among the numerous IDPs that interact with LC8, many contain multiple LC8-binding sites. Although it is established that LC8 forms parallel IDP duplexes with some partners, such as nucleoporin Nup159 and dynein intermediate chain, the molecular details of these interactions and LC8's interactions with other diverse partners remain largely uncharacterized. LC8 dimers could bind in either a paired "in-register" or a heterogeneous off-register manner to any of the available sites on a multivalent partner. Here, using NMR chemical shift perturbation, analytical ultracentrifugation, and native electrospray ionization MS, we show that LC8 forms a predominantly in-register complex when bound to an IDP domain of the multivalent regulatory protein ASCIZ. Using saturation transfer difference NMR, we demonstrate that at substoichiometric LC8 concentrations, the IDP domain preferentially binds to one of the three LC8 recognition motifs. Further, the differential dynamic behavior for the three sites and the size of the fully bound complex confirmed an in-register complex. Dynamics measurements also revealed that coupling between sites depends on the linker length separating these sites. These results identify linker length and motif specificity as drivers of in-register binding in the multivalent LC8-IDP complex assembly and the degree of compositional and conformational heterogeneity as a promising emerging mechanism for tuning of binding and regulation.

Altered Protein Dynamics and Increased Aggregation of Human γS-Crystallin Due to Cataract-Associated Deamidations.

Deamidation is a major age-related modification in the human lens that is highly prevalent in crystallins isolated from the insoluble fraction of cataractous lenses and also causes protein aggregation in vitro. However, the mechanism by which deamidation causes proteins to become insoluble is not known because only subtle structural changes were observed in vitro. We have identified Asn14 and Asn76 of γS-crystallin as highly deamidated in insoluble proteins isolated from aged lenses. These sites are on the surface of the N-terminal domain and were mimicked by replacing the Asn with Asp residues in order to generate recombinant human γS and deamidated mutants. Both N14D and N76D had increased light scattering compared to wild-type γS (WT) and increased aggregation during thermal-induced denaturation. Aggregation was enhanced by oxidized glutathione, suggesting deamidation may increase susceptibility to form disulfide bonds. These changes were correlated to changes in protein dynamics determined by NMR spectroscopy. Heteronuclear NMR spectroscopy was used to measure amide hydrogen exchange and 15N relaxation dynamics to identify regions with increased dynamics compared to γS WT. Residue-specific changes in solvent accessibility and dynamics were both near and distant from the sites of deamidation, suggesting that deamidation had both local and global effects on the protein structure at slow (ms to s) and fast (µs to ps) time scales. Thus, a potential mechanism for γS deamidation-induced insolubilization in cataractous lenses is altered dynamics due to local regions of unfolding and increased flexibility in both the N- and C-terminal domains particularly at surface helices. This conformational flexibility increases the likelihood of aggregation, which would be enhanced in the oxidizing cytoplasm of the aged and cataractous lens. The NMR data combined with the in vivo insolubility and in vitro aggregation findings support a model that deamidation drives changes in protein dynamics that facilitate protein aggregation associated with cataracts.

NMR Analysis of the Interactions and Conformational Plasticity of Dynein Intermediate Chain.

Cytoplasmic dynein complexes play crucial roles in intracellular transport of cellular organelles. While the motor domain of dynein is well characterized by techniques such as X-ray crystallography and cryo-electron microscopy (Cryo-EM), structural representations of dynein usually include only the more packed and easily resolved regions and omit the long flexible and poorly structured regions. One such flexible region is the N-terminal half of the intermediate chain (IC), which contains almost 300 amino acids that are predicted to be disordered. This level of disorder makes IC impossible to study by X-ray crystallography and Cryo-EM, but amenable to study by solution nuclear magnetic resonance (NMR), a powerful technique that can elucidate residue-specific information in a dynamic ensemble of structures, and transient binding interactions of associated proteins. Here, we describe the methods we use to characterize flexible and disordered proteins including protein expression, purification, sample preparation, and NMR data acquisition and analysis.

RNA structure and multiple weak interactions balance the interplay between RNA binding and phase separation of SARS-CoV-2 nucleocapsid.

The nucleocapsid (N) protein of SARS-CoV-2 binds viral RNA, condensing it inside the virion, and phase separating with RNA to form liquid-liquid condensates. There is little consensus on what differentiates sequence-independent N-RNA interactions in the virion or in liquid droplets from those with specific genomic RNA (gRNA) motifs necessary for viral function inside infected cells. To identify the RNA structures and the N domains responsible for specific interactions and phase separation, we use the first 1,000 nt of viral RNA and short RNA segments designed as models for single-stranded and paired RNA. Binding affinities estimated from fluorescence anisotropy of these RNAs to the two-folded domains of N (the NTD and CTD) and comparison to full-length N demonstrate that the NTD binds preferentially to single-stranded RNA, and while it is the primary RNA-binding site, it is not essential to phase separation. Nuclear magnetic resonance spectroscopy identifies two RNA-binding sites on the NTD: a previously characterized site and an additional although weaker RNA-binding face that becomes prominent when binding to the primary site is weak, such as with dsRNA or a binding-impaired mutant. Phase separation assays of nucleocapsid domains with double-stranded and single-stranded RNA structures support a model where multiple weak interactions, such as with the CTD or the NTD's secondary face promote phase separation, while strong, specific interactions do not. These studies indicate that both strong and multivalent weak N-RNA interactions underlie the multifunctional abilities of N.

Linker Length Drives Heterogeneity of Multivalent Complexes of Hub Protein LC8 and Transcription Factor ASCIZ.

LC8, a ubiquitous and highly conserved hub protein, binds over 100 proteins involved in numerous cellular functions, including cell death, signaling, tumor suppression, and viral infection. LC8 binds intrinsically disordered proteins (IDPs), and although several of these contain multiple LC8 binding motifs, the effects of multivalency on complex formation are unclear. Drosophila ASCIZ has seven motifs that vary in sequence and inter-motif linker lengths, especially within subdomain QT2-4 containing the second, third, and fourth LC8 motifs. Using isothermal-titration calorimetry, analytical-ultracentrifugation, and native mass-spectrometry of QT2-4 variants, with methodically deactivated motifs, we show that inter-motif spacing and specific motif sequences combine to control binding affinity and compositional heterogeneity of multivalent duplexes. A short linker separating strong and weak motifs results in stable duplexes but forms off-register structures at high LC8 concentrations. Contrastingly, long linkers engender lower cooperativity and heterogeneous complexation at low LC8 concentrations. Accordingly, two-mers, rather than the expected three-mers, dominate negative-stain electron-microscopy images of QT2-4. Comparing variants containing weak-strong and strong-strong motif combinations demonstrates sequence also regulates IDP/LC8 assembly. The observed trends persist for trivalent ASCIZ subdomains: QT2-4, with long and short linkers, forms heterogeneous complexes, whereas QT4-6, with similar mid-length linkers, forms homogeneous complexes. Implications of linker length variations for function are discussed.

Multivalency, autoinhibition, and protein disorder in the regulation of interactions of dynein intermediate chain with dynactin and the nuclear distribution protein.

As the only major retrograde transporter along microtubules, cytoplasmic dynein plays crucial roles in the intracellular transport of organelles and other cargoes. Central to the function of this motor protein complex is dynein intermediate chain (IC), which binds the three dimeric dynein light chains at multivalent sites, and dynactin p150Glued and nuclear distribution protein (NudE) at overlapping sites of its intrinsically disordered N-terminal domain. The disorder in IC has hindered cryo-electron microscopy and X-ray crystallography studies of its structure and interactions. Here we use a suite of biophysical methods to reveal how multivalent binding of the three light chains regulates IC interactions with p150Glued and NudE. Using IC from Chaetomium thermophilum, a tractable species to interrogate IC interactions, we identify a significant reduction in binding affinity of IC to p150Glued and a loss of binding to NudE for constructs containing the entire N-terminal domain as well as for full-length constructs when compared to the tight binding observed with short IC constructs. We attribute this difference to autoinhibition caused by long-range intramolecular interactions between the N-terminal single α-helix of IC, the common site for p150Glued, and NudE binding, and residues closer to the end of the N-terminal domain. Reconstitution of IC subcomplexes demonstrates that autoinhibition is differentially regulated by light chains binding, underscoring their importance both in assembly and organization of IC, and in selection between multiple binding partners at the same site.

Motor proteins are the freight trains of the cell, transporting large molecular cargo from one location to another using an array of 'roads' known as microtubules. These hollow tubes are oriented, with one extremity (the plus-end) growing faster than the other (the minus-end). While over 40 different motor proteins travel towards the plus-end of microtubules, just one is responsible for moving cargo in the opposite direction. This protein, called dynein, performs a wide range of functions which must be carefully regulated, often through changes in the shape and interactions of various dynein segments. The intermediate chain is one of the essential subunits that form dynein, and it acts as a binding site for a range of molecular actors. In particular, it connects the three other dynein subunits (known as the light chains) to the dynein heavy chain containing the motor domain. It also binds to two non-dynein proteins: NudE, which helps to organise microtubules, and the p150^Glued region of dynactin, a protein required for dynein activity. Despite their distinct roles, p150^Glued and NudE attach to the same region of the intermediate chain, a highly flexible 'unstructured' segment which is difficult to study. How the binding of p150^Glued and NudE is regulated has therefore remained unsolved. In response, Jara et al. decided to investigate how the three dynein light chains may help to control interactions between the intermediate chain and non-dynein proteins. They used more stable versions of dynein, NudE and dynactin (from a fungus that grows at high temperatures) to produce the various subcomplexes formed by the intermediate chain, the three dynein light chains, and parts of p150^Glued and NudE. A suite of biophysical techniques was applied to study these structures, as they are challenging to capture using traditional approaches. This revealed that the unstructured region of the intermediate chain can fold back on itself, bringing together its two extremities; such folding blocks the p150^Glued and NudE binding site. This obstruction is cleared when the light chains bind to the intermediate chain, demonstrating how these three subunits can regulate dynein activity. In humans, mutations in dynein are associated with a range of serious neurological and muscular diseases. The work by Jara et al. brings new insight into the way this protein works; more importantly, it describes how to combine several biophysical techniques to study non-structured proteins, offering a blueprint that is likely to be relevant for a wide range of scientists.

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.

The Tail of Kinesin-14a in Giardia Is a Dual Regulator of Motility.

Kinesin-14s are microtubule-based motor proteins that play important roles in mitotic spindle assembly [1]. Ncd-type kinesin-14s are a subset of kinesin-14 motors that exist as homodimers with an N-terminal microtubule-binding tail, a coiled-coil central stalk (central stalk), a neck, and two identical C-terminal motor domains. To date, no Ncd-type kinesin-14 has been found to naturally exhibit long-distance minus-end-directed processive motility on single microtubules as individual homodimers. Here, we show that GiKIN14a from Giardia intestinalis [2] is an unconventional Ncd-type kinesin-14 that uses its N-terminal microtubule-binding tail to achieve minus-end-directed processivity on single microtubules over micrometer distances as a homodimer. We further find that although truncation of the N-terminal tail greatly reduces GiKIN14a processivity, the resulting tailless construct GiKIN14a-Δtail is still a minimally processive motor and moves its center of mass via discrete 8-nm steps on the microtubule. In addition, full-length GiKIN14a has significantly higher stepping and ATP hydrolysis rates than does GiKIN14a-Δtail. Inserting a flexible polypeptide linker into the central stalk of full-length GiKIN14a nearly reduces its ATP hydrolysis rate to that of GiKIN14a-Δtail. Collectively, our results reveal that the N-terminal tail of GiKIN14a is a de facto dual regulator of motility and reinforce the notion of the central stalk as a key mechanical determinant of kinesin-14 motility [3].

Multivalency regulates activity in an intrinsically disordered transcription factor.

The transcription factor ASCIZ (ATMIN, ZNF822) has an unusually high number of recognition motifs for the product of its main target gene, the hub protein LC8 (DYNLL1). Using a combination of biophysical methods, structural analysis by NMR and electron microscopy, and cellular transcription assays, we developed a model that proposes a concerted role of intrinsic disorder and multiple LC8 binding events in regulating LC8 transcription. We demonstrate that the long intrinsically disordered C-terminal domain of ASCIZ binds LC8 to form a dynamic ensemble of complexes with a gradient of transcriptional activity that is inversely proportional to LC8 occupancy. The preference for low occupancy complexes at saturating LC8 concentrations with both human and Drosophila ASCIZ indicates that negative cooperativity is an important feature of ASCIZ-LC8 interactions. The prevalence of intrinsic disorder and multivalency among transcription factors suggests that formation of heterogeneous, dynamic complexes is a widespread mechanism for tuning transcriptional regulation.

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

The mechanism by which the inflammatory enzyme prostaglandin H(2) 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 (NO(x)). Purified PGHS-1 was stabilized when solubilized in beta-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.