Helical Folding Research Articles

Mutation‐Dependent Capacity of the ER Proteostasis Network to Quality Control Disease‐Causing Collagen Misfolding

Osteogenesis imperfecta (OI) is an inherited disease most commonly caused by autosomal dominant mutations in collagen type‐I. OI‐causing mutations are found in both the triple helical region and the C‐propeptide region of collagen‐I. Mutations in the triple helical region cause disruption to triple helical folding, stability, and structure. Mutations in the C‐propeptide region, the C‐terminal nucleation domain of collagen‐I, disrupt the initial assembly steps in collagen folding. Currently, the mechanism by which the cellular protein folding machinery recognizes and processes these types of misfolded variants of collagen‐I is not well‐understood. Here, we report how the endoplasmic reticulum (ER) differentially processes normal and misfolding, OI‐causing collagen‐I variants. We employed state‐of‐the‐art, quantitative mass spectrometry‐based proteomic methods to determine the full complement of proteostasis network components that differentially engage wild‐type (wt) and several OI‐causing mutants of collagen‐I. We identified >75 putative proteins that might play a role in collagen‐I proteostasis, in addition to the currently known collagen‐I interactors. Interestingly, we found that while there is no significant difference in the interactomes of wild‐type (wt) and triple‐helical mutant collagen‐I variants, mutant C‐propeptide collagen‐I is well‐recognized by a number of components of the ER proteostasis network. These results indicate that the cell can specifically recognize collagen C‐propeptide misfolding, but has limited capacity to address triple‐helix domain misfolding. Our findings contribute to an understanding of how the proteostasis network reacts to different types of collagen folding defects and maintains proper collagen folding and secretion.

Structural Investigation of Hybrid Peptide Foldamers Composed of α-Dipeptide Equivalent β-Oxy-δ5 -amino Acids.

Due to their equivalent lengths, δ-amino acids can serve as surrogates of α-dipeptides. However, δ-amino acids with proteinogenic side chains have not been well studied because of synthetic difficulties and because of their insolubility in organic solvents. Recently we reported the spontaneous supramolecular gelation of δ-peptides composed of β(O)-δ5 -amino acids. Here, we report the incorporation of β(O)-δ5 -amino acids as guests into the host α-helix, α,γ-hybrid peptide 12-helix and their single-crystal conformations. In addition, we studied the solution conformations of hybrid peptides composed of 1:1 alternating α and β(O)-δ5 -amino acids. In contrast to the control α-helix structures, the crystal structure of peptides with β(O)-δ5 -amino acids exhibit α-helical conformations consisting of both 13- and 10-membered H-bonds. The α,δ-hybrid peptide adopted mixed 13/11-helix conformation in solution with alternating H-bond directionality. Crystal-structure analysis revealed that the α,γ4 -hybrid peptide accommodated the guest β(O)-δ5 -amino acid without significant deviation to the overall helix folding. The results reported here emphasize that β(O)-δ5 -amino acids with proteinogenic side chains can be accommodated into regular α-helix or 12-helix as guests without much deviation of the overall helix folding of the peptides.

On the Role of the Solvent Environment in the Folding and Unfolding of Amphipathic Helices

On the Role of the Solvent Environment in the Folding and Unfolding of Amphipathic Helices

Development of Helical Aromatic Amide Foldamers with a Diphenylacetylene Backbone.

We designed and synthesized aromatic polyamides with a diphenylacetylene backbone, α-DPA and β-DPA, bearing (S)-α- and (S)-β-methyl-substituted triethyleneglycol (TEG) side chains, respectively, and examined their conformations in solution. Both polymers exhibit strong, solvent polarity-dependent circular dichroism spectra, which indicated that they take helical conformations in low-polarity solvents. The spectra were mirror images, depending on the chiral position of the side chains. Thus, the polyamide α-DPA bearing (S)-α-methyl-substituted TEG groups takes a left-handed helical conformation, while the polyamide β-DPA with (S)-β-methyl-substituted TEG groups takes a right-handed helical conformation. The difference in the screw sense of α-DPA and β-DPA would be caused by the steric interaction between the main chain and the side chain, as observed in poly(p-benzamide) possessing (S)-β-methyl-substituted TEG side chains (β-PA) because the large cavity of the helical structure of DPA would disturb the solvophobically induced helical folding. Detailed conformational analyses of the oligoamides 6-12 with β-methyl-substituted TEG groups were conducted. Theoretical calculations indicated that the oligoamides with β-methyl-substituted TEG groups exist in a helical conformation with a cavity of 7 Å in diameter. The 1H NMR spectra of the oligomers revealed interactions with small anions such as chloride and acetate anions and with pyridinium cations.

Is the E. coli Homolog of the Formate/Nitrite Transporter Family an Anion Channel? A Computational Study

Formate/nitrite transporters (FNTs) selectively transport monovalent anions and are found in prokaryotes and lower eukaryotes. They play a significant role in bacterial growth and act against the defense mechanism of infected hosts. Because FNTs do not occur in higher animals, they are attractive drug targets for many bacterial diseases. Phylogenetic analysis revealed that they can be classified into eight subgroups, two of which belong to the uncharacterized YfdC-α and YfdC-β groups. Experimentally determined structures of FNTs belonging to different phylogenetic groups adopt the unique aquaporin-like hourglass helical fold. We considered the formate channel from Vibrio cholerae, the hydrosulphide channel from Clostridium difficile, and the uncharacterized channel from Escherichia coli (EcYfdC) to investigate the mechanism of transport and selectivity. Using equilibrium molecular dynamics and umbrella sampling studies, we determined temporal channel radius profiles, permeation events, and potential of mean force profiles of different substrates with the conserved central histidine residue in protonated or neutral form. Unlike the formate channel from V. cholerae and the hydrosulphide channel from C. difficile, molecular dynamics studies showed that the formate substrate was unable to enter the vestibule region of EcYfdC. Absence of a conserved basic residue and presence of acidic residues in the vestibule regions, conserved only in YfdC-α, were found to be responsible for high energy barriers for the anions to enter EcYfdC. Potential of mean force profiles generated for ammonia and ammonium ion revealed that EcYfdC can transport neutral solutes and could possibly be involved in the transport of cations analogous to the mechanism proposed for ammonium transporters. Although YfdC members belong to the FNT family, our studies strongly suggest that EcYfdC is not an anion channel. Absence or presence of specific charged residues at particular positions makes EcYfdC selective for neutral or possibly cationic substrates. Further experimental studies are needed to get a definitive answer to the question of the substrate selectivity of EcYfdC. This provides an example of membrane proteins from the same family transporting substrates of different chemical nature.

Strengthening Peptoid Helicity through Sequence Site-Specific Positioning of Amide cis-Inducing NtBu Monomers.

The synthesis of biomimetic helical secondary structures is sought after for the construction of innovative nanomaterials and applications in medicinal chemistry such as the development of protein-protein interaction modulators. Peptoids, a sequence-defined family of oligomers, enable a peptidomimetic strategy, especially considering the easily accessible monomer diversity and peptoid helical folding propensity. However, cis-trans isomerization of the backbone tertiary amides may impair the peptoid's adoption of stable secondary structures, notably the all-cis polyproline I-like helical conformation. Here, we show that cis-inducing NtBu achiral monomers strategically positioned within chiral sequences may reinforce the degree of peptoid helicity, although with a reduced content of chiral side chains. The design principles presented here will undoubtedly help achieve more conformationally stable helical peptoids with desired functions.

Exploring the Ability of Cyclic Peptides to Target SAM Domains: A Computational and Experimental Study

Sterile alpha motif (SAM) domains are protein interaction modules with a helical fold. SAM-SAM interactions often adopt the mid-loop (ML)/end-helix (EH) model, in which the C-terminal helix and adjacent loops of one SAM unit (EH site) bind the central regions of another SAM domain (ML site). Herein, an original strategy to attack SAM-SAM associations is reported. It relies on the design of cyclic peptides that target a region of the SAM domain positioned at the bottom side of the EH interface, which is thought to be important for the formation of a SAM-SAM complex. This strategy has been preliminarily tested by using a model system of heterotypic SAM-SAM interactions involving the erythropoietin-producing hepatoma kinase A2 (EphA2) receptor and implementing a multidisciplinary plan made up of computational docking studies, experimental interaction assays (by NMR spectroscopy and surface plasmon resonance techniques) and conformational analysis (by NMR spectroscopy and circular dichroism). This work further highlights how only a specific balance between flexibility and rigidity may be needed to generate modulators of SAM-SAM interactions.

Lowering the culture temperature corrects collagen abnormalities caused by HSP47 gene knockout

Heat shock protein 47 (HSP47) is an endoplasmic reticulum (ER)-resident molecular chaperone that specifically recognizes triple helical portions of procollagens. The chaperone function of HSP47 is indispensable in mammals, and hsp47-null mice show an embryonic lethal phenotype accompanied by severe abnormalities in collagen-based tissue structures. Two leading hypotheses are currently accepted for the molecular function of HSP47 as a procollagen-specific chaperone. One is facilitation of procollagen folding by stabilizing thermally unstable triple helical folding intermediates, and the other is inhibition of procollagen aggregation or lateral association in the ER. The aim of this study was to elucidate the functional essence of this unique chaperone using fibroblasts established from hsp47−/− mouse embryos. When the cells were cultured at 37 °C, various defects in procollagen biosynthesis were observed, such as accumulation in the ER, over-modifications including prolyl hydroxylation, lysyl hydroxylation, and further glycosylation, and unusual secretion of type I collagen homotrimer. All defects were corrected by culturing the cells at a lower temperature of 33 °C. These results indicated that lowering the culture temperature compensated for the loss of HSP47. This study elucidated that HSP47 stabilizes the elongating triple helix of procollagens, which is otherwise unstable at the body temperature of mammals.

Accelerated Molecular Dynamics Simulation for Helical Proteins Folding in Explicit Water.

In this study, we examined the folding processes of eight helical proteins (2I9M, TC5B, 1WN8, 1V4Z, 1HO2, 1HLL, 2KFE, and 1YYB) at room temperature using the explicit solvent model under the AMBER14SB force field with the accelerated molecular dynamics (AMD) and traditional molecular dynamics (MD), respectively. We analyzed and compared the simulation results obtained by these two methods based on several aspects, such as root mean square deviation (RMSD), native contacts, cluster analysis, folding snapshots, free energy landscape, and the evolution of the radius of gyration, which showed that these eight proteins were successfully and consistently folded into the corresponding native structures by AMD simulations carried out at room temperature. In addition, the folding occurred in the range of 40~180 ns after starting from the linear structures of the eight proteins at 300 K. By contrast, these stable folding structures were not found when the traditional molecular dynamics (MD) simulation was used. At the same time, the influence of high temperatures (350, 400, and 450 K) is also further investigated. Study found that the simulation efficiency of AMD is higher than that of MD simulations, regardless of the temperature. Of these temperatures, 300 K is the most suitable temperature for protein folding for all systems. To further investigate the efficiency of AMD, another trajectory was simulated for eight proteins with the same linear structure but different random seeds at 300 K. Both AMD trajectories reached the correct folded structures. Our result clearly shows that AMD simulation are a highly efficient and reliable method for the study of protein folding.

Sharp kinking of a coiled-coil in MutS allows DNA binding and release.

DNA mismatch repair (MMR) corrects mismatches, small insertions and deletions in DNA during DNA replication. While scanning for mismatches, dimers of MutS embrace the DNA helix with their lever and clamp domains. Previous studies indicated generic flexibility of the lever and clamp domains of MutS prior to DNA binding, but whether this was important for MutS function was unknown. Here, we present a novel crystal structure of DNA-free Escherichia coli MutS. In this apo-structure, the clamp domains are repositioned due to kinking at specific sites in the coiled-coil region in the lever domains, suggesting a defined hinge point. We made mutations at the coiled-coil hinge point. The mutants made to disrupt the helical fold at the kink site diminish DNA binding, whereas those made to increase stability of coiled-coil result in stronger DNA binding. These data suggest that the site-specific kinking of the coiled-coil in the lever domain is important for loading of this ABC-ATPase on DNA.

Diverse Structural Conversion and Aggregation Pathways of Alzheimer's Amyloid-β (1-40).

Complex amyloid aggregation of amyloid-β (1-40) (Aβ1-40) in terms of monomer structures has not been fully understood. Herein, we report the microscopic mechanism and pathways of Aβ1-40 aggregation with macroscopic viewpoints through tuning its initial structure and solubility. Partial helical structures of Aβ1-40 induced by low solvent polarity accelerated cytotoxic Aβ1-40 amyloid fibrillation, while predominantly helical folds did not aggregate. Changes in the solvent polarity caused a rapid formation of β-structure-rich protofibrils or oligomers via aggregation-prone helical structures. Modulation of the pH and salt concentration transformed oligomers to protofibrils, which proceeded to amyloid formation. We reveal diverse molecular mechanisms underlying Aβ1-40 aggregation with conceptual energy diagrams and propose that aggregation-prone partial helical structures are key to inducing amyloidogenesis. We demonstrate that context-dependent protein aggregation is comprehensively understood using the macroscopic phase diagram, which provides general insights into differentiation of amyloid formation and phase separation from unfolded and folded structures.

Interaction of SNARE Mimetic Peptides with Lipid bilayers: Effects of Secondary Structure, Bilayer Composition and Lipid Anchoring

The coiled-coil forming peptides ‘K’ enriched in lysine and ‘E’ enriched in glutamic acid have been used as a minimal SNARE mimetic system for membrane fusion. Here we describe atomistic molecular dynamics simulations to characterize the interactions of these peptides with lipid bilayers for two different compositions. For neutral phosphatidylcholine (PC)/phosphatidylethanolamine (PE) bilayers the peptides experience a strong repulsive barrier against adsorption, also observed in potential of mean force (PMF) profiles calculated with umbrella sampling. For peptide K, a minimum of −12 kBT in the PMF provides an upper bound for the binding free energy whereas no stable membrane bound state could be observed for peptide E. In contrast, the electrostatic interactions with negatively charged phosphatidylglycerol (PG) lipids lead to fast adsorption of both peptides at the head-water interface. Experimental data using fluorescently labeled peptides confirm the stronger binding to PG containing bilayers. Lipid anchors have little effect on the peptide-bilayer interactions or peptide structure, when the peptide also binds to the bilayer in the absence of a lipid anchor. For peptide E, which does not bind to the PC bilayer without a lipid anchor, the presence of such an anchor strengthens the electrostatic interactions between the charged side chains and the zwitterionic head-groups and leads to a stabilization of the peptide’s helical fold by the membrane.

The Preparation of Enantiopure [6]- and [7]Helicenes from Binaphthanol.

A general and efficient synthetic methodology for the preparation of enantio- and diastereopure [6]-, and [7]helicenes is developed. Commercially available chiral binaphthanols are utilized to generate the arylene-vinylene precursors, which undergo helical folding via photocyclization to give enantio- and diastereopure [6]-, and [7]helicenes. These optically pure helicenes could be easily obtained via silica gel column chromatography without the use of expensive HPLC or chiral resolution reagents. The configurations and structures of these helicenes are confirmed by CD spectra and X-ray crystallographic analysis. This work provides a new method for preparation of enantiopure helicenes.

Hydrophobic Moieties Bestow Fast-Folding and Hyperstability on Collagen Triple Helices.

Trans amide bonds and fast cis- trans isomerization of Xaa-Pro bonds are crucial for the stability and folding rate of collagen, the most abundant protein in mammals. Here, we explored the effect of pendant hydrophobic moieties on the folding and stability of collagen triple helices. Kinetic studies with a series of collagen model peptides showed that a local hydrophobic environment accelerates cis- trans isomerization to an extent that thermally induced unfolding and folding of the collagen triple helix take place at the same speed. Thermal denaturation studies revealed that the hydrophobic appendages provide hyperstable collagen triple helices ( Tm = 70 °C).

Photoactivatable Hsp47: A Tool to Regulate Collagen Secretion and Assembly.

Collagen is the most abundant structural protein in mammals and is crucial for the mechanical integrity of tissues. Hsp47, an endoplasmic reticulum resident collagen‐specific chaperone, is involved in collagen biosynthesis and plays a fundamental role in the folding, stability, and intracellular transport of procollagen triple helices. This work reports on a photoactivatable derivative of Hsp47 that allows regulation of collagen biosynthesis within mammalian cells using light. Photoactivatable Hsp47 contains a non‐natural light‐responsive tyrosine (o‐nitro benzyl tyrosine (ONBY)) at Tyr383 position of the protein sequence. This mutation renders Hsp47 inactive toward collagen binding. The inactive, photoactivatable protein is easily uptaken by cells within a few minutes of incubation, and accumulated at the endoplasmic reticulum via retrograde KDEL receptor‐mediated uptake. Upon light exposure, the photoactivatable Hsp47 turns into functional Hsp47 in situ. The increased intracellular concentration of Hsp47 results in stimulated secretion of collagen. The ability to promote collagen synthesis on demand, with spatiotemporal resolution, and in diseased state cells is demonstrated in vitro. It is envisioned that photoactivatable Hsp47 allows unprecedented fundamental studies of collagen biosynthesis, matrix biology, and inspires new therapeutic concepts in biomedicine and tissue regeneration.

Myxococcus xanthus truncated globin HbO: in silico analysis and functional characterization.

Truncated globins are 20-40 amino acids shorter than full length globins. Till date, globins have been characterized predominantly from bacteria involved in pathogenicity, nitrogen fixation and photosynthesis, where they are implicated in bacterial virulence within the host, protection of nitrogenase from oxygen inactivation and prevention of oxidative damage to the photosynthetic machinery respectively. Myxococcus xanthus, the model myxobacterium, is an obligate aerobe with a multicellular stage in its life cycle where cells encounter oxygen limitation. This work was undertaken to investigate the potential role of the truncated globin in M. xanthus. To examine the role of globins in this unique group of bacteria, the gene coding for a putative truncated globin (HbO) was identified in the genome of M. xanthus DK 1622. The sequence analysis by bioinformatics approaches revealed that HbO from M. xanthus (Mx-HbO) likely adopts a 2-on-2 alpha helical fold of the truncated globins. The gene coding for Mx-HbO was cloned and its expression in E. coli imparted reddish tinge to the cells. The spectral analysis confirmed it to be a functional globin. The expression of Mx-HbO in the heterologous host improved its growth, resulting in the attainment of higher cell density in culture. The transcript of Mx-hbO was induced threefold in the host cells when grown under low aeration condition as compared to the cells grown under high aeration condition. In M. xanthus, an obligate aerobe, where cell growth accompanies swarming, there is a higher density of cells in the middle of the swarm. Our results suggest that Mx-HbO is a functional globin and could facilitate the growth of cells facing oxygen deprivation, the condition prevailing in the middle of the swarm.

Glycosaminoglycans Induce Amyloid Self-Assembly of a Peptide Hormone by Concerted Secondary and Quaternary Conformational Transitions.

Amyloids are polypeptide supramolecular assemblies that have been historically associated with numerous pathologies. Nonetheless, recent studies have identified many amyloid structures that accomplish vital physiological functions. Interestingly, amyloid fibrils, either pathological or functional, have been reported to be consistently associated with other biomolecules such as RNA and glycosaminoglycans (GAGs). These linear polyanions, RNA and GAGs, have also demonstrated an inherent ability to accelerate and/or promote amyloid formation. GAGs, including heparan sulfate, are highly charged polysaccharides that may have essential roles in the storage of peptide hormones in the form of amyloids. In this study, we evaluated the ability of sulfated GAGs to promote the self-assembly of the peptide (neuro)hormone PACAP27 and investigated the secondary and quaternary conformational transitions associated with the amyloidogenic process. PACAP27 readily self-assembled into insoluble, α-helix-rich globular particulates in the presence of sulfated GAGs, which gradually condensed and disappeared as nontoxic β-sheet-rich amyloid fibrils were formed. By designing a PACAP27 derivative for which helical folding was hindered, we observed that the α-helix-to-β-sheet conformational transition within the amorphous particulates constitutes the rate-limiting step of primary nucleation events. The proposed mechanism of GAG-induced self-assembly within insoluble particulates appears to be fundamentally different from usual amyloidogenic systems, which commonly implicates the formation of soluble prefibrillar proteospecies. Overall, this study provides new insights into the mechanistic details involved in the formation of functional amyloids catalyzed by polyanions, such as the assembly of nuclear amyloid bodies and the storage of peptide hormones.

Disorder-to-helix conformational conversion of the human immunomodulatory peptide LL-37 induced by antiinflammatory drugs, food dyes and some metabolites.

The human antimicrobial and immunomodulatory peptide LL-37 is ubiquitously expressed and secreted by epithelial cells of mucosal surfaces including the gastrointestinal tract, the primary absorption site of orally administered drugs and food components. Besides antimicrobial properties, LL-37 also contributes to the pathophysiology of various diseases such as ulcerative colitis, Crohn's disease and cancer. Non-covalent association of antiinflammatory drugs, porphyrin pigments, bile salts and food dyes to the peptide was uncovered and evaluated by circular dichroism (CD) spectroscopy. These agents induce the disorder-to-order conformational transition of the natively unstructured LL-37 leading to its helical folding. Even in the presence of chloride ions, when LL-37 is partially folded, the inducers were able to rise the α-helix content. CD titration data indicated positive cooperativity between the ligand molecules accommodated to the peptide chain resulting in multimeric complexes with apparent dissociation constants ranged from 2 to 500 μM. Computational docking suggested the prominent role of the Lys8-Arg19 segment in the accommodation of small molecules, governed principally by salt bridges and H-bonding. Since pleiotropic biological functions of LL-37 are strongly conformation-dependent, it could be anticipated that folding inducer compounds may modulate its in vivo actions and also of related cationic peptides.

PS Membrane Asymmetry Influences the Folding and Insertion of a Transmembrane Helix

The plasma membrane (PM) has an asymmetric distribution of lipids between the inner and outer leaflets of its bilayer. A lipid of special interest in eukaryotic cells is the negatively charged phosphatidylserine (PS). In healthy cells, PS is actively sequestered to the inner leaflet of the PM but can redistribute to the outer leaflet when the cell is damaged or at the onset of apoptosis. However, membranes contain also proteins. Marginally hydrophobic membrane proteins contain acidic residues in their transmembrane sequence and can experience topological transitions after membrane association. The pH low insertion peptide (pHLIP), which undergoes a topological reorientation and inserts into the membrane at low pH, as its name implies, is a well-characterized model for studying these transitions. Although it is known that the inclusion of PS in symmetric vesicles affects the membrane insertion process of pHLIP by lowering the pH midpoint of insertion, it is unclear how PS asymmetry affects these topological transitions. Here, we studied pHLIP's topology using freely-floating asymmetric phosphatidylcholine (PC)/PS vesicles with PS enriched in the inner leaflet. We found that the protocol to create asymmetric vesicles had to be modified due to the inclusion of PS. We used Annexin V labeled with an Alexa 568 fluorophore as a new way of quantifying PS asymmetry. For pHLIP, membrane insertion was affected by the surface charge difference between bilayer leaflets because of the asymmetric distribution of charged lipids. We thus conclude that lipid asymmetry can have consequences for the behavior of membrane-associated proteins. A corollary is that model studies using symmetric bilayers to mimic the PM may fail to capture important details of protein-membrane interactions.

Conformational Sampling of the pH Low Insertion Peptide is Tuned by pH

The pH Low Insertion Peptide (pHLIP) is a 36 residue, pH-sensitive membrane-active peptide that is coiled both in solution and when bound to a membrane at physiological pH. Upon acidification, pHLIP folds into an α-helix and inserts unidirectionally into the bilayer. Thus, pHLIP could act as a potential vector for targeted drug delivery to or diagnostic imaging of tissues associated with acidosis, such as cancer or heart ischemia. However, without a detailed description of the biophysical interactions of pHLIP that lead to function, it will be difficult to design variants with increased targeting abilities. A recent study utilizing pH probes on cancerous cells has determined an increased radius of extracellular space is affected by acidosis [1], and therefore a peptide approaching the bilayer would experience an acidic environment before interacting with the membrane. With this in mind, we employed constant pH molecular dynamics (MD) simulations to determine potential changes in conformation of pHLIP as it transitions into the acidic extracellular space. Surprisingly, we found that a single amino acid residue, D31, has a noticeably higher pKa than what is expected from the experimentally determined pKa of aspartic acid residues. In addition, we observe formation of a stable helical fold from D14 to T18 at pH values below 4.5, suggesting pHLIP has the potential to sample conformations (i.e., α-helices) that facilitate partitioning into the membrane while still in solution. Our results provide a unique and essential perspective on the behavior of pHLIP as it transitions into acidic microenvironments such as the extracellular region of cancerous cells. [1] Luo (2018) Anal. Chem. 90:5803-5809.