Folding Motif Research Articles

Oligomerization of the Nrdp1 E3 Ubiquitin Ligase Is Necessary for Efficient Autoubiquitination but Not ErbB3 Ubiquitination

Overexpression of the ErbB3 receptor tyrosine kinase protein in breast and other cancers contributes to tumor malignancy and therapeutic resistance. The RBCC/TRIM family RING finger E3 ubiquitin ligase Nrdp1 mediates the ubiquitination of ErbB3 in normal mammary epithelial cells to facilitate receptor degradation and suppress steady-state receptor levels. Post-transcriptional loss of Nrdp1 in patient breast tumors allows ErbB3 overexpression and receptor contribution to tumor progression, and elevated lability through autoubiquitination contributes to the observed loss of Nrdp1 in tumors relative to normal tissue. To begin to understand the mechanisms underlying Nrdp1 protein self-regulation through lability, we investigated the structural determinants required for efficient autoubiquitination and ErbB3 ubiquitination. Using mutagenesis, chemical cross-linking, size exclusion chromatography, and native polyacrylamide gel electrophoresis, we demonstrate that Nrdp1 self-associates into a stable oligomeric complex in cells. Deletion of its coiled-coil domain abrogates oligomerization but does not affect Nrdp1-mediated ErbB3 ubiquitination or degradation. On the other hand, the presence of the coiled-coil domain is necessary for efficient Nrdp1 autoubiquitination via a trans mechanism, indicating that Nrdp1 ubiquitination of its various targets is functionally separable. Finally, a GFP fusion of the coiled-coil domain stabilizes Nrdp1 and potentiates ErbB3 ubiquitination and degradation. These observations point to a model whereby the coiled-coil domain plays a key role in regulating Nrdp1 lability by promoting its assembly into an oligomeric complex, and raise the possibility that inhibition of ligase oligomerization via its coiled-coil domain could be of therapeutic benefit to breast cancer patients by restoring Nrdp1 protein.

Rosetta Structural Modeling of Tarantula Toxin Binding to Voltage Sensors

Arachnids produce inhibitory cystine knot (ICK) peptide toxins that are potent allosteric modulators of ion channel voltage sensors. This study investigates the structural basis of voltage sensor toxin interaction. Guangxitoxin-1E (GxTX) and Scodra griseipes toxin (SgTX) inhibit Kv2.1 channels by binding to the third segment of the voltage sensor (S3b), but their sequences share only the cystine residues required for their folding motif. Using Rosetta structural modeling we constructed theoretical complexes of Kv2.1 with GxTX and SgTX. These toxins were docked to the α-helical tarantula toxin receptor site on the S3b region of the activated Kv2.1 voltage sensor paddle. These amphiphilic toxins partition into lipid bilayers and modulate channels by interacting with extracellular solution and membrane lipids as well as their receptor site. To better understand the importance of solution and lipid contacts, docking simulations were preformed in both aqueous and membrane-like environments. In aqueous environments, the complexes identified energetically favorable interfaces with the receptor site. Toxin docking with an implicit membrane yielded convergent structures with SgTX, but similar complexes with GxTX did not find energetic minima. The interaction surfaces in these membrane embedded models of SgTX compare favorably with key residues identified by experimental alanine scans. Voltage clamp recordings and fluorescent measurements of toxin binding to Kv2.1 reveal that GxTX has a greatly weakened affinity for activated voltage sensors. Our results are consistent with GxTX having a lower affinity for activated voltage sensors than SgTX. We propose the binding site for GxTX, but not SgTX, is occluded when voltage sensors are activated.

DNA bending in the synaptic complex in V(D)J recombination: turning an ancestral transpososome upside down.

In all jawed vertebrates RAG (recombination activating gene) recombinase orchestrates V(D)J recombination in B and T lymphocyte precursors, assembling the V, D and J germline gene segments into continuous functional entities which encode the variable regions of their immune receptors. V(D)J recombination is the process by which most of the diversity of our specific immune receptors is acquired and is thought to have originated by domestication of a transposon in the genome of a vertebrate. RAG acts similarly to the cut and paste transposases, by first binding two recombination signal DNA sequences (RSSs), which flank the two coding genes to be adjoined, in a process called synaptic or paired complex (PC) formation. At these RSS-coding borders, RAG first nicks one DNA strand, then creates hairpins, thus cleaving the duplex DNA at both RSSs. Although RAG reaction mechanism resembles that of insect mobile element transposases and RAG itself can inefficiently perform intramolecular and intermolecular integration into the target DNA, inside the nuclei of the developing lymphocytes transposition is extremely rare and is kept under proper surveillance. Our review may help understand how RAG synaptic complex organization prevents deleterious transposition. The phosphoryl transfer reaction mechanism of RNAseH-like fold DDE motif enzymes, including RAG, is discussed accentuating the peculiarities described for various transposases from the light of their available high resolution structures (Tn5, Mu, Mos1 and Hermes). Contrasting the structural 3D organization of DNA in these transpososomes with that of the RSSs-DNA in RAG PC allows us to propose several clues for how evolutionarily RAG may have become “specialized” in recombination versus transposition.

G-Quadruplex DNA Folding and Dynamics within Duplex DNA

The helix is the classical view of genomic DNA structure. Recent findings suggest that alternate folding motifs can play an important role in gene expression. One example is the G-Quadruplex, a single stranded DNA sequence composed of four repeat sequences of two or more G's each separated by varying spacer sequences. This structure has been suggested to affect the expression of several important genes in cancer development (c-MYC, c-KIT, VEGF)[1].Recently, immune assay based experiments have shown that G-Quadruplexes are stably found throughout the genome [2]. The dynamic properties underlying the interaction between the G-Quadruplex and its single stranded DNA complement have yet to be characterized. Preliminary studies suggest that these structures are capable of forming in response to specific cellular actions and are persistent in a biologically relevant time scale.To determine sequence dependent properties of G-Quadruplexes, we utilized a combination of Single Molecule FRET microscopy and small molecule binding fluorescence assays. We investigated biophysical properties such as stability, folding patterns, and structure persistence. These properties will help elucidate the biological roles of specific genomic quadruplexes. This knowledge will aid in interpreting the roles of the many G-Quadruplex associated proteins and may assist in the development of the next generation of chemotherapeutic agents.[1]. Patel, D et al. (2007) Nucleic Acids Research UK 35(22):7429-7455.[2]. Lam, EYN et al. (2013) Nature Communications USA 4:1796.

Fluorinated amino acids in amyloid formation: a symphony of size, hydrophobicity and α-helix propensity

Fluorinated amino acids can have dramatic effects on protein stability and protein–protein interactions due to the unique stereoelectronic properties of fluorine. Previous approaches to assessing their properties have mainly focused on helical systems, even though fluoro-amino acids are known to exhibit lower intrinsic helix propensities than their hydrocarbon analogues. Fluorination of specific β-sheet positions within globular proteins has been shown to have a stabilizing effect, suggesting that fluorinated amino acids may generally be well suitable for modulating non-helical structures. Still, fluorinated amino acids have rarely been studied in amyloid forming peptides, which take on a characteristically high cross-β-sheet content. Here, we examine the substitution of natural amino acids within an amyloid forming model peptide by amino acids that contain different stoichiometries of fluorine in their side chains. This approach enables a systematic evaluation of the impact of fluorine on amyloid formation. We have investigated the impact of size, hydrophobicity and secondary structure propensities of the fluorinated amino acids on the amyloid formation process. The structure of the model peptide is based on an engineered coiled coil folding motif that was designed to provide an α-helical starting structure that can fold into β-sheet rich amyloids under controlled conditions. Substitution with fluorinated amino acids was accomplished for two neighboring valine residues that play a key role in the structural transition. The resulting peptides show an unexpected folding behavior as a consequence of the interplay of stereoelectronic effects, helix propensity, hydrophobicity and position of the particular substitution within the amyloid forming system.

Conformational analysis of helical aminoisobutyric acid (Aib) oligomers bearing C-terminal ester Schellman motifs.

The effect of Schellman motifs on the adoption of stable 310 helical conformations in a series of aminoisobutyric (Aib) oligomers has been studied in the solid state and solution. The destabilising effect of the Schellman motif (a local inversion of helical screw-sense due to a C-terminal ester residue) was quantified in the solid state using X-ray crystallography through analysis of the torsion angles and their deviation from those observed in an ideal 310 helix. Investigation of the intramolecular hydrogen-bonding interactions in the solid state led to the identification of a fully extended C5 conformation in one oligomer, which is a novel folding motif for Aib oligomers. The effect of ester groups with differing steric demands on intermolecular hydrogen-bonding contacts in the solid state was also ascertained. In solution, the adoption of a 310 conformation in Aib oligomers appeared to be more finely tuned, depending on a number of factors, including chain length and the steric demands of the C-terminal destabilising Schellman motif.

Comprehensive Analysis of Gly-Leu-Gly-Gly-Lys Peptide Dication Structures and Cation-Radical Dissociations Following Electron Transfer: From Electron Attachment to Backbone Cleavage, Ion–Molecule Complexes, and Fragment Separation

Experimental data from ion mobility measurements and electron transfer dissociation were combined with extensive computational analysis of ion structures and dissociation energetics for Gly-Leu-Gly-Gly-Lys cations and cation radicals. Experimental and computational collision cross sections of (GLGGK + 2H)(2+) ions pointed to a dominant folding motif that is represented in all low free-energy structures. The local folding motifs were preserved in several fragment ions produced by electron transfer dissociation. Gradient optimizations of (GLGGK + 2H)(+•) cation-radicals revealed local energy minima corresponding to distonic zwitterionic structures as well as aminoketyl radicals. Both of these structural types can isomerize to low-energy tautomers that are protonated at the radical-containing amide group forming a new type of intermediates, -C(•)O(-)NH2(+)- and -C(•)(OH)NH2(+)-, respectively. Extensive mapping with B3LYP, M06-2X, and MP2(frozen core) calculations of the potential energy surface of the ground doublet electronic state of (GLGGK + 2H)(+•) provided transition-state and dissociation energies for backbone cleavages of the N-Cα and amide C-N bonds leading to ion-molecule complexes. The complexes can undergo facile prototropic migrations that are catalyzed by the Lys ammonium group and isomerize enolimine c-type fragments to the more stable amide tautomers. In contrast, interfragment hydrogen atom migrations in the complexes were found to have relatively high transition energies and did not compete with fragment separation. The extensive analysis of the intermediate and transition-state energies led to the conclusion that the observed dissociations cannot proceed competitively on the same potential energy surface. The reactive intermediates for the dissociations originate from distinct electronic states that are accessed by electron transfer.

Kar3Vik1 Mechanochemistry Is Inhibited by Mutation or Deletion of the C Terminus of the Vik1 Subunit

Force production by kinesins has been linked to structural rearrangements of the N and C termini of their motor domain upon nucleotide binding. In recent crystal structures, the Kar3-associated protein Vik1 shows unexpected homology to these conformational states even though it lacks a nucleotide-binding site. This conservation infers a degree of commonality in the function of the N- and C-terminal regions during the mechanochemical cycle of all kinesins and kinesin-related proteins. We tested this inference by examining the functional effects on Kar3Vik1 of mutating or deleting residues in Vik1 that are involved in stabilizing the C terminus against the core and N terminus of the Vik1 motor homology domain (MHD). Point mutations at two moderately conserved residues near the Vik1 C terminus impaired microtubule gliding and microtubule-stimulated ATP turnover by Kar3Vik1. Deletion of the seven C-terminal residues inhibited Kar3Vik1 motility much more drastically. Interestingly, none of the point mutants seemed to perturb the ability of Kar3Vik1 to bind microtubules, whereas the C-terminal truncation mutant did. Molecular dynamics simulations of these C-terminal mutants showed distinct root mean square fluctuations in the N-terminal region of the Vik1 MHD that connects it to Kar3. Here, the degree of motion in the N-terminal portion of Vik1 highly correlated with that in the C terminus. These observations suggest that the N and C termini of the Vik1 MHD form a discrete folding motif that is part of a communication pathway to the nucleotide-binding site of Kar3.

Water mediation is essential to nucleation of β-turn formation in peptide folding motifs.

Water-mediated bond formation: The structure of the peptide GPG-NH2 has been investigated in aqueous solution to understand the role of water in the formation of a β-turn. Using a combination of neutron diffraction enhanced by isotopic substitution, NMR spectroscopy, and computer simulations, it was found that water is an essential component to initiate folding in solution.

Water Mediation Is Essential to Nucleation of β‐Turn Formation in Peptide Folding Motifs

Wasser-vermittelte Bindungsbildung: Die Struktur des GPG-NH2-Peptids (siehe Bild) wurde in wässriger Lösung untersucht, um die Rolle von Wasser bei der Bildung einer β-Schleife verstehen zu können. Mit einer Kombination aus Neutronendiffraktometrie, NMR-Spektroskopie und Computersimulationen konnte gezeigt werden, dass Wasser die Faltung des Peptids in Lösung initiiert.

Archaeal Ubiquitin-like SAMP3 is Isopeptide-linked to Proteins via a UbaA-dependent Mechanism

SAMP1 and SAMP2 are ubiquitin-like proteins that function as protein modifiers and are required for the production of sulfur-containing biomolecules in the archaeon Haloferax volcanii. Here we report a novel small archaeal modifier protein (named SAMP3) with a β-grasp fold and C-terminal diglycine motif characteristic of ubiquitin that is functional in protein conjugation in Hfx. volcanii. SAMP3 conjugates were dependent on the ubiquitin-activating E1 enzyme homolog of archaea (UbaA) for synthesis and were cleaved by the JAMM/MPN+ domain metalloprotease HvJAMM1. Twenty-three proteins (28 lysine residues) were found to be isopeptide-linked to the C-terminal carboxylate of SAMP3, and 331 proteins were reproducibly found associated with SAMP3 in a UbaA-dependent manner based on tandem mass spectrometry (MS/MS) analysis. The molybdopterin (MPT) synthase large subunit homolog MoaE, found samp3ylated at conserved active site lysine residues in MS/MS analysis, was also shown to be covalently bound to SAMP3 by immunoprecipitation and tandem affinity purifications. HvJAMM1 was demonstrated to catalyze the cleavage of SAMP3 from MoaE, suggesting a mechanism of controlling MPT synthase activity. The levels of samp3ylated proteins and samp3 transcripts were found to be increased by the addition of dimethyl sulfoxide to aerobically growing cells. Thus, we propose a model in which samp3ylation is covalent and reversible and controls the activity of enzymes such as MPT synthase. Sampylation of MPT synthase may govern the levels of molybdenum cofactor available and thus facilitate the scavenging of oxygen prior to the transition to respiration with molybdenum-cofactor-containing terminal reductases that use alternative electron acceptors such as dimethyl sulfoxide. Overall, our study of SAMP3 provides new insight into the diversity of functional ubiquitin-like protein modifiers and the network of ubiquitin-like protein targets in Archaea.

Impact of Vibrational Entropy on the Stability of Unsolvated Peptide Helices with Increasing Length

Helices are a key folding motif in protein structure. The question of which factors determine helix stability for a given polypeptide or protein is an ongoing challenge. Here we use van-der-Waals-corrected density functional theory to address a part of this question in a bottom-up approach. We show how intrinsic helical structure is stabilized with length and temperature for a series of experimentally well-studied unsolvated alanine-based polypeptides, Ac-Alan-LysH(+). By exhaustively exploring the conformational space of these molecules, we find that helices emerge as the preferred structure in the length range n = 4-8 not just due to enthalpic factors (hydrogen bonds and their cooperativity, van der Waals dispersion interactions, electrostatics) but importantly also by a vibrational entropic stabilization over competing conformers at room temperature. The stabilization is shown to be due to softer low-frequency vibrational modes in helical conformers than in more compact ones. This observation is corroborated by including anharmonic effects explicitly through ab initio molecular dynamics and generalized by testing different terminations and considering larger helical peptide models.

Circular permutation of the Trp-cage: fold rescue upon addition of a hydrophobic staple

The Trp-cage, at 20 residues in length, is generally acknowledged as the smallest fully protein-like folding motif. Linking the termini by a two-residue unit and excising one residue affords circularly permuted sequences that adopt the same structure. This represents the first successful circular permutation of any fold of less than 50-residue length. As was observed for the original topology, a hydrophobic staple near the chain termini is required for enhanced fold stability.

Synthesis and solution-state dynamics of donor–acceptor oligorotaxane foldamers

We describe in detail a strategy for creating foldamers in which interactions between mechanically interlocked components dictate the single-molecule assembly of a folded secondary structure. This unique folding motif is based on a flexible polyether dumbbell bearing 1,5-dioxynaphthalene (DNP) donors, which folds its way through a series of cyclobis(paraquat-p-phenylene) (CBPQT4+) acceptor rings in a serpentine fashion to enable extended donor–acceptor (D–A) stacking between DNP and the electron-poor 4,4′-bipyridinium (BIPY2+) units in CBPQT4+. These oligorotaxanes can be prepared in a wide range of sizes, with molecular weights up to >15 000 Da, on account of novel one-pot reactions we developed to generate the necessary oligo-DNP precursors. The product distributions from the final kinetically controlled stoppering reactions are highly biased towards oligorotaxanes in which approximately half of the DNP units are encircled by rings, a fact which can be rationalized if the dominant solution-state structures of the pseudorotaxane precursors reflect the solid-state superstructures of analogous compounds, which express 50% recognition site occupancy because of their proclivity to pack into continuous D–A–D–A stacks. The presence of well-defined folded structures in solution have been confirmed by 1H NMR spectroscopy in CD3CN. Moreover, we discovered an empirical selection rule forbidding CBPQT4+ rings to occupy adjacent DNP sites, which elegantly explains both the product distributions and the 1H NMR spectra. Depending on their adherence to this selection rule, all of the oligorotaxanes belong to one of three families: whereas ‘Confused’ oligorotaxanes adopt multiple translational isomers that satisfy the rule and ‘Frustrated’ species cannot obey it at all, members of the ‘Happy’ family each express only one rule-compliant ‘Goldilocks’ isomer. The NMR spectra of these oligorotaxanes also shed light on their dynamics; rapid 180° rotations of DNP units cause pairs of heterotopic BIPY2+ protons in the accompanying CBPQT4+ rings to exchange sites, giving rise to time-averaged signals. This process, which we term ‘superrotation’, will apply much more generally to other mechanically interlocked systems.

Lamins in development, tissue maintenance and stress

Lamins are nuclear intermediate filament proteins. They provide mechanical stability, organize chromatin and regulate transcription, replication, nuclear assembly and nuclear positioning. Recent studies provide new insights into the role of lamins in development, differentiation and tissue response to mechanical, reactive oxygen species and thermal stresses. These studies also propose the existence of separate filament networks for A- and B-type lamins and identify new roles for the different networks. Furthermore, they show changes in lamin composition in different cell types, propose explanations for the more than 14 distinct human diseases caused by lamin A and lamin C mutations and propose a role for lamin B1 in these diseases.

Formation of α-Helical Nanofibers by Mixing β-Structured and α-Helical Coiled Coil Peptides

The helical coiled coil is a well-studied folding motif that can be used for the design of nanometer-sized bioinspired fibrous structures with potential applications as functional materials. A two-component system of coiled coil based model peptides is investigated, which forms, under acidic conditions, uniform, hundreds of nanometers long, and ~2.6 nm thick trimeric α-helical fibers. In the absence of the other component and under the same solvent conditions, one model peptide forms β-sheet-rich amyloid fibrils and the other forms stable trimeric α-helical coiled coils, respectively. These observations reveal that the complementary interactions driving helical folding are much stronger here than those promoting the intermolecular β-sheet formation. The results of this study are important in the context of amyloid inhibition but also open up new avenues for the design of novel fibrous peptidic materials.

TFIID TAF6-TAF9 Complex Formation Involves the HEAT Repeat-containing C-terminal Domain of TAF6 and Is Modulated by TAF5 Protein

The general transcription factor TFIID recognizes specifically the core promoter of genes transcribed by eukaryotic RNA polymerase II, nucleating the assembly of the preinitiation complex at the transcription start site. However, the understanding in molecular terms of TFIID assembly and function remains poorly understood. Histone fold motifs have been shown to be extremely important for the heterodimerization of many TFIID subunits. However, these subunits display several evolutionary conserved noncanonical features when compared with histones, including additional regions whose role is unknown. Here we show that the conserved additional C-terminal region of TFIID subunit TAF6 can be divided into two domains: a small middle domain (TAF6M) and a large C-terminal domain (TAF6C). Our crystal structure of the TAF6C domain from Antonospora locustae at 1.9 Å resolution reveals the presence of five conserved HEAT repeats. Based on these data, we designed several mutants that were introduced into full-length human TAF6. Surprisingly, the mutants affect the interaction between TAF6 and TAF9, suggesting that the formation of the complex between these two TFIID subunits do not only depend on their histone fold motifs. In addition, the same mutants affect even more strongly the interaction between TAF6 and TAF9 in the context of a TAF5-TAF6-TAF9 complex. Expression of these mutants in HeLa cells reveals that most of them are unstable, suggesting their poor incorporation within endogenous TFIID. Taken together, our results suggest that the conserved additional domains in histone fold-containing subunits of TFIID and of co-activator SAGA are important for the assembly of these complexes.

Pan‐Selective Aptamers for the Family of Small GTPases

Pan‐Selective Aptamers for the Family of Small GTPases

Structural Basis of Transcriptional Gene Silencing Mediated by Arabidopsis MOM1

Shifts between epigenetic states of transcriptional activity are typically correlated with changes in epigenetic marks. However, exceptions to this rule suggest the existence of additional, as yet uncharacterized, layers of epigenetic regulation. MOM1, a protein of 2,001 amino acids that acts as a transcriptional silencer, represents such an exception. Here we define the 82 amino acid domain called CMM2 (Conserved MOM1 Motif 2) as a minimal MOM1 fragment capable of transcriptional regulation. As determined by X-ray crystallography, this motif folds into an unusual hendecad-based coiled-coil. Structure-based mutagenesis followed by transgenic complementation tests in plants demonstrate that CMM2 and its dimerization are effective for transcriptional suppression at chromosomal loci co-regulated by MOM1 and the siRNA pathway but not at loci controlled by MOM1 in an siRNA–independent fashion. These results reveal a surprising separation of epigenetic activities that enable the single, large MOM1 protein to coordinate cooperating mechanisms of epigenetic regulation.

The d′--d--d′ Vertical Triad Is Less Discriminating Than the a′--a--a′ Vertical Triad in the Antiparallel Coiled-Coil Dimer Motif

Elucidating relationships between the amino-acid sequences of proteins and their three-dimensional structures, and uncovering non-covalent interactions that underlie polypeptide folding, are major goals in protein science. One approach toward these goals is to study interactions between selected residues, or among constellations of residues, in small folding motifs. The α-helical coiled coil has served as a platform for such studies because this folding unit is relatively simple in terms of both sequence and structure. Amino acid side chains at the helix-helix interface of a coiled coil participate in so-called "knobs-into-holes" (KIH) packing whereby a side chain (the knob) on one helix inserts into a space (the hole) generated by four side chains on a partner helix. The vast majority of sequence-stability studies on coiled-coil dimers have focused on lateral interactions within these KIH arrangements, for example, between an a position on one helix and an a' position of the partner in a parallel coiled-coil dimer, or between a--d' pairs in an antiparallel dimer. More recently, it has been shown that vertical triads (specifically, a'--a--a' triads) in antiparallel dimers exert a significant impact on pairing preferences. This observation provides impetus for analysis of other complex networks of side-chain interactions at the helix-helix interface. Here, we describe a combination of experimental and bioinformatics studies that show that d'--d--d' triads have much less impact on pairing preference than do a'--a--a' triads in a small, designed antiparallel coiled-coil dimer. However, the influence of the d'--d--d' triad depends on the lateral a'--d interaction. Taken together, these results strengthen the emerging understanding that simple pairwise interactions are not sufficient to describe side-chain interactions and overall stability in antiparallel coiled-coil dimers; higher-order interactions must be considered as well.