Assembly Of Signaling Complexes Research Articles

Redefining the TIR domain: From Axon Degeneration to Innate Immunity and Beyond

Abstract The Toll/Interleukin-1 Receptor (TIR) domain is an evolutionary ancient protein domain, present in numerous receptors and adaptor proteins, and is the signature-signaling domain of Toll-Like Receptors (TLRs). In animals, these TIR domains generally serve as scaffolds that promote the assembly of signaling complexes to trigger activation of pro-inflammatory cytokines and other defense-related products. In plants, TIR domain proteins are known to mediate disease resistance against pathogens, and trigger hypersensitive cell death. However, their mechanism of action has remained elusive. In bacteria, TIR domains have been associated with virulence and defense against some viruses. Here, we redefine the canonical scaffolding function of the TIR domain by showing that TIR domains can possess intrinsic enzymatic activity, and constitute a family of enzymes that cleave the essential metabolic cofactor Nicotinamide Adenine Dinucleotide (NAD+). We identify the TIR domain of SARM1 as the founding member of the TIR NADase family that triggers axon self-destruction upon axonal injury. We show that the TIR enzymatic activity in conserved in bacteria, and archaea, where in bacteria this NADase activity has recently been linked to defense against viruses. Finally, we extend our findings to plant innate immunity, and show that plant TIR-domain Immune Receptors trigger cell death and transduce recognition of pathogens into an immune response via this conserved enzymatic activity. Altogether, our findings establish TIR domain proteins as a new family of metabolic enzymes, and we posit that the scaffolding function in TLR signaling likely represents a repurposing of this evolutionary ancient enzymatic function.

Regulation of the Drosophila Imd pathway by signaling amyloids

Fruit flies elicit effective defense responses against numerous microbes. The responses against Gram-negative bacteria are mediated by the Imd pathway, an evolutionarily conserved NF-κB pathway recognizing meso-diaminopimelic acid (DAP)-type peptidoglycan from bacterial cell walls. Several reviews already provide a detailed view of ligand recognition and signal transduction during Imd signaling, but the formation and regulation of the signaling complex immediately downstream of the peptidoglycan-sensing receptors is still elusive. In this review, we focus on the formation of the Imd amyloidal signaling center and post-translational modifications in the assembly and disassembly of the Imd signaling complex.

MAVS polymers smaller than 80 nm induce mitochondrial membrane remodeling and interferon signaling.

Double‐stranded RNA (dsRNA) is a potent proinflammatory signature of viral infection and is sensed primarily by RIG‐I‐like receptors (RLRs). Oligomerization of RLRs following binding to cytosolic dsRNA activates and nucleates self‐assembly of the mitochondrial antiviral‐signaling protein (MAVS). In the current signaling model, the caspase recruitment domains of MAVS form helical fibrils that self‐propagate like prions to promote signaling complex assembly. However, there is no conclusive evidence that MAVS forms fibrils in cells or with the transmembrane anchor present. We show here with super‐resolution light microscopy that MAVS activation by dsRNA induces mitochondrial membrane remodeling. Quantitative image analysis at imaging resolutions as high as 32 nm shows that in the cellular context, MAVS signaling complexes and the fibrils within them are smaller than 80 nm. The transmembrane domain of MAVS is required for its membrane remodeling, interferon signaling, and proapoptotic activities. We conclude that membrane tethering of MAVS restrains its polymerization and contributes to mitochondrial remodeling and apoptosis upon dsRNA sensing.

Targeting KPNB1 overcomes TRAIL resistance by regulating DR5, Mcl-1 and FLIP in glioblastoma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine with potential anticancer effect, but innate and adaptive TRAIL resistance in majority of cancers limit its clinical application. Karyopherin β1 (KPNB1) inhibition in cancer cells has been reported to abrogate the nuclear import of TRAIL receptor DR5 and facilitate its localization on the cell surface ready for TRAIL stimulation. However, our study reveals a more complicated mechanism. Genetic or pharmacological inhibition of KPNB1 potentiated TRAIL-induced apoptosis selectively in glioblastoma cells mainly by unfolded protein response (UPR). First, it augmented ATF4-mediated DR5 expression and promoted the assembly of death-inducing signaling complex (DISC). Second, it freed Bax and Bak from Mcl-1. Third, it downregulated FLIPL and FLIPS, inhibitors of caspase-8 cleavage, partly through upregulating ATF4–induced 4E-BP1 expression and disrupting the cap-dependent translation initiation. Meanwhile, KPNB1 inhibition-induced undesirable autophagy and accelerated cleaved caspase-8 clearance. Inhibition of autophagic flux maintained cleaved caspase-8 and aggravated apoptosis induced by KPNB1 inhibitor plus TRAIL, which were abolished by caspase-8 inhibitor. These results unveil new molecular mechanism for optimizing TRAIL-directed therapeutic efficacy against cancer.

The demise of catalysis, but new functions arise: pseudoenzymes as the phoenixes of the protein world

Pseudoenzymes are noncatalytic homologues of enzymes and are found in most enzyme families. Although lacking catalytic activity and sometimes referred to as 'dead' enzymes, they instead resemble phoenixes because the loss of a catalytic function during evolution was associated with the development of vital new functions. They are important in regulating the activity and location of catalytically active homologues, scaffolding the assembly of signaling complexes, and regulating transcription or translation. They are key actors in cell proliferation and differentiation, proteostasis, and many other biochemical pathways and processes. They perform their functions in diverse ways, but many retain some aspects of the function of their catalytically active homologues. In some pseudoenzymes, their functions are very different from other members of their protein families, suggesting some arose from ancient moonlighting proteins during evolution. Much less is known about pseudoenzymes than their catalytically active counterparts, but a growing appreciation of their key roles in many important biochemical processes and signaling pathways has led to increased investigation in recent years. It is clear that there is still much more to learn about the structures, functions, and cellular roles of these phoenix-like proteins.

TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between members of the tumor necrosis factor (TNF) ligand superfamily and their receptors. Recent evidence indicated that TNF-α-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation via a TRAF6-dependent signaling pathway; but paradoxically, it inhibits RANK ligand (RANKL)-induced osteoclast differentiation. Although a number of signaling pathways were linked to the RANK and osteoclastogenesis, it is not known how TRAIL regulates RANK signaling. In this study, we demonstrate that TRAIL regulates RANK-induced osteoclastogenesis in terms of the assembly of lipid raft-associated signaling complexes. RANKL stimulation induced recruitment of TRAF6, c-Src, and DAP-12 into lipid rafts. However, the RANKL-induced assembly of lipid raft-associated signaling complexes and TRAF6 recruitment was abolished in the presence of TRAIL. TRAIL-induced dissociation of RANKL-induced lipid raft signaling complexes was reversed by treatment with TRAIL receptor (TRAIL-R) siRNA or an anti-TRAIL-R blocking antibody, indicating that TRAIL mediates suppression of RANKL-induced lipid raft signaling via interactions with TRAIL-R. Finally, we demonstrated that TRAIL suppressed inflammation-induced bone resorption and osteoclastogenesis in a collagen-induced arthritis (CIA) rat animal model. Our results provide a novel apoptosis-independent role of TRAIL in regulating RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment.

Measuring Ultra-Weak Protein Self-Association by Non-ideal Sedimentation Velocity.

Ultra-weak self-association can govern the macroscopic solution behavior of concentrated macromolecular solutions ranging from food products to pharmaceutical formulations and the cytosol. For example, it can promote dynamic assembly of multi-protein signaling complexes, lead to intracellular liquid–liquid phase transitions, and seed crystallization or pathological aggregates. Unfortunately, weak self-association is technically extremely difficult to study, as it requires very high protein concentrations where short intermolecular distances cause strongly correlated particle motion. Additionally, protein samples near their solubility limit in vitro frequently show some degree of polydispersity. Here we exploit the strong mass-dependent separation of assemblies in the centrifugal field to study ultra-weak binding, using a sedimentation velocity technique that allows us to determine particle size distributions while accounting for colloidal hydrodynamic interactions and thermodynamic non-ideality (Chaturvedi, S. K.; et al. Nat. Commun.2018, 9, 4415; DOI: 10.1038/s41467-018-06902-x). We show that this approach, applied to self-associating proteins, can reveal a time-average association state for rapidly reversible self-associations from which the free energy of binding can be derived. The method is label-free and allows studying mid-sized proteins at millimolar protein concentrations in a wide range of solution conditions. We examine the performance of this method with hen egg lysozyme as a model system, reproducing its well-known ionic-strength-dependent weak self-association. The application to chicken γS-crystallin reveals weak monomer–dimer self-association with KD = 24 mM, corresponding to a standard free energy change of approximately −9 kJ/mol, which is a large contribution to the delicate balance of forces ensuring eye lens transparency.

Caspase recruitment domains for proteininteractions in cellular signaling (Review).

The caspase recruitment domain (CARD), a well-known protein interaction module, belongs to the death domain (DD) superfamily, which includes DDs, death effector domains, and pyrin domains. The DD superfamily mediates the protein interactions necessary for apoptosis and immune cell signaling pathways. Among these domains, the CARD has been studied extensively as it mediates important cellular signaling events that are associated with various human diseases including cancer, neuro-degenerative diseases and immune disorders. Homo-type and hetero-type CARD-CARD interactions mediate the formation of large signaling complexes, including caspase-activating complexes and downstream signaling complexes. The present review summarizes and discusses the results of structural studies of various CARDs and their complexes. These studies shed light on the mechanisms that control the assembly and disassembly of signaling complexes and provide an improved understanding of cellular signaling processes.

Scribble: A master scaffold in polarity, adhesion, synaptogenesis, and proliferation.

Key events ranging from cell polarity to proliferation regulation to neuronal signaling rely on the assembly of multiprotein adhesion or signaling complexes at particular subcellular sites. Multidomain scaffolding proteins nucleate assembly and direct localization of these complexes, and the protein Scribble and its relatives in the LAP protein family provide a paradigm for this. Scribble was originally identified because of its role in apical-basal polarity and epithelial integrity in Drosophila melanogaster It is now clear that Scribble acts to assemble and position diverse multiprotein complexes in processes ranging from planar polarity to adhesion to oriented cell division to synaptogenesis. Here, we explore what we have learned about the mechanisms of action of Scribble in the context of its multiple known interacting partners and discuss how this knowledge opens new questions about the full range of Scribble protein partners and their structural and signaling roles.

Structural biology of G protein-coupled receptor signaling complexes.

G protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors that mediate numerous cell signaling pathways, and are targets of more than one-third of clinical drugs. Thanks to the advancement of novel structural biology technologies, high-resolution structures of GPCRs in complex with their signaling transducers, including G-protein and arrestin, have been determined. These 3D complex structures have significantly improved our understanding of the molecular mechanism of GPCR signaling and provided a structural basis for signaling-biased drug discovery targeting GPCRs. Here we summarize structural studies of GPCR signaling complexes with G protein and arrestin using rhodopsin as a model system, and highlight the key features of GPCR conformational states in biased signaling including the sequence motifs of receptor TM6 that determine selective coupling of G proteins, and the phosphorylation codes of GPCRs for arrestin recruitment. We envision the future of GPCR structural biology not only to solve more high-resolution complex structures but also to show stepwise GPCR signaling complex assembly and disassembly and dynamic process of GPCR signal transduction.

Cooperative assembly of a four-molecule signaling complex formed upon T cell antigen receptor activation

The T cell antigen receptor encounters foreign antigen during the immune response. Receptor engagement leads to activation of specific protein tyrosine kinases, which then phosphorylate multiple enzymes and adapter proteins. One such enzyme, phospholipase-Cγ1, is responsible for cleavage of a plasma membrane lipid substrate, a phosphoinositide, into two second messengers, diacylglycerol, which activates several enzymes including protein kinase C, and an inositol phosphate, which induces intracellular calcium elevation. In T cells, phospholipase-Cγ1 is recruited to the plasma membrane as part of a four-protein complex containing three adapter molecules. We have used recombinant proteins and synthetic phosphopeptides to reconstitute this quaternary complex in vitro. Extending biophysical tools to study concurrent interactions of the four protein components, we demonstrated the formation and determined the composition of the quaternary complex using multisignal analytical ultracentrifugation, and we characterized the thermodynamic driving forces of assembly by isothermal calorimetry. We demonstrate that the four proteins reversibly associate in a circular arrangement of binding interfaces, each protein interacting with two others. Three interactions are of high affinity, and the fourth is of low affinity, with the assembly of the quaternary complex exhibiting significant enthalpy-entropy compensation as in an entropic switch. Formation of this protein complex enables subsequent recruitment of additional molecules needed to activate phospholipase-Cγ1. Understanding the formation of this complex is fundamental to full characterization of a central pathway in T cell activation. Such knowledge is critical to developing ways in which this pathway can be selectively inhibited.

IFITM3-Mediated Regulation of Cell Membrane Dynamics Is Essential for Malignant B-Cell Transformation

Background & Hypothesis: B cell receptor (BCR) signaling and oncogenic tyrosine kinases that mimic BCR-signaling in B-lineage leukemia and lymphoma depend on assembly of membrane proximal signaling complexes. Signalosomes in normal BCR- and oncogene (e.g. BCR-ABL1, RAS-pathway lesions) signal transduction are recruited to phospholipid anchors in lipid rafts. The robustness of these complexes depends on cholesterol accumulation in lipid rafts. Here we identified the interferon-induced transmembrane protein IFITM3 as a central regulator of cholesterol in lipid rafts.Results: IFITM3 is mostly localized to endosomal compartments. By antagonizing VAP-A and oxysterol-binding protein 1 (OSBP1), IFITM3 promotes cholesterol accumulation and solidifies the endosomal membrane. This mechanism is particular important in anti-viral immunity, to “trap”intraluminal viral particles for lysosomal degradation. In B-cells, IFITM3 can translocate to the cell membrane and form a complex with the BCR and its co-receptors CD19, CD81 and CD21. While the functional significance of membrane expression of IFITM3 on B-cells was not known, we found that higher IFITM3 mRNA levels at the time of diagnosis represents a strong predictor of poor clinical outcome for children (COG P9906; P=0.006; n=207) and adults (ECOG E2993; P=0.014; n=215) with B-ALL. In addition, higher than median IFITM3 mRNA levels at the time of diagnosis were associated with a higher risk of relapse and positive MRD status at the end of induction chemotherapy in B-ALL and other B-cell malignancies. Interestingly, IFITM3 is a transcriptional target and strongly repressed by IKZF1 (Ikaros) a potent tumor suppressor in B- ALL and high IFITM3 mRNA levels represents a biomarker for patients with IKZF1-deletion.While its membrane-topology can vary in different cell types, we found that IFITM3 functions as a dual-pass transmembrane protein in tight association with CD19 and the Iga and Igb signaling chains of the BCR in B-ALL and B-cell lymphoma cells. To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1 or oncogenic NRASG12D. Strikingly, deletion of IFITM3 resulted in destabilization of lipid rafts, loss of CD19 surface expression and loss of PI3K signaling. Ifitm3-/- leukemia cells could not sustain oncogenic signaling from BCR-ABL1 or oncogenic NRASG12D and failed to initiate fatal leukemia in transplant recipient mice. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and increased propensity to apoptosis.In mechanistic studies, we identified type II transmembrane topology for IFITM3 at plasma membrane with extracellular C and intracellular N terminus which interacted with CD19, LYN, SYK, PI3K and AKT (see schematic, left). Disruption of endocytic motif (20YEML23) by substitution of Tyr20 to Phe induced IFITM3 gain of function and forced accumulation of IFITM3 on the cell membrane, constitutive CD19-PI3K signaling, intracellular calcium mobilization, homotypic cellular aggregation and massively increased proliferation of pre-B ALL cells (see schematic, right). Conversely, inducible overexpression of IKZF1 transcriptionally silenced IFITM3, resulting in loss of IFITM3 expression, reduction of lipid rafts and impairment of membrane-associated oncogenic signaling. Through Filipin-based cholesterol staining, we found Ifitm3-/- pre-B cells have reduced levels of cholesterol in lipid rafts, which causes disruption of lipid rafts formation, as reflected by decreased levels of ganglioside GM1. Notably, the homeostatic cholesterol fluidity by presence of IFITM3 on plasma membrane was also required for initiation of B- and T cell receptor signaling in mature B- and T cell lymphoma to induce Ca2+ mobilization.Conclusions: These findings identify novel role of the viral immunity IFITM3 surface receptor as a central regulator of cell membrane cholesterol fluidity and critical mediator of sustained oncogenic tyrosine kinase (BCR-ABL1) and RAS (NRASG12D) signaling in B cell malignancies. In promoting cholesterol aggregates in lipid rafts, IFITM3 protects healthy individuals from potentially lethal viral infections, but also enables oncogenic signaling by providing a robust membrane scaffold for tyrosine kinase and RAS-pathway oncogenes. DisclosuresWiita:Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

ADAP is an upstream regulator that precedes SLP-76 at sites of TCR engagement and stabilizes signaling microclusters.

Antigen recognition by the T cell receptor (TCR) directs the assembly of essential signaling complexes known as SLP-76 (also known as LCP2) microclusters. Here, we show that the interaction of the adhesion and degranulation-promoting adaptor protein (ADAP; also known as FYB1) with SLP-76 enables the formation of persistent microclusters and the stabilization of T cell contacts, promotes integrin-independent adhesion and enables the upregulation of CD69. By analyzing point mutants and using a novel phospho-specific antibody, we show that Y595 is essential for normal ADAP function, that virtually all tyrosine phosphorylation of ADAP is restricted to a Y595-phosphorylated (pY595) pool, and that multivalent interactions between the SLP-76 SH2 domain and its binding sites in ADAP are required to sustain ADAP phosphorylation. Although pY595 ADAP enters SLP-76 microclusters, non-phosphorylated ADAP is enriched in protrusive actin-rich structures. The pre-positioning of ADAP at the contact sites generated by these structures favors the retention of nascent SLP-76 oligomers and their assembly into persistent microclusters. Although ADAP is frequently depicted as an effector of SLP-76, our findings reveal that ADAP acts upstream of SLP-76 to convert labile, Ca2+-competent microclusters into stable adhesive junctions with enhanced signaling potential.

POLAR-guided signalling complex assembly and localization drive asymmetric cell division.

Stomatal cell lineage is an archetypal example of asymmetric cell division (ACD), which is necessary for plant survival1-4. In Arabidopsis thaliana, the GLYCOGEN SYNTHASE KINASE3 (GSK3)/SHAGGY-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2) phosphorylates both the mitogen-activated protein kinase (MAPK) signalling module5,6 and its downstream target, the transcription factor SPEECHLESS (SPCH)7, to promote and restrict ACDs, respectively, in the same stomatal lineage cell. However, the mechanisms that balance these mutually exclusive activities remain unclear. Here we identify the plant-specific protein POLAR as a stomatal lineage scaffold for a subset of GSK3-like kinases that confines them to the cytosol and subsequently transiently polarizes them within the cell, together with BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), before ACD. As a result, MAPK signalling is attenuated, enabling SPCH to drive ACD in the nucleus. Moreover, POLAR turnover requires phosphorylation on specific residues, mediated by GSK3. Our study reveals a mechanism by which the scaffolding protein POLAR ensures GSK3 substrate specificity, and could serve as a paradigm for understanding regulation of GSK3 in plants.

Understanding early TLR signaling through the Myddosome.

TLRs are expressed on the plasma and endosomal membranes of innate immune cells acting as sensors of foreign and inherent danger signals that threaten the host. Upon activation, TLRs facilitate the assembly of large intracellular oligomeric signaling complexes, termed Myddosomes, which initiate key signal transduction pathways to elicit critical inflammatory immune responses. The formation of the Myddosome is integral for TLR signaling; however, the molecular mechanisms controlling its formation, disassembly, and the subsequent proximal signaling events remain to be clearly defined. In this review, we present a brief overview of TLR signal transduction pathways, summarize the current understanding of the Myddosome and the proteins that comprise its structure, including MyD88 and members of the IL-1 receptor-associated kinase (IRAK) family. Finally, we will discuss recent advances and open questions regarding early TLR signaling in the context of the Myddosome complex.

Postsynaptic localization and regulation of AMPA receptors and Cav1.2 by β2 adrenergic receptor/PKA and Ca2+/CaMKII signaling.

The synapse transmits, processes, and stores data within its tiny space. Effective and specific signaling requires precise alignment of the relevant components. This review examines current insights into mechanisms of AMPAR and NMDAR localization by PSD-95 and their spatial distribution at postsynaptic sites to illuminate the structural and functional framework of postsynaptic signaling. It subsequently delineates how β2 adrenergic receptor (β2 AR) signaling via adenylyl cyclase and the cAMP-dependent protein kinase PKA is organized within nanodomains. Here, we discuss targeting of β2 AR, adenylyl cyclase, and PKA to defined signaling complexes at postsynaptic sites, i.e., AMPARs and the L-type Ca2+ channel Cav1.2, and other subcellular surface localizations, the role of Akinase anchor proteins, the physiological relevance of the spatial restriction of corresponding signaling, and their interplay with signal transduction by the Ca2+- and calmodulin-dependent kinase CaMKII How localized and specific signaling by cAMP occurs is a central cellular question. The dendritic spine constitutes an ideal paradigm for elucidating the dimensions of spatially restricted signaling because of their small size and defined protein composition.

Molecular basis of dimerization of initiator caspase was revealed by crystal structure of caspase-8 pro-domain.

The assembly of death-inducing signaling complex (DISC) for activation of initiator caspase is a key step for the receptor-mediated apoptosis signaling. Many death effector domain (DED)-containing proteins are involved in DISC assembly and controlling. One of the main DISC component, caspase-8, contains DED and DED-mediated dimerization and oligomerization in the DISC is critical for the activation of this initiator caspase. There have been intensive studies to understand DED-mediated dimerization and oligomerization for the DISC assembly but no clear answer has been provided and there are many controversial arguments. Here, we suggested novel dimerization process of tandem DED of caspase-8 with crystallographic study.

Non-canonical Activation of the DNA Sensing Adaptor STING by ATM and IFI16 Mediates NF-κB Signaling after Nuclear DNA Damage

SummaryDNA damage can be sensed as a danger-associated molecular pattern by the innate immune system. Here we find that keratinocytes and other human cells mount an innate immune response within hours of etoposide-induced DNA damage, which involves the DNA sensing adaptor STING but is independent of the cytosolic DNA receptor cGAS. This non-canonical activation of STING is mediated by the DNA binding protein IFI16, together with the DNA damage response factors ATM and PARP-1, resulting in the assembly of an alternative STING signaling complex that includes the tumor suppressor p53 and the E3 ubiquitin ligase TRAF6. TRAF6 catalyzes the formation of K63-linked ubiquitin chains on STING, leading to the activation of the transcription factor NF-κB and the induction of an alternative STING-dependent gene expression program. We propose that STING acts as a signaling hub that coordinates a transcriptional response depending on its mode of activation.

Regulation of IL-1 signaling by the decoy receptor IL-1R2.

The pleiotropic cytokine IL-1 mediates its biological functions via association with the signaling receptor IL-1R1. Despite an apparent simplicity in IL-1 signaling activation, multiple negative regulators have been identified. The decoy receptor IL-1R2 (also known as CD121b) can suppress IL-1 maturation, sequester its active forms or hinder the signaling complex assembly. IL-1R2 is differentially expressed among numerous cell types and displays cis- and trans- modes of action. In this review, we link different forms of IL-1R2 (membrane-bound (mIL-1R2), secreted (sIL-1R2), shedded (shIL-1R2), cytoplasmic, and intracellular domain (IL-1R2ICD) restricted) with their ability to interfere with IL-1, thereby regulating immune responses. We also discuss the intriguing possible function of IL-1R2 as a transcriptional regulator. Finally, we summarize the known impact of IL-1R2 in disease pathogenesis and discuss its potential role in treatment of inflammatory conditions.

Controllable activation of nanoscale dynamics in a disordered protein alters binding kinetics

The phosphorylation of specific residues in a flexible disordered activation loop yields precise control of signal transduction. One paradigm is the phosphorylation of S339/S340 in the intrinsically disordered tail of the multi-domain scaffolding protein NHERF1, which affects the intracellular localization and trafficking of NHERF1 assembled signaling complexes. Using neutron spin echo spectroscopy (NSE), we show salt-concentration-dependent excitation of nanoscale motion at the of the C-terminal tail in the phosphomimic S339D/S340D mutant. The tip of the whip that is unleashed is near the S339/S340 phosphorylation site and flanks the hydrophobic Ezrin-binding motif. The kinetic association rate constant of the binding of the S339D/S340D mutant to the FERM domain of Ezrin is sensitive to buffer salt concentration, correlating with the excited nanoscale dynamics. The results suggest that electrostatics modulates the activation of nanoscale dynamics of an intrinsically disordered protein, controlling the binding kinetics of signaling partners. NSE can pinpoint the nanoscale dynamics changes in a highly specific manner.