A structurally dynamic N-terminal region drives function of the staphylococcal peroxidase inhibitor (SPIN).

The heme-containing enzyme myeloperoxidase (MPO) is critical for optimal antimicrobial activity of human neutrophils. We recently discovered that the bacterium Staphylococcus aureus expresses a novel immune evasion protein, called SPIN, that binds tightly to MPO, inhibits MPO activity, and contributes to bacterial survival following phagocytosis. A co-crystal structure of SPIN bound to MPO suggested that SPIN blocks substrate access to the catalytic heme by inserting an N-terminal ß-hairpin into the MPO active-site channel. Here, we describe a series of experiments that more completely define the structure/function relationships of SPIN. Whereas the SPIN N terminus adopts a ß-hairpin confirmation upon binding to MPO, the solution NMR studies presented here are consistent with this region of SPIN being dynamically structured in the unbound state. Curiously, whereas the N-terminal ß-hairpin of SPIN accounts for ∼55% of the buried surface area in the SPIN-MPO complex, its deletion did not significantly change the affinity of SPIN for MPO but did eliminate the ability of SPIN to inhibit MPO. The flexible nature of the SPIN N terminus rendered it susceptible to proteolytic degradation by a series of chymotrypsin-like proteases found within neutrophil granules, thereby abrogating SPIN activity. Degradation of SPIN was prevented by the S. aureus immune evasion protein Eap, which acts as a selective inhibitor of neutrophil serine proteases. Together, these studies provide insight into MPO inhibition by SPIN and suggest possible functional synergy between two distinct classes of S. aureus immune evasion proteins.

Complete non-proline backbone resonance assignments of the S. aureus neutrophil serine protease inhibitor, EapH1.

The S. aureus extracellular adherence protein (Eap) and its homologs, EapH1 and EapH2, serve roles in evasion of the human innate immune system. EapH1 binds with high-affinity and inhibits the neutrophil azurophilic granule proteases neutrophil elastase, cathepsin-G and proteinase-3. Previous structural studies using X-ray crystallography have shown that EapH1 binds to neutrophil elastase and cathepsin-G using a globally similar binding mode. However, whether the same holds true in solution is unknown and whether the inhibitor experiences dynamic changes following binding remains uncertain. To facilitate solution-phase structural and biochemical studies of EapH1 and its complexes with neutrophil granule proteases, we have characterized EapH1 by multidimensional NMR spectroscopy. Here we report a total of 100% of the non-proline backbone resonance assignments of EapH1 with BMRB accession number 50,304.

Structural transformation and physical properties of a hydrogel-forming peptide studied by NMR, transmission electron microscopy, and dynamic rheometer.

Peptide-based hydrogels are attractive biological materials. Study of their self-assembly pathways from their monomer structures is important not only for undertaking the rational design of peptide-based materials, but also for understanding their biological functions and the mechanism of many human diseases relative to protein aggregation. In this work, we have monitored the conformation, morphological, and mechanical properties of a hydrogel-forming peptide during hydrogelation in different dimethylsulfoxide (DMSO)/H(2)O solutions. The peptide shows nanofiber morphologies in DMSO/H(2)O solution with a ratio lower than 4:1. Increased water percentage in the solution enhanced the hydrogelation rate and gel strength. One-dimensional and two-dimensional proton NMR and electron microscopy studies performed on the peptide in DMSO/H(2)O solution with different ratios indicate that the peptide monomer tends to adopt a more helical structure during the hydrogelation as the DMSO/H(2)O ratio is reduced. Interestingly, at the same DMSO/H(2)O ratio, adding Ca(2+) not only promotes peptide hydrogelation and gel strength, but also leads to special shear-thinning and recovery properties of the hydrogel. Without changing the peptide conformation, Ca(2+) binds to the charged Asp residues and induces the change of interfiber interactions that play an important role in hydrogel properties.

Ecdysis triggering hormone peptide in the African malaria mosquito Anopheles gambiae: The peptide structure for receptor activation.

Infections by mosquito-borne diseases represent one of the leading causes of death in third world countries. The rapid progression of resistance to conventional insecticide causes a significant threat to the highly efficient preventive methods currently in place. Insect neuropeptidergic system offers potential targets to control the insect vectors. The essential roles of the neuropeptide ecdysis triggering hormone (ETH) in insect development and reproduction led us to attempt understanding of the fundamentals of the biochemical interaction between ETH and its receptor in the African malaria mosquito Anopheles gambiae. One of two ETH peptides of the African malaria mosquito (AgETH1), a small peptide hormone with 17 amino acid residues (SESPGFFIKLSKSVPRI-NH2 ), was studied to elucidate its molecular structure. N-termini deletions and mutations of conserved amino acids in the ligand revealed the critical residues for the receptor activation. The solution structure of AgETH1 using 2D 1 H-1 H nuclear magnetic resonance (NMR) spectroscopy and nuclear overhauser effect (NOE) derived constraints revealed a short alpha helix between residues 3S and 11S. The NMR solution structure of AgETH1 will be of significant assistance for designing a new class of insecticidal compounds that acts on the AgETH receptor aiming for in silico docking studies.

1H, 15N, and 13C backbone resonance assignments of the C4b-binding region from the S. aureus extracellular adherence protein.

The Extracellular Adherence Protein (Eap) from Staphylococcus aureus is a potent inhibitor of the classical and lectin pathways of the complement system. Previous studies have shown that Eap binds with nanomolar affinity to complement component C4b and prevents C4b binding the pro-protease, C2, thereby inhibiting formation of the pro-C3 convertase shared by the classical and lectin pathways (Woehl et al. in J Immunol 193:6161-6171, 2014). The C4b-binding and complement-inhibitory properties of Eap from S. aureus strain Mu50 lie within the two C terminal-most Eap domains (i.e. Eap34) (Woehl et al. J Immunol 193:6161-6171, 2014). Interestingly, Eap34 binds C4b with an apparent KD that is nearly 100-fold tighter than that of either Eap3 or Eap4 alone (Woehl et al. in Protein Sci 26:1595-1608, 2017). This suggests that linking these two domains into a single molecule is a significant determinant of Eap function. To better understand this property at the structural level, we undertook a solution NMR study of the ~ 23 kDa Eap34 protein. In this communication, we report that greater than 98% of the total non-proline backbone residues have been assigned. These data have been deposited in the BMRB database under the accession number 50210.

Structural characterization of two pore-forming peptides: consequences of introducing a C-terminal tryptophan.

Synthetic channel-forming peptides that can restore chloride conductance across epithelial membranes could provide a novel treatment of channelopathies such as cystic fibrosis. Among a series of 22-residue peptides derived from the second transmembrane segment of the glycine receptor alpha(1)-subunit (M2GlyR), p22-S22W (KKKKP ARVGL GITTV LTMTT QW) is particularly promising with robust membrane insertion and assembly. The concentration to reach one-half maximal short circuit current is reduced to 45 +/- 6 microM from that of 210 +/- 70 microM of peptide p22 (KKKKP ARVGL GITTV LTMTT QS). However, this is accompanied with nearly 50% reduction in conductance. Toward obtaining a molecular level understanding of the channel activities, we combine information from solution NMR, existing biophysical data, and molecular modeling to construct atomistic models of the putative pentameric channels of p22 and p22-S22W. Simulations in membrane bilayers demonstrate that these structural models, even though highly flexible, are stable and remain adequately open for ion conductance. The membrane-anchoring tryptophan residues not only rigidify the whole channel, suggesting increased stability, but also lead to global changes in the pore profile. Specifically, the p22-S22W pore has a smaller opening on average, consistent with lower measured conductance. Direct observation of several incidences of chloride transport suggests several qualitative features of how these channels might selectively conduct anions. The current study thus helps to rationalize the functional consequences of introducing a single C-terminal tryptophan. Availability of these structural models also paves the way for future work to rationally modify and improve M2GlyR-derived peptides toward potential peptide-based channel replacement therapy.

Morphology and structural properties of pH-responsive amphiphilic peptides.

Protein nanomaterials at the peptide level have shown great potential for medical applications. Peptides change their morphological conformation because of changes in self-assembly properties when they are exposed to changes in solvent composition or pH. Two 15-residue peptide sequences, KhK (KKKFLIVIGSIIKKK) and Alternating Kh (KFLKKIVKIGKKSII), were designed for the purpose of determining the role of peptide sequence on solution morphology and conformation. KhK solutions exhibited a random coil to helical transition when solvent conditions were changed from water to a trifluorethanol/water solution at acidic pH. Alternating Kh solutions, however, demonstrated primarily random coil character under similar solvent and pH conditions as determined by circular dichroism spectroscopy and 2D-1H-1H nuclear magnetic resonance spectroscopy. At basic pH, circular dichroism spectroscopy and nuclear magnetic resonance spectroscopy analysis demonstrated that random coil character increased at basic pH for KhK, whereas Alternating Kh exhibited an increase in beta-sheet character. Further analysis by transmission electron microscopy showed marked differences in the peptide solution morphology. Peptide particle aggregation and fiber formation were significantly affected by solvent composition and pH values for both peptide sequences.

Dietary flavonoid fisetin binds human SUMO1 and blocks sumoylation of p53.

Flavonoids are plant-derived compounds that occur abundantly in fruits and vegetables and have been shown to possess potent anti-cancer, antioxidant, and anti-inflammatory properties. However, their direct targets and molecular mechanism of action are not well characterized, hampering exploitation of the beneficial properties of flavonoids for drug development. Small ubiquitin-related modifier 1 (SUMO1) is attached to target proteins as part of a post-translational modification system implicated in a myriad of cellular processes from nuclear trafficking to transcriptional regulation. Using a combination of surface plasmon resonance, differential scanning fluorimetry and fluorescence quenching studies, we provide evidence for direct binding of the dietary flavonoid fisetin to human SUMO1. Our NMR chemical shift perturbation analyses reveal that binding to fisetin involves four conserved amino acid residues (L65, F66, E67, M82) previously shown to be important for conjugation of SUMO1 to target proteins. In vitro sumoylation experiments indicate that fisetin blocks sumoylation of tumor suppressor p53, consistent with fisetin negatively affecting post-translational modification and thus the biological activity of p53. A series of differential scanning fluorimetry experiments suggest that high concentrations of fisetin result in destabilization and unfolding of SUMO1, presenting a molecular mechanism by which flavonoid binding affects its activity. Overall, our data establish a novel direct interaction between fisetin and SUMO1, providing a mechanistic explanation for the ability of fisetin to modulate multiple key signaling pathways inside cells.

Backbone resonance assignments of innate immune evasion protein EapH2 from the S. aureus.

Staphylococcus aureus is a ubiquitous and persistent pathogen of humans and livestock. The bacterium disrupts the host's innate immune system's ability to recognize and clear bacteria with optimal efficiency by expressing a wide variety of virulence proteins. Two single domain protein homologs (EapH1, EapH2) of the extracellular adherence protein (Eap) have been reported. Eap is a multidomain protein that participates in various protein-protein interactions that inhibit the innate immune response, including both the complement and Neutrophil Serine Proteases (NSPs). EapH1 and EapH2 are also inhibitors of NSPs (Stapels et al., Proc Natl Acad Sci 111:13187-13192, 2014), but lack the ability to inhibit the classical, and lectin pathways of the complement activation system (Woehl et al., J Immunol 193:6161-6171, 2014). We continue the characterization of Eap domains, here with the experiments on EapH2, we acquired a series of 2D and 3D NMR spectra of EapH2 in solution. We completed 99% of expected non-proline backbone 1H, 15N, and 13C resonance assignments of EapH2 and predicted secondary structure via the TALOS-N server. The assignment data have been deposited in the BMRB data bank under Accession Number 27540.

1H, 15N, and 13C resonance assignments of the third domain from the S. aureus innate immune evasion protein Eap.

Staphylococcus aureus is a widespread and persistent pathogen of humans and livestock. The bacterium expresses a wide variety of virulence proteins, many of which serve to disrupt the host's innate immune system from recognizing and clearing bacteria with optimal efficiency. The extracellular adherence protein (Eap) is a multidomain protein that participates in various protein-protein interactions that inhibit the innate immune response, including both the complement system (Woehl et al in J Immunol 193:6161-6171, 2014) and Neutrophil Serine Proteases (NSPs) (Stapels et al in Proc Natl Acad Sci USA 111:13187-13192, 2014). The third domain of Eap, Eap3, is an ~ 11 kDa protein that was recently shown to bind complement component C4b (Woehl et al in Protein Sci 26:1595-1608, 2017) and therefore play an essential role in inhibiting the classical and lectin pathways of complement (Woehl et al in J Immunol 193:6161-6171, 2014). Since structural characterization of Eap3 is still incomplete, we acquired a series of 2D and 3D NMR spectra of Eap3 in solution. Here we report the backbone and side-chain 1H, 15N, and 13C resonance assignments of Eap3 and its predicted secondary structure via the TALOS-N server. The assignment data have been deposited in the BMRB data bank under accession number 27087.

Backbone and side-chain 1H, 15N, and 13C resonance assignments of a novel Staphylococcal inhibitor of myeloperoxidase.

The bacterium Staphylococcus aureus produces an array of anti-inflammatory molecules that prevent the innate immune system from recognizing it as a pathogen and clearing it from the host. In the acute phase of inflammation, our immune system relies on neutrophils to clear invading bacteria. Recently, novel classes of secreted proteins from S. aureus, including the Extracellular Adherence Protein (EAP) family (Stapels et al., Proc Natl Acad Sci USA 111:13187-13192, 2014) and the Staphylococcal Peroxidase Inhibitor (SPIN), (unpublished work) have been identified as highly selective inhibitors acting on Neutrophil Serine Proteases (NSPs) and myeloperoxidase (MPO) respectively. SPIN is a protein found only in Staphylococci, with no sequence homology to any known proteins. Solution NMR structural studies of SPIN are therefore expected to provide a deeper understanding of its interaction with MPO. In this study, we report the backbone and side-chain 1H, 15N, and 13C resonance assignments of SPIN. Furthermore, using the chemical shifts of these resonances, we predicted the secondary structure of SPIN in solution via the TALOS-N server. The assignment data has been deposited in the BMRB data bank under Accession No. 27069.

Solution Structure and Expression Profile of an Insect Cytokine: Manduca sexta Stress Response Peptide-2.

Manduca sexta stress response peptide-2 (SRP2) is predicted to be a 25-residue peptide (FGVKDGKCPSGRVRRLGICVPDDDY), which may function as an insect cytokine to regulate immune responses. Produced as an inactive precursor, endogenous proSRP2 is probably converted to active SRP2 by limited proteolysis in response to invading pathogens, along with prophenoloxidase and pro-Spätzle activation. In addition to immunity, SRP2 may control head morphogenesis or other developmental processes in the lepidopteran insect. We have examined the profiles of SRP2 gene expression in terms of immune induction capacity, tissue specificity, and developmental changes. To gain insights into its functions, we chemically synthesized SRP2, injected the peptide solution into naïve larvae, and detected significant up-regulation of several antimicrobial peptide genes. We determined the 3D molecular structure in solution of SRP2 by two-dimensional 1H-1H NMR spectroscopy. SRP2 has an ordered structure, which is composed of two short ß-strands at regions R12 - R15 and I18 - V20, one type-I' ß-turn at region R15 - I18, and a half turn at region C8 - S10 in its welldefined core stabilized by a covalent disulfide bond between C8 and C19. The secondary and tertiary structures are further stabilized by hydrogen bonds. Possible relationships between the structure and function are also discussed.

Membrane Interacting Peptides: A Review.

Membrane interacting peptides of natural or synthetic origins serve a variety of biological purposes. They have been extensively studied for their involvement in immunity, diseases, and for their potential as medical therapeutics and research tools. In this review membrane interacting peptides are categorized into four groups according to their function: antimicrobial peptides, cell-penetrating peptides, channel forming peptides and amyloid peptides. A historical overview of the development, their functional mechanisms, and recent advances are presented for each of the groups. Considerable research is still devoted to this field of study and in this report a representative sample of the latest studies is presented. A set of common features among peptide groups emerges as the understanding of their action mechanisms increase. The possibility of a membrane centric general model for peptide-membrane interaction is also discussed. This review seeks to provide a unifying view of the field and promote the interaction between research groups working on peptides that have so far been studied as belonging to completely different fields.

(1)H, (15)N, and (13)C resonance assignments of Staphylococcus aureus extracellular adherence protein domain 4.

The pathogenic bacterium Staphylococcus aureus has evolved to actively evade many aspects of the human innate immune system by expressing a series of secreted inhibitory proteins. Among these, the extracellular adherence protein (Eap) has been shown to inhibit the classical and lectin pathways of the complement system. By binding to complement component C4b, Eap is able to inhibit formation of the CP/LP C3 pro-convertase. Secreted full-length, mature Eap consists of four ~98 residue domains, all of which adopt a similar beta-grasp fold, and are connected through a short linker region. Through multiple biochemical approaches, it has been determined that the third and fourth domains of Eap are responsible for C4b binding. Here we report the backbone and side-chain resonance assignments of the 11.3 kDa fourth domain of Eap. The assignment data has been deposited in the BMRB database under the accession number 26726.

Lignins of bioenergy crops: a review?

Lignin provides structural support, a mechanical barrier against microbial infestation and facilitates movement of water inside plant systems. It is the second most abundant natural polymer in the terrestrial environments and possesses unique routes for the production of bulk and specialty chemicals with aromatic/phenolic skeletons. The commercial applications of lignin are limited and it is often recognized for its negative impact on the biochemical conversion of lignocellulosic biomass to fuels and chemicals. Understanding of the structure of lignin monomers and their interactions among themselves, as well as with carbohydrate polymers in biomass, is vital for the development of innovative biomass deconstruction processes and thereby valorization of all biopolymers of lignocellulosic residues, including lignin. In this paper, we review the major energy crops and their lignin structure, as well as the recent developments in biomass lignin characterization, with special focus on 1D and 2D Nuclear Magnetic Resonance (NMR) techniques.

Measuring experiential avoidance and psychological inflexibility: The Spanish version of the Acceptance and Action Questionnaire - II.

BACKGROUND: Experiential avoidance and psychological inflexibility have been recently found to be important constructs related to a wide range of psychological disorders and quality of life. The current study presents psychometric and factor structure data concerning the Spanish translation of a general measure of both constructs: the Acceptance and Action Questionnaire - II (AAQ-II). METHOD: Six samples, with a total of 712 participants, from several independent studies were analyzed. RESULTS: Data were very similar to the ones obtained in the original AAQ-II version. The internal consistency across the different samples was good (between a= .75 and a= .93). The differences between clinical and nonclinical samples were statistically significant and the overall factor analysis yielded to a one-factor solution. The AAQ-II scores were significantly related to general psychopathology and quality of life measures. CONCLUSIONS: This Spanish translation of the AAQ-II emerges as a reliable and valid measure of experiential avoidance and psychological inflexibility.

The central kink region of fowlicidin-2, an alpha-helical host defense peptide, is critically involved in bacterial killing and endotoxin neutralization.

Fowlicidins are a group of newly identified chicken cathelicidin host defense peptides. We have shown that the putatively mature fowlicidin-2 of 31 amino acid residues possesses potent antibacterial and lipopolysaccharide (LPS)- neutralizing activities, but with a noticeable toxicity to mammalian cells. As a first step in exploring the structure-activity relationships of fowlicidin-2, in this study we determined its tertiary structure by nuclear magnetic resonance spectroscopy. Unlike the majority of cathelicidins, which are composed of a predominant alpha-helix with a short hinge sequence near the center, fowlicidin-2 consists of 2 well-defined alpha-helical segments (residues 6-12 and 23-27) connected by a long extensive kink (residues 13-20) induced by proline. To further investigate the functional significance of each of these structural components, several N- and C-terminal deletion analogs of fowlicidin-2 were synthesized and analyzed for their antibacterial, cytotoxic and LPS-neutralizing activities. Our results indicated that neither the N- nor C-terminal alpha-helix alone is sufficient to confer any function. Rather, fowlicidin-2(1-18) and fowlicidin-2(15-31), 2 alpha-helical segments with inclusion of the central cationic kink region, retained substantial capacities to kill bacteria and neutralize the LPS-induced proinflammatory response, relative to the parent peptide. More desirably, these 2 peptide analogs showed substantially reduced toxicity to human erythrocytes and epithelial cells, indicative of improved potential as antibacterial and antisepsis agents. To our knowledge, fowlicidin-2 is the first alpha-helical cathelicidin, with the central kink region shown to be critically important in killing bacteria and neutralizing LPS.