Structure–function relationships in histidine-rich antimicrobial peptides from Atlantic cod

Gad-1 and Gad-2 are antimicrobial peptide (AMP) sequences encoded by paralogous genes. They are rich in histidine, which suggests that their activity might be pH-dependent. We examined their structure–function relationships with a view to learning how to improve AMP therapeutic ratios. Activity assays with Gram-negative bacteria and cancer cell lines demonstrate that Gad-2 is substantially more active at slightly acidic pH than it is at neutral pH. By contrast, the activity of Gad-1 at lower pH is similar to its activity at pH 7. Circular dichroism spectra indicate that the greater functional plasticity of Gad-2 correlates with a greater structural plasticity; Gad-2's percent helicity varies dramatically with altered pH and lipid environment. Interestingly, Gad-2's highest levels of helicity do not correspond to the conditions where it is most active. High resolution solution NMR structures were determined in SDS micelles at pH 5, conditions that induce an intermediate level of helicity in the peptides. Gad-1 is more helical than Gad-2, with both peptides exhibiting the greatest helical tendencies in their central region and lowest helicity in their N-termini. The high resolution structures suggest that maximum activity relies on the appropriate balance between an N-terminal region with mixed hydrophobic/hydrophilic structure features and an amphipathic central and C-terminal region. Taken together with previous studies, our results suggest that to improve the therapeutic ratio of AMPs, consideration should be given to including sequential histidine-pairs, keeping the overall charge of the peptide modest, and retaining a degree of structural plasticity and imperfect amphipathicity.     

Structure–function relationships of epoxide hydrolases and their potential use in biocatalysis

Background

Chiral epoxides and diols are important synthons for manufacturing fine chemicals and pharmaceuticals. The epoxide hydrolases (EC 3.3.2.-) catalyze the hydrolytic ring opening of epoxides producing the corresponding vicinal diol. Several isoenzymes display catalytic properties that position them as promising biocatalytic tools for the generation of enantiopure epoxides and diols.

Scope of review

This review focuses on the present data on enzyme structure and function in connection to biocatalytic applications. Available data on biocatalysis employed for purposes of stereospecific ring opening, to produce chiral vicinal diols, and kinetic resolution regimes, to achieve enantiopure epoxides, are discussed and related to results gained from structure–activity studies on the enzyme catalysts. More recent examples of the concept of directed evolution of enzyme function are also presented.

Major conclusions

The present understanding of structure–activity relationships in epoxide hydrolases regarding chemical catalysis is strong. With the ongoing research, a more detailed view of the factors that influence substrate specificities and stereospecificities is expected to arise. The already present use of epoxide hydrolases in synthetic applications is expected to expand as new enzymes are being isolated and characterized. Refined methodologies for directed evolution of desired catalytic and physicochemical properties may further boost the development of novel and useful biocatalysts.

General significance

The catalytic power of enzymes provides new possibilities for efficient, specific and sustainable technologies to be developed for production of useful chemicals.     

Structure–function relationships in HIV-1 Nef

The accessory Nef protein of HIV and SIV is essential for viral pathogenesis, yet it is perplexing in its multitude of molecular functions. In this review we analyse the structure–function relationships of motifs recently proposed to play roles in aspects of Nef modification, signalling and trafficking, and thereby to impinge on the ability of the virus to survive in, and to manipulate, its cellular host. Based on the full-length structure assembly of HIV Nef, we correlate surface accessibility with secondary structure elements and sequence conservation. Motifs involved in Nef-mediated CD4 and MHC I downregulation are located in flexible regions of Nef, suggesting that the formation of the transient trafficking complexes involved in these processes depends on the recognition of primary sequences. In contrast, the interaction sites for signalling molecules that contain SH3 domains or the p21-activated kinases are associated with the well folded core domain, suggesting the recognition of highly structured protein surfaces.     

Structure–function relationships in the bifunctional ferrisiderophore FpvA receptor from Pseudomonas aeruginosa

FpvA is the primary outer membrane transporter required for iron acquisition via the siderophore pyoverdine (Pvd) in Pseudomonas aeruginosa. FpvA, like other ferrisiderophore transporters, consists of a membrane-spanning β-barrel occluded by a plug domain. The β-strands of the barrel are connected by large extracellular loops and periplasmic turns. Like some other TonB-dependent transporters, FpvA has a periplasmic domain involved in a signalling cascade that regulates expression of genes required for ferrisiderophore transport. Here, the structures of FpvA in different loading states are analysed in light of mutagenesis data. This analysis highlights the roles of different protein domains in Pvd-Fe uptake and the signalling cascade and reveals a strong correlation between Pvd-Fe transport and activation of the signalling cascade. It is likely that conclusions drawn for FpvA will be relevant to other TonB-dependent ferrisiderophore transport and signalling proteins.     

Structure–function relationships in human d-amino acid oxidase

Since d-amino acids were identified in mammals, d-serine has been one of the most extensively studied “unnatural amino acids”. This brain-enriched transmitter-like molecule plays a pivotal role in the human central nervous system by modulating the activity of NMDA receptors. Physiological levels of d-serine are required for normal brain development and function; thus, any alterations in neuromodulator concentrations might result in NMDA receptor dysfunction, which is known to be involved in several pathological conditions, including neurodegeneration(s), epilepsy, schizophrenia, and bipolar disorder. In the brain, the concentration of d-serine stored in cells is defined by the activity of two enzymes: serine racemase (responsible for both the synthesis and degradation) and d-amino acid oxidase (which catalyzes d-serine degradation). Both enzymes emerged recently as new potential therapeutic targets for NMDA receptor-related diseases. In this review we have focused on human d-amino acid oxidase and provide an extensive overview of the biochemical and structural properties of this flavoprotein and their functional significance. Furthermore, we discuss the mechanisms involved in modulating enzyme activity and stability with the aim to substantiate the pivotal role of d-amino acid oxidase in brain d-serine metabolism in physiological and pathological conditions and to highlight its great significance for novel drug design/development.     

Structure–function relationships of the antigenicity of mycolic acids in tuberculosis patients

Cell wall mycolic acids (MA) from Mycobacterium tuberculosis (M.tb) are CD1b presented antigens that can be used to detect antibodies as surrogate markers of active TB, even in HIV coinfected patients. The use of the complex mixtures of natural MA is complicated by an apparent antibody cross-reactivity with cholesterol. Here firstly we report three recombinant monoclonal scFv antibody fragments in the chicken germ-line antibody repertoire, which demonstrate the possibilities for cross-reactivity: the first recognized both cholesterol and mycolic acids, the second mycolic acids but not cholesterol, and the third cholesterol but not mycolic acids. Secondly, MA structure is experimentally interrogated to try to understand the cross-reactivity. Unique synthetic mycolic acids representative of the three main functional classes show varying antigenicity against human TB patient sera, depending on the functional groups present and on their stereochemistry. Oxygenated (methoxy- and keto-) mycolic acid was found to be more antigenic than alpha-mycolic acids. Synthetic methoxy-mycolic acids were the most antigenic, one containing a trans-cyclopropane apparently being somewhat more antigenic than the natural mixture. Trans-cyclopropane-containing keto- and hydroxy-mycolic acids were also found to be the most antigenic among each of these classes. However, none of the individual synthetic mycolic acids significantly and reproducibly distinguished the pooled serum of TB positive patients from that of TB negative patients better than the natural mixture of MA. This argues against the potential to improve the specificity of serodiagnosis of TB with a defined single synthetic mycolic acid antigen from this set, although sensitivity may be facilitated by using a synthetic methoxy-mycolic acid.     

Ferritin iron uptake and release. Structure–function relationships

Iron uptake and release by ferritin molecules of different iron contents show similar profiles. These are discussed in relation to the structure of the ferritin molecule. Two models of iron uptake and release are considered. One involves iron oxidation–reduction sites on the protein. The other allows direct interaction of reagents with the iron-core crystallites. It is concluded that the second model accounts better for the experimental results presented now and in previous publications.     

SIR–nucleosome interactions: Structure–function relationships in yeast silent chromatin

Discrete regions of the eukaryotic genome assume a heritable chromatin structure that is refractory to gene expression, referred to as heterochromatin or “silent” chromatin. Constitutively silent chromatin is found in subtelomeric domains in a number of species, ranging from yeast to man. In addition, chromatin-dependent repression of mating type loci occurs in both budding and fission yeasts, to enable sexual reproduction. The silencing of chromatin in budding yeast is characterized by an assembly of Silent Information Regulatory (SIR) proteins—Sir2, Sir3 and Sir4—with unmodified nucleosomes. Silencing requires the lysine deacetylase activity of Sir2, extensive contacts between Sir3 and the nucleosome, as well as interactions among the SIR proteins, to generate the Sir2–3–4 or SIR complex. Results from recent structural and reconstitution studies suggest an updated model for the ordered assembly and organization of SIR-dependent silent chromatin in yeast. Moreover, studies of subtelomeric gene expression reveal the importance of subtelomeric silent chromatin in the regulation of genes other than the silent mating type loci. This review covers recent advances in this field.     

Structure–activity relationships of lipopolysaccharide sequestration in N-alkylpolyamines

We have previously shown that simple N-acyl or N-alkyl polyamines bind to and sequester Gram-negative bacterial lipopolysaccharide, affording protection against lethality in animal models of endotoxicosis. Several iterative design-and-test cycles of SAR studies, including high-throughput screens, had converged on compounds with polyamine scaffolds which have been investigated extensively with reference to the number, position, and length of acyl or alkyl appendages. However, the polyamine backbone itself had not been explored sufficiently, and it was not known if incremental variations on the polymethylene spacing would affect LPS-binding and neutralization properties. We have now systematically explored the relationship between variously elongated spermidine [NH₂–(CH₂)₃–NH–(CH₂)₄–NH₂] and norspermidine [NH₂–(CH₂)₃–NH–(CH₂)₃–NH₂] backbones, with the N-alkyl group being held constant at C₁₆ in order to examine if changing the spacing between the inner secondary amines may yield additional SAR information. We find that the norspermine-type compounds consistently showed higher activity compared to corresponding spermine homologues.     

Stability of microsclerotial inoculum of Colletotrichum truncatum encapsulated in wheat flour–kaolin granules

Maintaining adequate viability of microorganisms in products for biocontrol is critically important for commercial reasons. Microsclerotia (MS) of the mycoherbicide agent, Colletotrichum truncatum, are its hardy, over-wintering fungal structures. Microsclerotial inoculum at 2, 7, and 23 MS/granule were matrix-encapsulated in wheat flour–kaolin granules (Pesta), in which the flour provided gluten for the matrix and a food base for the fungus. Pesta granules were dried to a water activity of 0.18–0.29. After storage for 52 weeks at 25°C, granules containing 7 and 23 MS were 100% viable and granules with 2 MS were 95% viable. Granules with 7 MS were 50% viable after 36 weeks at 35°C. Pesta granules (440 granules/g) with conidial inoculum at 3.3×105 c.f.u./g were less storage-stable than granules at the 2 MS/granule level. At all MS inoculum levels, granules stored for up to 2years produced 108 c.f.u./g in vitro when incubated on water agar. High water activity was detrimental to long-term viability. In the greenhouse, 7 MS/granule samples controlled 94% of hemp sesbania when incorporated into the soil pre-planting. The strategy of encapsulation of the naturally stable C. truncatum MS and drying to a favourable water activity led to excellent shelf-life for a live biocontrol agent.     

Structure–function relationships of molluscan troponin T revealed by limited proteolysis

Molluscan troponin regulates muscle contraction through a novel Ca²⁺-dependent activating mechanism associated with Ca²⁺-binding to the C-terminal domain of troponin C. To elucidate the further details of this regulation, we performed limited chymotryptic digestion of the troponin complex from akazara scallop striated muscle. The results indicated that troponin T is very susceptible to the protease, compared to troponin C or troponin I. The cleavage occurred at the C-terminal extension, producing an N-terminal 33-kDa fragment and a C-terminal 6-kDa fragment. This extension is conserved in various invertebrate troponin T proteins, but not in vertebrate troponin T. A ternary complex composed of the 33-kDa fragment of troponin T, troponin I, and troponin C could be separated from the 6-kDa troponin T fragment by gel filtration. This complex did not show any Ca²⁺-dependent activation of the Mg-ATPase activity of rabbit-actomyosin–scallop-tropomyosin. In addition, the actin–tropomyosin-binding affinity of this complex was significantly decreased with increasing Ca²⁺ concentration. These results indicate that the C-terminal extension of molluscan troponin T plays a role in anchoring the troponin complex to actin–tropomyosin filaments and is essential for regulation.     

Structure–function relationships in ShKT domain peptides: ShKT-Ts1 from the sea anemone Telmatactis stephensoni

Diverse structural scaffolds have been described in peptides from sea anemones, with the ShKT domain being a common scaffold first identified in ShK toxin from Stichodactyla helianthus. ShK is a potent blocker of voltage-gated potassium channels (K_V1.x), and an analog, ShK-186 (dalazatide), has completed Phase 1 clinical trials in plaque psoriasis. The ShKT domain has been found in numerous other species, but only a tiny fraction of ShKT domains has been characterized functionally. Despite adopting the canonical ShK fold, some ShKT peptides from sea anemones inhibit K_V1.x, while others do not. Mutagenesis studies have shown that a Lys–Tyr (KY) dyad plays a key role in K_V1.x blockade, although a cationic residue followed by a hydrophobic residue may also suffice. Nevertheless, ShKT peptides displaying an ShK-like fold and containing a KY dyad do not necessarily block potassium channels, so additional criteria are needed to determine whether new ShKT peptides might show activity against potassium channels. In this study, we used a combination of NMR and molecular dynamics (MD) simulations to assess the potential activity of a new ShKT peptide. We determined the structure of ShKT-Ts1, from the sea anemone Telmatactis stephensoni, examined its tissue localization, and investigated its activity against a range of ion channels. As ShKT-Ts1 showed no activity against K_V1.x channels, we used MD simulations to investigate whether solvent exposure of the dyad residues may be informative in rationalizing and potentially predicting the ability of ShKT peptides to block K_V1.x channels. We show that either a buried dyad that does not become exposed during MD simulations, or a partially exposed dyad that becomes buried during MD simulations, correlates with weak or absent activity against K_V1.x channels. Therefore, structure determination coupled with MD simulations, may be used to predict whether new sequences belonging to the ShKT family may act as potassium channel blockers.     

Structure–function relationships of two paralogous single-stranded DNA-binding proteins from Streptomyces coelicolor: implication of SsbB in chromosome segregation during sporulation

The linear chromosome of Streptomyces coelicolor contains two paralogous ssb genes, ssbA and ssbB. Following mutational analysis, we concluded that ssbA is essential, whereas ssbB plays a key role in chromosome segregation during sporulation. In the ssbB mutant, ∼30% of spores lacked DNA. The two ssb genes were expressed differently; in minimal medium, gene expression was prolonged for both genes and significantly upregulated for ssbB. The ssbA gene is transcribed as part of a polycistronic mRNA from two initiation sites, 163 bp and 75 bp upstream of the rpsF translational start codon. The ssbB gene is transcribed as a monocistronic mRNA, from an unusual promoter region, 73 bp upstream of the AUG codon. Distinctive DNA-binding affinities of single-stranded DNA-binding proteins monitored by tryptophan fluorescent quenching and electrophoretic mobility shift were observed. The crystal structure of SsbB at 1.7 Å resolution revealed a common OB-fold, lack of the clamp-like structure conserved in SsbA and previously unpublished S-S bridges between the A/B and C/D subunits. This is the first report of the determination of paralogous single-stranded DNA-binding protein structures from the same organism. Phylogenetic analysis revealed frequent duplication of ssb genes in Actinobacteria, whereas their strong retention suggests that they are involved in important cellular functions.     

Structure–function characterization of the recombinant aspartic proteinase A1 from Arabidopsis thaliana

Aspartic proteinases (APs) are involved in several physiological processes in plants, including protein processing, senescence, and stress response and share many structural and functional features with mammalian and microbial APs. The heterodimeric aspartic proteinase A1 from Arabidopsis thaliana (AtAP A1) was the first acid protease identified in this model plant, however, little information exists regarding its structure function characteristics. Circular dichroism analysis indicated that recombinant AtAP A1 contained an higher α-helical content than most APs which was attributed to the presence of a sequence known as the plant specific insert in the mature enzyme. rAtAP A1 was stable over a broad pH range (pH 3–8) with the highest stability at pH 5–6, where 70–80% of the activity was retained after 1 month at 37 °C. Using calorimetry, a melting point of 79.6 °C was observed at pH 5.3. Cleavage profiles of insulin β-chain indicated that the enzyme exhibited a higher specificity as compared to other plant APs, with a high preference for the Leu₁₅–Tyr₁₆ peptide bond. Molecular modeling of AtAP A1 indicated that exposed histidine residues and their interaction with nearby charged groups may explain the pH stability of rAtAP A1.     

Structure–function correlations in tyrosinases

Tyrosinases are metalloenzymes belonging to the type-3 copper protein family which contain two copper ions in the active site. They are found in various prokaryotes as well as in plants, fungi, arthropods, and mammals and are responsible for pigmentation, wound healing, radiation protection, and primary immune response. Tyrosinases perform two sequential enzymatic reactions: hydroxylation of monophenols and oxidation of diphenols to form quinones which polymerize spontaneously to melanin. Two other members of this family are catechol oxidases, which are prevalent mainly in plants and perform only the second oxidation step, and hemocyanins, which lack enzymatic activity and are oxygen carriers. In the last decade, several structures of plant and bacterial tyrosinases were determined, some with substrates or inhibitors, highlighting features and residues which are important for copper uptake and catalysis. This review summarizes the updated information on structure–function correlations in tyrosinases along with comparison to other type-3 copper proteins.     

Structure–function analysis of cytochromes P450 2B

In the last 4 years, breakthroughs were made in the field of P450 2B (CYP2B) structure–function through determination of one ligand-free and two inhibitor-bound X-ray crystal structures of CYP2B4, which revealed many of the structural features required for binding ligands of different size and shape. Large conformational changes of several plastic regions of CYP2B4 can dramatically reshape the active site of the enzyme to fit the size and shape of the bound ligand without perturbing the overall P450 fold. Solution biophysical studies using isothermal titration calorimetry (ITC) have revealed the large difference in the thermodynamic parameters of CYP2B4 in binding inhibitors of different ring chemistry and side chains. Other studies have revealed that the effects of site-specific mutations on steady-state kinetic parameters and mechanism-based inactivation are often substrate dependent. These findings agree with the structural data that the enzymes adopt different conformations to bind various ligands. Thus, the substrate specificity of an individual enzyme is determined not only by active site residues but also non-active site residues that modulate conformational changes that are important for substrate access and rearrangement of the active site to accommodate the bound substrate.     

Structure–activity relationships of lipopolysaccharide sequestration in guanylhydrazone-bearing lipopolyamines

The toxicity of Gram-negative bacterial endotoxin (lipopolysaccharide, LPS) resides in its structurally highly conserved glycolipid component called lipid A. Our major goal has been to develop small-molecules that would sequester LPS by binding to the lipid A moiety, so that it could be useful for the prophylaxis or adjunctive therapy of Gram-negative sepsis. We had previously identified in rapid-throughput screens several guanylhydrazones as potent LPS binders. We were desirous of examining if the presence of the guanylhydrazone (rather than an amine) functionality would afford greater LPS sequestration potency. In evaluating a congeneric set of guanylhydrazone analogues, we find that C₁₆ alkyl substitution is optimal in the N-alkylguanylhydrazone series; a homospermine analogue with the terminal amine N-alkylated with a C₁₆ chain with the other terminus of the molecule bearing an unsubstituted guanylhydrazone moiety is marginally more active, suggesting very slight, if any, steric effects. Neither C₁₆ analogue is significantly more active than the N-C₁₆-alkyl or N-C₁₆-acyl compounds that we had characterized earlier, indicating that basicity of the phosphate-recognizing cationic group, is not a determinant of LPS sequestration activity.     

Accumulation of multiple-repeat starch-binding domains (SBD2–SBD5) does not reduce amylose content of potato starch granules

This study investigates whether it is possible to produce an amylose-free potato starch by displacing the amylose enzyme, granule-bound starch synthase I (GBSSI), from the starch granule by engineered, high-affinity, multiple-repeat family 20 starch-binding domains (SBD2, SBD3, SBD4, and SBD5). The constructs were introduced in the amylose-containing potato cultivar (cv. Kardal), and the starches of the resulting transformants were compared with those of SBD2-expressing amylose-free (amf) potato clones. It is shown that a correctly sized protein accumulated in the starch granules of the various transformants. The amount of SBD accumulated in starch increased progressively from SBD to SBD3; however, it seemed as if less SBD4 and SBD5 was accumulated. A reduction in amylose content was not achieved in any of the transformants. However, it is shown that SBDn expression can affect physical processes underlying granule assembly, in both genetic potato backgrounds, without altering the primary structure of the constituent starch polymers and the granule melting temperature. Granule size distribution of the starches obtained from transgenic Kardal plants were similar to those from untransformed controls, irrespective of the amount of SBDn accumulated. In the amf background, granule size is severely affected. In both the Kardal and amf background, apparently normal oval-shaped starch granules were composed of multiple smaller ones, as evidenced from the many “Maltese crosses” within these granules. The results are discussed in terms of different binding modes of SBD.     

Structure–function relationships of the peptide Paulistine: A novel toxin from the venom of the social wasp Polybia paulista

Background

The peptide Paulistine was isolated from the venom of wasp Polybia paulista. This peptide exists under a natural equilibrium between the forms: oxidised — with an intra-molecular disulphide bridge; and reduced — in which the thiol groups of the cysteine residues do not form the disulphide bridge. The biological activities of both forms of the peptide are unknown up to now.

Methods

Both forms of Paulistine were synthesised and the thiol groups of the reduced form were protected with the acetamidemethyl group [Acm-Paulistine] to prevent re-oxidation. The structure/activity relationships of the two forms were investigated, taking into account the importance of the disulphide bridge.

Results

Paulistine has a more compact structure, while Acm-Paulistine has a more expanded conformation. Bioassays reported that Paulistine caused hyperalgesia by interacting with the receptors of lipid mediators involved in the cyclooxygenase type II pathway, while Acm-Paullistine also caused hyperalgesia, but mediated by receptors involved in the participation of prostanoids in the cyclooxygenase type II pathway.

Conclusion

The acetamidemethylation of the thiol groups of cysteine residues caused small structural changes, which in turn may have affected some physicochemical properties of the Paulistine. Thus, the dissociation of the hyperalgesy from the edematogenic effect when the actions of Paulistine and Acm-Paulistine are compared to each other may be resulting from the influence of the introduction of Acm-group in the structure of Paulistine.

General significance

The peptides Paulistine and Acm-Paulistine may be used as interesting tools to investigate the mechanisms of pain and inflammation in future studies.