Protein Segments Research Articles

FragFit: a web-application for interactive modeling of protein segments into cryo-EM density maps.

Cryo-electron microscopy (cryo-EM) is a standard method to determine the three-dimensional structures of molecular complexes. However, easy to use tools for modeling of protein segments into cryo-EM maps are sparse. Here, we present the FragFit web-application, a web server for interactive modeling of segments of up to 35 amino acids length into cryo-EM density maps. The fragments are provided by a regularly updated database containing at the moment about 1 billion entries extracted from PDB structures and can be readily integrated into a protein structure. Fragments are selected based on geometric criteria, sequence similarity and fit into a given cryo-EM density map. Web-based molecular visualization with the NGL Viewer allows interactive selection of fragments. The FragFit web-application, accessible at http://proteinformatics.de/FragFit, is free and open to all users, without any login requirements.

Screening and selection of artificial riboswitches

Synthetic riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules, and a challenge to select this engineered response requires robust screening tools. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer library with a randomized expression platform followed by in vivo selection and screening. In order to determine response to analyte, we developed a dual-color reporter comprising elements of the E. coli fimbriae phase variation system: recombinase FimE controlled by a synthetic riboswitch and an invertible DNA segment (fimS) containing a constitutively active promoter placed between two fluorescent protein genes. Without an analyte, the fluorescent reporter constitutively expressed green fluorescent protein (GFPa1). Addition of the analyte initiated translation of fimE causing unidirectional inversion of the fimS segment and constitutive expression of red fluorescent protein (mKate2). The dual color reporter system can be used to select and to optimize artificial riboswitches in E. coli cells. In this work, the enriched library of aptamers incorporated into the riboswitch architecture reduces the sequence search space by offering a higher percentage of potential ligand binders. The study was designed to produce structure switching aptamers, a necessary feature for riboswitch function and efficiently quantify this function using the dual color reporter system.

A lipid-binding loop of botulinum neurotoxin serotypes B, DC and G is an essential feature to confer their exquisite potency.

The exceptional toxicity of botulinum neurotoxins (BoNTs) is mediated by high avidity binding to complex polysialogangliosides and intraluminal segments of synaptic vesicle proteins embedded in the presynaptic membrane. One peculiarity is an exposed hydrophobic loop in the toxin’s cell binding domain HC, which is located between the ganglioside- and protein receptor-binding sites, and that is particularly pronounced in the serotypes BoNT/B, DC, and G sharing synaptotagmin as protein receptor. Here, we provide evidence that this HC loop is a critical component of their tripartite receptor recognition complex. Binding to nanodisc-embedded receptors and toxicity were virtually abolished in BoNT mutants lacking residues at the tip of the HC loop. Surface plasmon resonance experiments revealed that only insertion of the HC loop into the lipid-bilayer compensates for the entropic penalty inflicted by the dual-receptor binding. Our results represent a new paradigm of how BoNT/B, DC, and G employ ternary interactions with a protein, ganglioside, and lipids to mediate their extraordinary neurotoxicity.

Breaking the Rules

Breaking the Rules

CNNH_PSS: protein 8-class secondary structure prediction by convolutional neural network with highway

BackgroundProtein secondary structure is the three dimensional form of local segments of proteins and its prediction is an important problem in protein tertiary structure prediction. Developing computational approaches for protein secondary structure prediction is becoming increasingly urgent.ResultsWe present a novel deep learning based model, referred to as CNNH_PSS, by using multi-scale CNN with highway. In CNNH_PSS, any two neighbor convolutional layers have a highway to deliver information from current layer to the output of the next one to keep local contexts. As lower layers extract local context while higher layers extract long-range interdependencies, the highways between neighbor layers allow CNNH_PSS to have ability to extract both local contexts and long-range interdependencies. We evaluate CNNH_PSS on two commonly used datasets: CB6133 and CB513. CNNH_PSS outperforms the multi-scale CNN without highway by at least 0.010 Q8 accuracy and also performs better than CNF, DeepCNF and SSpro8, which cannot extract long-range interdependencies, by at least 0.020 Q8 accuracy, demonstrating that both local contexts and long-range interdependencies are indeed useful for prediction. Furthermore, CNNH_PSS also performs better than GSM and DCRNN which need extra complex model to extract long-range interdependencies. It demonstrates that CNNH_PSS not only cost less computer resource, but also achieves better predicting performance.ConclusionCNNH_PSS have ability to extracts both local contexts and long-range interdependencies by combing multi-scale CNN and highway network. The evaluations on common datasets and comparisons with state-of-the-art methods indicate that CNNH_PSS is an useful and efficient tool for protein secondary structure prediction.

Human insulin fibrillogenesis in the presence of epigallocatechin gallate and melatonin: Structural insights from a biophysical approach

Fibrillogenesis of monomeric human insulin in the presence or absence of (-)-epigallocatechin-3-gallate and melatonin was here investigated using a multi-technique approach. Results from Raman and Infrared spectroscopy pointed out that a high content of intermolecular β-sheet aggregates is formed after long-term incubation. However, near UV experiments, Dynamic Light Scattering, Thioflavin-T fluorescence measurements and Atomic Force Microscopy revealed that the kinetics from native-to-fibrillar state of insulin is hampered only in the presence of (-)-epigallocatechin-3-gallate. Molecular dynamic simulations indicated that this compound binds near the B11–B18 protein segment, where hydrophobic residues responsible for the beginning of cooperative aggregation are located. Such a preferential binding region is not recognized by melatonin, a highly mobile molecule, which indeed does not affect fibril formation. The results of the present study demonstrate that (-)-epigallocatechin-3-gallate interferes with the insulin nucleation phase, giving rise to amorphous aggregates in the early stages of the aggregation process.

Co-translational Folding Intermediate Dictates Membrane Targeting of the Signal Recognition Particle Receptor

Much of our knowledge on the function of proteins is deduced from their mature, folded states. However, it is unknown whether partially synthesized nascent protein segments can execute biological functions during translation and whether their premature folding states matter. A recent observation that a nascent chain performs a distinct function, co-translational targeting in vivo, has been made with the Escherichia coli signal recognition particle receptor FtsY, a major player in the conserved pathway of membrane protein biogenesis. FtsY functions as a membrane-associated entity, but very little is known about the mode of its targeting to the membrane. Here we investigated the underlying structural mechanism of the co-translational FtsY targeting to the membrane. Our results show that helices N2–4, which mediate membrane targeting, form a stable folding intermediate co-translationally that greatly differs from its fold in the mature FtsY. These results thus resolve a long-standing mystery of how the receptor targets the membrane even when deleted of its alleged membrane targeting sequence. The structurally distinct targeting determinant of FtsY exists only co-translationally. Our studies will facilitate further efforts to seek cellular factors required for proper targeting and association of FtsY with the membrane. Moreover, the results offer a hallmark example for how co-translational nascent intermediates may dictate biological functions.

The Variable Salt Dependence of Mini‐Inteins from Haloquadratum walsbyi

Protein splicing is a post‐translational modification by which a segment of protein, known as an intein, self‐catalyzes its excision from the protein (exteins) while simultaneously ligating the two flanking exteins. Inteins can be found in many types of archaebacteria, including halophiles, which thrive in environments with high salt concentrations. Previous results have shown that mini inteins of Haloquadratum walsbyi, such as those interrupting the cdc21 protein, require high concentrations of salt to splice. One intein that interrupts the DNA Gyrase B splices in E. coli, whereas similar inteins have required high salt incubation in vitro for splicing. The Hwa cdc21a, cdc21c, and PolIIb inteins were likely present in the most recent halophile ancestor as they are monophyletic within the halobacteria, the PolIIa and cdc21b inteins are polyphyletic, and the GyrB intein is likely related to inteins from bacteria. It is hypothesized that the GyrB intein has more recently invaded the halobacteria, therefore making the intein less stable to unfolding in the presence of salt when compared to earlier invading inteins, which many have adapted to high salt environments and are dependent on high salt concentrations for activity.Support or Funding InformationThis work was supported by the National Science Foundation (grants MCB‐1244089 and MCB‐1517138 to KVM) and the Dreyfus Foundation (KVM).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Probing the Function of Apolipoprotein A‐I Using Chimera Proteins

Human apolipoprotein A‐I (apoA‐I) is a 243 residue, 28 kDa protein that consists of an N‐terminal (NT) helix bundle domain and a less structured C‐terminal (CT) domain. ApoA‐I is the major protein component in high‐density lipoprotein (HDL) and is known to play an important role in reverse cholesterol transport. HDL promotes the removal of excess cholesterol from peripheral tissues to the liver. The CT domain of apoA‐I has a high affinity for phospholipids and initiates lipid binding, whereas the NT domain harbors the site of lecithin‐cholesterol acyltransferase activity. A recent study showed that removal of residues 231–243 led to a decrease in lipid binding affinity. Other studies have suggested that segments in the NT domain, residues 1–43 and 44–65, initiate lipid binding. Thus, conflicting data exists regarding the location of high‐affinity lipid binding in apoA‐I. A chimera protein was recently designed in which the CT domain of apoA‐I was attached to insect apolipophorin (apoLp‐III). The insect apolipoprotein acquired apoA‐I like properties, including high affinity lipid binding. Thus, this system allows for the identification of apoA‐I segments that have a high affinity for lipid surfaces. To identify potential lipid binding regions within apoA‐I, three chimeric constructs were designed using L. migratoria apoLp‐III. Residues 1–43 or 44–65 of apoA‐I were attached to the NT end of apoLp‐III. In addition, residues 231–243 of apoA‐I were attached to the CT end of apoLp‐III. The three DNA constructs were inserted into the pET‐20b (+) expression vector through restriction enzymes HindIII and NdeI. Site‐directed mutagenesis was conducted to introduce two cysteines into apoLp‐III at positions Thr‐20 and Ala‐149. The two cysteines lock the protein in an inactive state under oxidizing conditions to prevent lipid binding. This enables to study the lipid binding properties of the attached apoA‐I segments in the chimera proteins. Proteins are currently being expressed in Escherichia coli BL21(DE3) pLysS cells and will be purified through nickel‐affinity and size‐exclusion chromatography. The lipid binding properties will be assessed by the ability to solubilize phospholipid vesicles.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exploiting a Phage-Bacterium Interaction System as a Molecular Switch to Decipher Macromolecular Interactions in the Living Cell.

Pathogenicity islands of Staphylococcus aureus are under the strong control of helper phages, where regulation is communicated at the gene expression level via a family of specific repressor proteins. The repressor proteins are crucial to phage-host interactions and, based on their protein characteristics, may also be exploited as versatile molecular tools. The Stl repressor from this protein family has been recently investigated and although the binding site of Stl on DNA was recently discovered, there is a lack of knowledge on the specific protein segments involved in this interaction. Here, we develop a generally applicable system to reveal the mechanism of the interaction between Stl and its cognate DNA within the cellular environment. Our unbiased approach combines random mutagenesis with high-throughput analysis based on the lac operon to create a well-characterized gene expression system. Our results clearly indicate that, in addition to a previously implicated helix-turn-helix segment, other protein moieties also play decisive roles in the DNA binding capability of Stl. Structural model-based investigations provided a detailed understanding of Stl:DNA complex formation. The robustness and reliability of our novel test system were confirmed by several mutated Stl constructs, as well as by demonstrating the interaction between Stl and dUTPase from the Staphylococcal ϕ11 phage. Our system may be applied to high-throughput studies of protein:DNA and protein:protein interactions.

On the Mechanism of Flow Sensing by Guinea Pig Carotid Endothelial Cells

It is known that luminal facing endothelial G‐protein coupled receptors (GPCRs) are affected by flow. In human blood vessels bradykinin produces a relaxing effect whereas in guinea pig its effect is vasoconstrictive and affected by flow.Objetiveto study the mechanism by which flow is sensed by endothelial cells.Methodswe have used guinea pig carotid arteries to evaluate the effect of flow on the contractile response to bradykinin and cultured carotid endothelial cells to know the expression pattern of p‐SRC and p‐ERK in response to flow.ResultsWe have found that in guinea pig carotid artery the flow‐dependent vasoconstrictive effect of bradykinin is augmented by 5 min luminal exposure to heparinase or alfa‐chymotrypsin enzymes suggesting that the flow sensor of the bradykinin receptor (BR) is associated to a carbohydrate residue and/or a protein segment of the receptor present in the extracellular face of the plasma membrane. By looking at the available molecular structure of the BR we have identified a segment of the N‐terminus that could be acting as the flow sensor by a very simple physical mechanism. On the other hand, by using carotid cultured endothelial cells we have found that flow decreases the expression of p‐SRC protein and does not change the p‐ERK expression.Conclusionsthe mechanism by which flow is sensed by endothelial cells is still on debate. However, one mechanism for sensing the flow could be located at the extracellular segment of the protein receptor. The N‐terminus segment of the BR could move depending on the flow velocity and, as a consequence, increasing or decreasing the availability of the BR to its ligand.Support or Funding InformationSupported by Conacyt 223350 to RET.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Origins of the Mechanochemical Coupling of Peptide Bond Formation to Protein Synthesis.

Mechanical forces acting on the ribosome can alter the speed of protein synthesis, indicating that mechanochemistry can contribute to translation control of gene expression. The naturally occurring sources of these mechanical forces, the mechanism by which they are transmitted 10 nm to the ribosome's catalytic core, and how they influence peptide bond formation rates are largely unknown. Here, we identify a new source of mechanical force acting on the ribosome by using in situ experimental measurements of changes in nascent-chain extension in the exit tunnel in conjunction with all-atom and coarse-grained computer simulations. We demonstrate that when the number of residues composing a nascent chain increases, its unstructured segments outside the ribosome exit tunnel generate piconewtons of force that are fully transmitted to the ribosome's P-site. The route of force transmission is shown to be through the nascent polypetide's backbone, not through the wall of the ribosome's exit tunnel. Utilizing quantum mechanical calculations we find that a consequence of such a pulling force is to decrease the transition state free energy barrier to peptide bond formation, indicating that the elongation of a nascent chain can accelerate translation. Since nascent protein segments can start out as largely unfolded structural ensembles, these results suggest a pulling force is present during protein synthesis that can modulate translation speed. The mechanism of force transmission we have identified and its consequences for peptide bond formation should be relevant regardless of the source of the pulling force.

The Un(f)told Story of General Anesthesia.

Inhalational anesthetics are routinely employed in clinical practice to accomplish general anesthesia. Concerns have recently emerged regarding the deleterious impact of these volatile agents on cognitive performance, immune functions, and tumor recurrence and metastasis. These agents have been shown to modify the gene-expression pattern as well as cell signaling in tumor cells, but the underlying molecular mechanisms remain a matter of conjecture. Regulatory/signaling proteins either of cytosolic or membrane origin abundantly contain intrinsically disordered sequences, the conformational pliability of which is pivotal in their biological functions. It is well known that chloroform (an anesthetic itself), trifluoroethanol, hexafluoroisopropanol, and related haloalcohols markedly affect the structure of disordered proteins and protein regions by inducing folding, misfolding, or even aggregation. Taking into consideration the physicochemical similarities and protein interaction modes of these volatile solvents and inhaled anesthetics, it is postulated that administration of these drugs can also modify the secondary structure of disordered protein segments. Accordingly, pharmacological effects of anesthetics may, at least in part, be mediated by conformational perturbations of intrinsic disorder-based regulatory protein networks of cells.

Solution Structures of Engineered Vault Particles

Prior crystal structures of the vault have provided clues of its structural variability but are non-conclusive due to crystal packing. Here, we obtained vaults by engineering at the N terminus of rat major vault protein (MVP) an HIV-1 Gag protein segment and determined their near-atomic resolution (∼4.8Å) structures in a solution/non-crystalline environment. The barrel-shaped vaults in solution adopt two conformations, 1 and 2, both with D39 symmetry. From the N to C termini, each MVP monomer has three regions: body, shoulder, and cap. While conformation 1 is identical to one of the crystal structures, the shoulder in conformation 2 is translocated longitudinally up to 10Å, resulting in an outward-projected cap. Our structures clarify the structural discrepancies in the body region in the prior crystallography models. The vault's drug-delivery potential is highlighted by the internal disposition and structural flexibility of its Gag-loaded N-terminal extension at the barrel waist of the engineered vault.

Atomic-level evidence for packing and positional amyloid polymorphism by segment from TDP-43 RRM2.

Proteins in the fibrous amyloid state are a major hallmark of neurodegenerative disease. Understanding the multiple conformations, or polymorphs, of amyloid proteins at the molecular level is a challenge of amyloid research. Here, we detail the wide range of polymorphs formed by a segment of human TAR DNA-binding protein 43 (TDP-43) as a model for the polymorphic capabilities of pathological amyloid aggregation. Using X-ray diffraction, microelectron diffraction (MicroED) and single-particle cryo-EM, we show that the 247DLIIKGISVHI257 segment from the second RNA-recognition motif (RRM2) forms an array of amyloid polymorphs. These associations include seven distinct interfaces displaying five different symmetry classes of steric zippers. Additionally, we find that this segment can adopt three different backbone conformations that contribute to its polymorphic capabilities. The polymorphic nature of this segment illustrates at the molecular level how amyloid proteins can form diverse fibril structures.

Both positional and chemical variables control in vitro proteolytic cleavage of a presenilin ortholog

Mechanistic details of intramembrane aspartyl protease (IAP) chemistry, which is central to many biological and pathogenic processes, remain largely obscure. Here, we investigated the in vitro kinetics of a microbial intramembrane aspartyl protease (mIAP) fortuitously acting on the renin substrate angiotensinogen and the C-terminal transmembrane segment of amyloid precursor protein (C100), which is cleaved by the presenilin subunit of γ-secretase, an Alzheimer disease (AD)-associated IAP. mIAP variants with substitutions in active-site and putative substrate-gating residues generally exhibit impaired, but not abolished, activity toward angiotensinogen and retain the predominant cleavage site (His-Thr). The aromatic ring, but not the hydroxyl substituent, within Tyr of the catalytic Tyr-Asp (YD) motif plays a catalytic role, and the hydrolysis reaction incorporates bulk water as in soluble aspartyl proteases. mIAP hydrolyzes the transmembrane region of C100 at two major presenilin cleavage sites, one corresponding to the AD-associated Aβ42 peptide (Ala-Thr) and the other to the non-pathogenic Aβ48 (Thr-Leu). For the former site, we observed more favorable kinetics in lipid bilayer-mimicking bicelles than in detergent solution, indicating that substrate-lipid and substrate-enzyme interactions both contribute to catalytic rates. High-resolution MS analyses across four substrates support a preference for threonine at the scissile bond. However, results from threonine-scanning mutagenesis of angiotensinogen demonstrate a competing positional preference for cleavage. Our results indicate that IAP cleavage is controlled by both positional and chemical factors, opening up new avenues for selective IAP inhibition for therapeutic interventions.

Atomic structures of low-complexity protein segments reveal kinked β sheets that assemble networks.

Subcellular membraneless assemblies are a reinvigorated area of study in biology, with spirited scientific discussions on the forces between the low-complexity protein domains within these assemblies. To illuminate these forces, we determined the atomic structures of five segments from protein low-complexity domains associated with membraneless assemblies. Their common structural feature is the stacking of segments into kinked β sheets that pair into protofilaments. Unlike steric zippers of amyloid fibrils, the kinked sheets interact weakly through polar atoms and aromatic side chains. By computationally threading the human proteome on our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, and keratins, which are known to form networks and localize to membraneless assemblies.

POTENCI: prediction of temperature, neighbor and pH-corrected chemical shifts for intrinsically disordered proteins.

Chemical shifts contain important site-specific information on the structure and dynamics of proteins. Deviations from statistical average values, known as random coil chemical shifts (RCCSs), are extensively used to infer these relationships. Unfortunately, the use of imprecise reference RCCSs leads to biased inference and obstructs the detection of subtle structural features. Here we present a new method, POTENCI, for the prediction of RCCSs that outperforms the currently most authoritative methods. POTENCI is parametrized using a large curated database of chemical shifts for protein segments with validated disorder; It takes pH and temperature explicitly into account, and includes sequence-dependent nearest and next-nearest neighbor corrections as well as second-order corrections. RCCS predictions with POTENCI show root-mean-square values that are lower by 25-78%, with the largest improvements observed for 1Hα and 13C'. It is demonstrated how POTENCI can be applied to analyze subtle deviations from RCCSs to detect small populations of residual structure in intrinsically disorder proteins that were not discernible before. POTENCI source code is available for download, or can be deployed from the URL http://www.protein-nmr.org .

UFD-2 is an adaptor-assisted E3 ligase targeting unfolded proteins

Muscle development requires the coordinated activities of specific protein folding and degradation factors. UFD-2, a U-box ubiquitin ligase, has been reported to play a central role in this orchestra regulating the myosin chaperone UNC-45. Here, we apply an integrative in vitro and in vivo approach to delineate the substrate-targeting mechanism of UFD-2 and elucidate its distinct mechanistic features as an E3/E4 enzyme. Using Caenorhabditis elegans as model system, we demonstrate that UFD-2 is not regulating the protein levels of UNC-45 in muscle cells, but rather shows the characteristic properties of a bona fide E3 ligase involved in protein quality control. Our data demonstrate that UFD-2 preferentially targets unfolded protein segments. Moreover, the UNC-45 chaperone can serve as an adaptor protein of UFD-2 to poly-ubiquitinate unfolded myosin, pointing to a possible role of the UFD-2/UNC-45 pair in maintaining proteostasis in muscle cells.

The Moonlighting Mechanisms of UNC-45 for Keeping Muscle Myosin in Shape

Muscle development requires the coordinated activities of specific protein folding and degradation factors. UNC-45, for example, is a highly specialized chaperone that orchestrates myosin folding and assembly. Structural and biochemical data from our lab demonstrated that UNC-45 can form linear protein chains offering multiple binding sites for co-working chaperones and client proteins. Accordingly, Hsp70 and Hsp90, which bind to the TPR domain of UNC-45, can act in concert and with defined periodicity on captured myosin molecules. The identified multi-chaperone complex represents a novel type of filament assembly factor coupling myosin folding with myofilament formation. Strikingly, UNC-45 does not only team-up with Hsp70/Hsp90, but also with UFD-2, a U-box ubiquitin ligase previously implicated in regulating UNC-45 levels. To better understand the UNC-45/UFD-2 interplay, we studied the substrate-targeting mechanism of UFD-2 and its distinct mechanistic features as an E4 ubiquitination enzyme (a so-called processivity factor, extending preformed Ub chains). Using Caenorhabditis elegans as model system, we demonstrate that UFD-2 is not regulating the protein levels of UNC-45 in muscle cells, but rather shows characteristic properties of a bona-fide E3 ligase involved in protein quality control. Our data demonstrate that UFD-2 preferentially targets unfolded protein segments. Moreover, the UNC-45 chaperone can serve as an adaptor protein of UFD-2 to poly-ubiquitinate unfolded myosin, pointing to a possible role of the UFD-2/UNC-45 pair in maintaining proteostasis in muscle cells. These data highlight the moonlighting function of UNC-45 in promoting both the folding and the degradation of myosin, depending on its partner proteins.