A single C-terminal residue controls SARS-CoV-2 spike trafficking and incorporation into VLPs.

The spike (S) protein of SARS-CoV-2 is delivered to the virion assembly site in the ER-Golgi Intermediate Compartment (ERGIC) from both the ER and cis-Golgi in infected cells. However, the relevance and modulatory mechanism of this bidirectional trafficking are unclear. Here, using structure-function analyses, we show that S incorporation into virus-like particles (VLP) and VLP fusogenicity are determined by coatomer-dependent S delivery from the cis-Golgi and restricted by S-coatomer dissociation. Although S mimicry of the host coatomer-binding dibasic motif ensures retrograde trafficking to the ERGIC, avoidance of the host-like C-terminal acidic residue is critical for S-coatomer dissociation and therefore incorporation into virions or export for cell-cell fusion. Because this C-terminal residue is the key determinant of SARS-CoV-2 assembly and fusogenicity, our work provides a framework for the export of S protein encoded in genetic vaccines for surface display and immune activation.

Acquired resistance to KRAS G12C small-molecule inhibitors via genetic/nongenetic mechanisms in lung cancer.

Inherent or acquired resistance to sotorasib poses a substantialt challenge for NSCLC treatment. Here, we demonstrate that acquired resistance to sotorasib in isogenic cells correlated with increased expression of integrin ß4 (ITGB4), a component of the focal adhesion complex. Silencing ITGB4 in tolerant cells improved sotorasib sensitivity, while overexpressing ITGB4 enhanced tolerance to sotorasib by supporting AKT-mTOR bypass signaling. Chronic treatment with sotorasib induced WNT expression and activated the WNT/ß-catenin signaling pathway. Thus, silencing both ITGB4 and ß-catenin significantly improved sotorasib sensitivity in tolerant, acquired, and inherently resistant cells. In addition, the proteasome inhibitor carfilzomib (CFZ) exhibited synergism with sotorasib by down-regulating ITGB4 and ß-catenin expression. Furthermore, adagrasib phenocopies the combination effect of sotorasib and CFZ by suppressing KRAS activity and inhibiting cell cycle progression in inherently resistant cells. Overall, our findings unveil previously unrecognized nongenetic mechanisms underlying resistance to sotorasib and propose a promising treatment strategy to overcome resistance.

Reversible switching between two common protein folds in a designed system using only temperature.

Naturally occurring metamorphic proteins have the ability to interconvert from one folded state to another through either a limited set of mutations or by way of a change in the local environment. Here, we show in a designed system that it is possible to switch reversibly between two of the most common monomeric folds employing only temperature changes. We demonstrate that a latent 3α state can be unmasked from an α/ß-plait topology with a single V90T amino acid substitution, populating both forms simultaneously. The equilibrium between these two states exhibits temperature dependence, such that the 3α state is predominant (>90%) at 5 °C, while the α/ß-plait fold is the major species (>90%) at 30 °C. We describe the structure and dynamics of these topologies, how mutational changes affect the temperature dependence, and the energetics and kinetics of interconversion. Additionally, we demonstrate how ligand-binding function can be tightly regulated by large amplitude changes in protein structure over a relatively narrow temperature range that is relevant to biology. The 3α/αß switch thus represents a potentially useful approach for designing proteins that alter their fold topologies in response to environmental triggers. It may also serve as a model for computational studies of temperature-dependent protein stability and fold switching.

Design and characterization of a protein fold switching network.

To better understand how amino acid sequence encodes protein structure, we engineered mutational pathways that connect three common folds (3α, ß-grasp, and α/ß-plait). The structures of proteins at high sequence-identity intersections in the pathways (nodes) were determined using NMR spectroscopy and analyzed for stability and function. To generate nodes, the amino acid sequence encoding a smaller fold is embedded in the structure of an ~50% larger fold and a new sequence compatible with two sets of native interactions is designed. This generates protein pairs with a 3α or ß-grasp fold in the smaller form but an α/ß-plait fold in the larger form. Further, embedding smaller antagonistic folds creates critical states in the larger folds such that single amino acid substitutions can switch both their fold and function. The results help explain the underlying ambiguity in the protein folding code and show that new protein structures can evolve via abrupt fold switching.

Intrinsically disordered proteins: Ensembles at the limits of Anfinsen's dogma.

Intrinsically disordered proteins (IDPs) are proteins that lack rigid 3D structure. Hence, they are often misconceived to present a challenge to Anfinsen's dogma. However, IDPs exist as ensembles that sample a quasi-continuum of rapidly interconverting conformations and, as such, may represent proteins at the extreme limit of the Anfinsen postulate. IDPs play important biological roles and are key components of the cellular protein interaction network (PIN). Many IDPs can interconvert between disordered and ordered states as they bind to appropriate partners. Conformational dynamics of IDPs contribute to conformational noise in the cell. Thus, the dysregulation of IDPs contributes to increased noise and "promiscuous" interactions. This leads to PIN rewiring to output an appropriate response underscoring the critical role of IDPs in cellular decision making. Nonetheless, IDPs are not easily tractable experimentally. Furthermore, in the absence of a reference conformation, discerning the energy landscape representation of the weakly funneled IDPs in terms of reaction coordinates is challenging. To understand conformational dynamics in real time and decipher how IDPs recognize multiple binding partners with high specificity, several sophisticated knowledge-based and physics-based in silico sampling techniques have been developed. Here, using specific examples, we highlight recent advances in energy landscape visualization and molecular dynamics simulations to discern conformational dynamics and discuss how the conformational preferences of IDPs modulate their function, especially in phenotypic switching. Finally, we discuss recent progress in identifying small molecules targeting IDPs underscoring the potential therapeutic value of IDPs. Understanding structure and function of IDPs can not only provide new insight on cellular decision making but may also help to refine and extend Anfinsen's structure/function paradigm.

Engineering subtilisin proteases that specifically degrade active RAS.

We describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

Protein conformational dynamics and phenotypic switching.

Intrinsically disordered proteins (IDPs) are proteins that lack rigid 3D structure but exist as conformational ensembles. Because of their structural plasticity, they can interact with multiple partners. The protein interactions between IDPs and their partners form scale-free protein interaction networks (PINs) that facilitate information flow in the cell. Because of their plasticity, IDPs typically occupy hub positions in cellular PINs. Furthermore, their conformational dynamics and propensity for post-translational modifications contribute to "conformational" noise which is distinct from the well-recognized transcriptional noise. Therefore, upregulation of IDPs in response to a specific input, such as stress, contributes to increased noise and, hence, an increase in stochastic, "promiscuous" interactions. These interactions lead to activation of latent pathways or can induce "rewiring" of the PIN to yield an optimal output underscoring the critical role of IDPs in regulating information flow. We have used PAGE4, a highly intrinsically disordered stress-response protein as a paradigm. Employing a variety of experimental and computational techniques, we have elucidated the role of PAGE4 in phenotypic switching of prostate cancer cells at a systems level. These cumulative studies over the past decade provide a conceptual framework to better understand how IDP conformational dynamics and conformational noise might facilitate cellular decision-making.

Peptide-MHC Binding Reveals Conserved Allosteric Sites in MHC Class I- and Class II-Restricted T Cell Receptors (TCRs).

T cells are vital for adaptive immune responses that protect against pathogens and cancers. The T cell receptor (TCR)-CD3 complex comprises a diverse αß TCR heterodimer in noncovalent association with three invariant CD3 dimers. The TCR is responsible for recognizing antigenic peptides bound to MHC molecules (pMHC), while the CD3 dimers relay activation signals to the T cell. However, the mechanisms by which TCR engagement by pMHC is transmitted to CD3 remain mysterious, although there is growing evidence that mechanosensing and allostery both play a role. Here, we carried out NMR analysis of a human autoimmune TCR (MS2-3C8) that recognizes a self-peptide from myelin basic protein presented by the MHC class II molecule HLA-DR4. We observed pMHC-induced NMR signal perturbations in MS2-3C8 that indicate long-range effects on TCR ß chain conformation and dynamics. Our results demonstrate that, in addition to expected changes in the NMR resonances of pMHC-contacting residues, perturbations extend to the Vß/Vα, Vß/Cß, and Cß/Cα interfacial regions. Moreover, the pattern of long-range perturbations is similar to that detected previously in the ß chains of two MHC class I-restricted TCRs, thereby revealing a common allosteric pathway among three unrelated TCRs. Molecular dynamics (MD) simulations predict similar pMHC-induced effects. Taken together, our results demonstrate that pMHC binding induces long-range allosteric changes in the TCR ß chain at conserved sites in both representative MHC class I- and class II-restricted TCRs, and that these sites may play a role in the transmission of signaling information.

Erratum: A Non-genetic Mechanism Involving the Integrin ß4/Paxillin Axis Contributes to Chemoresistance in Lung Cancer.

[This corrects the article DOI: 10.1016/j.isci.2020.101496.].

A Non-genetic Mechanism Involving the Integrin ß4/Paxillin Axis Contributes to Chemoresistance in Lung Cancer.

Tumor heterogeneity and cisplatin resistance are major causes of tumor relapse and poor survival. Here, we show that in lung cancer, interaction between paxillin (PXN) and integrin ß4 (ITGB4), components of the focal adhesion (FA) complex, contributes to cisplatin resistance. Knocking down PXN and ITGB4 attenuated cell growth and improved cisplatin sensitivity, both in 2D and 3D cultures. PXN and ITGB4 independently regulated expression of several genes. In addition, they also regulated expression of common genes including USP1 and VDAC1, which are required for maintaining genomic stability and mitochondrial function, respectively. Mathematical modeling suggested that bistability could lead to stochastic phenotypic switching between cisplatin-sensitive and resistant states in these cells. Consistently, purified subpopulations of sensitive and resistant cells re-created the mixed parental population when cultured separately. Altogether, these data point to an unexpected role of the FA complex in cisplatin resistance and highlight a novel non-genetic mechanism.

The structural basis of T-cell receptor (TCR) activation: An enduring enigma.

T cells are critical for protective immune responses to pathogens and tumors. The T-cell receptor (TCR)-CD3 complex is composed of a diverse αß TCR heterodimer noncovalently associated with the invariant CD3 dimers CD3ϵγ, CD3ϵδ, and CD3ζζ. The TCR mediates recognition of antigenic peptides bound to MHC molecules (pMHC), whereas the CD3 molecules transduce activation signals to the T cell. Whereas much is known about downstream T-cell signaling pathways, the mechanism whereby TCR engagement by pMHC is first communicated to the CD3 signaling apparatus, a process termed early T-cell activation, remains largely a mystery. In this review, we examine the molecular basis for TCR activation in light of the recently determined cryoEM structure of a complete TCR-CD3 complex. This structure provides an unprecedented opportunity to assess various signaling models that have been proposed for the TCR. We review evidence from single-molecule and structural studies for force-induced conformational changes in the TCR-CD3 complex, for dynamically-driven TCR allostery, and for pMHC-induced structural changes in the transmembrane and cytoplasmic regions of CD3 subunits. We identify major knowledge gaps that must be filled in order to arrive at a comprehensive model of TCR activation that explains, at the molecular level, how pMHC-specific information is transmitted across the T-cell membrane to initiate intracellular signaling. An in-depth understanding of this process will accelerate the rational design of immunotherapeutic agents targeting the TCR-CD3 complex.

Structural and Dynamical Order of a Disordered Protein: Molecular Insights into Conformational Switching of PAGE4 at the Systems Level.

Folded proteins show a high degree of structural order and undergo (fairly constrained) collective motions related to their functions. On the other hand, intrinsically disordered proteins (IDPs), while lacking a well-defined three-dimensional structure, do exhibit some structural and dynamical ordering, but are less constrained in their motions than folded proteins. The larger structural plasticity of IDPs emphasizes the importance of entropically driven motions. Many IDPs undergo function-related disorder-to-order transitions driven by their interaction with specific binding partners. As experimental techniques become more sensitive and become better integrated with computational simulations, we are beginning to see how the modest structural ordering and large amplitude collective motions of IDPs endow them with an ability to mediate multiple interactions with different partners in the cell. To illustrate these points, here, we use Prostate-associated gene 4 (PAGE4), an IDP implicated in prostate cancer (PCa) as an example. We first review our previous efforts using molecular dynamics simulations based on atomistic AWSEM to study the conformational dynamics of PAGE4 and how its motions change in its different physiologically relevant phosphorylated forms. Our simulations quantitatively reproduced experimental observations and revealed how structural and dynamical ordering are encoded in the sequence of PAGE4 and can be modulated by different extents of phosphorylation by the kinases HIPK1 and CLK2. This ordering is reflected in changing populations of certain secondary structural elements as well as in the regularity of its collective motions. These ordered features are directly correlated with the functional interactions of WT-PAGE4, HIPK1-PAGE4 and CLK2-PAGE4 with the AP-1 signaling axis. These interactions give rise to repeated transitions between (high HIPK1-PAGE4, low CLK2-PAGE4) and (low HIPK1-PAGE4, high CLK2-PAGE4) cell phenotypes, which possess differing sensitivities to the standard PCa therapies, such as androgen deprivation therapy (ADT). We argue that, although the structural plasticity of an IDP is important in promoting promiscuous interactions, the modulation of the structural ordering is important for sculpting its interactions so as to rewire with agility biomolecular interaction networks with significant functional consequences.

Peptide-MHC (pMHC) binding to a human antiviral T cell receptor induces long-range allosteric communication between pMHC- and CD3-binding sites.

T cells generate adaptive immune responses mediated by the T cell receptor (TCR)-CD3 complex comprising an αß TCR heterodimer noncovalently associated with three CD3 dimers. In early T cell activation, αß TCR engagement by peptide-major histocompatibility complex (pMHC) is first communicated to the CD3 signaling apparatus of the TCR-CD3 complex, but the underlying mechanism is incompletely understood. It is possible that pMHC binding induces allosteric changes in TCR conformation or dynamics that are then relayed to CD3. Here, we carried out NMR analysis and molecular dynamics (MD) simulations of both the α and ß chains of a human antiviral TCR (A6) that recognizes the Tax antigen from human T cell lymphotropic virus-1 bound to the MHC class I molecule HLA-A2. We observed pMHC-induced NMR signal perturbations in the TCR variable (V) domains that propagated to three distinct sites in the constant (C) domains: 1) the Cß FG loop projecting from the Vß/Cß interface; 2) a cluster of Cß residues near the Cß αA helix, a region involved in interactions with CD3; and 3) the Cα AB loop at the membrane-proximal base of the TCR. A biological role for each of these allosteric sites is supported by previous mutational and functional studies of TCR signaling. Moreover, the pattern of long-range, ligand-induced changes in TCR A6 revealed by NMR was broadly similar to that predicted by the MD simulations. We propose that the unique structure of the TCR ß chain enables allosteric communication between the TCR-binding sites for pMHC and CD3.

Structural metamorphism and polymorphism in proteins on the brink of thermodynamic stability.

The classical view of the structure-function paradigm advanced by Anfinsen in the 1960s is that a protein's function is inextricably linked to its three-dimensional structure and is encrypted in its amino acid sequence. However, it is now known that a significant fraction of the proteome consists of intrinsically disordered proteins (IDPs). These proteins populate a polymorphic ensemble of conformations rather than a unique structure but are still capable of performing biological functions. At the boundary, between well-ordered and inherently disordered states are proteins that are on the brink of stability, either weakly stable ordered systems or disordered but on the verge of being stable. In such marginal states, even relatively minor changes can significantly alter the energy landscape, leading to large-scale conformational remodeling. Some proteins on the edge of stability are metamorphic, with the capacity to switch from one fold topology to another in response to an environmental trigger (e.g., pH, temperature/salt, redox). Many IDPs, on the other hand, are marginally unstable such that small perturbations (e.g., phosphorylation, ligands) tip the balance over to a range of ordered, partially ordered, or even more disordered states. In general, the structural transitions described by metamorphic fold switches and polymorphic IDPs possess a number of common features including low or diminished stability, large-scale conformational changes, critical disordered regions, latent or attenuated binding sites, and expansion of function. We suggest that these transitions are, therefore, conceptually and mechanistically analogous, representing adjacent regions in the continuum of order/disorder transitions.

Prostate-Associated Gene 4 (PAGE4): Leveraging the Conformational Dynamics of a Dancing Protein Cloud as a Therapeutic Target.

Prostate cancer (PCa) is a leading cause of mortality and morbidity globally. While genomic alterations have been identified in PCa, in contrast to some other cancers, use of such information to personalize treatment is still in its infancy. Here, we discuss how PAGE4, a protein which appears to act both as an oncogenic factor as well as a metastasis suppressor, is a novel therapeutic target for PCa. Inhibiting PAGE4 may be a viable strategy for low-risk PCa where it is highly upregulated. Conversely, PAGE4 expression is downregulated in metastatic PCa and, therefore, reinstituting its sustained expression may be a promising option to subvert or attenuate androgen-resistant PCa. Thus, fine-tuning the levels of PAGE4 may represent a novel approach for personalized medicine in PCa.

PAGE4 and Conformational Switching: Insights from Molecular Dynamics Simulations and Implications for Prostate Cancer.

Prostate-associated gene 4 (PAGE4) is an intrinsically disordered protein implicated in prostate cancer. Thestress-response kinase homeodomain-interacting protein kinase 1 (HIPK1) phosphorylates two residues in PAGE4, serine 9 and threonine 51. Phosphorylation of these two residues facilitates the interaction of PAGE4 with activator protein-1 (AP-1) transcription factor complex to potentiate AP-1's activity. In contrast, hyperphosphorylation of PAGE4 by CDC-like kinase 2 (CLK2) attenuates this interaction with AP-1. Small-angleX-ray scattering and single-molecule fluorescence resonance energy transfer measurements have shown that PAGE4 expands upon hyperphosphorylation and that this expansion is localized to its N-terminal half. To understand the interactions underlying this structural transition, we performed molecular dynamics simulations using Atomistic AWSEM, a multi-scale molecular model that combines atomistic and coarse-grained simulation approaches. Our simulations show that electrostatic interactions drive transient formation of an N-terminal loop, the destabilization of which accounts for the dramatic change in size upon hyperphosphorylation. Phosphorylation also changes the preference of secondary structure formation of the PAGE4 ensemble, which leads to a transition between states that display different degrees of disorder. Finally, we construct a mechanism-based mathematical model that allows us to capture the interactions ofdifferent phosphoforms of PAGE4 with AP-1 and its downstream target, the androgen receptor (AR)-a key therapeutic target in prostate cancer. Our model predicts intracellular oscillatory dynamics of HIPK1-PAGE4, CLK2-PAGE4, and AR activity, indicating phenotypic heterogeneity in an isogenic cell population. Thus, conformational switching of PAGE4 may potentially affect the efficiency of therapeutically targeting AR activity.

Phenotypic Plasticity, Bet-Hedging, and Androgen Independence in Prostate Cancer: Role of Non-Genetic Heterogeneity.

It is well known that genetic mutations can drive drug resistance and lead to tumor relapse. Here, we focus on alternate mechanisms-those without mutations, such as phenotypic plasticity and stochastic cell-to-cell variability that can also evade drug attacks by giving rise to drug-tolerant persisters. The phenomenon of persistence has been well-studied in bacteria and has also recently garnered attention in cancer. We draw a parallel between bacterial persistence and resistance against androgen deprivation therapy in prostate cancer (PCa), the primary standard care for metastatic disease. We illustrate how phenotypic plasticity and consequent mutation-independent or non-genetic heterogeneity possibly driven by protein conformational dynamics can stochastically give rise to androgen independence in PCa, and suggest that dynamic phenotypic plasticity should be considered in devising therapeutic dosing strategies designed to treat and manage PCa.

Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity.

Intrinsically disordered proteins (IDPs) that lack a unique 3D structure and comprise a large fraction of the human proteome play important roles in numerous cellular functions. Prostate-Associated Gene 4 (PAGE4) is an IDP that acts as a potentiator of the Activator Protein-1 (AP-1) transcription factor. Homeodomain-Interacting Protein Kinase 1 (HIPK1) phosphorylates PAGE4 at S9 and T51, but only T51 is critical for its activity. Here, we identify a second kinase, CDC-Like Kinase 2 (CLK2), which acts on PAGE4 and hyperphosphorylates it at multiple S/T residues, including S9 and T51. We demonstrate that HIPK1 is expressed in both androgen-dependent and androgen-independent prostate cancer (PCa) cells, whereas CLK2 and PAGE4 are expressed only in androgen-dependent cells. Cell-based studies indicate that PAGE4 interaction with the two kinases leads to opposing functions. HIPK1-phosphorylated PAGE4 (HIPK1-PAGE4) potentiates c-Jun, whereas CLK2-phosphorylated PAGE4 (CLK2-PAGE4) attenuates c-Jun activity. Consistent with the cellular data, biophysical measurements (small-angle X-ray scattering, single-molecule fluorescence resonance energy transfer, and NMR) indicate that HIPK1-PAGE4 exhibits a relatively compact conformational ensemble that binds AP-1, whereas CLK2-PAGE4 is more expanded and resembles a random coil with diminished affinity for AP-1. Taken together, the results suggest that the phosphorylation-induced conformational dynamics of PAGE4 may play a role in modulating changes between PCa cell phenotypes. A mathematical model based on our experimental data demonstrates how differential phosphorylation of PAGE4 can lead to transitions between androgen-dependent and androgen-independent phenotypes by altering the AP-1/androgen receptor regulatory circuit in PCa cells.

Prostate-associated gene 4 (PAGE4), an intrinsically disordered cancer/testis antigen, is a novel therapeutic target for prostate cancer.

Prostate-associated gene 4 (PAGE4) is a remarkably prostate-specific Cancer/Testis Antigen that is highly upregulated in the human fetal prostate and its diseased states but not in the adult normal gland. PAGE4 is an intrinsically disordered protein (IDP) that functions as a stress-response protein to suppress reactive oxygen species as well as prevent DNA damage. In addition, PAGE4 is also a transcriptional regulator that potentiates transactivation by the oncogene c-Jun. c-Jun forms the AP-1 complex by heterodimerizing with members of the Fos family and plays an important role in the development and pathology of the prostate gland, underscoring the importance of the PAGE4/c-Jun interaction. HIPK1, also a component of the stress-response pathway, phosphorylates PAGE4 at T51 which is critical for its transcriptional activity. Phosphorylation induces conformational and dynamic switching in the PAGE4 ensemble leading to a new cellular function. Finally, bioinformatics evidence suggests that the PAGE4 mRNA could be alternatively spliced resulting in four potential isoforms of the polypeptide alluding to the possibility of a range of conformational ensembles with latent functions. Considered together, the data suggest that PAGE4 may represent the first molecular link between stress and prostate cancer (PCa). Thus, pharmacologically targeting PAGE4 may be a novel opportunity for treating and managing patients with PCa, especially patients with low-risk disease.

Phosphorylation-induced Conformational Ensemble Switching in an Intrinsically Disordered Cancer/Testis Antigen.

Prostate-associated gene 4 (PAGE4) is an intrinsically disordered cancer/testis antigen that is up-regulated in the fetal and diseased human prostate. Knocking down PAGE4 expression results in cell death, whereas its overexpression leads to a growth advantage of prostate cancer cells (Zeng, Y., He, Y., Yang, F., Mooney, S. M., Getzenberg, R. H., Orban, J., and Kulkarni, P. (2011) The cancer/testis antigen prostate-associated gene 4 (PAGE4) is a highly intrinsically disordered protein. J. Biol. Chem. 286, 13985-13994). Phosphorylation of PAGE4 at Thr-51 is critical for potentiating c-Jun transactivation, an important factor in controlling cell growth, apoptosis, and stress response. Using NMR spectroscopy, we show that the PAGE4 polypeptide chain has local and long-range conformational preferences that are perturbed by site-specific phosphorylation at Thr-51. The population of transient turn-like structures increases upon phosphorylation in an ∼20-residue acidic region centered on Thr-51. This central region therefore becomes more compact and more negatively charged, with increasing intramolecular contacts to basic sequence motifs near the N and C termini. Although flexibility is decreased in the central region of phospho-PAGE4, the polypeptide chain remains highly dynamic overall. PAGE4 utilizes a transient helical structure adjacent to the central acidic region to bind c-Jun with low affinity in vitro. The binding interaction is attenuated by phosphorylation at Thr-51, most likely because of masking the effects of the more compact phosphorylated state. Therefore, phosphorylation of PAGE4 leads to conformational shifts in the dynamic ensemble, with large functional consequences. The changes in the structural ensemble induced by posttranslational modifications are similar conceptually to the conformational switching events seen in some marginally stable ("metamorphic") folded proteins in response to mutation or environmental triggers.