Acidic C-terminal Tail Research Articles

Structural basis for Retriever-SNX17 assembly and endosomal sorting

During endosomal recycling, Sorting Nexin 17 (SNX17) facilitates the transport of numerous membrane cargo proteins by tethering them to the Retriever complex. Despite its importance, the mechanisms underlying this interaction have remained elusive. Here, we provide biochemical, structural, cellular, and proteomic analyses of the SNX17-Retriever interaction. Our data reveal that SNX17 adopts an autoinhibited conformation in the basal state, with its FERM domain sequestering its C-terminal tail. The binding of cargo proteins to the FERM domain displaces the C-terminal tail through direct competition. The released tail engages with Retriever by binding to a highly conserved interface between its VPS35L and VPS26C subunits, as revealed by cryogenic electron microscopy (cryo-EM). Disrupting this interface impairs the Retriever-SNX17 interaction, subsequently affecting the recycling of SNX17-dependent cargoes and altering the composition of the plasma membrane proteome. Intriguingly, the SNX17-binding pocket on Retriever can be utilized by other ligands containing a consensus acidic C-terminal tail motif. Together, our findings uncover a mechanism underlying endosomal trafficking of critical cargo proteins and reveal how Retriever can potentially engage with other regulatory factors or be exploited by pathogens.

The acidic C-terminal tail of DNA Gyrase of Salmonella enterica serovar Typhi controls DNA relaxation in an acidic environment

The intracellular bacteria, Salmonella Typhi adapts to acidic conditions in the host cell by resetting the chromosomal DNA topology majorly controlled by DNA Gyrase, a Type II topoisomerase. DNA Gyrase forms a heterodimer A2B2 complex, which manages the DNA supercoiling and relaxation in the cell. DNA relaxation forms a part of the regulatory mechanism to activate the transcription of genes required to survive under hostile conditions. Acid-induced stress attenuates the supercoiling activity of the DNA Gyrase, resulting in DNA relaxation. Salmonella DNA becomes relaxed as the bacteria adapt to the acidified intracellular environment. Despite comprehensive studies on DNA Gyrase, the mechanism to control supercoiling activity needs to be better understood. A loss in supercoiling activity in E. coli was observed upon deletion of the non-conserved acidic C-tail of Gyrase A subunit. Salmonella Gyrase also contains an acidic tail at the C-terminus of Gyrase A, where its deletion resulted in reduced supercoiling activity compared to wild-type Gyrase. Interestingly, we also found that wild-type Gyrase compromises supercoiling activity at acidic pH 2–3, thereby causing DNA relaxation. The absence of a C-tail displayed DNA supercoiling to some extent between pH 2–9. Hence, the C-tail of Gyrase A might be one of the controlling factors that cause DNA relaxation in Salmonella at acidic pH conditions. We propose that the presence of the C-tail of GyraseA causes acid-mediated inhibition of the negative supercoiling activity of Gyrase, resulting in relaxed DNA that attracts DNA-binding proteins for controlling the transcriptional response.

Structural basis for the role of C-terminus acidic tail of Saccharomyces cerevisiae ubiquitin-conjugating enzyme (Rad6) in E3 ligase (Bre1) mediated recognition of histones

Ubiquitination of histone H2B on chromatin is key to gene regulation. E3 ligase Bre1 and E2 Rad6 in Saccharomyces cerevisiae associate together to catalyze mono-ubiquitination at histone H2BK123. Prior studies identified the role of a highly dynamic C-terminal acidic tail of Rad6 indispensable for H2BK123 mono-ubiquitination. However, the mechanistic basis for the Rad6-acidic tail role remained elusive. Using different structural and biophysical approaches, this study for the first time uncovers the direct role of Rad6-acidic tail in interaction with the Bre1 Rad6-Binding Domain (RBD) and recognition of histones surface to facilitate histone H2B mono-ubiquitination. A combination of NMR, SAXS, ITC, site-directed mutagenesis and molecular dynamics studies reveal that RBD domain of Bre1 interacts with Rad6 to stabilize the dynamics of acidic tail. This Bre1-RBD mediated stability in acidic tail of Rad6 could be one of the key factors for facilitating correct recognition of histone surface and ubiquitin-transfer at H2BK123. We provide biophysical evidence that Rad6-acidic tail and a positivity charged surface on histone H2B are involved in recognition of E2:Histones. Taken together, this study uncovers the mechanistic basis for the role of Rad6-acidic in Bre1-RBD mediated recognition of histone surface that ensure the histone H2B mono-ubiquitination.

Deinococcus lineage and Rad52 family-related protein DR0041 is involved in DNA protection and compaction

DR0041 ORF encodes an uncharacterized Deinococcus lineage protein. We earlier reported presence of DR0041 protein in DNA repair complexes of Ssb and RecA in Deinococcus radiodurans. Here, we systematically examined the role of DR0041 in DNA metabolism using various experimental methodologies including electrophoretic mobility assays, nuclease assays, strand exchange assays and transmission electron microscopy. Interaction between DR0041 and the C-terminal acidic tail of Ssb was assessed through co-expression and in vivo cross-linking studies. A knockout mutant was constructed to understand importance of DR0041 ORF for various physiological processes. Results highlight binding of DR0041 protein to single-stranded and double-stranded DNA, interaction with Ssb-coated single-stranded DNA without interference with RecA-mediated strand exchange, protection of DNA from exonucleases, and compaction of high molecular weight DNA molecules into tightly condensed forms. Bridging and compaction of sheared DNA by DR0041 protein might have implications in the preservation of damaged DNA templates to maintain genome integrity upon exposure to gamma irradiation. Our results suggest that DR0041 protein is dispensable for growth under standard growth conditions and following gamma irradiation but contributes to protection of DNA during transformation. We discuss the role of DR0041 protein from the perspective of protection of broken DNA templates and functional redundancy.

Phosphatidate phosphatase Pah1 contains a novel RP domain that regulates its phosphorylation and function in yeast lipid synthesis

The Saccharomyces cerevisiae PAH1-encoded phosphatidate (PA) phosphatase, which catalyzes the Mg2+-dependent dephosphorylation of PA to produce diacylglycerol, is one of the most highly regulated enzymes in lipid metabolism. The enzyme controls whether cells utilize PA to produce membrane phospholipids or the major storage lipid triacylglycerol. PA levels, which are regulated by the enzyme reaction, also control the expression of UASINO-containing phospholipid synthesis genes via the Henry (Opi1/Ino2-Ino4) regulatory circuit. Pah1 function is largely controlled by its cellular location, which is mediated by phosphorylation and dephosphorylation. Multiple phosphorylations sequester Pah1 in the cytosol and protect it from 20S proteasome-mediated degradation. The endoplasmic reticulum-associated Nem1-Spo7 phosphatase complex recruits and dephosphorylates Pah1 allowing the enzyme to associate with and dephosphorylate its membrane-bound substrate PA. Pah1 contains domains/regions that include the N-LIP and haloacid dehalogenase-like catalytic domains, N-terminal amphipathic helix for membrane binding, C-terminal acidic tail for Nem1-Spo7 interaction, and a conserved tryptophan within the WRDPLVDID domain required for enzyme function. Through bioinformatics, molecular genetics, and biochemical approaches, we identified a novel RP (regulation of phosphorylation) domain that regulates the phosphorylation state of Pah1. We showed that the ΔRP mutation results in a 57% reduction in the endogenous phosphorylation of the enzyme (primarily at Ser-511, Ser-602, and Ser-773/Ser-774), an increase in membrane association and PA phosphatase activity, but reduced cellular abundance. This work not only identifies a novel regulatory domain within Pah1 but emphasizes the importance of the phosphorylation-based regulation of Pah1 abundance, location, and function in yeast lipid synthesis.

Histone chaperone Nap1 dismantles an H2A/H2B dimer from a partially unwrapped nucleosome.

DNA translocases, such as RNA polymerases, inevitably collide with nucleosomes on eukaryotic chromatin. Upon these collisions, histone chaperones are suggested to facilitate nucleosome disassembly and re-assembly. In this study, by performing in vitro transcription assays and molecular simulations, we found that partial unwrapping of a nucleosome by an RNA polymerase dramatically facilitates an H2A/H2B dimer dismantling from the nucleosome by Nucleosome Assembly Protein 1 (Nap1). Furthermore, the results uncovered molecular mechanisms of Nap1 functions in which the highly acidic C-terminal flexible tails of Nap1 contribute to the H2A/H2B binding by associating with the binding interface buried and not accessible to Nap1 globular domains, supporting the penetrating fuzzy binding mechanism seemingly shared across various histone chaperones. These findings have broad implications for the mechanisms by which histone chaperones process nucleosomes upon collisions with translocases in transcription, histone recycling and nucleosomal DNA repair.

Molecular mechanism of H2A/H2B dismantling from a partially unwrapped nucleosome by Nap1.

During DNA transactions on chromatin such as transcription, replication, and DNA repair, RNA polymerases, replicative helicases, exonucleases, and so on inevitably collide with nucleosomes and unwrap nucleosomal DNA. Histones, components of a nucleosome, contain epigenetic information in the form of chemical modification, which regulates cell growth and fates. To maintain such information upon unwrapping, nucleosomes ahead of the DNA translocases are supposed to be temporarily disassembled. Histone chaperones are proteins modulating histone binding to DNA. However, the roles of histone chaperones in the nucleosome processing upon unwrapping remain unclear. In this study, we performed in vitro transcription assays and coarse-grained molecular dynamics simulations, showing that a histone chaperone, Nap1, dismantles H2A/H2B from a partially unwrapped nucleosome. Moreover, the simulation results reveal that highly acidic C-terminal tails of Nap1 bind to the exposed H2A/H2B via its binding interface not accessible to the Nap1 globular domain, leading to the H2A/H2B dismantling. These findings provide implications for the molecular mechanism by which histone chaperones process nucleosomes during DNA transactions on chromatin.

Structural insights into the activity regulation of full-length non-structural protein 1 from SARS-CoV-2

Non-structural protein 1 (Nsp1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major virulence factor and thus an attractive drug target. The last 33 amino acids of Nsp1 have been shown to bind within the mRNA entry tunnel of the 40S ribosomal subunit, shutting off host gene expression. Here, we report the solution-state structure of full-length Nsp1, which features an α/β fold formed by a six-stranded, capped β-barrel-like globular domain (N-terminal domain [NTD]), flanked by short N-terminal and long C-terminal flexible tails. The NTD has been found to be critical for 40S-mediated viral mRNA recognition and promotion of viral gene expression. We find that in free Nsp1, the NTD mRNA-binding surface is occluded by interactions with the acidic C-terminal tail, suggesting a mechanism of activity regulation based on the interplay between the folded NTD and the disordered C-terminal region. These results are relevant for drug-design efforts targeting Nsp1.

Functional mapping of the N-terminal region of the yeast moonlighting protein Sis2/Hal3 reveals crucial residues for Ppz1 regulation.

The function of the Saccharomyces cerevisiae Ppz1 phosphatase is controlled by its inhibitory subunit Hal3. Hal3 is a moonlighting protein, which associates with Cab3 to form a decarboxylase involved in the CoA biosynthetic pathway. Hal3 is composed by a conserved core PD region, required for both Ppz1 regulation and CoA biosynthesis, a long N-terminal extension, and an acidic C-terminal tail. Cab3 has a similar structure, but it is not a Ppz1 inhibitor. We show here that deletion or specific mutations in a short region of the N-terminal extension of Hal3 compromise its function as a Ppz1 inhibitor in vivo and in vitro without negatively affecting its ability to interact with the phosphatase. This study defines a R-K-X(3) -VTFS- sequence whose presence explains the unexpected ability of Cab3 (but not Hal3) to regulate Ppz1 function in Candida albicans. This sequence is conserved in a subset of fungi and it could serve to estimate the relevance of Hal3 or Cab3 proteins in regulating fungal Ppz enzymes. We also show that the removal of the motif moderately affects both Ppz1 intracellular relocalization and counteraction of toxicity in cells overexpressing the phosphatase. Thus, our work contributes to our understanding of the regulation of Ppz phosphatases, which are determinants for virulence in some pathogenic fungi.

The C-Terminal Acidic Tail Modulates the Anticancer Properties of HMGB1.

Energy metabolism reprogramming was recently listed as a hallmark of cancer. In this process, the switch from pyruvate kinase isoenzyme type M1 to pyruvate kinase isoenzyme type M2 (PKM2) is believed to play a crucial role. Interestingly, the activity of the active form of PKM2 can efficiently be inhibited by the high-mobility group box 1 (HMGB1) protein, leading to a rapid blockage of glucose-dependent aerobic respiration and cancer cell death. HMGB1 is a member of the HMG protein family. It contains two DNA-binding HMG-box domains and an acidic C-terminal tail capable of positively or negatively modulating its biological properties. In this work, we report that the deletion of the C-terminal tail of HMGB1 increases its activity towards a large panel of cancer cells without affecting the viability of normal immortalized fibroblasts. Moreover, in silico analysis suggests that the truncated form of HMGB1 retains the capacity of the full-length protein to interact with PKM2. However, based on the capacity of the cells to circumvent oxidative phosphorylation inhibition, we were able to identify either a cytotoxic or cytostatic effect of the proteins. Together, our study provides new insights in the characterization of the anticancer activity of HMGB1.

Spatiotemporal modulations in heterotypic condensates of prion and \u03b1-synuclein control phase transitions and amyloid conversion

Biomolecular condensation via liquid-liquid phase separation of proteins and nucleic acids is associated with a range of critical cellular functions and neurodegenerative diseases. Here, we demonstrate that complex coacervation of the prion protein and α-synuclein within narrow stoichiometry results in the formation of highly dynamic, reversible, thermo-responsive liquid droplets via domain-specific electrostatic interactions between the positively-charged intrinsically disordered N-terminal segment of prion and the acidic C-terminal tail of α-synuclein. The addition of RNA to these coacervates yields multiphasic, vesicle-like, hollow condensates. Picosecond time-resolved measurements revealed the presence of transient electrostatic nanoclusters that are stable on the nanosecond timescale and can undergo breaking-and-making of interactions on slower timescales giving rise to a liquid-like behavior in the mesoscopic regime. The liquid-to-solid transition drives a rapid conversion of complex coacervates into heterotypic amyloids. Our results suggest that synergistic prion-α-synuclein interactions within condensates provide mechanistic underpinnings of their physiological role and overlapping neuropathological features.

Alterations of the Mdm2 C-Terminus Differentially Impact Its Function In Vivo.

This in vivo study highlights that alterations to the C-terminus of Mdm2 perturb its regulation of the tumor suppressor p53.

Identification of amphioxus protein disulfide isomerase as both an enzyme and an immunocompotent factor

Previous studies have shown that protein disulfide isomerase (PDI), a member of the thioredoxin (TRX) superfamily, are broadly associated with immune responses in a variety of animals. However, it remains largely unknown about the direct roles of PDIs during a bacterial infection. In this study, we identified the presence of a single pdi gene in the amphioxus Branchiostoma japonicum, Bjpdi. The deduced protein BjPDI is structurally characterized by the presence of four Trx-like domains in the order of a, b, b’ and a’ and a short acidic C-terminal tail, that are characteristic of PDIs. We demonstrated that rBjPDI displayed both thiol reductase and disulfide bond isomerase activities, indicating comparability of BjPDI with PDIs in term of enzymatic activities. We also showed that rBjPDI induced bacterial agglutination and exhibited a lectin-like activity capable of binding both bacteria (E. coli and S. aureus) and their signature molecules LPS and LTA. Furthermore, BjPDI could kill S. aureus via inducing membrane depolarization and intracellular ROS production in vitro, and treatment of amphioxus with a blocking anti-PDI antibody in vivo markedly reduced the survival rate of amphioxus following attack by S. aureus. Collectively, our study demonstrates that amphioxus protein disulfide isomerase acts as both an enzyme and an immunocompotent factor, and reports the specific function and mode of action of PDIs in immune responses.

DNA Polymerase B1 Binding Protein 1 Is Important for DNA Repair by Holoenzyme PolB1 in the Extremely Thermophilic Crenarchaeon Sulfolobus acidocaldarius

DNA polymerase B1 (PolB1) is a member of the B-family DNA polymerase family and is a replicative DNA polymerase in Crenarchaea. PolB1 is responsible for the DNA replication of both the leading and lagging strands in the thermophilic crenarchaeon Sulfolobus acidocaldarius. Recently, two subunits, PolB1-binding protein (PBP)1 and PBP2, were identified in Saccharolobus solfataricus. Previous in vitro studies suggested that PBP1 and PBP2 influence the core activity of apoenzyme PolB1 (apo-PolB1). PBP1 contains a C-terminal acidic tail and modulates the strand-displacement synthesis activity of PolB1 during the synthesis of Okazaki fragments. PBP2 modestly enhances the DNA polymerase activity of apo-PolB1. These subunits are present in Sulfolobales, Acidilobales, and Desulfurococcales, which belong to Crenarchaea. However, it has not been determined whether these subunits are essential for the activity of apo-PolB1. In this study, we constructed a pbp1 deletion strain in S. acidocaldarius and characterized its phenotypes. However, a pbp2 deletion strain was not obtained, indicating that PBP2 is essential for replication by holoenzyme PolB1. A pbp1 deletion strain was sensitive to various types of DNA damage and exhibited an increased mutation rate, suggesting that PBP1 contribute to the repair or tolerance of DNA damage by holoenzyme PolB1. The results of our study suggest that PBP1 is important for DNA repair by holoenzyme PolB1 in S. acidocaldarius.

α-Synuclein emerges as a potent regulator of VDAC-facilitated calcium transport

Voltage-dependent anion channel (VDAC) is the most ubiquitous channel at the mitochondrial outer membrane, and is believed to be the pathway for calcium entering or leaving the mitochondria. Therefore, understanding the molecular mechanisms of how VDAC regulates calcium influx and efflux from the mitochondria is of particular interest for mitochondrial physiology. When the Parkinson’s disease (PD) related neuronal protein, alpha-synuclein (αSyn), is added to the reconstituted VDAC, it reversibly and partially blocks VDAC conductance by its acidic C-terminal tail. Using single-molecule VDAC electrophysiology of reconstituted VDAC we now demonstrate that, at CaCl2 concentrations below 150 mM, αSyn reverses the channel’s selectivity from anionic to cationic. Importantly, we find that the decrease in channel conductance upon its blockage by αSyn is hugely overcompensated by a favorable change in the electrostatic environment for calcium, making the blocked state orders-of-magnitude more selective for calcium and thus increasing its net flux. -Our findings with higher calcium concentrations also demonstrate that the phenomenon of “charge inversion” is taking place at the level of a single polypeptide chain. Measurements of ion selectivity of three VDAC isoforms in CaCl2 gradient show that VDAC3 exhibits the highest calcium permeability among them, followed by VDAC2 and VDAC1, thus pointing to isoform-dependent physiological function. Mutation of the E73 residue – VDAC1 purported calcium binding site – shows that there is no measurable effect of the mutation in either open or αSyn-blocked VDAC1 states. Our results confirm VDACs involvement in calcium signaling and reveal a new regulatory role of αSyn, with clear implications for both normal calcium signaling and PD-associated mitochondrial dysfunction.

The role of high mobility group protein B3 (HMGB3) in tumor proliferation and drug resistance.

The high mobility group protein B (HMGB) family (including HMGB1, HMGB2, HMGB3, and HMGB4) can regulate the mechanisms of DNA replication, transcription, recombination, and repair, and act as cytokines to mediate responses to infection, injury, and inflammation. HMGB1/2/3 has a high similarity in sequence and structure, while HMGB4 has no acidic C-terminal tail. Among them, HMGB3 can regulate the self-renewal and differentiation of normal hematopoietic stem cell population, but the decrease of its expression is easy to induce leukemia. Up-regulation of its expression promotes tumor development and chemotherapy resistance through a variety of mechanisms, and non-coding RNA can regulate to promote tumor cell proliferation, invasion, and migration and inhibit cancer cell apoptosis.

The influence of various regions of the FOXP2 sequence on its structure and DNA-binding function.

FOX proteins are a superfamily of transcription factors which share a DNA-binding domain referred to as the forkhead domain. Our focus is on the FOXP subfamily members, which are involved in language and cognition amongst other things. The FOXP proteins contain a conserved zinc finger and a leucine zipper motif in addition to the forkhead domain. The remainder of the sequence is predicted to be unstructured and includes an acidic C-terminal tail. In the present study, we aim to investigate how both the structured and unstructured regions of the sequence cooperate so as to enable FOXP proteins to perform their function. We do this by studying the effect of these regions on both oligomerisation and DNA binding. Structurally, the FOXP proteins appear to be comparatively globular with a high proportion of helical structure. The proteins multimerise via the leucine zipper, and the stability of the multimers is controlled by the unstructured interlinking sequence including the acid rich tail. FOXP2 is more compact than FOXP1, has a greater propensity to form higher order oligomers, and binds DNA with stronger affinity. We conclude that while the forkhead domain is necessary for DNA binding, the affinity of the binding event is attributable to the leucine zipper, and the unstructured regions play a significant role in the specificity of binding. The acid rich tail forms specific contacts with the forkhead domain which may influence oligomerisation and DNA binding, and therefore the acid rich tail may play an important regulatory role in FOXP transcription.

High-Throughput Screening to Identify Inhibitors of SSB-Protein Interactions.

The bacterial single-stranded DNA-binding protein (SSB) uses an acidic C-terminal tail to interact with over a dozen proteins, acting as a genome maintenance hub. These SSB-protein interactions are essential, as mutations to the C-terminal tail that disrupt these interactions are lethal in Escherichia coli. While the roles of individual SSB-protein interactions have been dissected with mutational studies, small-molecule inhibitors of these interactions could serve as valuable research tools and have potential as novel antimicrobial agents. This chapter describes a high-throughput screening campaign used to identify inhibitors of SSB-protein interactions. A screen targeting the PriA-SSB interface from Klebsiella pneumoniae is presented as an example, but the methods may be adapted to target nearly any SSB interaction.

Structural Analysis of Calreticulin, an Endoplasmic Reticulum-Resident Molecular Chaperone.

Calreticulin (Calr) is an endoplasmic reticulum (ER) chaperone involved in protein quality control, Ca2+ regulation and other cellular processes. The structure of Calr is unusual, reflecting different functions of the protein: a proline-rich β-hairpin arm and an acidic C-terminal tail protrude from a globular core, composed of a β-sheet sandwich and an α-helix. The arm and tail interact in the presence of Ca2+ and cover the upper β-sheet, where a carbohydrate-binding site gives the chaperone glycoprotein affinity. At the edge of the carbohydrate-binding site is a conserved, strained disulphide bridge, formed between C106 and C137 of human Calr, which lies in a polypeptide-binding site. The lower β-sheet has several conserved residues, comprised of a characteristic triad, D166-H170-D187, Tyr172 and the free C163. In addition to its role in the ER, Calr translocates to the cell surface upon stress and functions as an immune surveillance marker. In some myeloproliferative neoplasms, the acidic Ca2+-binding C-terminal tail is transformed into a polybasic sequence.

Cleavage of HMGB1 by Proteolytic Enzymes Associated with Inflammatory Conditions

Extracellular HMGB1 acts as an alarmin in multiple autoimmune diseases. While its release and functions have been extensively studied, there is a substantial lack of knowledge regarding HMGB1 regulation at the site of inflammation. Herein we show that enzymes present in arthritis-affected joints process HMGB1 into smaller peptides in vitro. Gel electrophoresis, Western blotting and mass spectrometry analyses indicate cleavage sites for human neutrophil elastase, cathepsin G, and matrix metalloproteinase 3 within the HMGB1 structure. While human neutrophil elastase and matrix metalloproteinase 3 might alter the affinity of HMGB1 to its receptors by cleaving the acidic C-terminal tail, cathepsin G rapidly and completely degraded the alarmin. Contrary to a previous report we demonstrate that HMGB1 is not a substrate for dipeptidyl peptidase IV. We also provide novel information regarding the presence of these proteases in synovial fluid of juvenile idiopathic arthritis patients. Correlation analysis of protease levels and HMGB1 levels in synovial fluid samples did not, however, reveal any direct relationship between the recorded levels. This study provides knowledge of proteolytic processing of HMGB1 relevant for the regulation of HMGB1 during inflammatory disease.