Brendon Conlan

Circular proteins and mechanisms of cyclization

Cyclization via head‐to‐tail linkage of the termini of a peptide chain occurs in only a small percentage of proteins, but engenders the resultant cyclic proteins with exceptional stability. The mechanisms involved are poorly understood and this review attempts to summarize what is known of the events that lead to cyclization. Cyclic proteins are found in both prokaryotic and eukaryotic species. The prokaryotic circular proteins include the bacteriocins and pilins. The eukaryotic circular proteins in mammals include the theta defensins, found in rhesus macaques, and the retrocyclins. Two types of cyclic proteins have been found in plants, the sunflower trypsin inhibitor and the larger, more prolific, group known as cyclotides. The cyclotides from Oldenlandia affinis, the plant in which these cyclotides were first discovered, are processed by an asparaginyl endopeptidase which is a cysteine protease. Cysteine proteases are commonly associated with transpeptidation reactions, which, for suitable substrates can lead to cyclization events. These proteases cleave an amide bond and form an acyl enzyme intermediate before nucleophilic attack by the amine group of the N‐terminal residue to form a peptide bond, resulting in a cyclic peptide.

Efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase

Cyclotides are diverse plant backbone cyclized peptides that have attracted interest as pharmaceutical scaffolds, but fundamentals of their biosynthetic origin remain elusive. Backbone cyclization is a key enzyme-mediated step of cyclotide biosynthesis and confers a measure of stability on the resultant cyclotide. Furthermore, cyclization would be desirable for engineered peptides. Here we report the identification of four asparaginyl endopeptidases (AEPs), proteases implicated in cyclization, from the cyclotide-producing plant Oldenlandia affinis. We recombinantly express OaAEP1b and find it functions preferably as a cyclase by coupling C-terminal cleavage of propeptide substrates with backbone cyclization. Interestingly, OaAEP1b cannot cleave at the N-terminal site of O. affinis cyclotide precursors, implicating additional proteases in cyclotide biosynthesis. Finally, we demonstrate the broad utility of this enzyme by cyclization of peptides unrelated to cyclotides. We propose that recombinant OaAEP1b is a powerful tool for use in peptide engineering applications where increased stability of peptide products is desired.

The evolution of Photosystem II: insights into the past and future

This article attempts to address the molecular origin of Photosystem II (PSII), the central component in oxygenic photosynthesis. It discusses the possible evolution of the relevant cofactors needed for splitting water into molecular O2 with respect to the following functional domains in PSII: the reaction center (RC), the oxygen evolving complex (OEC), and the manganese stabilizing protein (MSP). Possible ancestral sources of the relevant cofactors are considered, as are scenarios of how these components may have been brought together to produce the intermediate steps in the evolution of PSII. Most importantly, the driving forces that maintained these intermediates for continued adaptation are considered. We then apply our understanding of the evolution of PSII to the bioengineering of a water oxidizing catalyst for utilization of solar energy.

Photo-catalytic oxidation of a di-nuclear manganese centre in an engineered bacterioferritin 'reaction centre'

Photosynthesis involves the conversion of light into chemical energy through a series of electron transfer reactions within membrane-bound pigment/protein complexes. The Photosystem II (PSII) complex in plants, algae and cyanobacteria catalyse the oxidation of water to molecular O2. The complexity of PSII has thus far limited attempts to chemically replicate its function. Here we introduce a reverse engineering approach to build a simple, light-driven photo-catalyst based on the organization and function of the donor side of the PSII reaction centre. We have used bacterioferritin (BFR) (cytochrome b1) from Escherichia coli as the protein scaffold since it has several, inherently useful design features for engineering light-driven electron transport. Among these are: (i.) a di-iron binding site; (ii.) a potentially redox-active tyrosine residue; and (iii.) the ability to dimerise and form an inter-protein heme binding pocket within electron tunnelling distance of the di-iron binding site. Upon replacing the heme with the photoactive zinc-chlorin e6 (ZnCe6) molecule and the di-iron binding site with two manganese ions, we show that the two Mn ions bind as a weakly coupled di-nuclear Mn2II,II centre, and that ZnCe6 binds in stoichiometric amounts of 1:2 with respect to the dimeric form of BFR. Upon illumination the bound ZnCe6 initiates electron transfer, followed by oxidation of the di-nuclear Mn centre possibly via one of the inherent tyrosine residues in the vicinity of the Mn cluster. The light dependent loss of the MnII EPR signals and the formation of low field parallel mode Mn EPR signals are attributed to the formation of MnIII species. The formation of the MnIII is concomitant with consumption of oxygen. Our model is the first artificial reaction centre developed for the photo-catalytic oxidation of a di-metal site within a protein matrix which potentially mimics water oxidation centre (WOC) photo-assembly.

Insights into processing and cyclization events associated with biosynthesis of the cyclic peptide kalata B1

Background: Production of insecticidal and nematocidal cyclic peptides is inefficient in transgenic plants. Results: Efficient cyclization requires cleavage of the N-terminal propeptide from the mature cyclotide domain and a C terminus containing a binding motif. Conclusion: The cyclization motif has Asn at position P1, a small amino acid at position P1′, and a Leu at position P2′. Significance: Understanding substrate requirements will help produce cyclotides in transgenic plants. Plant cyclotides are the largest family of gene-encoded cyclic proteins. They act as host defense molecules to protect plants and are promising candidates as insecticidal and nematocidal agents in agriculture. For this promise to be realized a greater understanding of the post-translational processing of these proteins is needed. Cyclotides are cleaved from precursor proteins with subsequent ligation of the N and C termini to form a continuous peptide backbone. This cyclization step is inefficient in transgenic plants and our work aims to shed light on the specificity requirements at the excision sites for cyclic peptide production. Using the prototypic cyclotide kalata B1 (kB1) expressed from the Oak1 gene, MALDI-TOF mass spectrometry was used to examine the cyclization efficiency when mutants of the Oak1 gene were expressed in transgenic Nicotiana benthamiana. Cleavage at the N terminus of the cyclotide domain occurs rapidly with no strict specificity requirements for amino acids at the cleavage site. In contrast, the C-terminal region of the cyclotide domain in the P2, P1, P1′, and P2′ positions is highly conserved and only specific amino acids can occupy these positions. The cyclization reaction requires an Asn at position P1 followed by a small amino acid (Ala, Gly, Ser) at the P1′ position. The P2′ position must be filled by Leu or Ile; in their absence an unusual post-translational modification occurs. Substitution of the P2′ Leu with Ala leads to hydroxylation of the neighboring proline. Through mutational analysis this novel proline hydroxylation motif was determined to be Gly-Ala-Pro-Ser.

The changing of the guard: the Pto/Prf receptor complex of tomato and pathogen recognition

One important model for disease resistance is the Prf recognition complex of tomato, which responds to different bacterial effectors. Prf incorporates a protein kinase called Pto as its recognition domain that mimics effector virulence targets, and activates resistance after interaction with specific effectors. Recent findings show that this complex is oligomeric, and reveal how this impacts mechanism. Oligomerisation brings two or more kinases into proximity, where they can phosphorylate each other after effector perception. Effector attack on one kinase activates another in trans, constituting a molecular trap for the effector. Oligomerisation of plant resistance proteins may be a general concept that broadens pathogen recognition and restricts the ability of pathogens to evolve virulence.

Engineering model proteins for Photosystem II function

Our knowledge of Photosystem II and the molecular mechanism of oxygen production are rapidly advancing. The time is now ripe to exploit this knowledge and use it as a blueprint for the development of light-driven catalysts, ultimately for the splitting of water into O2 and H2. In this article, we outline the background and our approach to this technological application through the reverse engineering of Photosystem II into model proteins.

Subcellular targeting and biosynthesis of cyclotides in plant cells

PREMISE OF THE STUDY The cyclotide kalata B1 is found in the leaves of Oldenlandia affinis and is a potent insecticidal and nematocidal molecule. This peptide is cleaved from a precursor protein, Oak1, and ligation of the N- and C-termini occurs to form a continuous peptide backbone. The subcellular location of the excision and cyclization reactions is unknown, and there is debate as to which enzyme catalyzes the event. To determine where in the plant cell Oak1 is processed, we prepared constructs encoding GFP (green fluorescent protein) linked to the cyclotide precursor Oak1. METHODS The GFP constructs were transiently expressed in the leaves of Nicotiana benthamiana, and GFP fluorescence was observed in living cells using confocal microscopy. A Fei Mao (FM) styryl dye was infiltrated into whole leaves that were still growing and expressing GFP constructs, enabling the plasma membrane and the tonoplast to be highlighted for visualization of the vacuole in living cells. KEY RESULTS The full length Oak1 precursor directed GFP to the vacuole, suggesting that excision and cyclization of the cyclotide domain occurs in the vacuole where the cyclotides are then stored. The N-terminal propeptide and N-terminal repeat of Oak1 were both sufficient to target GFP to the vacuole, although the C-terminal propeptide, which is essential for cyclization, was not a targeting signal. CONCLUSIONS The vacuolar location of cyclotides supports our hypothesis that the vacuolar processing enzyme, asparaginyl endoproteinase, has a pivotal role in excision and cyclization from cyclotide precursors.

The N-terminal domain of the tomato immune protein Prf contains multiple homotypic and Pto kinase interaction sites.

Background: Plant immune proteins display complex conformations. Results: The Prf N-terminal domain forms a homo-dimer, has two binding sites for Pto kinase, and interacts with the Prf leucine-rich repeats domain. Conclusion: The Prf N-terminal domain coordinates multiple domain interactions to control the activity of the immune complex. Significance: Additional resolution is supplied to the Prf-Pto complex. Resistance to Pseudomonas syringae bacteria in tomato (Solanum lycopersicum) is conferred by the Prf recognition complex, composed of the nucleotide-binding leucine-rich repeats protein Prf and the protein kinase Pto. The complex is activated by recognition of the P. syringae effectors AvrPto and AvrPtoB. The N-terminal domain is responsible for Prf homodimerization, which brings two Pto kinases into close proximity and holds them in inactive conformation in the absence of either effector. Negative regulation is lost by effector binding to the catalytic cleft of Pto, leading to disruption of its P+1 loop within the activation segment. This change is translated through Prf to a second Pto molecule in the complex. Here we describe a schematic model of the unique Prf N-terminal domain dimer and its interaction with the effector binding determinant Pto. Using heterologous expression in Nicotiana benthamiana, we define multiple sites of N domain homotypic interaction and infer that it forms a parallel dimer folded centrally to enable contact between the N and C termini. Furthermore, we found independent binding sites for Pto at either end of the N-terminal domain. Using the constitutively active mutant ptoL205D, we identify a potential repression site for Pto in the first ∼100 amino acids of Prf. Finally, we find that the Prf leucine-rich repeats domain also binds the N-terminal region, highlighting a possible mechanism for transfer of the effector binding signal to the NB-LRR regulatory unit (consisting of a central nucleotide binding and C-terminal leucine-rich repeats).

Circular micro-proteins and mechanisms of cyclization

Transpeptidation reactions result in the formation of new peptide bonds and this can occur between two separate peptides or within the one peptide. These reactions are catalyzed by enzymes and when the N- and C-terminus of the one peptide are joined it results in the formation of cyclic proteins. Cyclization via head-to-tail linkage of the termini of a peptide chain occurs in only a small percentage of proteins but gives the resultant cyclic proteins exceptional stability. The mechanisms are not well understood and this review documents what is known of the events that lead to cyclization. Gene encoded cyclic proteins are found in both prokaryotic and eukaryotic species. The prokaryotic circular proteins include the bacteriocins and pilins. The eukaryotic circular proteins in mammals include the θ-defensins and retrocyclins. Small cyclic proteins are also found in fungi and a large range of cyclic proteins are expressed in cyanobacteria. Three types of cyclic proteins have been found in plants, the small cyclic proteins of 5-12 amino acids, the cyclic proteins from sunflower which are made up of 12-14 amino acids, and the larger group known as cyclotides which contain 28-37 amino acids. Three classes of enzymes are able to catalyse transpeptidation reactions, these include the cysteine, serine and threonine proteases. However only cysteine and serine proteases have been documented to cyclize proteins. The cyclotides from Oldenlandia affinis, the plant in which cyclotides were first discovered, are processed by an asparaginyl endopeptidase which is a cysteine protease. These proteases cleave an amide bond and form an acyl enzyme intermediate before nucleophilic attack of the amine group of the N-terminal residue to form a peptide bond, resulting in a cyclic peptide.