Β-helix Protein Research Articles

Cotranslational Folding of a Pentarepeat β-Helix Protein

It is becoming increasingly clear that many proteins start to fold cotranslationally before the entire polypeptide chain has been synthesized on the ribosome. One class of proteins that a priori would seem particularly prone to cotranslational folding is repeat proteins, that is, proteins that are built from an array of nearly identical sequence repeats. However, while the folding of repeat proteins has been studied extensively in vitro with purified proteins, only a handful of studies have addressed the issue of cotranslational folding of repeat proteins. Here, we have determined the structure and studied the cotranslational folding of a β-helix pentarepeat protein from the human pathogen Clostridium botulinum—a homolog of the fluoroquinolone resistance protein MfpA—using an assay in which the SecM translational arrest peptide serves as a force sensor to detect folding events. We find that cotranslational folding of a segment corresponding to the first four of the eight β-helix coils in the protein produces enough force to release ribosome stalling and that folding starts when this unit is ~35 residues away from the P-site, near the distal end of the ribosome exit tunnel. An additional folding transition is seen when the whole PENT moiety emerges from the exit tunnel. The early cotranslational formation of a folded unit may be important to avoid misfolding events in vivo and may reflect the minimal size of a stable β-helix since it is structurally homologous to the smallest known β-helix protein, a four-coil protein that is stable in solution.

The structure of rice weevil pectin methylesterase.

Rice weevils (Sitophilus oryzae) use a pectin methylesterase (EC 3.1.1.11), along with other enzymes, to digest cell walls in cereal grains. The enzyme is a right-handed β-helix protein, but is circularly permuted relative to plant and bacterial pectin methylesterases, as shown by the crystal structure determination reported here. This is the first structure of an animal pectin methylesterase. Diffraction data were collected to 1.8 Å resolution some time ago for this crystal form, but structure solution required the use of molecular-replacement techniques that have been developed and similar structures that have been deposited in the last 15 years. Comparison of the structure of the rice weevil pectin methylesterase with that from Dickeya dandantii (formerly Erwinia chrysanthemi) indicates that the reaction mechanisms are the same for the insect, plant and bacterial pectin methylesterases. The similarity of the structure of the rice weevil enzyme to the Escherichia coli lipoprotein YbhC suggests that the evolutionary origin of the rice weevil enzyme was a bacterial lipoprotein, the gene for which was transferred to a primitive ancestor of modern weevils and other Curculionidae. Structural comparison of the rice weevil pectin methylesterase with plant and bacterial enzymes demonstrates that the rice weevil protein is circularly permuted relative to the plant and bacterial molecules.

A new family of β-helix proteins with similarities to the polysaccharide lyases.

Microorganisms that degrade biomass produce diverse assortments of carbohydrate-active enzymes and binding modules. Despite tremendous advances in the genomic sequencing of these organisms, many genes do not have an ascribed function owing to low sequence identity to genes that have been annotated. Consequently, biochemical and structural characterization of genes with unknown function is required to complement the rapidly growing pool of genomic sequencing data. A protein with previously unknown function (Cthe_2159) was recently isolated in a genome-wide screen using phage display to identify cellulose-binding protein domains from the biomass-degrading bacterium Clostridium thermocellum. Here, the crystal structure of Cthe_2159 is presented and it is shown that it is a unique right-handed parallel β-helix protein. Despite very low sequence identity to known β-helix or carbohydrate-active proteins, Cthe_2159 displays structural features that are very similar to those of polysaccharide lyase (PL) families 1, 3, 6 and 9. Cthe_2159 is conserved across bacteria and some archaea and is a member of the domain of unknown function family DUF4353. This suggests that Cthe_2159 is the first representative of a previously unknown family of cellulose and/or acid-sugar binding β-helix proteins that share structural similarities with PLs. Importantly, these results demonstrate how functional annotation by biochemical and structural analysis remains a critical tool in the characterization of new gene products.

Structure and properties of the C-terminal β-helical domain of VgrG protein from Escherichia coli O157

The bacterial Type 6 secretion system (T6SS) translocates protein toxins (also called effectors) from the cytosol of a T6SS-carrying cell to a target cell by a syringe-like supramolecular complex resembling a contractile tail of bacteriophages. Valine-glycine repeat protein G (VgrG) proteins, which are the homologues of the gp27-gp5 (gene product) cell puncturing complex of bacteriophage T4, are considered to be located at the attacking tip of the bacterial T6SS apparatus. Here, we over-expressed six VgrG proteins from pathogenic Escherichia coli O157 and CFT073 strains. Purified VgrG1 of E. coli O157 and c3393 of E. coli CFT073 form trimer in solution and are rich in β-structure. We also solved the crystal structure of a trypsin-resistant C-terminal fragment of E. coli O157 VgrG1 (VgrG1C(G561)) at 1.95 Å resolution. VgrG1C(G561) forms a three-stranded antiparallel β-helix which is structurally similar to the β-helix domain of the central spike protein (gp138) of phi92 phage, indicating a possible evolutional relationship. Comparison of four different three-stranded β-helix proteins shows how their amino acid composition determines the protein fold.

The K5 Lyase KflA Combines a Viral Tail Spike Structure with a Bacterial Polysaccharide Lyase Mechanism

K5 lyase A (KflA) is a tail spike protein (TSP) encoded by a K5A coliphage, which cleaves K5 capsular polysaccharide, a glycosaminoglycan with the repeat unit [-4)-betaGlcA-(1,4)- alphaGlcNAc(1-], displayed on the surface of Escherichia coli K5 strains. The crystal structure of KflA reveals a trimeric arrangement, with each monomer containing a right-handed, single-stranded parallel beta-helix domain. Stable trimer formation by the intertwining of strands in the C-terminal domain, followed by proteolytic maturation, is likely to be catalyzed by an autochaperone as described for K1F endosialidase. The structure of KflA represents the first bacteriophage tail spike protein combining polysaccharide lyase activity with a single-stranded parallel beta-helix fold. We propose a catalytic site and mechanism representing convergence with the syn-beta-elimination site of heparinase II from Pedobacter heparinus.

Invivo folding efficiencies for mutants of the P22 tailspike β-helix protein correlate with predicted stability changes

Parallel β-helices are among the simplest repetitive structural elements in proteins. The folding behavior of β-helix proteins has been studied intensively, also to gain insight on the formation of amyloid fibrils, which share the parallel β-helix as a central structural motif. An important system for investigating β-helix folding is the tailspike protein from the Salmonella bacteriophage P22. The central domain of this protein is a right-handed parallel β-helix with 13 windings. Extensive mutational analyses of the P22 tailspike protein have revealed two main phenotypes: temperature-sensitive-folding (tsf) mutations that reduce the folding efficiency at elevated temperatures, and global suppressor (su) mutations that increase the tailspike folding efficiency. A central question is whether these phenotypes can be understood from changes in the protein stability induced by the mutations. Experimental determination of the protein stability is complicated by the nearly irreversible trimerization of the folded tailspike protein. Here, we present calculations of stability changes with the program FoldX, focusing on a recently published extensive data set of 145 singe-residue alanine mutants. We find that the calculated stability changes are correlated with the experimentally measured in vivo folding efficiencies. In addition, we determine the free-energy landscape of the P22 tailspike protein in a nucleation–propagation model to explore the folding mechanism of this protein, and obtain a processive folding route on which the protein nucleates in the N-terminal region of the helix.

Large deformation and fracture mechanics of a beta-helical protein nanotube: Atomistic and continuum modeling

We report molecular dynamics (MD) simulation and atomistically informed continuum modeling of the nanomechanics of the β-helix protein motif, a key building block of amyloids associated with Alzheimer’s, Huntington’s and prion diseases, forming a nanotube-like protein structure. We find that the β-helix structure is extremely extensible and can sustain tensile deformation up to 800% engineering strain without rupture of the covalently bonded protein backbone. Our atomistic simulation results reveal that the instantaneous strength of the tube is proportional to the rate of H-bond rupture, providing a link between the dynamics of hydrogen bond rupture and the mechanical signature of the protein structure. This finding proves that concurrent as opposed to sequential breaking of bonds leads to higher mechanical resistance, corroborating earlier results found in studies of β-sheet protein domains. Inspired by the beta-helical domain of the needle-like cell puncture device of bacteriophage T4, we carry out compressive loading simulations of the β-helix, and show that this protein motif can withstand extremely large compressive loads, far exceeding the tensile strength. Different mechanical deformation modes under compressive loading are summarized in a deformation map. Our findings illustrate the potential of the β-helix protein motif as an inspiration for nano-scale materials applications, ranging from stiff nanotubes to self-assembling peptide based fibers inspired by amyloids.

Parallel β-Helix Proteins Required for Accurate Capsule Polysaccharide Synthesis and Virulence in the Yeast Cryptococcus neoformans

The principal capsular polysaccharide of the opportunistic fungal pathogen Cryptococcus neoformans consists of an alpha-1,3-linked mannose backbone decorated with a repeating pattern of glucuronyl and xylosyl side groups. This structure is critical for virulence, yet little is known about how the polymer, called glucuronoxylomannan (GXM), is faithfully synthesized and assembled. We have generated deletions in two genes encoding predicted parallel beta-helix repeat proteins, which we have designated PBX1 and PBX2. Deletion of either gene results in a dry-colony morphology, clumpy cells, and decreased capsule integrity. Two-dimensional nuclear magnetic resonance spectroscopy of purified GXM from the mutants indicated that both the wild-type GXM structure and novel, aberrant linkages were present. Carbohydrate composition and linkage analysis determined that these aberrant structures are correlated with the incorporation of terminal glucose residues that are not found in wild-type capsule polysaccharide. We conclude that Pbx1 and Pbx2 are required for the fidelity of GXM synthesis and may be involved in editing incorrectly added glucose residues. PBX1 and PBX2 knockout mutants showed severely attenuated virulence in a murine inhalation model of cryptococcosis. Unlike acapsular strains, these mutant strains induced delayed symptoms of cryptococcosis, though the infected animals eventually contained the infection and recovered.

Use of Constrained Synthetic Amino Acids in β-Helix Proteins for Conformational Control

A highly constrained amino acid has been introduced in the turn region of a beta-helix to increase the conformational stability of the native fold for nanotechnological purposes. The influence of this specific amino acid replacement in the final organization of beta-helix motifs has been evaluated by combining ab initio first-principles calculations on model systems and molecular dynamics simulations of entire peptide segments. The former methodology, which has been applied to a sequence containing three amino acids, has been used to develop adjusted templates. Calculations indicated that 1-amino-2,2-diphenylcyclopropanecarboxylic acid, a constrained cyclopropane analogue of phenylalanine, exhibits a strong tendency to form and promote folded conformations. On the other hand, molecular dynamics simulations are employed to probe the ability of such a synthetic amino acid to enhance the conformational stability of the beta-helix motif, which is the first requirement for further protein nanoengineering. A highly regular segment from a naturally occurring beta-helix protein was selected as a potential nanoconstruct module. Simulations of wild type and mutated segments revealed that the ability of the phenylalanine analogue to nucleate turn conformations enhances the conformational stability of the beta-helix motif in isolated peptide segments.

Structural and Spectroscopic Studies Shed Light on the Mechanism of Oxalate Oxidase

Oxalate oxidase (EC 1.2.3.4) catalyzes the conversion of oxalate and dioxygen to hydrogen peroxide and carbon dioxide. In this study, glycolate was used as a structural analogue of oxalate to investigate substrate binding in the crystalline enzyme. The observed monodentate binding of glycolate to the active site manganese ion of oxalate oxidase is consistent with a mechanism involving C-C bond cleavage driven by superoxide anion attack on a monodentate coordinated substrate. In this mechanism, the metal serves two functions: to organize the substrates (oxalate and dioxygen) and to transiently reduce dioxygen. The observed structure further implies important roles for specific active site residues (two asparagines and one glutamine) in correctly orientating the substrates and reaction intermediates for catalysis. Combined spectroscopic, biochemical, and structural analyses of mutants confirms the importance of the asparagine residues in organizing a functional active site complex.

2P557 Artificial three strand β-helix protein as a nano tube(53. Bioengineering,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

2P557 Artificial three strand β-helix protein as a nano tube(53. Bioengineering,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Energy landscape of amyloidogenic peptide oligomerization by parallel-tempering molecular dynamics simulation: Significant role of Asn ladder

Recent evidence suggests that amyloidogenic oligomers may be the toxic species in cell cultures. Thus, it is crucial to understand their structure and oligomerization mechanism in atomistic detail. By employing tens of fast central processing units and an advanced phase-space sampling algorithm, parallel-tempering molecular dynamics, we have explored the energy landscape of amyloidogenic peptide oligomerization in explicit water. A pentapeptide, DFNKF, derived from human calcitonin and its mutant, DFAKF, was simulated with a total simulation time of approximately 500 ns. The detailed oligomerization process of a DFNKF parallel beta-sheet formation at 300 K has been characterized. The assembly of a parallel beta-sheet from the amorphous state mainly occurs via a "bottleneck" channel where the interstrand Asn-Asn stacking is the major interaction. The interactions of Asn-Asn stacking include both backbone and side-chain hydrogen bonds. The Asn-Asn interactions work like "glue" by sticking the DFNKF strands together and assist the "on-pathway" oligomerization. The Asn-Asn stacking observed here is similar to the Asn ladder commonly found in globular beta-helix proteins. A control run shows that when Asn is mutated to Ala, the stability and population of the DFAKF parallel beta-sheet is decreased. Furthermore, our in vitro mutagenesis experiments show that the ability of DFAKF peptides to form amyloid fibrils is significantly reduced, in agreement with the simulations. Knowledge of the energy landscape of oligomerization may provide hints for rational drug design, preventing amyloid-associated diseases.

Natural beta-sheet proteins use negative design to avoid edge-to-edge aggregation.

The fact that natural beta-sheet proteins are usually soluble but that fragments or designs of beta structure usually aggregate suggests that natural beta proteins must somehow be designed to avoid this problem. Regular beta-sheet edges are dangerous, because they are already in the right conformation to interact with any other beta strand they encounter. We surveyed edge strands in a large sample of all-beta proteins to tabulate features that could protect against further beta-sheet interactions. beta-barrels, of course, avoid edges altogether by continuous H-bonding around the barrel cylinder. Parallel beta-helix proteins protect their beta-sheet ends by covering them with loops of other structure. beta-propeller and single-sheet proteins use a combination of beta-bulges, prolines, strategically placed charges, very short edge strands, and loop coverage. beta-sandwich proteins favor placing an inward-pointing charged side chain on one of the edge strands where it would be buried by dimerization; they also use bulges, prolines, and other mechanisms. One recent beta-hairpin design has a constrained twist too great for accommodation into a larger beta-sheet, whereas some beta-sheet edges are protected by the bend and reverse twist produced by an Lbeta glycine. All free edge strands were seen to be protected, usually by several redundant mechanisms. In contrast, edge strands that natively form beta H-bonded dimers or rings have long, regular stretches without such protection. These results are relevant to understanding how proteins may assemble into beta-sheet amyloid fibers, and they are especially applicable to the de novo design of beta structure. Many edge-protection strategies used by natural proteins are beyond our current abilities to constrain by design, but one possibility stands out as especially useful: a single charged side chain near the middle of what would ordinarily be the hydrophobic side of the edge beta strand. This minimal negative-design strategy changes only one residue, requires no backbone distortion, and is easy to design. The accompanying paper [Wang, W. & Hecht, M. H. (2002) Proc. Natl. Acad. Sci. USA 99, 2760-2765] makes use of the inward-pointing charge strategy with great success, turning highly aggregated beta-sandwich designs into soluble monomers.

The stability, structural organization, and denaturation of pectate lyase C, a parallel beta-helix protein.

Pectate lyase C (pelC) was the first protein in which the parallel beta-helix structure was recognized. The unique features of parallel beta-helix-containing proteins-a relatively simple topology and unusual interactions among side chains-make pelC an interesting protein to study with respect to protein folding. In this paper, we report studies of the unfolding equilibrium of pelC. PelC is unfolded reversibly by gdn-HCl at pH 7 and 5, as monitored by far- and near-UV CD and fluorescence. The coincidence of these spectroscopically detected transitions is consistent with a two-state transition at pH 7, but the three probes are not coincident at pH 5. No evidence was found for a loosely folded intermediate in the transition region at pH 5. At pH 7, the for unfolding is 12.2 kcal/mol, with the midpoint of the transition at 0.99 M gdn-HCl and m = 12.3 kcal/(mol.M). Thus, pelC is unusually stable and has an m value that is much larger than for typical globular proteins. Thermal denaturation of pelC has been studied by differential scanning calorimetry (DSC) and by CD. Although thermal denaturation is not reversible, valid thermodynamic data can be obtained for the unfolding transition. DeltaH(van't Hoff)/DeltaH(cal) is less than 1 for pHs between 5 and 8, with a maximum value of 0.91 at pH 7 decreasing to 0.85 at pH 8 and to 0.68 at pH 5. At all pHs studied, the excess heat capacity can be deconvoluted into two components corresponding to two-state transitions that are nearly coincident at pH 7, but deviate more at higher and lower pH. Thus, pelC appears to consist of two domains that interact strongly and unfold in a cooperative fashion at pH 7, but the cooperativity decreases at higher and lower pH. The crystal structure of pelC shows no obvious domain structure, however.

Do Parallel β-Helix Proteins Have a Unique Fourier Transform Infrared Spectrum?

Several polypeptides have been found to adopt an unusual domain structure known as the parallel β-helix. These domains are characterized by parallel β-strands, three of which form a single parallel β-helix coil, and lead to long, extended β-sheets. We have used ATR-FTIR (attenuated total reflectance-fourier transform infrared spectroscopy) to analyze the secondary structure of representative examples of this class of protein. Because the three-dimensional structures of parallel β-helix proteins are unique, we initiated this study to determine if there was a corresponding unique FTIR signal associated with the parallel β-helix conformation. Analysis of the amide I region, emanating from the carbonyl stretch vibration, reveals a strong absorbance band at 1638 cm −1 in each of the parallel β-helix proteins. This band is assigned to the parallel β-sheet structure. However, components at this frequency are also commonly observed for β-sheets in many classes of globular proteins. Thus we conclude that there is no unique infrared signature for parallel β-helix structure. Additional contributions in the 1638 cm −1 region, and at lower frequencies, were ascribed to hydrogen bonding between the coils in the loop/turn regions and amide side-chain interactions, respectively. A 13-residue peptide that forms fibrils and has been proposed to form β-helical structure was also examined, and its FTIR spectrum was compared to that of the parallel β-helix proteins.

Structure and Evolution of Parallel β-Helix Proteins

Three bacterial pectate lyases, a pectin lyase fromAspergillus niger,the structures of rhamnogalacturonase A fromAspergillus aculeatus,RGase A, and the P22-phage tailspike protein, TSP, display the right-handed parallel β-helix architecture first seen in pectate lyase. The lyases have 7 complete coils while RGase A and TSP have 11 and 12, respectively. Each coil contains three β-strands and three turn regions named PB1, T1, PB2, T2, PB3, and T3 in their order of occurrence. The lyases have homologous sequences but RGase A and TSP do not show obvious sequence homology either to the lyases or to each other. However, the structural similarities between all these molecules are so extensive that divergence from a common ancestor is much more probable than convergence to the same fold. The region PB2-T2-PB3 is the best conserved region in the lyases and shows the clearest structural similarity. Not only is the pleating and the direction of the hydrogen bonding in the sheets conserved, but so is the unusual αL-conformation turn between the two sheets. However, the overall shape, the position of long loops, a conserved α-helix that covers the amino-terminal end of the parallel β-helix and stacks of residues in αR-conformation at the start of PB1 all suggest a common ancestor. The functional similarity, that the enzymes all bind α-galactose containing polymers at an equivalent site involving PB1 and its two flanking turn regions, further supports divergent evolution. We suggest that the stacking of the coils and the unusual near perpendicular junction of PB2 and PB3 make the parallel β-helix fold especially likely to maintain similar main chain conformations during divergent evolution even after all vestige of similarity in primary structure has vanished.

X-ray diffraction and far-UV CD studies of filaments formed by a leucine-rich repeat peptide: structural similarity to the amyloid fibrils of prions and Alzheimer's disease β-protein

The development of neuro-degenerative diseases often involves amyloidosis, that is the formation of polymeric fibrillar structures from normal cellular proteins or peptides. For example, in Alzheimer's disease, a 42 amino acid peptide processed from the amyloid precursor protein forms filaments with a β-sheet structure. Because of this, the structure and dynamics of polymeric peptide filaments is of considerable interest. We showed previously that a 23 amino acid peptide constituting a single leucine-rich repeat (LRRN) polymerises spontaneously in solution to form long filaments of a β-sheet structure, a property similar to that of Alzheimer's β-amyloid and prion peptides. Here we report that a variant of LRRN in which a highly conserved asparagine residue is replaced by aspartic acid does not form either filaments or β structure. By contrast, a variant which replaces this asparagine residue with glutamine forms filaments ultrastructurally indistinguishable from those of LRRN. Electron micrographs of LRRN filaments show that many consist of two interleaved strands which appear to have a ribbon-like morphology. X-ray diffraction patterns from oriented LRRN fibres reveal that they are composed of long β-sheet arrays, with the interstrand hydrogen bonding parallel to the filament axis. This `cross-β' structure is similar to that adopted by β-amyloid and prion derived fibres. Taken together, these results indicate that the LRR filaments are stabilised by inter- or intra-strand hydrogen bonded interactions comparable to the asparagine ladders of β-helix proteins or the `glutamine zippers' of poly-glutamine peptides. We propose that similar stabilising interactions may underlie a number of characterised predispositions to neuro-degenerative diseases that are caused by mutations to amide residues. Our finding that amyloid-like filaments can form from a peptide motif not at present correlated with degenerative disease suggests that a propensity for β-filament formation is a common feature of protein sub-domains.