Prokaryotic Ubiquitin-Like Protein Pup Is Intrinsically Disordered

The prokaryotic ubiquitin-like protein Pup targets substrates for degradation by the Mycobacterium tuberculosis proteasome through its interaction with Mpa, an ATPase that is thought to abut the 20S catalytic subunit. Ubiquitin, which is assembled into a polymer to similarly signal for proteasomal degradation in eukaryotes, adopts a stable and compact structural fold that is adapted into other proteins for diverse biological functions. We used NMR spectroscopy to demonstrate that, unlike ubiquitin, the 64-amino-acid protein Pup is intrinsically disordered with small helical propensity in the C-terminal region. We found that the Pup:Mpa interaction involves an extensive contact surface that spans S21-K61 and that the binding is in the “slow exchange” regime on the NMR time scale, thus demonstrating higher affinity than most ubiquitin:ubiquitin receptor pairs. Interestingly, during the titration experiment, intermediate Pup species were observable, suggesting the formation of one or more transient state(s) upon binding. Moreover, Mpa selected one configuration for a region undergoing chemical exchange in the free protein. These findings provide mechanistic insights into Pup's functional role as a degradation signal.     

The Dnmt1 Intrinsically Disordered Domain Regulates Genomic Methylation During Development

The DNMT1 cytosine methyltransferase enzyme contains a large ∼300-aa intrinsically disordered domain (IDD) that we previously showed regulated DNA methylation patterns in mouse ES cells. Here we generated seven mouse lines with different mutations in the IDD. Homozygous mutant mice of five lines developed normally, with normal levels of methylation on both imprinted and nonimprinted DNA sequences. The other two lines, however, had alterations in imprinted and/or nonimprinted (global) DNA methylation appearing during embryonic development. Embryos of one line expressing a DNMT1 variant containing a 6-aa rat orthologous sequence in the IDD maintained imprinted methylation, showed very reduced levels of global methylation and occasionally completed fetal development. These in vivo studies demonstrate that at least two DNMT1-dependent methylation processes can be distinguished during fetal development. One process maintains the bulk of genomic methylation on nonimprinted sequences. The other process maintains methylation on a much smaller class of sequences including but not limited to gametic differentially methylated domains (gDMDs) that transmit essential imprinted parent-specific methylation for embryonic development.     

Mechanism of Assembly of the Non-Covalent Spectrin Tetramerization Domain from Intrinsically Disordered Partners

Interdomain interactions of spectrin are critical for maintenance of the erythrocyte cytoskeleton. In particular, “head-to-head” dimerization occurs when the intrinsically disordered C-terminal tail of β-spectrin binds the N-terminal tail of α-spectrin, folding to form the “spectrin tetramer domain”. This non-covalent three-helix bundle domain is homologous in structure and sequence to previously studied spectrin domains. We find that this tetramer domain is surprisingly kinetically stable. Using a protein engineering Φ-value analysis to probe the mechanism of formation of this tetramer domain, we infer that the domain folds by the docking of the intrinsically disordered β-spectrin tail onto the more structured α-spectrin tail.     

The Lifestyle Switch Protein Bd0108 of Bdellovibrio bacteriovorus Is an Intrinsically Disordered Protein

Bdellovibrio bacteriovorus is a δ-proteobacterium that preys upon Salmonella spp., E. coli, and other Gram-negative bacteria. Bdellovibrio can grow axenically (host-independent, HI, rare and mutation-driven) or subsist via a predatory lifecycle (host-dependent, HD, the usual case). Upon contact with prey, B. bacteriovorus enters the host periplasm from where it slowly drains the host cytosol of nutrients for its own replication. At the core of this mechanism is a retractile pilus, whose architecture is regulated by the protein Bd0108 and its interaction with the neighboring gene product Bd0109. Deletion of bd0108 results in negligible pilus formation, whereas an internal deletion (the one that instigates host-independence) causes mis-regulation of pilus length. These mutations, along with a suite of naturally occurring bd0108 mutant strains, act to control the entry to HI growth. To further study the molecular mechanism of predatory regulation, we focused on the apparent lifecycle switch protein Bd0108. Here we characterize the solution structure and dynamics of Bd0108 using nuclear magnetic resonance (NMR) spectroscopy complemented with additional biophysical methods. We then explore the interaction between Bd0108 and Bd0109 in detail utilizing isothermal titration calorimetry (ITC) and NMR spectroscopy. Together our results demonstrate that Bd0108 is an intrinsically disordered protein (IDP) and that the interaction with Bd0109 is of low affinity. Furthermore, we observe that Bd0108 retains an IDP nature while binding Bd0109. From our data we conclude that Bdellovibrio bacteriovorus utilizes an intrinsically disordered protein to regulate its pilus and control predation signaling.     

Leukemia Fusion Target AF9 Is an Intrinsically Disordered Transcriptional Regulator that Recruits Multiple Partners via Coupled Folding and Binding

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Highlights? The AF9 AHD is intrinsically disordered ? The AHD recruits AF4, BCoR, Dot1L, and hPC3 by coupled folding and binding ? AF9 binding partners compete for binding to a common site ? Dynamics of the AF4-AF9 complex may facilitate exchange between partners     

Entropy Hotspots for the Binding of Intrinsically Disordered Ligands to a Receptor Domain

Proline-rich motifs (PRMs) are widely used for mediating protein-protein interactions with weak binding affinities. Because they are intrinsically disordered when unbound, conformational entropy plays a significant role for the binding. However, residue-level differences of the entropic contribution in the binding of different ligands remain not well understood. We use all-atom molecular dynamics simulation and the maximal information spanning tree formalism to analyze conformational entropy associated with the binding of two PRMs, one from the Abl kinase and the other from the nonstructural protein 1 of the 1918 Spanish influenza A virus, to the N-terminal SH3 (nSH3) domain of the CrkII protein. Side chains of the stably folded nSH3 experience more entropy change upon ligand binding than the backbone, whereas PRMs involve comparable but heterogeneous entropy changes among the backbone and side chains. In nSH3, two conserved nonpolar residues forming contacts with the PRM experience the largest side-chain entropy loss. In contrast, the C-terminal charged residues of PRMs that form polar contacts with nSH3 experience the greatest side-chain entropy loss, although their “fuzzy” nature is attributable to the backbone that remains relatively flexible. Thus, residues that form high-occupancy contacts between nSH3 and PRM do not reciprocally contribute to entropy loss. Furthermore, certain surface residues of nSH3 distal to the interface with PRMs gain entropy, indicating a nonlocal effect of ligand binding. Comparing between the PRMs from cAbl and nonstructural protein 1, the latter involves a larger side-chain entropy loss and forms more contacts with nSH3. Consistent with experiments, this indicates stronger binding of the viral ligand at the expense of losing the flexibility of side chains, whereas the backbone experiences less entropy loss. The entropy “hotspots” as identified in this study will be important for tuning the binding affinity of various ligands to a receptor.     

The Intrinsically Disordered Domain of the Antitoxin Phd Chaperones the Toxin Doc against Irreversible Inactivation and Misfolding

The toxin Doc from the phd/doc toxin-antitoxin module targets the cellular translation machinery and is inhibited by its antitoxin partner Phd. Here we show that Phd also functions as a chaperone, keeping Doc in an active, correctly folded conformation. In the absence of Phd, Doc exists in a relatively expanded state that is prone to dimerization through domain swapping with its active site loop acting as hinge region. The domain-swapped dimer is not capable of arresting protein synthesis in vitro, whereas the Doc monomer is. Upon binding to Phd, Doc becomes more compact and is secured in its monomeric state with a neutralized active site.     

Tyrosinase Degradation Is Prevented when EDEM1 Lacks the Intrinsically Disordered Region

EDEM1 is a mannosidase-like protein that recruits misfolded glycoproteins from the calnexin/calreticulin folding cycle to downstream endoplasmic reticulum associated degradation (ERAD) pathway. Here, we investigate the role of EDEM1 in the processing of tyrosinase, a tumour antigen overexpressed in melanoma cells. First, we analyzed and modeled EDEM1 major domains. The homology model raised on the crystal structures of human and Saccharomyces cerevisiae ER class I α1,2-mannosidases reveals that the major mannosidase domain located between aminoacids 121-598 fits with high accuracy. We have further identified an N-terminal region located between aminoacids 40-119, predicted to be intrinsically disordered (ID) and susceptible to adopt multiple conformations, hence facilitating protein-protein interactions. To investigate these two domains we have constructed an EDEM1 deletion mutant lacking the ID region and a triple mutant disrupting the glycan-binding domain and analyzed their association with tyrosinase. Tyrosinase is a glycoprotein partly degraded endogenously by ERAD and the ubiquitin proteasomal system. We found that the degradation of wild type and misfolded tyrosinase was enhanced when EDEM1 was overexpressed. Glycosylated and non-glycosylated mutants co-immunoprecipitated with EDEM1 even in the absence of its intact mannosidase-like domain, but not when the ID region was deleted. In contrast, calnexin and SEL 1L associated with the deletion mutant. Our data suggest that the ID region identified in the N-terminal end of EDEM1 is involved in the binding of glycosylated and non-glycosylated misfolded proteins. Accelerating tyrosinase degradation by EDEM1 overexpression may lead to an efficient antigen presentation and enhanced elimination of melanoma cells.     

The Intrinsically Disordered N-terminal Domain of Thymidylate Synthase Targets the Enzyme to the Ubiquitin-independent Proteasomal Degradation Pathway

The ubiquitin-independent proteasomal degradation pathway is increasingly being recognized as important in regulation of protein turnover in eukaryotic cells. One substrate of this pathway is the pyrimidine biosynthetic enzyme thymidylate synthase (TS; EC 2.1.1.45), which catalyzes the reductive methylation of dUMP to form dTMP and is essential for DNA replication during cell growth and proliferation. Previous work from our laboratory showed that degradation of TS is ubiquitin-independent and mediated by an intrinsically disordered 27-residue region at the N-terminal end of the molecule. In the current study we show that this region, in cooperation with an α-helix formed by the next 15 residues, functions as a degron, i.e. it is capable of destabilizing a heterologous protein to which it is fused. Comparative analysis of the primary sequence of TS from a number of mammalian species revealed that the N-terminal domain is hypervariable among species yet is conserved with regard to its disordered nature, its high Pro content, and the occurrence of Pro at the penultimate site. Characterization of mutant proteins showed that Pro-2 protects the N terminus against N^α-acetylation, a post-translational process that inhibits TS degradation. However, although a free amino group at the N terminus is necessary, it is not sufficient for degradation of the polypeptide. The implications of these findings to the proteasome-targeting function of the N-terminal domain, particularly with regard to its intrinsic flexibility, are discussed.     

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and its interaction with the human chaperone cyclophilin A are both targets for highly potent and promising antiviral drugs that are in the late stages of clinical development. Despite its high interest in regards to the development of drugs to counteract the worldwide HCV burden, NS5A is still an enigmatic multifunctional protein poorly characterized at the molecular level. NS5A is required for HCV RNA replication and is involved in viral particle formation and regulation of host pathways. Thus far, no enzymatic activity or precise molecular function has been ascribed to NS5A that is composed of a highly structured domain 1 (D1), as well as two intrinsically disordered domains 2 (D2) and 3 (D3), representing half of the protein. Here, we identify a short structural motif in the disordered NS5A-D2 and report its NMR structure. We show that this structural motif, a minimal Pro³¹⁴–Trp³¹⁶ turn, is essential for HCV RNA replication, and its disruption alters the subcellular distribution of NS5A. We demonstrate that this Pro-Trp turn is required for proper interaction with the host cyclophilin A and influences its peptidyl-prolyl cis/trans isomerase activity on residue Pro³¹⁴ of NS5A-D2. This work provides a molecular basis for further understanding of the function of the intrinsically disordered domain 2 of HCV NS5A protein. In addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function.     

The Desmoglein-Specific Cytoplasmic Region Is Intrinsically Disordered in Solution and Interacts with Multiple Desmosomal Protein Partners

The desmoglein-specific cytoplasmic region (DSCR) is a conserved region of unknown structure and function that uniquely defines the desmoglein family of cell adhesion molecules. It is the site of caspase cleavage during apoptosis, and its mutation is linked to cardiomyopathy. Here, we reveal that a 276-residue DSCR construct of human desmoglein 1 is intrinsically disordered and forms an interaction hub for desmosomal proteins. In solution, it contains 6.5% helical and 10.3% β-strand structure based on circular dichroism spectroscopy. A single monomeric state with a predominantly unfolded structure is found by size-exclusion chromatography and analytical ultracentrifugation. Thermal stability assays and nuclear magnetic resonance spectroscopy reveal a nonglobular structure under a range of solution conditions. However, the introduction of detergent micelles increases structure to 18% helical and 16% β-strand character, suggesting an inducible structure. The DSCR exhibits weak but specific interactions with plakoglobin, the plakin domain of desmoplakin, plakophilin 1, and the cytoplasmic domain of desmocollin 1. The desmoglein 1 membrane proximal region also interacts with all four DSCR ligands, strongly with plakoglobin and plakophilin and more weakly with desmoplakin and desmocollin 1. Thus, the DSCR is an intrinsically disordered functional domain with an inducible structure that, along with the membrane proximal region, forms a flexible scaffold for cytoplasmic assembly at the desmosome.     

Conformational Recognition of an Intrinsically Disordered Protein

There is a growing interest in understanding the properties of intrinsically disordered proteins (IDPs); however, the characterization of these states remains an open challenge. IDPs appear to have functional roles that diverge from those of folded proteins and revolve around their ability to act as hubs for protein-protein interactions. To gain a better understanding of the modes of binding of IDPs, we combined statistical mechanics, calorimetry, and NMR spectroscopy to investigate the recognition and binding of a fragment from the disordered protein Gab2 by the growth factor receptor-bound protein 2 (Grb2), a key interaction for normal cell signaling and cancer development. Structural ensemble refinement by NMR chemical shifts, thermodynamics measurements, and analysis of point mutations indicated that the population of preexisting bound conformations in the free-state ensemble of Gab2 is an essential determinant for recognition and binding by Grb2. A key role was found for transient polyproline II (PPII) structures and extended conformations. Our findings are likely to have very general implications for the biological behavior of IDPs in light of the evidence that a large fraction of these proteins possess a specific propensity to form PPII and to adopt conformations that are more extended than the typical random-coil states.