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bioRxiv2017

Two distinct DNA sequences recognized by transcription factors represent enthalpy and entropy optima

Most transcription factors (TFs) can bind to a population of sequences closely related to a single optimal site. However, some TFs can bind to two distinct sequences that represent two local optima in the Gibbs free energy of binding ({Delta}G). To determine the molecular mechanism behind this effect, we solved the structures of human HOXB13 and CDX2 bound to their two optimal DNA sequences, CAATAAA and TCGTAAA. Thermodynamic analyses by isothermal titration calorimetry revealed that both sites were bound with similar {Delta}G. However, the interaction with the CAA sequence was driven by change in enthalpy ({Delta}H), whereas the TCG site was bound with similar affinity due to smaller loss of entropy ({Delta}S). The common presence of at least two local optima is general to all macromolecular interactions, as {Delta}G depends on two partially independent variables {Delta}H and {Delta}S according to the central equation of thermodynamics, {Delta}G = {Delta}H - T{Delta}S.

biochemistry↗

bioRxiv2017

Allosteric activation dictates PRC2 activity independent of its recruitment to chromatin

PRC2 is a therapeutic target for several types of cancers currently undergoing clinical trials. Its activity is regulated by a positive feedback loop whereby its terminal enzymatic product, H3K27me3, is specifically recognized and bound by an aromatic cage present in its EED subunit. The ensuing allosteric activation of the complex stimulates H3K27me3 deposition on chromatin. Here, we report a step-wise feedback mechanism entailing key residues within distinctive interfacing motifs of EZH2 or EED that are found mutated in cancers and/or Weaver syndrome. PRC2 harboring these EZH2 or EED mutants manifest little activity in vivo but, unexpectedly, exhibited similar chromatin association as wild-type PRC2, indicating an uncoupling of PRC2 activity and recruitment. With genetic and chemical tools, we further demonstrated that targeting allosteric activation overrode the gain-of-function effect of EZH2Y646X oncogenic mutations. These results revealed critical implications to the regulation and biology of PRC2 and a novel vulnerability in tackling PRC2-addicted cancers.

biochemistry↗

bioRxiv2017

OGT (O-GlcNAc Transferase) selectively modifies multiple residues unique to lamin A

The LMNA gene encodes lamins A and C with key roles in nuclear structure, signaling, chromatin organization, and genome integrity. Mutations in LMNA cause >12 diseases, termed laminopathies. Lamins A and C are identical for their first 566 residues. However, they form distinct filaments in vivo with apparently distinct roles. We report that lamin A is O-GlcNAc modified in human hepatoma (Huh7) cells and in mouse liver. In vitro assays with purified OGT enzyme showed robust O-GlcNAcylation of recombinant mature lamin A tails (residues 385-646), with no detectable modification of lamin B1, lamin C, or progerin ({Delta}50) tails. Using mass spectrometry, we identified 11 O-GlcNAc sites in a sweet spot unique to lamin A, with up to seven sugars per peptide. Most sites were unpredicted by current algorithms. Double-mutant (S612A/T643A) lamin A tails were still robustly O-GlcNAc-modified at seven sites. By contrast, O-GlcNAcylation was undetectable on tails bearing deletion {Delta}50, which causes Hutchinson-Gilford progeria syndrome, and greatly reduced by deletion {Delta}35, suggesting this region is required for substrate recognition or modification by OGT in vitro. These results suggest OGT, an essential protein and master regulator, regulates partners or function(s) unique to lamin A that are lost in progeria.

biochemistry↗

bioRxiv2017

In vivo cross-linking and transmembrane helix dynamics support a non-piston model of signaling within E. coli EnvZ

In Gram-negative bacteria, porins span the outer membrane and control the influx of several prominent groups of antibiotics. Thus, it should not be surprising that expression of these porins is often altered in clinical isolates exhibiting multidrug resistance (MDR). The major regulator of porin expression in Escherichia coli is EnvZ, a canonical sensor histidine kinase (SHK). It allosterically processes periplasmic interactions with MzrA and cytoplasmic osmosensing into a single unified change in the ratio of its kinase and phosphatase activities. Unfortunately, the role of the EnvZ transmembrane domain (TMD) in bidirectional communication of these signals remains not well understood. Here, we employed in vivo sulfhydryl-reactivity to probe the dynamics of the TM2 helices and demonstrate that upon stimulus perception, only the region proximal to the periplasm undergoes conformational rearrangement. Furthermore, in silico coarse-grained molecular dynamics (CG-MD) simulations with aromatically tuned variants of EnvZ TM2 demonstrate the existence of both tilting and azimuthal rotational components to transmembrane communication while ruling out piston-type repositioning of TM2. Finally, in contrast to a similar analysis of TM1, we identified position-specific mutants possessing a \"flipped\" phenotype by dual-color fluorescent reporter analysis suggesting that both the periplasmic and cytoplasmic ends of TM2 are critical for maintenance of EnvZ signal output. Taken together, these data strongly support that EnvZ employs a non-piston-type mechanism during transmembrane communication. We conclude by discussing these results within the context of allosteric processing by EnvZ and propose that these results can be used to predict and classify transmembrane communication by various SHKs.\n\nImportanceThe EnvZ sensor histidine kinase serves as the major regulator of porin expression within Escherichia coli. A long-standing question is how stimulus perception by a bacterial receptor on one side of a biological membrane is transmitted to the opposite side of the membrane. To address this question, we monitored the dynamics of the transmembrane domain of EnvZ in vivo and coupled these results with in silico simulations of membrane-embedded EnvZ transmembrane domains. Taken together, these results demonstrate that detection of osmotic stress by the cytoplasmic domain of EnvZ results in non-piston communication across the inner membrane of E. coli. Thus, in addition to understanding how EnvZ regulates porin balance and antibiotic influx, these results contribute to answering the long-standing question of how transmembrane communication is performed by bacterial receptors. Our work concludes with a framework that correlates receptor domain composition and signal transduction mechanisms that could be employed by other research groups on their particular receptors of interest.

biochemistry↗

bioRxiv2017

HSP90 inhibitors disrupt a transient HSP90-HSF1 interaction and identify a noncanonical model of HSP90-mediated HSF1 regulation

Heat shock factor 1 (HSF1) initiates a broad transcriptional response to proteotoxic stress while also mediating a cancer-specific transcriptional program. HSF1 is thought to be regulated by molecular chaperones, including Heat Shock Protein 90 (HSP90). HSP90 is proposed to sequester HSF1 in unstressed cells, but visualization of this interaction in vivo requires protein crosslinking. In this report, we show that HSP90 binding to HSF1 depends on HSP90 conformation and is only readily visualized for the ATP-dependent, N-domain dimerized chaperone, a conformation only rarely sampled by mammalian HSP90. We have used this mutationally fixed conformation to map HSP90 binding sites on HSF1. Further, we show that ATP-competitive, N-domain targeted HSP90 inhibitors disrupt this interaction, resulting in the increased duration of HSF1 occupancy of the hsp70 promoter and significant prolongation of both the constitutive and heat-induced HSF1 transcriptional activity. While our data do not support a role for HSP90 in sequestering HSF1 monomers to suppress HSF1 transcriptional activity, our findings do identify a noncanonical role for HSP90 in providing dynamic modulation of HSF1 activity by participating in removal of HSF1 trimers from heat shock elements in DNA, thus terminating the heat shock response.

biochemistry↗

bioRxiv2017

Conservation of specificity in two low-specificity proteins

S100 proteins bind linear peptide regions of target proteins and modulate their activity. The peptide binding interface, however, has remarkably low specificity and can interact with many target peptides. It is not clear if the interface discriminates targets in a biological context, or whether biological specificity is achieved exclusively through external factors such as subcellular localization. To discriminate these possibilities, we used an evolutionary biochemical approach to trace the evolution of paralogs S100A5 and S100A6. We first used isothermal titration calorimetry to study the binding of a collection of peptides with diverse sequence, hydrophobicity, and charge to human S100A5 and S100A6. These proteins bound distinct, but overlapping, sets of peptide targets. We then studied the peptide binding properties of S100A5 and S100A6 orthologs sampled from across five representative amniote species. We found that the pattern of binding specificity was conserved along all lineages, for the last 320 million years, despite the low specificity of each protein. We next used Ancestral Sequence Reconstruction to determine the binding specificity of the last common ancestor of the paralogs. We found the ancestor bound the whole set of peptides bound by modern S100A5 and S100A6 proteins, suggesting that paralog specificity evolved by subfunctionalization. To rule out the possibility that specificity is conserved because it is difficult to modify, we identified a single historical mutation that, when reverted in human S100A5, gave it the ability to bind an S100A6-specific peptide. These results indicate that there are strong evolutionary constraints on peptide binding specificity, and that, despite being able to bind a large number of targets, the specificity of S100 peptide interfaces is indeed important for the biology of these proteins.

biochemistry↗

bioRxiv2017

Mapping of the Fondaparinux Binding Site of JR-FL gp120 by High Resolution Hydroxyl Radical Protein Footprinting and Computational Docking

The adhesion of HIV gp120 antigen to human cells is modulated in part by interactions with heparan sulfate. The HXB2 strain of gp120 has been shown to interact with heparin primarily through the V3 loop, although other domains including the C-terminal domain were also implicated. However, the JR-FL strain (representative of CCR5-interacting strains that make up newest infections) was shown to have a drastically lowered affinity to heparin due to the loss of several basic residues in the V3 loop, and deletion of the V3 loop in JR-FL gp120 was shown to abrogate some, but not all, heparin binding. Here, we use high resolution hydroxyl radical protein footprinting to measure the changes in protein surface oxidation levels that result from the binding of a model heparin fragment (fondaparinux). Protection in both the V3 loop and the N-terminus of JR-FP gp120 is observed. The well-defined composition of fondaparinux allowed us to perform docking simulations, which showed two clusters of fondaparinux binding: the V3 loop, and a domain consisting of the N- and C-termini. Together, the experimental and theoretical results indicate the heparin/heparan sulfate binding sites on JR-FL gp120 and the efficient interaction of fondaparinux, a widely exploited therapeutic carbohydrate, on gp120.

biochemistry↗

bioRxiv2017

Visualization of ligand-induced transmembrane signalling in the full-length human insulin receptor

Using glycosylated full-length human insulin receptor reconstituted into lipid nanodiscs, we show that insulin binding to the dimeric receptor converts its ectodomains from an inverted U-shaped to a T-shaped conformation. This unprecedented structural rearrangement of the ectodomains propagates to the transmembrane domains, which are well separated in the inactive conformation, but come together upon insulin binding, allowing autophosphorylation of the cytoplasmic kinase domains.

biochemistry↗

bioRxiv2017

Programmable RNA recognition using a CRISPR-associated Argonaute

Argonaute proteins (Agos) are present in all domains of life. While the physiological function of eukaryotic Agos in regulating gene expression is well documented, the biological roles of many of their prokaryotic counterparts remain enigmatic. In some bacteria, Agos are associated with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci and use non-canonical 5-hydroxyled guide RNAs (gRNAs) for nucleic acid targeting. Here we show that using 5-bromo-2'-deoxyuridine (BrdU) as the 5 nucleotide of gRNAs stabilizes in vitro reconstituted CRISPR-associated Marinitoga piezophila Argonaute-gRNA complexes (MpAgo RNPs) and significantly improves their specificity and affinity for RNA targets. Using reconstituted MpAgo RNPs with 5-BrdU modified gRNAs, we mapped the seed region of the gRNA, and identified the nucleotides of the gRNA that play the most significant role in targeting specificity. We also show that these MpAgo RNPs can be programmed to distinguish between substrates that differ by a single nucleotide, using permutations at the 6th and 7th positions in the gRNA. Using these specificity features, we employed MpAgo RNPs to detect specific Adenosine to Inosine edited RNAs in a complex mixture. These findings broaden our mechanistic understanding of the interactions of Argonautes with guide and substrate RNAs, and demonstrate that MpAgo RNPs with 5-BrdU modified gRNAs can be used as a highly-specific RNA-targeting platform to probe RNA biology.\n\nSIGNIFICANCEArgonaute proteins are present in bacteria, archaea and eukaryotes. They play an important role in a wide range of biological processes, from transcriptional and translational gene expression regulation to defense against viruses and silencing of mobile genetic elements. Here we present mechanistic insights into the interactions of the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) with its guide RNA (gRNA) and RNA substrates. By modifying the 5-nucleotide of the gRNA, we demonstrate that MpAgo-gRNA complexes (RNPs) are easily programmable, have high affinity to fully complementary RNA substrates, and can discriminate by over 300 fold between substrates that differ by only a single nucleotide. These MpAgo RNPs should be useful for probing endogenous RNAs in living cells.

biochemistry↗

bioRxiv2017

A toolbox of anti-mouse and rabbit IgG secondary nanobodies

Polyclonal anti-IgG secondary antibodies are essential tools for many molecular biology techniques and diagnostic tests. Their animal-based production is, however, a major ethical problem. Here, we introduce a sustainable alternative, namely nanobodies against all mouse IgG subclasses and rabbit IgG. They can be produced at large scale in E. coli and could thus make secondary antibody-production in animals obsolete. Their recombinant nature allows fusion with affinity tags or reporter enzymes as well as efficient maleimide chemistry for fluorophore-coupling. We demonstrate their superior performance in Western Blotting, both in peroxidase- and fluorophore-linked form. Their site-specific labeling with multiple fluorophores creates bright imaging reagents for confocal and super-resolution microscopy with much smaller label displacement than traditional secondary antibodies. They also enable simpler and faster immunostaining protocols and even allow multi-target localization with primary IgGs from the same species and of the same class.

biochemistry↗

bioRxiv2017

Molecular basis for the recognition of the human AAUAAA polyadenylation signal

Nearly all eukaryotic messenger RNA precursors must undergo cleavage and polyadenylation at their 3'-end for maturation. A crucial step in this process is the recognition of the AAUAAA polyadenylation signal (PAS), and the molecular mechanism of this recognition has been a long-standing problem. Here we report the cryo-electron microscopy structure of a quaternary complex of human CPSF-160, WDR33, CPSF-30 and an AAUAAA RNA at 3.4 [A] resolution. Strikingly, the AAUAAA PAS assumes an unusual conformation that allows this short motif to be bound directly by both CPSF-30 and WDR33. The A1 and A2 bases are recognized specifically by zinc finger 2 (ZF2) of CPSF-30 and the A4 and A5 bases by ZF3. Interestingly, the U3 and A6 bases form an intramolecular Hoogsteen base pair and directly contact WDR33. CPSF-160 functions as an essential scaffold and pre-organizes CPSF-30 and WDR33 for high-affinity binding to AAUAAA. Our findings provide an elegant molecular explanation for how PAS sequences are recognized for mRNA 3'-end formation.

biochemistry↗

bioRxiv2017

Distinct stimulatory mechanisms regulate the catalytic activity of Polycomb Repressive Complex 2 (PRC2)

The maintenance of gene expression patterns during metazoan development is carried out, in part, by the actions of the Polycomb Repressive Complex 2 (PRC2). PRC2 catalyzes mono-, di-and trimethylation of histone H3 at lysine 27 (H3K27), with H3K27me2/3 being strongly associated with silenced genes. We demonstrate that EZH1 and EZH2, the two mutually exclusive catalytic subunits of PRC2, are differentially activated by various mechanisms. While both PRC2-EZH1 and PRC2-EZH2 are able to catalyze monomethylation, only PRC2-EZH2 is strongly activated by allosteric modulators and specific chromatin substrates to catalyze di-and trimethylation of H3K27. However, we also show that a PRC2 associated protein, AEBP2, can stimulate the activity of both complexes through a mechanism independent of and additive to allosteric activation. These results have strong implications regarding the cellular requirements for and accompanying adjustments in PRC2 activity, given the difference in the expression of EZH1 and EZH2 upon cellular differentiation.

biochemistry↗

bioRxiv2017

Hijacking of Multiple Phospholipid Biosynthetic Pathways and Induction of Membrane Biogenesis by a Picornaviral 3CD Protein

RNA viruses induce specialized membranous structures for use in genome replication. These structures are often referred to as replication organelles (ROs). ROs exhibit distinct lipid composition relative to other cellular membranes. In many picornaviruses, phosphatidylinositol-4-phosphate (PI4P) is a marker of the RO. Studies to date indicate that the viral 3A protein hijacks a PI4 kinase to induce PI4P by a mechanism unrelated to the cellular pathway, which requires Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1, GBF1, and ADP ribosylation factor 1, Arf1. Here we show that a picornaviral 3CD protein is sufficient to induce synthesis of not only PI4P but also phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylcholine (PC). Synthesis of PI4P requires GBF1 and Arf1. We identified 3CD derivatives: 3CDm and 3CmD, that we used to show that distinct domains of 3CD function upstream of GBF1 and downstream of Arf1 activation. These same 3CD derivatives still supported induction of PIP2 and PC, suggesting that pathways and corresponding mechanisms used to induce these phospholipids are distinct. Phospholipid induction by 3CD is localized to the perinuclear membrane, the outcome of which is the proliferation of membranes in this area of the cell. We conclude that a single viral protein can serve as a master regulator of cellular phospholipid and membrane biogenesis, likely by commandeering normal cellular pathways.\n\nAUTHOR SUMMARYPicornaviruses replicate their genomes in association with host membranes. Early during infection, existing membranes are used but remodeled to contain a repertoire of lipids best suited for virus multiplication. Later, new membrane synthesis occurs, which requires biosynthesis of phosphatidylcholine in addition to the other more specialized lipids. We have learned that a single picornaviral protein is able to induce membrane biogenesis and decorate these membranes with some of the specialized lipids induced by the virus. A detailed mechanism of induction has been elucidated for one of these lipids. The ability of a single viral protein to commandeer host pathways that lead to membrane biogenesis was unexpected. This discovery reveals a new target for antiviral therapy with the potential to completely derail all aspects of the viral lifecycle requiring membrane biogenesis.

biochemistry↗

bioRxiv2017

Proteome-wide analysis of phospho-regulated PDZ domain interactions through phosphomimetic proteomic peptide phage display

We report phosphomimetic proteomic peptide-phage display, a powerful large-scale method for finding ligands of short linear motif binding domains that simultaneously pinpoint functional Ser/Thr phosphosites in three steps. First, we computationally designed an oligonucleotide library encoding all human C-terminal peptides containing known or predicted Ser/Thr phosphosites and phosphomimetic variants thereof. Second, we incorporated these oligonucleotides into a phage library. Third, we screened the six PDZ (PSD-95/Dlg/ZO-1) domains of Scribble and DLG1 for binding and identified known and novel ligands from the human proteome, and whether these interactions may be regulated by ligand phosphorylation. We demonstrate that the Scribble PDZ domains preferentially bind to ligands with phosphomimetic mutations at two distinct positions, and show that the equilibrium dissociation constant for Scribble PDZ1 with the C-terminal peptide of RPS6KA2 is enhanced over four-fold by phosphorylation. We elucidate the molecular determinants of phosphopeptide binding through NMR structure determination and mutational analysis. Finally, we discuss the role of Ser/Thr phosphorylation as a switching mechanism of PDZ domain interactions.

biochemistry↗

bioRxiv2017

HDAC11 regulates type I interferon signaling through defatty-acylation of SHMT2

The smallest histone deacetylase (HDAC) and the only class IV HDAC member, HDAC11, is reported to regulate immune activation and tumorigenesis, yet its physiological function is largely unknown. Here we identify HDAC11 as an efficient lysine defatty-acylase that is >10,000-fold more efficient than its deacetylase activity. Through proteomics studies, we identified SHMT2 as a defatty-acylation substrate of HDAC11. HDAC11-catalyzed defatty-acylation did not affect the enzymatic activity of SHMT2. Instead, it affects the ability of SHMT2 to regulate type I interferon receptor ubiquitination and internalization. Correspondingly, HDAC11 depletion increased type I interferon signaling in both cell culture and mice. This study is the first time a zinc-dependent HDAC is found to have an activity that is much more efficient than the corresponding deacetylase activity. The finding expands the physiological functions of HDAC11 and protein lysine fatty acylation, and opens up opportunities to develop HDAC11-specific inhibitors as therapeutics to modulate immune responses.

biochemistry↗

bioRxiv2017

Histone Deacetylase 11 is an ε-N-Myristoyllysine Hydrolase

Histone deacetylase (HDAC) enzymes are important regulators of diverse biological function, including gene expression, rendering them potential targets for intervention in a number of diseases, with a handful of compounds approved for treatment of certain hematologic cancers. Among the human zinc-dependent HDACs, the most recently discovered member, HDAC11, is the only member assigned to subclass IV, the smallest protein, and the least well understood with regards to biological function. Here we show that HDAC11 cleaves long chain acyl modifications on lysine side chains with remarkable efficiency compared to acetyl groups. We further show that several common types of HDAC inhibitors, including the approved drugs romidepsin and vorinostat, do not inhibit this enzymatic activity. Macrocyclic hydroxamic acid-containing peptides, on the other hand, potently inhibit HDAC11 demyristoylation activity. These findings should be taken carefully into consideration in future investigations of the biological function of HDAC11 and will serve as a foundation for the development of selective chemical probes targeting HDAC11.

biochemistry↗

bioRxiv2017

Successful aggregation of Tau protein labelled on its native cysteines

The formation of fibrillar tangles of the Tau protein is crucial in the development of Alzheimers disease. Biophysical methods based on labelling of the cysteines of Tau with fluorescence dyes would allow to study fibril formation with an internal eye. However, the two native cysteines of Tau at positions 291 and 322 are located in the repeat domain, which is involved in forming the fibrils. The contribution of both cysteines to this process is unclear. Here we show that blocking natural cysteines using large fluorescent dyes does not interfere with Tau fibrillation so that FRET can be used to follow structural changes during the process. We anticipate that cysteine-labelled Tau enables following structural rearrangements during fibril formation in detail. This may also allow to monitor the effect of drugs, small molecules and proteins on the process.

biochemistry↗

bioRxiv2017

HDAC11 is a fatty-acid deacylase

Histone deacetylase 11 (HDAC11) is a sole member of the class IV HDAC subfamily with negligible intrinsic deacetylation activity. Here we report in vitro profiling of HDAC11 deacylase activities, and our data unequivocally show that the enzyme efficiently removes acyl moieties spanning 8-18 carbons from the side chain nitrogen of the lysine residue of a peptidic substrate. Additionally, N-linked lipoic acid and biotin are removed by the enzyme, although with lower efficacy. Catalytic efficiencies toward dodecanoylated and myristoylated peptides exceed 70,000 M-1s-1 making HDAC11 the most proficient fatty acid deacylase of the HDAC family. Interestingly, HDAC11 is strongly inhibited by free myristic, palmitic and stearic acids with inhibition constants of 6.5 {micro}M, 0.9 {micro}M, and 1.6 {micro}M, respectively. At the same time, its deacylase activity is stimulated more than 2.5-fold by both palmitoyl-coenzyme A and myristoyl-coenzyme A, pointing toward metabolic control of the enzymatic activity by fatty acid metabolites. Our data reveal novel enzymatic activity of HDAC11 that can, in turn, facilitate the uncovering of additional biological functions of the enzyme as well as the design of isoform-specific HDAC inhibitors.

biochemistry↗