Intrinsically Disordered Linker and Plasma Membrane‐Binding Motif Sort Ist2 and Ssy1 to Junctions

Membrane junctions or contact sites are close associations of lipid bilayers of heterologous organelles. Ist2 is an endoplasmic reticulum (ER)‐resident transmembrane protein that mediates associations between the plasma membrane (PM) and the cortical ER (cER) in baker's yeast. We asked the question what structure in Ist2 bridges the up to 30 nm distance between the PM and the cER and we noted that the region spacing the transmembrane domain from the cortical sorting signal interacting with the PM is predicted to be intrinsically disordered (ID). In Ssy1, a protein that was not previously described to reside at membrane junctions, we recognized a domain organization similar to that in Ist2. We found that the localization of both Ist2 and Ssy1 at the cell periphery depends on the presence of a PM‐binding domain, an ID linker region of sufficient length and a transmembrane domain that most probably resides in the ER. We show for the first time that an ID amino acid domain bridges adjacent heterologous membranes. The length and flexibility of ID domains make them uniquely eligible for spanning large distances, and we suggest that this domain structure occurs more frequently in proteins that mediate the formation of membrane contact sites.      

Residual Structure Accelerates Binding of Intrinsically Disordered ACTR by Promoting Efficient Folding upon Encounter

Intrinsically disordered proteins (IDPs) often fold into stable structures upon specific binding. The roles of residual structure of unbound IDPs in coupling binding and folding have been under much debate. While many studies emphasize the importance of conformational flexibility for IDP recognition, it was recently demonstrated that stabilization the N-terminal helix of intrinsically disordered ACTR accelerated its binding to another IDP, NCBD of the CREB-binding protein. To understand how enhancing ACTR helicity accelerates binding, we derived a series of topology-based coarse-grained models that mimicked various ACTR mutants with increasing helical contents and reproduced their NCBD binding affinities. Molecular dynamics simulations were then performed to sample hundreds of reversible coupled binding and folding transitions. The results show that increasing ACTR helicity does not alter the baseline mechanism of synergistic folding, which continues to follow “extended conformational selection” with multiple stages of selection and induced folding. Importantly, these coarse-grained models, while only calibrated based on binding thermodynamics, recapitulate the observed kinetic acceleration with increasing ACTR helicity. However, the residual helices do not enhance the association kinetics via more efficient seeding of productive collisions. Instead, they allow the nonspecific collision complexes to evolve more efficiently into the final bound and folded state, which is the primary source of accelerated association kinetics. Meanwhile, reduced dissociation kinetics with increasing ACTR helicity can be directly attributed to smaller entropic cost of forming the bound state. Altogether, this study provides important mechanistic insights into how residual structure may modulate thermodynamics and kinetics of IDP interactions.     

Uncoupling the Folding and Binding of an Intrinsically Disordered Protein

The relationship between helical stability and binding affinity was examined for the intrinsically disordered transactivation domain of the myeloblastosis oncoprotein, c-Myb, and its ordered binding partner, KIX. A series of c-Myb mutants was designed to either increase or decrease helical stability without changing the binding interface with KIX. This included a complimentary series of A, G, P, and V mutants at three non-interacting sites. We were able to use the glycine mutants as a reference state and show a strong correlation between binding affinity and helical stability. The intrinsic helicity of c-Myb is 21%, and helicity values of the mutants ranged from 8% to 28%. The c-Myb helix is divided into two conformationally distinct segments. The N-terminal segment, from K291–L301, has an average helicity greater than 60% and the C-terminal segment, from S304–L315, has an average helicity less than 10%. We observed different effects on binding when these two segments were mutated. Mutants in the N-terminal segment that increased helicity had no effect on the binding affinity to KIX, while helix destabilizing glycine and proline mutants reduced binding affinity by more than 1 kcal/mol. Mutants that either increased or decreased helical stability in the C-terminal segment had almost no effect on binding. However, several of the mutants reveal the presence of multiple conformations accessible in the bound state based on changes in enthalpy and linkage analysis of binding free energies. These results may explain the high level of sequence identity (> 90%), even at non-interacting sites, for c-Myb homologues.     

HIV-1 ancestry primordial expansions of RRE and RRE related sequences.

The human T-lymphotropic retroviruses HTLV-I and HIV-1/-2 share a complementary patchwork homology in which the RRE regions of HIV-1 and -2 cover 78.1% of a 169 nucleotide (nt) region (PX1,2) positioned precisely between open reading frames PX I and PX II of ATLV (HTLV-I). The sequence character of the PX1,2 region is shown to be influenced by a primordial expansion, CTC2T, originating several hundred nucleotides upstream. A second primordial expansion, AGCU(U/C), is identified and shown to represent 52.1% of the HIV-1 RRE region. It is argued that prior to the present AIDS pandemic the efficiency of the rev receptor was enhanced by an ancestral recombination event.     

Macromolecular Crowding Induces a Binding Competent Transient Structure in Intrinsically Disordered Gab1

Intrinsically disordered proteins (IDPs) are an important class of proteins which lack tertiary structure elements. Their dynamic properties can depend on reversible post-translational modifications and the complex cellular milieu, which provides a crowded environment. Both influences the thermodynamic stability and folding of globular proteins as well as the conformational plasticity of IDPs. Here we investigate the intrinsically disordered C-terminal region (amino acids 613–694) of human Grb2-associated binding protein 1 (Gab1), which binds to the disease-relevant Src homolog region 2 (SH2) domain-containing protein tyrosine phosphatase SHP2 (PTPN11). This binding is mediated by phosphorylation at Tyr 627 and Tyr 659 in Gab1. We characterize induced structure in Gab1_613–694 and binding to SHP2 by NMR, CD and ITC under non-crowding and crowding conditions, employing chemical and biological crowding agents and compare the results of the non-phosphorylated and tyrosine phosphorylated C-terminal Gab1 fragment. Our results show that under crowding conditions pre-structured motifs in two distinct regions of Gab1 are formed whereas phosphorylation has no impact on the dynamics and IDP character. These structured regions are identical to the binding regions towards SHP2. Therefore, biological crowders could induce some SHP2 binding capacity. Our results therefore indicate that high concentrations of macromolecules stabilize the preformed or excited binding state in the C-terminal Gab1 region and foster the binding to the SH2 tandem motif of SHP2, even in the absence of tyrosine phosphorylation.     

Molecular recognition of a branched peptide with HIV-1 Rev Response Element (RRE) RNA

Interaction of HIV-1 rev response element (RRE) RNA with its cognate protein, Rev, is critical for HIV-1 replication. Understanding the mode of interaction between RRE RNA and ligands at the binding site can facilitate RNA molecular recognition as well as provide a strategy for developing anti-HIV therapeutics. Our approach utilizes branched peptides as a scaffold for multivalent binding to RRE IIB (high affinity rev binding site) with incorporation of unnatural amino acids to increase affinity via non-canonical interactions with the RNA. Previous high throughput screening of a 46,656-member library revealed several hits that bound RRE IIB RNA in the sub-micromolar range. In particular, the lead compound, 4B3, displayed a K_d value of 410?nM and demonstrated selectivity towards RRE. A ribonuclease protection assay revealed that 4B3 binds to the stem-loop structure of RRE IIB RNA, which was confirmed by SHAPE analysis with 234 nt long NL4-3 RRE RNA. Our studies further indicated interaction of 4B3 with both primary and secondary Rev binding sites.     

HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1 latency

Expression of the structural proteins of HIV-1 requires the direct interaction of the viral Rev trans-activator with its cis-acting RNA target sequence, the Rev response element or RRE. Here, we demonstrate that this specific RNA-binding event is, as expected, mediated by the conserved arginine-rich motif of Rev. However, we also show that amino acid residues located proximal to this basic domain that are critical for in vivo Rev function are dispensable for sequence-specific binding to the RRE. Instead, these sequences are required for the multimerization of Rev on the viral RRE target sequence. The observation that Rev function requires the sequential binding of multiple Rev molecules to the RRE provides a biochemical explanation for the observed threshold effect for Rev function in vivo and suggests a molecular model for the high incidence of latent infection by HIV-1.     

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.     

Limited Nucleotide Changes in the Rev Response Element (RRE) during HIV-1 Infection Alter Overall Rev-RRE Activity and Rev Multimerization

HIV-1 Rev and the Rev response element (RRE) enable a critical step in the viral replication cycle by facilitating the nuclear export of intron-containing mRNAs, yet their activities have rarely been analyzed in natural infections. This study characterized their genetic and functional variation in a small cohort of HIV-infected individuals. Multiple Rev and RRE sequences were obtained using single-genome sequencing (SGS) of plasma samples collected within 6 months after seroconversion and at a later time. This allowed the identification of cognate sequences that were linked in vivo in the same viral genome and acted together as a functional unit. Phylogenetic analyses of these sequences indicated that 4/5 infections were founded by a single transmission event. Rev and RRE variants from each time point were subjected to functional analysis as both cognate pairs and as individual components. While a range of Rev-RRE activities were seen, the activity of cognate pairs from a single time point clustered to a discrete level, which was termed the set point. In 3/5 patients, this set point changed significantly over the time period studied. In all patients, RRE activity was more sensitive to sequence variation than Rev activity and acted as the primary driver of the cognate set point. Selected patient RREs were also shown to have differences in Rev multimerization using gel shift binding assays. Thus, rather than acting as a simple on-off switch or maintaining a constant level of activity throughout infection, the Rev-RRE system can fluctuate, presumably to control replication.     

The HIV-1 Rev response element (RRE) adopts alternative conformations that promote different rates of virus replication

The HIV Rev protein forms a complex with a 351 nucleotide sequence present in unspliced and incompletely spliced human immunodeficiency virus (HIV) mRNAs, the Rev response element (RRE), to recruit the cellular nuclear export receptor Crm1 and Ran-GTP. This complex facilitates nucleo-cytoplasmic export of these mRNAs. The precise secondary structure of the HIV-1 RRE has been controversial, since studies have reported alternative structures comprising either four or five stem-loops. The published structures differ only in regions that lie outside of the primary Rev binding site. Using in-gel SHAPE, we have now determined that the wt NL4-3 RRE exists as a mixture of both structures. To assess functional differences between these RRE ‘conformers’, we created conformationally locked mutants by site-directed mutagenesis. Using subgenomic reporters, as well as HIV replication assays, we demonstrate that the five stem-loop form of the RRE promotes greater functional Rev/RRE activity compared to the four stem-loop counterpart.     

The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase

Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI). The Q motif, consisting of nine amino acids (GFXXPXPIQ) with an invariant glutamine (Q) residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase.     

Binding of dimeric aminoglycosides to the HIV-1 rev responsive element (RRE) RNA construct

Through a series of elegant fluorescence measurements, particularly through stopped-flow kinetic measurements, it was recently demonstrated that aminoglycoside antibiotics are able to bind to the HIV-1 Rev responsive element (RRE) RNA construct in more than a 1:1 stoichiometry (Lacourciere, K. A.; Stivers, J. T.; Marino, J. P. Biocheminstry 2000, 39, 5630). Here, we present the binding study results of dimeric neomycin ligands through fluorescence anisotropy studies, to the HIV-1 RRE RNA construct. The dimeric neomycin molecules are observed to be able to bind the HIV-1 RRE RNA construct approximately 17-fold higher when compared to the monomeric neomycin, lending evidence that there are indeed two or more neomycin binding sites within the HIV-1 RRE construct.     

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     

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.