Β-sheet Folding Research Articles

Linking the Sequence, Anti-Tumor Function, and Shared Structural Features of Class Ib Hydrophobins

Hydrophobins are low molecular weight (5-20 kDa) self-assembling proteins secreted by fungi that accumulate at hydrophobic-hydrophilic interfaces and are extremely surface-active. Hydrophobins may undergo structural rearrangement and oligomerize to form rodlets, which are an insoluble functional amyloid that coats fungal spores to act as a water repellent, facilitate dispersal into the air, and prevent immune recognition. Due to their biochemical properties hydrophobins are a target a wide range of potential commercial applications. Hydrohphobins might also find use as a cancer treatment as they have also been shown to have anti-tumor properties, although the precise mechanism of this action is unclear. To better understand which sequence characteristics determine hydrophobin properties, we are characterizing the structure and properties of class IB hydrophobins from various fungal sources. Target proteins from Serpula lacrymans (SL1), Wallemia ichthyophaga (WI1), and Phanerochaete carnosa (PC1) were chosen for study due to their different sequence composition. We expressed uniformly 13C/15N-labeled protein in E. coli and then purified it to homogeneity using Ni2+ affinity and RP-HPLC and determined the high-resolution structure using NMR spectroscopy. We discovered that each hydrophobin contains shared structural features despite their dissimilar sequence compositions. The core feature is a four strand anti-parallel β-sheet that is connected by three loop sequences (L1-L3). In all hydrophobins the β-sheet folds to form a β-barrel-like structure, however the first loop is disordered in WI1 while it is an a-helix in SL1 and PC1. Spectroscopic amyloid formation assays indicate that these hydrophobins have differing propensities to form rodlets and are using AFM to characterize rodlet morphology. We are also examining which sequence and structure characteristics of hydrophobins influence their anti-tumour activity. Overall, this work will provide a fundamental correlation between the structure, function, and anti-tumour properties of hydrophobins.

Clarifying the Copper Coordination Environment in a de Novo Designed Red Copper Protein.

Cupredoxins are copper-dependent electron-transfer proteins that can be categorized as blue, purple, green, and red depending on the spectroscopic properties of the Cu(II) bound forms. Interestingly, despite significantly different first coordination spheres and nuclearity, all cupredoxins share a common Greek Key β-sheet fold. We have previously reported the design of a red copper protein within a completely distinct three-helical bundle protein, α3DChC2. (1) While this design demonstrated that a β-barrel fold was not requisite to recapitulate the properties of a native cupredoxin center, the parent peptide α3D was not sufficiently stable to allow further study through additional mutations. Here we present the design of an elongated protein GRANDα3D (GRα3D) with Δ Gu = -11.4 kcal/mol compared to the original design's -5.1 kcal/mol. Diffraction quality crystals were grown of GRα3D (a first for an α3D peptide) and solved to a resolution of 1.34 Å. Examination of this structure suggested that Glu41 might interact with the Cu in our previously reported red copper protein. The previous bis(histidine)(cysteine) site (GRα3DChC2) was designed into this new scaffold and a series of variant constructs were made to explore this hypothesis. Mutation studies around Glu41 not only prove the proposed interaction, but also enabled tuning of the constructs' hyperfine coupling constant from 160 to 127 × 10-4 cm-1. X-ray absorption spectroscopy analysis is consistent with these hyperfine coupling differences being the result of variant 4p mixing related to coordination geometry changes. These studies not only prove that an Glu41-Cu interaction leads to the α3DChC2 construct's red copper protein like spectral properties, but also exemplify the exact control one can have in a de novo construct to tune the properties of an electron-transfer Cu site.

Triintsin, a human pathogenic fungus-derived defensin with broad-spectrum antimicrobial activity

Since there is a symbiotic and competitive relationship between microorganisms in the same ecological niche, fungal defensins have been found to be important resources for antimicrobial peptides. Here, a fungal defensin, triintsin, was characterized in a clinical isolate of Trichophyton interdigitale from a patient with onychomycosis. The comparison of its genomic and mRNA sequences showed the gene organization and structure of three coding exons separated by two introns. The precursor peptide of triintsin contained 85 amino acid residues, which were composed of three parts including an N-terminal signal domain of 21 residues, a pro-peptide of 47 residues that ended at lysine-arginine and a mature peptide of 38 residues at the C-terminus. The 3D-structure established by homology modeling revealed that triintsin presented a representative typical cysteine-stabilized α-helical and β-sheet fold. The reductive linear peptide of triintsin was obtained by chemical synthesis. After cyclization to form three pairs of disulfide bonds, the oxidative-type peptide displayed broad-spectrum antimicrobial activity against both gram-positive and gram-negative bacteria but also showed anti-fungal activity. Moreover, triintsin can effectively inhibit the growth of clinical strains. Altogether, the peptide is a human pathogenic fungus-derived defensin with broad-spectrum antimicrobial activity.

Hydrogen Bond Surrogate Stabilization of β-Hairpins.

Peptide secondary and tertiary structure motifs frequently serve as inspiration for the development of protein-protein interaction (PPI) inhibitors. While a wide variety of strategies have been used to stabilize or imitate α-helices, similar strategies for β-sheet stabilization are more limited. Synthetic scaffolds that stabilize reverse turns and cross-strand interactions have provided important insights into β-sheet stability and folding. However, these templates occupy regions of the β-sheet that might impact the β-sheet's ability to bind at a PPI interface. Here, we present the hydrogen bond surrogate (HBS) approach for stabilization of β-hairpin peptides. The HBS linkage replaces a cross-strand hydrogen bond with a covalent linkage, conferring significant conformational and proteolytic resistance. Importantly, this approach introduces the stabilizing linkage in the buried β-sheet interior, retains all side chains for further functionalization, and allows efficient solid-phase macrocyclization. We anticipate that HBS stabilization of PPI β-sheets will enhance the development of β-sheet PPI inhibitors and expand the repertoire of druggable PPIs.

Protein Folding Cooperativity and Thermodynamic Barriers of the Simplest β-Sheet Fold: A Survey of WW Domains.

Theory and experiments have shown that microsecond folding proteins exhibit characteristic thermodynamic properties that reflect the limited cooperativity of folding over marginal barriers (downhill folding). Those studies have mostly focused on proteins with large α-helical contents and small size, which tend to be the fastest folders. A key open question is whether such properties are also present in the fastest all-β proteins. We address this issue by investigating the unfolding thermodynamics of a collection of WW domains as representatives of the simplest β-sheet fold. WW domains are small microsecond folders, although they do not fold as fast as their α-helical counterparts. In previous work on the NEDD4-WW4 domain, we reported deviations from two-state thermodynamics that were less apparent and thus suggestive of an incipient downhill scenario. Here we investigate the unfolding thermodynamics of four other WW domains (NEDD4-WW3, YAP65-WW1(L30K), FBP11-WW1, and FBP11-WW2) by performing all of the thermodynamic tests for downhill folding that have been previously developed on α-helical proteins. This set of five WW domains shares low sequence identity and include examples from two specificity classes, thus providing a comprehensive survey. Thermodynamic analysis of the four new WW domains consistently reveals all of the properties of downhill folding equilibria, which are in all cases more marked than what we found before in NEDD4-WW4. Our results show that fast-folding all-β proteins do share limited cooperativity and gradual unfolding thermodynamics with fast α-helical proteins and suggest that the free energy barrier to folding of natural proteins is mostly determined by size and fold topology and much less by the specific amino acid sequence.

Crystal structure of human Acinus RNA recognition motif domain.

Acinus is an abundant nuclear protein involved in apoptosis and splicing. It has been implicated in inducing apoptotic chromatin condensation and DNA fragmentation during programmed cell death. Acinus undergoes activation by proteolytic cleavage that produces a truncated p17 form that comprises only the RNA recognition motif (RRM) domain. We have determined the crystal structure of the human Acinus RRM domain (AcRRM) at 1.65 Å resolution. It shows a classical four-stranded antiparallel β-sheet fold with two flanking α-helices and an additional, non-classical α-helix at the C-terminus, which harbors the caspase-3 target sequence that is cleaved during Acinus activation. In the structure, the C-terminal α-helix partially occludes the potential ligand binding surface of the β-sheet and hypothetically shields it from non-sequence specific interactions with RNA. Based on the comparison with other RRM-RNA complex structures, it is likely that the C-terminal α-helix changes its conformation with respect to the RRM core in order to enable RNA binding by Acinus.

The Chlamydia trachomatis PmpD adhesin forms higher order structures through disulphide-mediated covalent interactions.

Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial pathogen, and the leading cause of infectious blindness worldwide. We have recently shown that immunization with the highly conserved antigenic passenger domain of recombinant Ct polymorphic membrane protein D (rPmpD) is protective in the mouse model of Ct genital tract infection, and previously, that ocular anti-rPmpD antibodies are elicited following vaccination. However, the mechanisms governing the assembly and structure-function relationship of PmpD are unknown. Here, we provide a biophysical analysis of this immunogenic 65 kDa passenger domain fragment of PmpD. Using differential cysteine labeling coupled with LC-MS/MS analysis, we show that widespread intra- and intermolecular disulphide interactions play important roles in the preservation of native monomeric secondary structure and the formation of higher-order oligomers. While it has been proposed that FxxN and GGA(I, L,V) repeat motifs in the Pmp21 ortholog in Chlamydia pneumoniae mediate self-interaction, no such role has previously been identified for cysteine residues in chlamydial Pmps. Further characterisation reveals that oligomeric proteoforms and rPmpD monomers adopt β–sheet folds, consistent with previously described Gram-negative bacterial type V secretion systems (T5SSs). We also highlight adhesin-like properties of rPmpD, showing that both soluble rPmpD and anti-rPmpD serum from immunized mice abrogate binding of rPmpD-coated beads to mammalian cells in a dose-dependent fashion. Hence, our study provides further evidence that chlamydial Pmps may function as adhesins, while elucidating yet another important mechanism of self-association of bacterial T5SS virulence factors that may be unique to the Chlamydiaceae.

Structure and Dynamics of Membrane Proteins from Solid-State NMR.

Solid-state nuclear magnetic resonance (SSNMR) spectroscopy elucidates membrane protein structures and dynamics in atomic detail to yield mechanistic insights. By interrogating membrane proteins in phospholipid bilayers that closely resemble biological membranes, SSNMR spectroscopists have revealed ion conduction mechanisms, substrate transport dynamics, and oligomeric interfaces of seven-transmembrane helix proteins. Research has also identified conformational plasticity underlying virus-cell membrane fusions by complex protein machineries, and β-sheet folding and assembly by amyloidogenic proteins bound to lipid membranes. These studies collectively show that membrane proteins exhibit extensive structural plasticity to carry out their functions. Because of the inherent dependence of NMR frequencies on molecular orientations and the sensitivity of NMR frequencies to dynamical processes on timescales from nanoseconds to seconds, SSNMR spectroscopy is ideally suited to elucidate such structural plasticity, local and global conformational dynamics, protein-lipid and protein-ligand interactions, and protonation states of polar residues. New sensitivity-enhancement techniques, resolution enhancement by ultrahigh magnetic fields, and the advent of 3D and 4D correlation NMR techniques are increasingly aiding these mechanistically important structural studies.

A relay FRET event in a designed trichromophoric pentapeptide containing an o-,m-aromatic-amino acid scaffold.

The concept of a relay FRET event is established in a designed trichromophoric pentapeptide containing an o-,m-aromatic amino acid scaffold in the backbone as a novel β-turn mimetic β-sheet folding nucleator. This system would find application in studying fundamental processes involving interbiomolecular interactions in chemical biology.

Invasive fungi-derived defensins kill drug-resistant bacterial pathogens

Fungi-derived defensins are a class of antimicrobial peptides with therapeutic potential due to their high antibacterial efficacy and low toxicity. Based on the genomic strategy, we have identified 68 fungal defensin-like peptides (fDLPs) in five new genera, including Trichosporon, Apophysomyces, Lichtheimia, Beauveria and Scedosporium and characterized a new synthetic defensin (scedosporisin) from an invasive fungus. It was active against Gram-positive bacteria but not active against negative bacteria. Importantly, it killed several clinical resistant isolates such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci at low molecular concentrations. Scedosporisin showed low hemolysis and cytotoxicity and high serum stability. The killing kinetics of scedosporisin-2 against a clinical isolate of MRSA showed that it killed the bacteria more rapidly than that of vancomycin. Homology modeling analysis show that scedosporisin adopted a typical cysteine stabilized α-helical and β-sheet fold with a local hydrophobic patch. Scedosporisin significantly improved the survival rate of mice in the peritonitis model. This work has greatly expanded the library of fDLPs, and successfully selected leading molecules for antimicrobial drug reserves.

Synthesis and β-sheet propensity of constrained N-amino peptides

The stabilization of β-sheet secondary structure through peptide backbone modification represents an attractive approach to protein mimicry. Here, we present strategies toward stable β-hairpin folds based on peptide strand N-amination. Novel pyrazolidinone and tetrahydropyridazinone dipeptide constraints were introduced via on-resin Mitsunobu cyclization between α-hydrazino acid residues and a serine or homoserine side chain. Acyclic and cyclic N-amino peptide building blocks were then evaluated for their effect on β-hairpin stability in water using a GB1-derived model system. Our results demonstrate the strong β-sheet stabilizing effect of the peptide N-amino substituent, and provide useful insights into the impact of covalent dipeptide constraint on β-sheet folding.

Designed Heme-Cage β-Sheet Miniproteins.

The structure and function of naturally occurring proteins are governed by a large number of amino acids (≥100). The design of miniature proteins with desired structures and functions not only substantiates our knowledge about proteins but can also contribute to the development of novel applications. Excellent progress has been made towards the design of helical proteins with diverse functions. However, the development of functional β-sheet proteins remains challenging. Herein, we describe the construction and characterization of four-stranded β-sheet miniproteins made up of about 19 amino acids that bind heme inside a hydrophobic binding pocket or "heme cage" by bis-histidine coordination in an aqueous environment. The designed miniproteins bound to heme with high affinity comparable to that of native heme proteins. Atomic-resolution structures confirmed the presence of a four-stranded β-sheet fold. The heme-protein complexes also exhibited high stability against thermal and chaotrope-induced unfolding.

Short Synthetic β-Sheet Antimicrobial Peptides for the Treatment of Multidrug-Resistant Pseudomonas aeruginosa Burn Wound Infections.

Pseudomonas aeruginosa is often implicated in burn wound infections; its inherent drug resistance often renders these infections extremely challenging to treat. This is further compounded by the problem of emerging drug resistance and the dearth of novel antimicrobial drug discovery in recent years. In the perennial search for effective antimicrobial compounds, the authors identify short synthetic β-sheet folding peptides, IRIKIRIK (IK8L), IRIkIrIK (IK8-2D), and irikirik (IK8D) as prime candidates owing to their high potency against Gram-negative bacteria. In this study, the peptides are first assayed against 20 clinically isolated multidrug-resistant P. aeruginosa strains in comparison with the conventional antibiotics imipenem and ceftazidime, and IK8L is demonstrated to be the most effective. IK8L also exhibits superior antibacterial killing kinetics compared to imipenem and ceftazidime. From transmission electron microscopy, confocal microscopy, and protein release analyses, IK8L shows membrane-lytic antimicrobial mechanism. Repeated use of IK8L does not induce drug resistance, while the bacteria develop resistance against the antibiotics after several times of treatment at sublethal doses. Analysis of mouse blood serum chemistry reveals that peptide does not induce systemic toxicity. The potential utility of IK8L in the in vivo treatment of P. aeruginosa-infected burn wounds is further demonstrated in a mouse model.

Peptide N-Amination Supports β-Sheet Conformations.

The conformational heterogeneity of backbone N-substituted peptides limits their ability to adopt stable secondary structures. Herein, we describe a practical synthesis of backbone aminated peptides that readily adopt β-sheet folds. Data derived from model N-amino peptides suggest that extended conformations are stabilized through cooperative steric, electrostatic, and hydrogen-bonding interactions.

Delineating residues for haemolytic activities of snake venom cardiotoxin 1 from Naja naja as probed by molecular dynamics simulations and in vitro validations

Cardiotoxins (CTXs) are single polypeptide chain consisting of 59–62 amino acids with four disulfide bridges and globular proteins of simple β-sheet folds. The CTXs are one of principal toxic components causing haemolysis and damaging various cells and belong to three-finger toxin (TFT) superfamily of snake venoms. However, there is no natural or synthetic small molecular inhibitor to the protein toxins to date. In the present study, modes of interaction of cardiotoxin 1 (CTX1) from Indian cobra (Naja naja) with heterogeneous erythrocyte membrane (EM) model system have been extensively examined by using all-atom molecular dynamics (MD) simulations in near physiological conditions and comprehensive analyses of the MD data revealed two distinct principal regions (‘head groove’ and ‘loop groove’) of the protein toxin for establishing structural interactions with the EM system. Moreover, combined analyses of data from high-throughput virtual screening of NCI small molecular database, in vitro haemolytic assays for top-hits of the chemical compounds against crude venom of Naja naja and as well CTXs purified from the venom and pharmacokinetic examinations on the chemical compounds retarding haemolytic activities of CTXs suggested that Etidronic acid and Zoledronic acid are promising prototypic chemical inhibitors to CTXs of snake venoms.

Site-Specific Dynamics of β-Sheet Peptides with (D) Pro-Gly Turns Probed by Laser-Excited Temperature-Jump Infrared Spectroscopy.

Turn residues and side-chain interactions play an important role for the folding of β-sheets. We investigated the conformational dynamics of a three-stranded β-sheet peptide ((D) P(D) P) and a two-stranded β-hairpin (WVYY-(D) P) by time-resolved temperature-jump (T-jump) infrared spectroscopy. Both peptide sequences contain (D) Pro-Gly residues that favor a tight β-turn. The three-stranded β-sheet (Ac-VFITS(D) PGKTYTEV(D) PGOKILQ-NH2 ) is stabilized by the turn sequences, whereas the β-hairpin (SWTVE(D) PGKYTYK-NH2 ) folding is assisted by both the turn sequence and hydrophobic cross-strand interactions. Relaxation times after the T-jump were monitored as a function of temperature and occur on a sub-microsecond time scale, (D) P(D) P being faster than WVYY-(D) P. The Xxx-(D) Pro tertiary amide provides a detectable IR band, allowing us to probe the dynamics site-specifically. The relative importance of the turn versus the intrastrand stability in β-sheet formation is discussed.

Foldameric probes for membrane interactions by induced β-sheet folding

Design strategies were devised for α/β-peptide foldameric analogues of the antiangiogenic anginex with the goal of mimicking the diverse structural features from the unordered conformation to a folded β-sheet in response to membrane interactions. Structure-activity relationships were investigated in the light of different β-sheet folding levels.

Mesomartoxin, a new Kv1.2-selective scorpion toxin interacting with the channel selectivity filter

Venom-derived neurotoxins are ideal probes for the investigation of structure–function relationship of ion channels and promising scaffolds for the design of ion channel-targeted drug leads as well. The discovery of highly selective toxins against a specific channel subtype facilitates the development of drugs with reduced side effects. Here, we describe the systemic characterization of a new scorpion short-chain K+ channel blocker from Mesobuthus martensii, termed mesomartoxin (MMTX). MMTX is synthesized as a precursor comprising a signal peptide and a mature peptide of 29 residues. Nuclear magnetic resonance analysis confirmed that recombinant MMTX adopts a typical cysteine-stabilized α-helical and β-sheet fold. Electrophysiological experiments showed that MMTX exhibits high affinity for the Drosophila Shaker K+ channel but differential selectivity on different members of the rat voltage-gated K+ channel (Kv) family, with nanomolar affinity (IC50=15.6nM) for rKv1.2, micromolar affinity for rKv1.3 (IC50=12.5μM) and no activity on rKv1.1 at >50μM. Site-directed mutagenesis of the channel pore identified a key site located on the selectivity filter of the pore, which is directly implicated in toxin binding and controls target's selectivity of the toxin. Given a key role of Kv1.2 in epilepsy, MMTX might serve as a potential drug lead for the disease.

Fold modulating function: bacterial toxins to functional amyloids.

Many bacteria produce cytolytic toxins that target host cells or other competing microbes. It is well known that environmental factors control toxin expression, however, recent work suggests that some bacteria manipulate the fold of these protein toxins to control their function. The β-sheet rich amyloid fold is a highly stable ordered aggregate that many toxins form in response to specific environmental conditions. When in the amyloid state, toxins become inert, losing the cytolytic activity they display in the soluble form. Emerging evidence suggest that some amyloids function as toxin storage systems until they are again needed, while other bacteria utilize amyloids as a structural matrix component of biofilms. This amyloid matrix component facilitates resistance to biofilm disruptive challenges. The bacterial amyloids discussed in this review reveal an elegant system where changes in protein fold and solubility dictate the function of proteins in response to the environment.

An amino acid code for β-sheet packing structure

To understand the relationship between protein sequence and structure, this work extends the knob-socket model in an investigation of β-sheet packing. Over a comprehensive set of β-sheet folds, the contacts between residues were used to identify packing cliques: sets of residues that all contact each other. These packing cliques were then classified based on size and contact order. From this analysis, the two types of four-residue packing cliques necessary to describe β-sheet packing were characterized. Both occur between two adjacent hydrogen bonded β-strands. First, defining the secondary structure packing within β-sheets, the combined socket or XY:HG pocket consists of four residues i, i+2 on one strand and j, j+2 on the other. Second, characterizing the tertiary packing between β-sheets, the knob-socket XY:H+B consists of a three-residue XY:H socket (i, i+2 on one strand and j on the other) packed against a knob B residue (residue k distant in sequence). Depending on the packing depth of the knob B residue, two types of knob-sockets are found: side-chain and main-chain sockets. The amino acid composition of the pockets and knob-sockets reveal the sequence specificity of β-sheet packing. For β-sheet formation, the XY:HG pocket clearly shows sequence specificity of amino acids. For tertiary packing, the XY:H+B side-chain and main-chain sockets exhibit distinct amino acid preferences at each position. These relationships define an amino acid code for β-sheet structure and provide an intuitive topological mapping of β-sheet packing.