Insights into the mechanism of membrane fusion induced by the plant defense element, plant-specific insert.

In plants, many natural defense mechanisms include cellular membrane fusion as a way to resist infection by external pathogens. Several plant proteins mediate membrane fusion, but the detailed mechanism by which they promote fusion is less clear. Understanding this process could provide valuable insights into these proteins' physiological functions and guide bioengineering applications (i.e. the design of antimicrobial proteins). The plant-specific insert (PSI) from Solanum tuberosum can help reduce certain pathogen attack via membrane fusion. To gain new insights into the process of PSI-induced membrane fusion, a combined approach of NMR, FRET, and in silico studies was used. Our results indicate that (i) under acidic conditions, the PSI experiences a monomer-dimer equilibrium, and the dimeric PSI induces membrane fusion below a certain critical pH; (ii) after fusion, the PSI resides in a highly dehydrated environment with limited solvent accessibility, suggesting its capability in reducing repulsive dehydration forces between liposomes to facilitate fusion; and (iii) as shown by molecular dynamics simulations, the PSI dimer can bind stably to membrane surfaces and can bridge liposomes in close proximity, a critical step for the membrane fusion. In summary, this study provides new and unique insights into the mechanisms by which the PSI and similar proteins induce membrane fusion.

The prosegment catalyzes native folding of Plasmodium falciparum plasmepsin II.

Plasmepsin II is a malarial pepsin-like aspartic protease produced as a zymogen containing an N-terminal prosegment domain that is removed during activation. Despite structural similarities between active plasmepsin II and pepsin, their prosegments adopt different conformations in the respective zymogens. In contrast to pepsinogen, the proplasmepsin II prosegment is 80 residues longer, contains a transmembrane region and is non-essential for recombinant expression in an active form, thus calling into question the prosegment's precise function. The present study examines the role of the prosegment in the folding mechanism of plasmepsin II. Both a shorter (residues 77-124) and a longer (residues 65-124) prosegment catalyze plasmepsin II folding at rates more than four orders of magnitude faster compared to folding without prosegment. Native plasmepsin II is kinetically trapped and requires the prosegment both to catalyze folding and to shift the folding equilibrium towards the native conformation. Thus, despite low sequence identity and distinct zymogen conformations, the folding landscapes of plasmepsin II and pepsin, both with and without prosegment, are qualitatively identical. These results imply a conserved and unusual feature of the pepsin-like protease topology that necessitates prosegment-assisted folding.

Comparative structure-function characterization of the saposin-like domains from potato, barley, cardoon and Arabidopsis aspartic proteases.

The present study characterized the aspartic protease saposin-like domains of four plant species, Solanum tuberosum (potato), Hordeum vulgare L. (barley), Cynara cardunculus L. (cardoon; artichoke thistle) and Arabidopsis thaliana, in terms of bilayer disruption and fusion, and structure pH-dependence. Comparison of the recombinant saposin-like domains revealed that each induced leakage of bilayer vesicles composed of a simple phospholipid mixture with relative rates Arabidopsis>barley>cardoon>potato. When compared for leakage of bilayer composed of a vacuole-like phospholipid mixture, leakage was approximately five times higher for potato saposin-like domain compared to the others. In terms of fusogenic activity, distinctions between particle size profiles were noted among the four proteins, particularly for potato saposin-like domain. Bilayer fusion assays in reducing conditions resulted in altered fusion profiles except in the case of cardoon saposin-like domain which was virtually unchanged. Secondary structure profiles were similar across all four proteins under different pH conditions, although cardoon saposin-like domain appeared to have higher overall helix structure. Furthermore, increases in Trp emission upon protein-bilayer interactions suggested that protein structure rearrangements equilibrated with half-times ranging from 52 to 120s, with cardoon saposin-like domain significantly slower than the other three species. Overall, the present findings serve as a foundation for future studies seeking to delineate protein structural features and motifs in protein-bilayer interactions based upon variability in plant aspartic protease saposin-like domain structures.

Structure and mechanism of the saposin-like domain of a plant aspartic protease.

Many plant aspartic proteases contain an additional sequence of ~100 amino acids termed the plant-specific insert, which is involved in host defense and vacuolar targeting. Similar to all saposin-like proteins, the plant-specific insert functions via protein-membrane interactions; however, the structural basis for such interactions has not been studied, and the nature of plant-specific insert-mediated membrane disruption has not been characterized. In the present study, the crystal structure of the saposin-like domain of potato aspartic protease was resolved at a resolution of 1.9 Å, revealing an open V-shaped configuration similar to the open structure of human saposin C. Notably, vesicle disruption activity followed Michaelis-Menten-like kinetics, a finding not previously reported for saposin-like proteins including plant-specific inserts. Circular dichroism data suggested that secondary structure was pH-dependent in a fashion similar to influenza A hemagglutinin fusion peptide. Membrane effects characterized by atomic force microscopy and light scattering indicated bilayer solubilization as well as fusogenic activity. Taken together, the present study is the first report to elucidate the membrane interaction mechanism of plant saposin-like domains whereby pH-dependent membrane interactions resulted in bilayer fusogenic activity that probably arose from a viral type pH-dependent helix-kink-helix motif at the plant-specific insert N terminus.

Improving the alkaline stability of pepsin through rational protein design using renin, an alkaline-stable aspartic protease, as a structural and functional reference.

The present study sought to identify the structural determinants of aspartic protease structural stability and activity at elevated pH. Various hypotheses have been published regarding the features responsible for the unusual alkaline structural stability of renin, however, few structure-function studies have verified these claims. Using pepsin as a model system, and renin as a template for functional and structural alkaline stability, a rational re-design of pepsin was undertaken to identify residues contributing to the alkaline instability of pepsin-like aspartic proteases in regards to both structure and function. We constructed 13 mutants based on this strategy. Among them, mutants D159 L and D60A led to an increase in activity at elevated pH levels (p ≤ 0.05) and E4V and H53F were shown to retain native-like structure at elevated pH (p ≤ 0.05). Previously suggested carboxyl groups Asp11, Asp118, and Glu13 were individually shown not to be responsible for the structural instability or lack of activity at neutral pH in pepsin. The importance of the ß-barrel to structural stability was highlighted as the majority of the stabilizing residues identified, and 39% of the weakly conserved residues in the N-terminal lobe, were located in ß-sheet strands of the barrel. The results of the present study indicate that alkaline stabilization of pepsin will require reduction of electrostatic repulsions and an improved understanding of the role of the hydrogen bonding network of the characteristic ß-barrel.

Roles of Plant-Specific Inserts in Plant Defense.

Ubiquitously expressed in plants, the plant-specific insert (PSI) of typical plant aspartic proteases (tpAPs) has been associated with plant development, stress response, and defense processes against invading pathogens. Despite sharing high sequence identity, structural studies revealed possible different mechanisms of action among species. The PSI induces signaling pathways of defense hormones in vivo and demonstrates broad-spectrum activity against phytopathogens in vitro. Recent characterization of the PSI-tpAP relationship uncovered novel, nonconventional intracellular protein transport pathways and improved tpAP production yields for industrial applications. In spite of research to date, relatively little is known about the structure-function relationships of PSIs. A comprehensive understanding of their biological roles may benefit plant protection strategies against virulent phytopathogens.

Comparative bioinformatic and structural analyses of pepsin and renin.

Pepsin, the archetypal pepsin-like aspartic protease, is irreversibly denatured when exposed to neutral pH conditions whereas renin, a structural homologue of pepsin, is fully stable and optimally active in the same conditions despite sharing highly similar enzyme architecture. To gain insight into the structural determinants of differential aspartic protease pH stability, the present study used comparative bioinformatic and structural analyses. In pepsin, an abundance of polar and aspartic acid residues were identified, a common trait with other acid-stable enzymes. Conversely, renin was shown to have increased levels of basic amino acids. In both pepsin and renin, the solvent exposure of these charged groups was high. Having similar overall acidic residue content, the solvent-exposed basic residues may allow for extensive salt bridge formation in renin, whereas in pepsin, these residues are protonated and serve to form stabilizing hydrogen bonds at low pH. Relative differences in structure and sequence in the turn and joint regions of the ß-barrel and ψ-loop in both the N- and C-terminal lobes were identified as regions of interest in defining divergent pH stability. Compared to the structural rigidity of renin, pepsin has more instability associated with the N-terminus, specifically the B/C connector. By contrast, renin exhibits greater C-terminal instability in turn and connector regions. Overall, flexibility differences in connector regions, and amino acid composition, particularly in turn and joint regions of the ß-barrel and ψ-loops, likely play defining roles in determining pH stability for renin and pepsin.

Protein Structure Insights into the Bilayer Interactions of the Saposin-Like Domain of Solanum tuberosum Aspartic Protease.

Many plant aspartic proteases contain a saposin-like domain whose principal functions are intracellular sorting and host defence. Its structure is characterised by helical segments cross-linked by three highly conserved cystines. The present study on the saposin-like domain of Solanum tuberosum aspartic protease revealed that acidification from inactive to active conditions causes dimerisation and a strand-to-helix secondary structure transition independent of bilayer interaction. Bilayer fusion was shown to occur under reducing conditions yielding a faster shift to larger vesicle sizes relative to native conditions, implying that a lower level structural motif might be bilayer-active. Characterisation of peptide sequences based on the domain's secondary structural regions showed helix-3 to be active (~4% of the full domain's activity), and mutation of its sole positively charged residue resulted in loss of activity and disordering of structure. Also, the peptides' respective circular dichroism spectra suggested that native folding within the full domain is dependent on surrounding structure. Overall, the present study reveals that the aspartic protease saposin-like domain active structure is an open saposin fold dimer whose formation is pH-dependent, and that a bilayer-active motif shared among non-saposin membrane-active proteins including certain plant defence proteins is nested within an overall structure essential for native functionality.

In silico insights into protein-protein interactions and folding dynamics of the saposin-like domain of Solanum tuberosum aspartic protease.

The plant-specific insert is an approximately 100-residue domain found exclusively within the C-terminal lobe of some plant aspartic proteases. Structurally, this domain is a member of the saposin-like protein family, and is involved in plant pathogen defense as well as vacuolar targeting of the parent protease molecule. Similar to other members of the saposin-like protein family, most notably saposins A and C, the recently resolved crystal structure of potato (Solanum tuberosum) plant-specific insert has been shown to exist in a substrate-bound open conformation in which the plant-specific insert oligomerizes to form homodimers. In addition to the open structure, a closed conformation also exists having the classic saposin fold of the saposin-like protein family as observed in the crystal structure of barley (Hordeum vulgare L.) plant-specific insert. In the present study, the mechanisms of tertiary and quaternary conformation changes of potato plant-specific insert were investigated in silico as a function of pH. Umbrella sampling and determination of the free energy change of dissociation of the plant-specific insert homodimer revealed that increasing the pH of the system to near physiological levels reduced the free energy barrier to dissociation. Furthermore, principal component analysis was used to characterize conformational changes at both acidic and neutral pH. The results indicated that the plant-specific insert may adopt a tertiary structure similar to the characteristic saposin fold and suggest a potential new structural motif among saposin-like proteins. To our knowledge, this acidified PSI structure presents the first example of an alternative saposin-fold motif for any member of the large and diverse SAPLIP family.

The zymogen of plasmepsin V from Plasmodium falciparum is enzymatically active.

Plasmepsin V, a membrane-bound aspartic protease present in Plasmodium falciparum, is involved in the export of malaria parasite effector proteins into host erythrocytes and therefore is a potential target for antimalarial drug development. The present study reports the bacterial recombinant expression and initial characterization of zymogenic and mature plasmepsin V. A 484-residue truncated form of proplasmepsin (Glu37-Asn521) was fused to a fragment of thioredoxin and expressed as inclusion bodies. Refolding conditions were optimized and zymogen was processed into a mature form via cleavage at the Asn80-Ala81 peptide bond. Mature plasmepsin V exhibited a pH optimum of 5.5-7.0 with Km and kcat of 4.6 µM and 0.24s(-1), respectively, at pH 6.0 using the substrate DABCYL-LNKRLLHETQ-E(EDANS). Furthermore, the prosegment of proplasmepsin V was shown to be nonessential for refolding and inhibition. Unexpectedly, unprocessed proplasmepsin V was enzymatically active with slightly reduced substrate affinity (∼ 2-fold), and similar pH optimum as well as turnover compared to the mature form. Both zymogenic and mature plasmepsin V were partially inhibited by pepstatin A as well as several KNI aspartic protease inhibitors while certain metals strongly inhibited activity. Overall, the present study provides the first report on the nonessentiality of the prosegment for plasmepsin V folding and activity, and therefore, subsequent characterization of its structure-function relationships of both zymogen and mature forms in the development of novel inhibitors with potential antimalarial activities is warranted.

Rational redesign of porcine pepsinogen containing an antimicrobial peptide.

A novel strategy for the controlled release and localization of bioactive peptides within digestive and immunity-related enzymes was developed. The N-terminus of porcine pepsinogen A was fused to the basic amino acid-rich region of bovine lactoferricin B termed 'tLfcB', a cationic antimicrobial/anticancer peptide. Recombinant tLfcB-porcine pepsinogen A was expressed in soluble form in Escherichia coli as a thioredoxin (Trx) fusion protein. Thioredoxin-tLfcB-porcine pepsinogen A was found to activate autocatalytically under acidic conditions. Recombinant pepsin A derived from the activation of the fusion protein had a catalytic rate and substrate affinity similar to that derived from the recombinant thioredoxin-porcine pepsinogen A control. Pepsin-treated thioredoxin-tLfcB-porcine pepsinogen A yielded increased antimicrobial activity against the Gram-negative bacteria E.coli relative to control suggesting that a second function (antimicrobial activity) was successfully engineered into a functional peptidase. The novel design strategy described herein presents a potential strategy for targeted delivery of antimicrobial or therapeutic peptides in transgenic organisms via re-engineering native proteins critical to plant and animal defense mechanisms.

The structure and function of Saccharomyces cerevisiae proteinase A.

Saccharomyces cerevisiae proteinase A (saccharopepsin; EC 3.4.23.25) is a member of the aspartic proteinase superfamily (InterPro IPR001969), which are proteolytic enzymes distributed among a variety of organisms. Targeted to the vacuole as a zymogen, its activation at acidic pH can occur by two different pathways, a one-step process to release mature proteinase A, involving the intervention of proteinase B, or a step-wise pathway via the autoactivation product known as pseudo-proteinase A. Once active, S. cerevisiae proteinase A is essential to the activities of other yeast vacuolar hydrolases, including proteinase B and carboxypeptidase Y. The mature enzyme is bilobal, with each lobe providing one of the two catalytically essential aspartic acid residues in the active site. The crystal structure of free proteinase A reveals that the flap loop assumes an atypical position, pointing directly into the S(1) pocket of the enzyme. With regard to hydrolysis, proteinase A has a preference for hydrophobic residues with Phe, Leu or Glu at the P1 position and Phe, Ile, Leu or Ala at P1', and is inhibited by IA(3), a natural and highly specific inhibitor produced by S. cerevisiae. This review is the first comprehensive review of S. cerevisiae PrA.

A C-terminal glycine suppresses production of pleurocidin as a fusion peptide in Escherichia coli.

The winter flounder (Pseudopleuronectes americanus) antimicrobial peptide pleurocidin was produced in Escherichia coli using a synthetic gene constructed by PCR. The gene expresses pleurocidin from pET21a fused to the C-terminus of an insoluble carrier peptide. Once expressed, the fusion peptide formed inclusion bodies in the cytoplasm that were collected, solubilized in guanidine-HCl, and chemically cleaved using hydroxylamine at a unique asparaginyl-glycyl dipeptide. This released recombinant pleurocidin (r-pleurocidin), which was purified using ultrafiltration followed by reverse phase chromatography. The r-pleurocidin peptide resolved as a single band (2.7 kDa) when analyzed by Tris-Tricine buffered SDS-PAGE, and its amino acid sequence was confirmed using tandem mass spectrometry. Extending the pleurocidin sequence with a C-terminal glycine (r-pleurocidin-G) suppressed production of the fusion peptide 15-fold. When pleurocidin was extended further to include aspartate (r-pleurocidin-GD), the same effect was observed, and when pleurocidin was extended with aspartate alone, no effect was observed. Expression of fusion peptide containing either r-pleurocidin-G or r-pleurocidin-GD with low concentrations of inductant caused E. coli to enter stationary phase prematurely, but did not affect overall growth rates. A partial production recovery of r-pleurocidin-G was achieved by inducing expression in stationary phase cells. We observed r-pleurocidin-G to have enhanced antimicrobial activity compared with r-pleurocidin, and we propose that this activity interferes with E. coli metabolism during expression. This antimicrobial effect is probably facilitated by residual solubility of the fusion peptide and by a C-terminal cap structure, which stabilizes the r-pleurocidin-G alpha-helix that is thought to be important for activity.

Recombinant expression and partial characterization of an active soluble histo-aspartic protease from Plasmodium falciparum.

Malaria aspartic proteases are attractive drug targets for the treatment of malaria, however, recombinant expression of active histo-aspartic proteinase (HAP) to facilitate its characterization has proven elusive. The present study reports on the first recombinant expression of soluble, active histo-aspartic proteinase from Plasmodium falciparum as a thioredoxin fusion protein. A truncated form of HAP (77p-451) was fused to thioredoxin in the pET32b(+) vector and the fusion protein (Trx-tHAP) was expressed in Escherichia coli Rosetta-gami B (DE3)pLysS. The fusion protein was partially purified from the culture medium using a combination of anion exchange and Ni(2+) affinity chromatography. Soluble tHAP was subsequently purified by enterokinase treatment and removal, followed by gel filtration chromatography. Although truncated HAP was incapable of autocatalytic activation, enterokinase digestion of partially purified fusion protein released the truncated prosegment yielding a mature form of tHAP (mtHAP). N-terminal sequencing of mtHAP indicated that enterokinase cleavage took place at Lys119-Ser120, four residues upstream of the native cleavage site (Gly123-Ser124). Initial activity tests showed that mtHAP was capable of hydrolyzing acid-denatured globin as well as cleavage of the synthetic substrate EDANS-CO-CH(2)-CH(2)-CO-ALERMFLSFP-Dap(DABCYL)-OH. Inhibition studies showed that the activity of mtHAP was completely inhibited by pepstatin A and to a lesser degree, PMSF. Using the synthetic substrate, mtHAP showed a pH optimum of 5.2, and Km=3.4 microM and kcat=1.6 x 10(-3)s(-1). The successful expression of active recombinant HAP from E. coli will accelerate the investigation of the structure-function relationships of HAP and facilitate the development of specific inhibitors with antimalarial activities.

N-terminal modifications increase the neutral-pH stability of pepsin.

A structure-function study was undertaken to determine the effects of N-terminal mutations in pepsin designed to introduce the Lys-X-Tyr motif and increase N-terminal flexibility. At pH 7.0, E7K/T12A/E13Q pepsin was inactivated more slowly compared to WT, whereas the mutants E7K and T12A/E13Q were not stabilized. Far-UV circular dichroism revealed that changes in secondary structure accompanied the inactivation process, and that the structural changes occurred at approximately the same rate as inactivation. All of the inactivated pepsin forms showed retention of substantial secondary structure, more than previously determined for pepsin denatured at pH 7.2 and 8.0, suggesting the presence of a structural intermediate at pH 7.0. The coupled mutations at positions 12 and 13 impacted the pH dependence of activity at pH 0.9, lowered affinity for a synthetic substrate, and lowered the turnover number. The introduction of Lys at position 7 apparently destabilized the interaction between prosegment-enzyme body as evidenced by activation at higher pH (>or= 4.0) compared to WT, but showed no change for pH dependence of activity, nor a statistically significant change in affinity for the synthetic substrate.