• Dual Boxing Programs L2272

Background Kexin-like proteinases are a subfamily of the subtilisin-like serine proteinases with multiple regulatory functions in eukaryotes. In the yeast Saccharomyces cerevisiae the Kex2 protein is biochemically well investigated, however, with the exception of a few well known proteins such as the α-pheromone precursors, killer toxin precursors and aspartic proteinase propeptides, very few substrates are known. Fungal kex2 deletion mutants display pleiotropic phenotypes that are thought to result from the failure to proteolytically activate such substrates. Conclusion Statistical analysis of the cleavage sites revealed extended subsite recognition of negatively charged residues in the P1', P2' and P4' positions, which is also reflected in construction of the respective binding pockets in the ScKex2 enzyme. Additionally, we provide evidence for the existence of structural constrains in potential substrates prohibiting proteolysis. Furthermore, by using purified Kex2 proteinases from S.

Cerevisiae, P. Albicans and C. Glabrata, we show that while the substrate specificity is generally conserved between organisms, the proteinases are still distinct from each other and are likely to have additional unique substrate recognition.

Site specific proteolysis is a common feature in protein maturation and plays a crucial role in activation of many enzymes and in the generation of peptide hormones. In the late secretory pathway of eukaryotic cells this mechanism is mainly mediated by kexin-like proteinases, a subfamily of the subtilisin-like serine proteinases. Multicellular eukaryotes possess a large family of these regulatory proteinases, termed prohormone or proprotein convertases.

While in mammals this family consists of at least seven members with tissue-specific expression patterns (most recently reviewed in ), fungi harbour only a single gene coding for a subtilisin-like serine proteinase with this activity. Originally identified in kex2 mutants of Saccharomyces cerevisiae lacking the ability to process the virally encoded killer toxin ( killer expression) the fungal Kex2 protein has since been implicated in several other proteolytic activation events, e.g. Pheromone maturation at lysine-arginine motifs.

Cerevisiae Kex2 protein has been the target of substantial biochemical and crystallographic (reviewed in ) research. Apart from S. Cerevisiae, a diverse spectrum of phenotypic descriptions has been published for a range of kex2 deletion mutants from other yeasts, such as Candida albicans , , C. Glabrata , Pichia pastoris , Schizosaccharomyces pombe , or Yarrowia lipolytica and moulds such as Aspergillus niger , A. Oryzae or Trichoderma reesei. The phenotypes of these deletion mutants include morphological changes that are thought to result from the lack of activity from cell-wall modifying enzymes, reduced virulence in the case of C. Albicans , hypersensitivity to antimycotic drugs that target cell wall or plasma membrane integrity in C.

Glabrata and inviability in S. In theory, the phenotypes of kex2 deletion mutants can be explained by the lack of processing events in substrate proteins rendering these dysfunctional, as in the case of the α-pheromone, where the lack of processing renders the kex2 mutant of S. Cerevisiae mating deficient.

Because of the localization of the Kex2 protein in the late trans Golgi network and an endocytic, prevacuolar compartment , it can be concluded that the target spectrum is limited to proteins attached to the cell surface, those proteins which are secreted into the environment or to the luminal domains of integral membrane proteins passing through these compartments. Accordingly, the phenotypes of kex2 mutants include the secretion of unprocessed protein precursors into the environment, e.g.

The secretory xylanase of T. However, these effects are blurred as the phenotypes observed from kex2 mutants may only be secondary effects themselves. Furthermore, missing Kex2-processing events may well be covered up by processing through other proteinases, such as the yapsins, a family of glycosylphosphatidylinositol (GPI) anchored aspartic proteinases ,. In the case of proteinase pro-peptides these events may also occur autocatalytically, as proposed for CaSap2. While there is a fair number of proteins that have been annotated as potential Kex2 targets and two earlier studies have predicted Kex2 targets , , the number of proteins for which experimental proof of cleavage by Kex2 exists, remains low.

Knowing the substrates of this proteinase would not only help to explain the phenotypes observed in fungal kex2 deletion mutants, but also provide insights into essential cellular regulatory mechanisms. We have aimed at providing an improved assembly of Kex2 target proteins and present first biochemical evidence for the processing of selected substrates, in particular from the human pathogenic yeasts C. Albicans and C. Furthermore, we provide evidence for extended subsite recognition in the P1'–P4' region.

By using recombinant Kex2 proteinases and potential substrate proteins from pathogenic and non-pathogenic yeasts, we show that the substrate specificity is generally conserved between organisms. However, our data also suggest that some Kex2 proteinases have additional unique substrates. Figure 1 Plasmid constructions for proteinase expression. (A) Schematic representation of the domain structure of fungal Kex2 proteins. Kex2 consists of a signal peptide, an autocatalytically removed pro-peptide, a catalytic domain, a structural P-domain, a transmembrane domain and finally a cytosolic domain containing sorting signals to the Golgi apparatus.

(B) For expression of C. Glabrata Kex2 the part of the gene fused with a C-terminal 6 × His tag was cloned into pPic3.5 using the BamHI and NotI restriction sites. (C) For expression of P. Pastoris Kex2 the part of the gene fused with a C-terminal 6 × His tag was cloned into pPic3.5 using the SnaBI and NotI restriction sites.

(D) For expression of C. Albicans Kex2 the part of the gene fused with a C-terminal 6 × His tag was cloned into pCIp10 using the HinDIII and NheI restriction sites. For the expression of the soluble forms of S.

Cerevisiae, C. Glabrata and P. Pastoris Kex2 enzymes the P. Pastoris expression system (Invitrogen) was used. The strain expressing S.

Cerevisiae Kex2 was a kind gift of Guy Boileau. For the expression of C. Glabrata and P. Pastoris Kex2 enzymes the 5' part of the gene coding for the luminal domain of the enzyme, including the native signal- and pro-peptide, plus a C-terminal 6 × His-tag were cloned into the pic3.5 vector (Figure and ) and transformed into P. Pastoris strain GS115. The transformants displaying the strongest extracellular proteolytic activity (ppCgKex2#12 and ppPpKex2#5) in test expressions were used for large-scale production of the enzymes.

Attempts to purify the C. Glabrata and P. Pastoris Kex2 enzymes via 6 × His-affinity chromatography were not successful, possibly due to burial of the epitope inside the protein. Thus, all three enzymes, including the one from S.

Cerevisiae, were purified to near homogeneity by a combination of anion exchange and size exclusion chromatography (Additional file ). Because several attempts to produce the intact, soluble form of Kex2 of C. Albicans in the Pichia system failed, ultimately the native host C. Albicans was used for production of this enzyme: the 5' part of the C. Albicans KEX2 gene coding for the luminal domain of the enzyme, again including the native signal- and pro-peptide as well as a C-terminal 6 × His tag was put under the control of the constitutive and strong promoter of the ACT1 gene, as described under Methods (Figure ). The linearized plasmid was transformed into C.

Albicans strain CAI4 and the transformant giving the strongest Kex2-like activity in the supernatant (CaAct1-Kex2#7) was used for further large scale production of the enzyme, as above. While we were able to produce the high Kex2 activity in supernatants, the efficiency of its purification remained low. Highest yields of enzyme were achieved using complex media including yeast extract and peptone, but this resulted in only impure enzyme preparations. However, the parental strain did not produce this activity (Additional file, Figure ). To avoid low-weight impurities in the enzyme preparations, which would have disguised product bands in further analytical experiments, the medium was passed over a 10 kDa size-exclusion column prior to the expression. In combination with the purification methods as outlined above this resulted in an enzyme preparation that contained only few other proteins and was devoid of low molecular weight contaminants. Activity testing of the enzyme preparations Prior to use, enzyme preparations were adjusted to a common activity of one nmol/min per μl proteinase added in a standard reaction setup with the chromogenic substrate Z-Tyr-Lys-Arg-pNA.

Neither preparations from a P. Pastoris negative control strain nor from the C. Albicans parental CAI4 strain displayed this activity (data not shown).

In addition we performed controls with the C. Albicans enzyme preparation to ascertain that the proteolytic activity was Kex2-dependent: The activity was indeed inhibited by PMSF, EDTA and ZnCl 2, but not by pepstatin A (Additional file, Figure ). To test whether the enzymes had similar properties we first tested the enzymes for optimal pH and temperature with the chromogenic substrate Z-Tyr-Lys-Arg-pNA. The optimal pH for all enzymes was between 7.2 and 7.4 (data not shown), as described earlier for the S.

Cerevisiae enzyme and this pH was therefore used throughout all further experiments. In contrast, the result for the optimal temperature was surprising: all enzymes showed an elevated activity at unphysiological temperatures from 40°C to 50°C (data not shown), at which none of the source organisms display optimal growth, if any. Nevertheless, all following experiments were carried out at 37°C, reflecting human body temperature, as our main focus lay on the enzymes of the human pathogenic fungi C. Albicans and C. Since the KEX2 gene of C. Albicans can complement the kex2 deletion in S. Cerevisiae and the KEX2 gene from S.

Cerevisiae can complement the loss of the KEX2-ortholog KRP1 in Schizosaccharomyces pombe , it is feasible to assume that these enzymes have similar to identical biological functions and biochemical properties. To show that this is also the case for the Kex2 proteinases from C. Glabrata and P. Pastoris, we tested whether all four proteinases cleaved the S. Cerevisiae α-mating pheromone, a natural and proven substrate of Kex2 from S. Cerevisiae, in a similar manner (Figure ): The α-mating pheromone precursor protein was purified using the pET100-D E. Free download facebook software for nokia n97 mini gold.

Coli expression system as described below for the other substrate proteins. Indeed, the four proteinases showed the same digestion pattern of the pheromone precursor into the expected fragments of the N-terminal 11 kDa peptide and peptides of 2–3 kDa size (Figure ).

Figure 2 Activity testing of the purified Kex2 enzymes. The proteinases were tested with a proteinacious model substrate, the α-pheromone of S. Digestion of the substrate protein (20 KDa) with the different proteinases resulted in the same expected pattern of products (11 and 2–3 KDa). Cerevisiae Kex2, Ca: C. Albicans Kex2, Cg: C.

Glabrata Kex2 and Pp: P. Pastoris Kex2. Prediction of potential Kex2 substrates Next, we developed a prediction method for potential Kex2 cleavage sites in substrate proteins to identify proteins from C. Glabrata or S. Cerevisiae for testing with the proteinases.

Earlier studies used very stringent search parameters and only looked in the N-terminal region of protein sequences. However, there is biochemical and biological evidence for processing of sites containing other amino acids in the P2 position as well as activity on C-terminal motifs in other organisms such as the chloroperoxidase CPO of A. Niger and on membrane proteins such as Kex2 itself. Therefore we included Golgi-luminal portions of transmembrane proteins as well as full-length sequences of soluble proteins into our search. ER-retained proteins were excluded, as they should not come into contact with Kex2. The predicted set of proteins was screened for potential cleavage sites using a position specific scoring matrix (PSSM) (Table, columns P4 to P1). This also allowed for a ranking of the sites found.

The matrix used for the prediction of Kex2 substrate proteins was derived from systematic biochemical and genetic data generated with the S. Cerevisiae enzyme ,. All proteins with potential Kex2 cleavage sites were aligned with orthologous proteins of other fungi. This allowed for investigation of conservation of the potential cleavage site between different proteins with similar biochemical properties. This search yielded a total of 467 cleavage sites in 297 individual proteins (112 from C. Albicans, 90 from C.

Glabrata and 95 from S. Cerevisiae) which presumably pass the Golgi compartment. Selected substrate groups with conserved sites are shown in Additional file. From the 297 predicted potential Kex2 substrate proteins we selected a total of 43 proteins (three of S. Cerevisiae, 26 of C.

Albicans and 14 of C. Glabrata) for heterologous expression in E. Coli (Additional file ).

These were chosen to cover a wide range of different cleavage sites and protein types and expressed using the TOPO-pET D100 system. The DNA fragments cloned were devoid of domains encoding signal peptides and putative GPI-anchor sequences.

Out of these selected proteins, we were able to express and purify thirteen from C. Albicans, ten from C. Glabrata and one from S. Cerevisiae (the α-pheromone mentioned above).

Since the majority of the chosen proteins accumulated as inclusion bodies, we converted these proteins into a soluble form by on-column refolding. To test for overall correct folding of the refolded proteins, we performed an activity test for the substrate CA5147, an acid phosphatase, which was the only protein with a known activity in this set. Indeed, we were able to confirm the activity of this protein and observe a maximum activity at pH 4.2–4.3 (Figure ) using para-nitrophenol phosphate as a substrate. This shows that at least some refolded protein assumes its native structure and can thus be used for specific proteolysis assays. Very few studies provided experimental evidence that predicted Kex2 cleavage sites in potential substrate proteins are in fact processed by Kex2 proteinases.

In order to determine susceptibility of the purified proteins to proteolytic processing by Kex2, all potential substrate proteins purified above were digested with each of the four proteinases. A selection of digestions is depicted in Figure. Based on the scores given by the algorithm, we expected most proteins to be cleaved. Indeed, we observed rapid cleavage at the predicted cleavage sites for 2/3 of the proteins.

This also included cleavage at sites with lower scores in polypeptide precursors (e.g. CaEce1, position 92, Additional file ). In contrast, some proteins were not cleaved even though they contained sites with high scores, such as CaCcw14 (see Additional file ).

Furthermore, one protein (CA0365) was cleaved very differently by the proteinases: while it was not cleaved at all by ScKex2, CaKex2 rapidly processed the precursor into peptide sized fragments, without any noteworthy appearance of intermediates under the standard reaction conditions (Figure ). A similar activity was observed with CgKex2 and PpKex2, while at a considerably slower rate of hydrolysis. Figure 4 Proteolytic digests of putative substrate proteins. (A) Recombinant substrate proteins were digested with each of the four proteinases. Control, Sc: S.

Cerevisiae Kex2, Ca: C. Albicans Kex2, Cg: C. Glabrata Kex2 and Pp: P. Pastoris Kex2. Potential fragment sizes are given in kDa underneath the names (vertical bars: potential cleavage sites).

All digests were visualized in silver stained gels, except CaSun41, where the N-terminal X-press epitope was detected in a Western blot. Proteins are digested at all major substrate sites found in the sequence and for most, intermediate products can be observed. Proteins not hydrolysed by Kex2 are not depicted (see text). Substrate CA0365 is not processed by ScKex2, but by all other proteinases, most efficiently by CaKex2. Proteins that were cleaved into fragments of the expected sizes were CA0365, CaEce1 (CA1402), CA1873, CA2974, CaPga17(CA4679), CaTos1 (CA2303), CaSun41 (CA0883), CgScw4 (CAGL0M13805g), CgSUN4 (CAGL0L05434g), CgPir1 (CAGL0M08492g), CgPry1 (CAGL0F05137g) and CgPry2 (CAGL0G07667g). Proteins that remained fully uncleaved were CaCcw14 (CA2942), CaPho11 (CA5147), CaRbt4 (CA0104), CaCrh1 (CA0375), the Plb-homolog CAGL0J11770g and the three proteins of unknown function CA1394, CAGL0H08910g and CAGL0A02277g.

Dual Boxing Programs L2272

The pattern of cleavage vs. Non-cleavage observed was not sufficiently explained by the score calculated from the prediction algorithm among the proteins tested.

Therefore, we inspected the amino acid distribution surrounding the investigated and other known cleavage sites for other patterns: indeed, a high overrepresentation of negatively charged (aspartic/glutamic acid) and small (alanine, valine, leucine) residues in the P1', P2' positions and a similar moderate overrepresentation in the P4' position was found, while positively charged residues were underrepresented at those substrate sites which were digested. In the case for the sites not cleaved, no over- or under-representation was observed (Figure ).

Figure 5 Statistical sequence analysis of predicted Kex2 cleavage sites. (A) sequence logo of cleaved sites and (B) of non-cleaved sites.

Position 5 of the logos corresponds to the P1 position in the substrate. Negatively charged residues (red) are overrepresented in processed substrate P1', P2' and P4' positions. Color key: red: negatively charged, blue: positively charged, black: apolar, green: polar.

Reflection of substrate recognition in proteinase structure Next, we asked whether the apparent preference for negatively charged residues in the P1'–P4' region of substrates digested by Kex2 proteins is reflected by the structure of the proteinases in the substrate binding cleft. Recently, a 3D model of the bacterial subtilisin kumamolisin of Bacillus novospec was published. The enzyme studied there was incapable of autoproteolytic activation thus retaining the pro-peptide. By superimposition with the coordinate sets of S. Cerevisiae Kex2 and Mus musculus furin we were able to investigate the potential substrate binding pockets in the P1' – P4' region (Figure ) as outlined by the intact pro-peptide cleavage site still bound into the substrate binding cleft. Indeed, the P4-P1 positions of the Kumamolisin pro-domain aligned with the known S4-S1 pockets of the enzymes (not shown), as well as the P1'–P3' positions with the S1'–S3' pockets predicted in the literature (Figure ).

Figure 6 Investigation of the three dimensional models of Kex2, furin and Kumamolisin for substrate binding properties. (A) Superimposed 3D coordinate sets for the three proteases reveal colocalization of the Kumamolisin propeptide residues with the predicted S1' and S2' binding pockets in furin and Kex2. A region identified for binding of the inhibitor Eglin-c (purple) is not involved in binding of the propeptide. (B) and (C) A potential S4' binding pocket is identified which is terminated by H369 in Kex2 and E262 in furin.

Numbering 'a' through 'E' refers to residues used in Table 2, which lists the respective binding pockets and references. The neighbouring S1' and S3' pockets are characterized by positive charges in ScKex2 (H213, H381) as well as in furin (R193, H194, H364), and both pockets may well accommodate aspartate or glutamate residues in the substrate. In furin, the excess charge possibly results in a stronger selection for negatively charged residues in the P1' position, but as the S2 pocket is directly adjacent to the S1' pocket, the lack of a positively charged P2 residue in furin substrates may compensate this effect.

The S2' pocket, located on the opposite side of the cleft, as well contains a terminal positive charge (R318 in ScKex2, R298 in furin) which would favour negatively charged residues in the P2' position. A potential P4' pocket was also identified (Figure and ). The P4' residue aligns between S363 and Q350 and extends towards E362 in the furin model (Figure ). The alignment with ScKex2 is of lesser quality in this region, but nevertheless a similarly built potential binding pocket is seen in the ScKex2 enzyme bordered by S380 and Y367 (Figure ). However, the equivalent to the negative terminal charge of E362 in furin would be the positive charge of H369 in ScKex2.

In summary, the structure of the enzymes explains the increased preference for negatively charged P1'–P4' residues in the substrates. Conservation of residues involved in substrate recognition. It is known from previous studies, that C. Albicans KEX2 can complement KEX2 in S. Cerevisiae and this gene in turn can complement the KEX2 ortholog KRP1 in S. Therefore, it must be concluded that the corresponding proteinases have similar substrate specificities and activities. Nevertheless, we have been able to show that at least in the case of one substrate (CA0365) the proteinases of S.

Cerevisiae and C. Albicans behave differently. To investigate whether this difference as well as the question whether or not the substrate specificity in general is the same in different fungi, we generated a sequence alignment of Kex2-orthologous proteins from fungi and furin-orthologous proteins from mammals (Figure ) and investigated the residues involved in substrate recognition (Table ) for their degree of conservation between the different species. Figure 7 Sequence Alignment of fungal Kex2-like proteins.

A protein sequence alignment of the residues involved in substrate specificity determination shows that the electrostatic properties of the binding regions are highly conserved. Red: positive charges, blue: negative charges, orange: polar residues, green: apolar residues,: Propeptide cleavage site, numbering 'a' through 'E' refers to residues used in Table 2, which lists the respective binding pockets and references. The S1 pocket (composed of positions p, t, u, x, y and C) is fully conserved and among fungi this is also true for the four negative charges of the S2 pocket (positions a, b, c and d). Interestingly, we observed for the S4 and the S1' position that the enzymes from Ascomycetales combine the charge-selective properties of the S. Cerevisiae Kex2 enzyme with those from the furin enzymes, and thus probably display the most discrete substrate recognition.

Among the Saccharomycetales the residues are conserved for the major subsites S4, S2, S1 and S1' with minor exceptions only. Differences are visible in subsites where there is no strong selection to or discrimination against substrate residues, such as the S5 pocket (positions q and r). The S2' pocket is generally positively charged, however, this charge is mediated by one histidine in either the v or the w position. In summary, it is seen, that the substrate selectivity among Saccharomycetales Kex2 enzymes is very conserved, and that there are no substitutions that would explain the differential processing of substrate CA0365 between the four proteinases. Therefore, the enzymes must discriminate their substrates either through further subsites or through processes independent of the primary sequence surrounding the cleavage site.

Relevance of substrate structural features for cleavage. During the in vitro cleavage experiments, we observed that proteins purified from the soluble fraction of E. Coli lysates were generally processed more efficiently than those purified and refolded from inclusion bodies. Therefore, we predicted that the three dimensional structure of the substrate and the exposure of the putative processing site on the protein surface is crucial for processing to occur. To investigate this further, we tested if sites that were readily cleaved in the native protein were still cleaved in a denatured form of the protein: two substrate proteins that were readily cleaved by ScKex2 (CaEce1 and CA1873) were heat denatured prior to addition of the ScKex2 proteinase (Figure ). As expected, both were cleaved less in the denatured form. This effect is more pronounced for CA1873 than for CaEce1, as CaEce1 contains seven equal cleavage sites and is thus generally more prone to processing than CA1873.

The reduced cleavage of partially denatured/refolded proteins can be explained by either inaccessibility of the site due to burial in the denatured structure or by the failure to form a specific secondary structure needed for processing. Figure 8 Relevance of proper folding for proteolysis. (A) Sites readily cleaved in the native protein (nat) are cleaved less in heat denatured protein (denat). Shown are the results for CaEce1 and CA1873. (B) Potential sites not cleaved in the native protein are cleaved when exposed to the environment by fusion between GST and GFP (see text). Also, we did not observe cleavage for all proteins with potentially good sites. Therefore, we tested if this was due to an uncleavable primary sequence or if there were structural constraints preventing cleavage: site 3 of CaCcw14 and site 1 of CA0365, were each fused between a GST and a GFP domain and so exposed to the solvent.

The GST-CA0365 -GFP fusion protein was not cleaved (Figure, lane 2), indicating that this sequence is not a substrate of ScKex2 and the non-cleavage of the full length protein is not due to structural constraints, as was expected due to the cleavage by the other three Kex2 enzymes. In contrast, the GST-CaCcw14 -GFP fusion protein was readily cleaved by ScKex2 (Figure, lane 5), demonstrating that this primary sequence reflects a good substrate and the non-cleavage in the full length protein must be due to structural constraints. This gives further evidence that accessibility and/or secondary structure of the cleavage site are essential for processing. The pleiotropic phenotype of fungal Kex2 deletion mutants is attributed to the lack of posttranslational, proteolytic activation of substrate proteins. Besides biochemical data describing the P4-P1 substrate recognition towards short peptides of the Saccharomyces cerevisiae enzyme, only very few data exist of substrate preferences of fungal Kex2 proteins.

Several proteins have been discussed as 'potential Kex2 substrates', however there is no experimental data confirming actual cleavage by Kex2, except for a few cases, e.g. Killer toxin, α-mating pheromones and proteinase propeptides.

In the present study, we have investigated cleavage of recombinant Kex2 proteinases on recombinant, potential Kex2 substrates in order to get a first insight into the possible substrate repertoire of these regulatory proteases. For heterologous production of soluble Kex2 enzymes, we selected the proteins from the two pathogenic fungi C.

Albicans and C. Glabrata, as the phenotypes of the respective deletion mutants include avirulence and increased susceptibility to antifungal compounds. In addition, we selected the well characterized S. Cerevisiae enzyme and the ortholog from Pichia pastoris, as this enzyme is often involved in the heterologous production of secretory proteins.

The Golgi-luminal domains of these four enzymes were expressed in the host P. Pastoris and purified from culture supernatant, except for Kex2 from C. Albicans, which was produced in C. Albicans itself, as it was not expressible in Pichia. The purified enzymes showed similar pH- and temperature dependencies: the optimal pH was found at pH 7.2, as reported for S. Cerevisiae Kex2 , but surprisingly maximum cleavage of the artificial substrate Z-TKR-pNA was observed at unphysiological temperatures ranging from 40°C to 55°C. The fact that the enzymes retain their catalytic activities at theses temperatures could reflect a stabilizing effect on the protein structure proposed for the P-domain of Kex2.

To identify new substrates of Kex2, we have searched the genomes of C. Glabrata and S. Cerevisiae, for secretory proteins containing potential cleavage sites. These were grouped into clusters by sequence similarity and based on the conservation of such sites selected for heterologous expression and in vitro cleavage testing by Kex2 enzymes (Additional file ). All four proteinases cleaved the S. Cerevisiae α-mating pheromone precursor in the same expected pattern, confirming the orthologous enzymatic activities of the proteins.

As it is known, that the C. Albicans and S. Pombe Kex2 proteins can complement the S. Cerevisiae Kex2 protein in vivo (30, 38), it was not surprising that almost all substrates were cleaved (or not cleaved) in an identical manner. However, one substrate (CA0365, Figure ) was differentially processed. This demonstrates that even though the proteins have very high sequence similarity they still have partially different substrate preferences.

Statistical sequence analysis of processed vs. Non-processed sites reveals an overrepresentation of negatively charged (aspartic/glutamic acid) or small residues in the P1', P2' and P4' positions, which has also been reported for substrates of the mammalian furin/PC proteinase family (Figure ). This finding is strengthened by the fact that a mutant of ScPir4, where the Kex2 cleavage site was changed from KR/D to KR/A failed to undergo processing. Previous biochemical analyses of substrate preference have focussed on the S1–S4 regions of the enzymes , , due to the nature of the substrates used in those studies. However, the solved three dimensional structures of S.

Cerevisiae and Mus musculus furin in complex with proteinaceous inhibitors such as Eglin-c have lead to the postulation of binding pockets also in the S1' and S2' regions. In order to identify further residues involved in substrate recognition in the S1'–S4' region, we have produced a structural alignment of S. Cerevisiae Kex2, M. Musculus furin and the bacterial Subtilisin-like proteinase kumamolisin of Bacillus novospec MN-32.

The latter structure was solved for an active-site mutated form of the protein, which still retained its propeptide. Due to the autocatalytic nature of the maturation process of subtilisin-like proteinases , the propeptide is the first substrate cleaved by the enzyme and should reflect an optimal substrate.

Indeed, the P1' residue of the Kumamolisin propeptide aligned with the predicted S1' binding pocket of the kexins (Figure ). In addition, we identified a potential S4 binding pocket, which in Kex2 terminates with the positively charged H369 (Figure ). A sequence alignment of residues involved in substrate recognition shows that these residues are generally very highly conserved among the enzymes investigated here (Figure ). Accordingly, there is no single residue that could explain the strong difference between ScKex2 and CaKex2 in cleavage of substrate CA0365. However, it is possible that a combination of such amino acid exchanges could generate such an effect.

In accordance with the experimental data, it is likely that the Kex2-ortholog enzymes of the Saccharomycetales exhibit a similar activity and the cleaved substrate pattern is comparable within these. However, for the enzymes from Ascomycetales it would be expected that they are more stringently selective for charged residues in the P4 and P1' position. In addition to the very important direct enzyme-substrate interactions outlined here, other parameters must influence substrate recognition by Kex2 proteinases: the reduced cleavage of heat denatured protein shows that a site must be properly folded to be accessible. This view is strongly supported by the fact, that a potentially preferred substrate (CaCcw14) remains uncleaved in its native context but becomes cleavable, when exposed to the proteinase in a fusion protein (Figure ). In our experiments 1/3 of the selected proteins remained uncleaved. Hence, to properly identify proteinase substrates, it is essential to include further parameters such as substrate structure in addition to primary sequence into the prediction algorithm.

Our data provide information beyond those previous data based on in silico predictions or assays with small peptides only. By using heterologous expressed proteases and substrates we were able to show the potential of each of the investigated Kex2 enzymes to digest selected putative substrates. However, further in vivo experiments are necessary in future studies to undoubtedly infer proteolytic maturation of these substrates. Aside from α-mating pheromone- and killer toxin precursors, the only previously experimentally proven Kex2 substrates are the glycolytic enzymes Exg1 of S. Cerevisiae and Xylanases of T.

Reesei , the aspartic proteinase CaSap2 , the structural cell wall Pir protein family and the hydrophobin Rep1 of Ustilago maydis. In our experiments we were able to confirm processing by Kex2 for the cell wall modulating enzymes CaSun41 (CA0883), CgScw4 (CAGL0M13805g) and CgSun4 (CAGL0L05434g) and for CgPir1, which had been predicted to be Kex2 substrates in earlier in silico searches ,.

Additionally, we observed in vitro cleavage for several proteins which have not previously been discussed as Kex2 substrates such as CaEce1, a group of Ops4-like proteins and two members of the Pry-protein family. In our tests three proteins of the 'plant pathogenicity related' Pry-protein family (CaRbt4, CgPry1 and CgPry2) were included. The proteins of this family contain a strongly conserved KR-motif (see Additional file ), but the proteins are not cleaved in a similar pattern: While CgPry1 is cleaved efficiently, CaRbt4 is not cleaved at all and CgPry2 only very slowly. It is therefore likely, that the conserved site of the Pry proteins is not cleaved in the fully native protein, and that processing of CgPry1 only takes place in the additional sites not present in the other two proteins. The major phenotype described for kex2 deletion mutants in Candida revolves around morphological defects of the cell wall and the resulting hypersensitivity to compounds interfering with the surface integrity. Several Kex2 target proteins directly interact with the fungal cell wall or are structural components thereof: the Pir proteins, glucanases such as Exg1, or proteins of the Sun/Scw family. While the direct consequence of failure to mature is not known for these proteins, the phenotypes of the respective deletion strains resemble those of kex2 deletion strains: mutants lacking cell wall localized glucanases such as ScExg1 or CaBgl2 and mutants lacking members of the Pir or the SUN-family show similar increased sensitivities towards several cell wall or membrane perturbing compounds ,.

Here it is interesting, that the Kex2 cleavage site is found in several but not in all glucanases. Additionally, Pir deletions result in the formation of cell aggregates , which is also be seen in the S.

Cerevisiae sun4 and C. Albicans sun41 deletion strains and are also observed in C. Glabrata kex2 deletion strains (data not shown). Furthermore, a S. Cerevisiae scw4/scw10 double mutant and a C. Albicans sun41 strain showed enlarged cells , a phenotype which can also be observed in the C. Glabrata kex2 mutant (data not shown).

Furthermore, calcofluor white stained C. Albicans kex2 cells show an abberant staining pattern , which would be in agreement with the potential changes in chitin deposition as seen from the abberant septum processing in C.

Albicans sun41 strains. The Kex2 cleavage site in Sun4- and Scw10-like proteins is preceded by an N-terminal stretch of positively charged amino acids, mainly histidines (see Additional file ).

This feature, which we termed 'His-Box', is also found in Tos1 proteins, only here it is located further inside the protein and is additionally preceded by another Kex2 cleavage site. It can be speculated that, if this motif was involved in cell wall attachment, processing would lead to differential localization of the mature protein, e.g. Secretion as observed in C. Albicans for Sun41 and Tos1. Besides explaining previously observed phenotypes, the identification of cleavage sites may yield additional functional information about a protein: the expression of CaEce1 is tightly associated with hyphae in C.

Albicans, but the deletion has no apparent effect on morphology and no function could be assigned to this protein. While there is no sequence homology, the polypeptide precursor structure of CaEce1, and also that of CA0365, resemble that of the repellent protein Rep1 of Ustilago maydis. The UmRep1 protein contains ten strongly conserved repeats separated by Kex2 cleavage sites and a longer terminal fragment with no similarity to the repeats (Figure ). CA0365 is shorter, with only three conserved repeats each containing another internal Kex2 cleavage site, but no terminal fragment. In CaEce1, the seven repeats are less conserved, but the longer, terminal fragment is present.

UmRep1 functions as a structural component of aerial hyphae and CaEce1 or CA0365 might play similar roles on the hyphae of C. All three proteins seem to have in common that a processing via Kex2 proteinases may be necessary for their proper biological function. Figure 9 Schematic representation of polypeptide- and Ops4-like substrates. Kex2 cleavage sites are represented by vertical bars. SP: Signal peptide, GPI: potential GPI anchor attachment site. The proteins are digested at all sites found (see Figure 4 and Figure 8).

A second group of proteins without assigned function identified as Kex2 substrates is the family of C. Albicans Ops4-like proteins, whose members are differentially regulated in white-opaque switching and mating. This family consists of CaOps4, CA2974, CA6162, CA1873 and CaPga17 (Figure ).

Albicans and S. Cerevisiae kex2 deletion mutants are mating deficient ,.

This has been attributed to the lack of processed α-mating pheromone, but if the above proteins are indeed involved in the mating process, the kex2 mating deficiency could be more severe than thought. In summary, our data show that fungal Kex2 proteinases are similar in their substrate activities but these substrates may have different functions according to the different biological backgrounds of the investigated fungi, including pathogenicity in humans.

In addition, the preferred processing sites of these substrates do not only depend on the amino acids surrounding the processing site, but also on other features such as three dimensional structure. Furthermore, Kex2 proteinases may have unique substrates whose processing sites are adapted to individual proteinases in each organism. Oligonucleotides Oligonucleotides (TIBMolBiol, Germany) used for cloning of expression vectors in this study are given in Additional file.

Heterologous proteinase expression in Pichia pastoris Candida glabrata and Pichia pastoris Kex2 enzymes were expressed using the Pichia expression system (Invitrogen) according to manufacturer's instructions. Briefly, the DNA coding for the Golgi-luminal part of the protein was PCR-amplified from genomic DNA with oligonucleotides containing terminal restriction sites ( BamHI/ NotI and SnaBI/ NotI, respectively) and a sequence for a C-terminal 6 × His-tag, cloned into the pic3.5 vector and transformed into Pichia strain GS115 using an optimized electroporation protocol. Transformants were screened by testing of enzymatic activity against the chromogenic substrate Z-Tyr-Lys-Arg-pNA (see below) in the supernatant of pilot expressions and the clone exhibiting maximum activity used for scale-up. For large-scale production, cells were grown in 500 ml buffered minimal glycerol medium at 30°C over night, harvested by centrifugation, washed and resuspended in 50 ml buffered minimal methanol medium. Maximum activity was detected after 16 h of growth, after which the culture supernatants were harvested and the recombinant enzymes purified as described below. Heterologous proteinase expression in Candida albicans The soluble form of C.

Albicans Kex2 was expressed in the native host, as several attempts of heterologous expression in P. Pastoris failed. The KEX2 gene was PCR-amplified from genomic DNA of C. Albicans with primers containing restriction sites ( HinDIII/ NheI) and the sequence for a 6 × His-tag, cloned into pCIp10 and thus put under control of the mainly constitutive C. Albicans ACT1 promoter. The plasmid was linearized with NcoI and transformed into C. Albicans strain CAI4 using the same protocol as above for Pichia transformation.

Transformants were selected on minimal medium and screened using the supernatant of 5 ml YPD (1% yeast extract, 2% peptone, 1% glucose) over night cultures for testing of enzymatic activity as above. For preparative expression, a 500 ml YPD culture was grown over night, the cells harvested, washed twice with 50 ml YPD, resuspended in 50 ml YPD and further cultivated at 30°C. Maximum Kex2 activity in the supernatant was observed after 12 h of growth at 30°C, after which the supernatant was collected and the recombinant protein purified as described below. YPD medium used for expression was previously freed from low molecular weight impurities by passing over a 10 kDa size exclusion Centricon-20 column (Millipore), Purification of secreted soluble Kex2 proteins To purify the recombinant enzymes, 50 ml sterile filtered expression culture supernatant were concentrated on a 30 kDa size-exclusion Centricon-20 column (Millipore) to a volume of approximately 1–2 ml, desalted using a PD-10 column (Amersham Biosciences) and eluent diluted to a volume of 20 ml into IAEX buffer (50 mM BisTris pH 4.5, 10 mM NaCl). This was loaded onto an HiTrap ANX FF anion exchange column (Amersham Biosciences), washed, and eluted with IAEX buffer containing 100 mM NaCl. The eluent was then again concentrated, the buffer changed into storage buffer (50 mM BisTris, pH 7.2 50% w/v glycerol) and the enzymes kept at -20°C.

Proteinase activity quantification Proteolytic activity of the purified enzymes was assayed using the chromogenic substrate Z-Tyr-Lys-Arg-pNA (Bachem, Switzerland) as described previously. Assays were done in buffer containing 50 mM BisTris (pH 7.2), 1 mM CaCl 2, 0.5 mM substrate in a total volume of 100 μl at 37°C. For the measurement of time kinetic data, the reaction was started by mixing 50 μl of solution containing the proteinase with 50 μl containing the substrate. The temperature gradient for optimal reaction temperature measurement was generated in a thermocycler (Biometra) and the reaction terminated by the addition of EDTA to a final concentration of 10 mM.

Liberation of p-nitroannilide (pNA) was measured at 405 nm in a spectrophotometer (Tecan). All measurements were calibrated against negative controls without proteinase and repeated at l.

The plant hormone jasmonate (JA) plays crucial roles in regulating plant responses to herbivorous insects and microbial pathogens and is an important regulator of plant growth and development –. Key mediators of JA signaling include MYC transcription factors, which are repressed by JAZ transcriptional repressors at the resting state. In the presence of active JA, JAZ proteins function as JA co-receptors by forming a hormone-dependent complex with COI1, the F-box subunit of an SCF-type ubiquitin E3 ligase –.

The hormone-dependent formation of the COI1–JAZ co-receptor complex leads to ubiquitination and proteasome-dependent degradation of JAZ repressors and release of MYC proteins from transcriptional repression. The mechanism by which JAZ proteins repress MYC transcription factors and how JAZ proteins switch between the repressor function in the absence of hormone and the co-receptor function in the presence of hormone remain enigmatic.

Here we show that Arabidopsis MYC3 undergoes pronounced conformational changes when bound to the conserved Jas motif of the JAZ9 repressor. The Jas motif, previously shown to bind to hormone as a partially unwound helix, forms a complete α-helix that displaces the N-terminal helix of MYC3 and becomes an integral part of the MYC N-terminal fold. In this position, the Jas helix competitively inhibits MYC3 interaction with the MED25 subunit of the transcriptional Mediator complex. Our study elucidates a novel molecular switch mechanism that governs the repression and activation of a major plant hormone pathway. Jas peptide forms extensive interactions with the JID–TAD surface in MYC3 Next, we transfected the MYC-responsive p JAZ2::GUS reporter together with wildtype and mutant MYC3 expression plasmids into Arabidopsis protoplasts. As shown in, mutant MYC3 proteins that were defective in interaction with multiple JAZ proteins were partially relieved in repression (i.e., increased reporter gene activity).

Moreover, the extent at which mutations compromised MYC3 interactions with JAZ proteins correlated with the increase in reporter gene activity and the magnitude of changes in reporter gene activity could be further accentuated by expressing MYC3 mutant proteins from the strong cauliflower mosaic virus 35S promoter in coi1-30 mutant protoplasts, in which all JAZ repressors are presumably stabilized. Together, these data validate the MYC3–JAZ9 complex structure and provide strong evidence that amino acid interactions identified in the MYC3–JAZ9 complex structure are important for MYC3 repression in planta. Mutational analysis of the JAZ9–MYC3 interaction The Jas motif is required for its repressor function through interaction with MYC but also for its co-receptor function through interaction with COI1. While the Jas JAZ9 peptide in the MYC3 complex formed a continuous helix , representing the rest state of JAZ, the Jas JAZ1 peptide in the previously determined COI1–JA-Ile–Jas co-receptor structure (PDB: 3OGL) adopted a bipartite conformation with an N-terminal part stretched to form a distinct loop region followed by a shorter C-terminal helix, as illustrated by the structural alignment in and.

In addition to the Jas JAZ9–MYC3 complex, we solved the structure of the Jas JAZ1–MYC3 complex. As shown in the structure alignment in, the Jas helices of JAZ9 and JAZ1 overlap very well, confirming the Jas conformational change between MYC-bound (resting stage) and COI1-bound (hormone-activated stage) is likely common in MYC interaction with different JAZ transcriptional repressors. Distinct conformations of the Jas helix in the COI1–JAZ coreceptor complex vs.

The JAZ–MYC complex In the Jas JAZ1–COI1 complex, the loop region of the Jas JAZ1 helix is formed by the five moderately conserved N-terminal amino acids of the Jas motif that directly interact with the JA-Ile hormone , and is required for Jas–JA-Ile–COI1 co-receptor complex formation. When we mutated the corresponding N-terminal amino acids of JAZ9 to alanine (JAZ9-4A and JAZ9-AA; ), JAZ9 lost interaction with COI1 in yeast two-hybrid assays, but not with MYC3 , consistent with the MYC3 complex structure. Mutations in the middle of the Jas motif (S226A-R234A) affected binding to both MYC3 and COI1, albeit to different degrees. In addition, residues that are C-terminal to the Jas motif enhance JAZ9 interaction with COI1 in yeast two-hybrid assays, but are not critical for its interaction with MYC3 , which is consistent with a previous study of JAZ2, JAZ3, and JAZ10 interactions with COI1 and MYC2. Together, these results indicate that COI1 and MYC3 potentially compete for binding to the central part of the Jas motif, but that COI1 makes additional critical interactions with JAZ9 outside of the MYC3-interacting region, including the previously unrecognized hormone-dependent unwinding of the N-terminal helix of the Jas motif. These additional interactions may allow COI1 to drive JAZ ubiquitination and dissociation of the extensive JAZ–MYC interaction upon JA-Ile stimulation. MED25 is a subunit of the Mediator complex that recruits RNA polymerase II to the promoters of JA responsive genes and is required for various JA responses, including Arabidopsis susceptibility to Pseudomonas syringae bacterial infection and JA-induced inhibition of Arabidopsis root growth.

We found that MYC3(44-238) also directly binds MED25 and that a fragment (aa540-680) encompassing the MED25 Activator Interaction Domain (ACID) is sufficient to bind to MYC3 , analogous to what has previously been reported for MYC2. Since the MYC3 TAD makes critical interactions with JAZ repressors and is required for MYC3–JAZ9 complex formation (, and ), we explored the intriguing possibility that MYC3 binding of JAZ9 and MED25 are mutually exclusive. To test this prediction in a defined system, we performed AlphaScreen interaction assays between MED25(407-680) and both MYC3(44-238) and MYC3(5-242) in the presence of increasing amounts of untagged Jas22 JAZ9 peptide. As shown in, the JAZ peptide competitively inhibited the MYC–MED25 interaction with an IC 50 of 420 nM MYC3(44-238) and 490 nM MYC3(5-242). We further tested competition in planta by transiently expressing combinations of tagged MED25, JAZ9, and MYC3 in Nicotiana tabacum leaves.

As shown in, co-immunoprecipitation of MED25 with MYC3 was strongly reduced upon co-expression of JAZ9. Together, these results demonstrate that the Jas motif of JAZ proteins and the ACID domain of MED25 likely bind to a shared MYC3 surface, and that JAZ repressors can compete the MYC3 interaction with MED25 (and possibly other coactivators) in vitro and in planta. Jas motif peptide competes the MYC3–MED25 interaction In the past decade, despite the identification of analogous hormone perception and transcriptional gene regulation that underpins several major hormone signal transduction pathways in plants, no crystal structures of the transcriptional repressor-transcription factor complexes have been solved. The crystal structure of the MYC-JAZ complex reported here therefore provides the first structural insight into the mechanism of transcriptional repression in plant hormone signaling. Our structural, biochemical, and in planta analyses suggest that JAZ repressors employ a novel dual repression mechanism, which involves not only epigenetic modifications of the target gene chromatin structure through TPL co-repressors, as demonstrated previously, but also direct inhibition of MYC binding to MED25 (and possibly other co-activators) as an integral part of a mechanism of preventing transcriptional activation of JA response genes. In addition, we discovered distinct JAZ conformations in the MYC–JAZ resting complex vs. The JAZ–COI1 hormone-activated complex, providing the first structural insight into the switch mechanism between transcriptional repression and hormone-dependent transcriptional activation in a major plant hormone signalling pathway.

Protein preparation Wild type MYC3(44-238) was expressed as a fusion protein with a cleavable N-terminal His6Sumo tag from a modified pSUMO (LifeSensors) expression vector. BL21 (DE3) cells transformed with the expression plasmid were grown in LB broth at 16 °C to an OD 600 of 1.0 and induced with 0.1 mM IPTG for 16 h.

Cells were harvested, resuspended in 100 ml extract buffer (20 mM Tris, pH 8.0, 200 mM NaCl, and 10% glycerol) per six liters of cells, and passed three times through a French Press with pressure set at 1,000 Pa. The lysate was centrifuged at 18,500 rpm in a Sorvall SS34 rotor for 30 min, and the supernatant was loaded on a 50 ml Nickel HP column. The column was washed with 600 ml of 10% buffer B (20 mM Tris, pH 8.0, 200 mM NaCl, 500 mM imidazole, and 10% glycerol) and eluted with 200 ml of 50% buffer B, followed by 100 ml of 100% buffer B. The eluted His6Sumo-MYC3(44-238) was dialyzed against extract buffer and cleaved overnight with SUMO protease at a protease/protein ratio of 1:1000 at 4°C. The cleaved His6Sumo tag was removed by passing through a 5 ml Nickel HP column, and the protein was further purified by chromatography though a HiLoad 26/60 Superdex 200 gel filtration column in 25 mM Tris, pH 8.0, 200 mM ammonium acetate, 1 mM dithiothreitol and 1 mM EDTA.

To prepare the protein-ligand complex, we mixed Jas22 JAZ1, Jas22 JAZ8, Jas22 JAZ9 or Jas22 JAZ12 peptides with purified MYC3(44-238) proteins at a 1.5:1 molar ratio. The expression and purification of MYC3(5-242) followed the same method as for MYC3(44-238) described above. To prepare the protein-ligand complex, we mixed Jas22 JAZ1, Jas22 JAZ8, Jas22 JAZ9 or Jas22 JAZ12 peptides with purified MYC3(5-242) proteins at a 1.5:1 molar ratio. To prepare MYC3(44-238) selenomethionyl (Se-Met) protein for phase determination, we followed the same methods as described previously. Purification of Se-Met MYC3(44-238) proteins followed the same protocol as for MYC3(44-238) native protein except that the procedure was performed more quickly to avoid protein oxidization. The MYC3(5-242)-Jas22 JAZ9 complex was constructed as a fusion protein with His6Sumo-MYC3(5-242) at the N-terminus and Jas22 JAZ9 at the C-terminus, separated by a flexible GSAGSAGSAGSA (4xGSA) linker His6Sumo-MYC3(5-242)-4xGSA-Jas22 JAZ9.

The expression and purification of the fusion protein followed the same methods as for MYC3(44-238). Small-scale purification of His6Sumo-tagged MYC2/3/4 protein fragments (including JID and TAD domain) for binding studies with Jas peptides followed the same methods as for MYC3(44-238), except that the His6Sumo tag was not removed. Small-scale purification of His6Sumo-tagged MED25 protein fragments (including the ACID domain) for binding studies with biotinylated MYC3(44-238) followed the same methods as for MYC3(44-238), except that the His6Sumo tag was not removed. To express and purify biotinylated MYC3(44-238) and MYC3(5-242) protein for binding studies and JAZ Jas competition assays , we followed the methods described previously. Crystallization The apo-MYC3(5-242) crystals were grown at 20 °C in sitting drops containing 0.2 µl of purified MYC3(5-242) protein at a concentration of 10 mg/ml and 0.2 µl of well solution containing 0.2 M magnesium chloride, 0.1 M Tris, pH 8.5, 30% (w/v) polyethylene glycol 4,000 for 3 days. The Se-Met MYC3 (44-238) crystals were grown at 20 °C in sitting drops containing 0.2 µl of the purified protein at a concentration of 15 mg/ml and 0.2 µl of well solution containing 0.2 M sodium chloride, 0.1 M Bis-Tris, pH 5.5, and 25% (w/v) polyethylene glycol 3,350. Crystals of about 100 µm in length appeared in 3 days.

The MYC3(44-238)–Jas22 JAZ9 complex crystals were grown at 20 °C in sitting drops containing 0.2 µl of the purified complex proteins at a concentration of 15 mg/ml and 0.2 µl of well solution containing 0.2 M magnesium chloride, 0.1 M Tris, pH 8.5, and 30% (w/v) polyethylene glycol 4,000 for 3 days. The MYC3(44-238)–Jas22 JAZ1 complex crystals were grown at 20 °C in sitting drops containing 0.2 µl of the purified complex proteins at a concentration of 15 mg/ml and 0.2 µl of well solution containing 3.5 M sodium formate. Crystals of about 80 µm in length appeared in 2 days. The MYC3(5-242)-Jas22 JAZ9 fusion protein crystals were grown at 20 °C in sitting drops containing 0.2 µl of the purified fusion proteins at a concentration of 15 mg/ml and 0.2 µl of well solution containing 0.2 M magnesium nitrate, 20% (w/v) polyethylene glycol 3,350. Crystals of about 100 µm in length appeared in 3 days.

All crystals were serially transferred to the well solution with 20% (v/v) ethylene glycol before flash freezing in liquid nitrogen. AlphaScreen luminescence proximity assays In vitro interactions between MYC3 and Jas peptides or MED25 fragments were assessed by luminescence–proximity AlphaScreen (Perkin Elmer) technology as described previously. Reactions contained 50 nM His6Sumo-MYC3 protein bound to nickel-acceptor beads and 50 nM synthesized biotinylated Jas peptides bound to streptavidin donor beads ( and ) or 50 nM His6Sumo-MED25-ACID protein bound to nickel-acceptor beads and 50 nM biotin-MYC3(44-238) bound to streptavidin donor beads. The results were based on an average of three experiments with standard errors typically less than 10% of the measurement. For the competition assay , non-biotinylated Jas22 JAZ9 peptide was added into the reaction at concentrations of 0, 5, 10, 100, 300, 1,000, 3,000, 10,000, 30,000 and 100,000 nM. The results were based on an average of three experiments with stand errors typically less than 10% of the measurement. Transient expression in tobacco leaves and Arabidopsis protoplasts For transient expression in tobacco leaves, coding sequences of JAZ9, MED25 and MYC3 were cloned into pJYP003, pJYP011 and pJYP018 (Yao and He, unpublished), respectively, to create p35S:3xHA-JAZ9, p35S:3xFLAG-MED25 and p35S:YFP-MYC3 fusion constructs, which were transfected as previously described.

Protein extracts were immunoprecipitated (IP) with an anti-YFP antibody and analyzed by western blot (WB) with HA, FLAG, or YFP antibodies as previously described. For transient expression in Arabidopsis mesophyll protoplasts, MYC3 (no stop codon) with or without its promoter (the 2-kb sequence upstream of the start codon) were PCR-amplified and cloned into pENTR-D/TOPO vector (Invitrogen) to create entry clones. Then, the MYC3 or pMYC3:MYC3 inserts were introduced into pSAT4A-DEST-Venus or pBR-DEST-Venus (Yao and He, unpublished) to create the p35S:MYC3-YFP or pMYC3-MYC3-YFP constructs. The JAZ2 promoter (the 2-kb sequence upstream of its start codon) was cloned into pBR-Gus (Yao and He, unpublished) to create p JAZ2:GUS reporter constructs. The transient expression assays using pBS-35S-Luc as transfection control followed a published protocol. A 35S::LUC reporter construct was co-transfected as control.

GUS activities were normalized to the luciferase activity. Root growth inhibition assay Arabidopsis wild-type (Col-0) and med25 ( pft1-2; SALK129555) seedlings were used for the root growth inhibition assay. Seeds were surface-sterilized, stratified at 4°C, and germinated on ½ strength MS agar plates containing 1 µM, 3 µM or 10 µM MeJA or 0.1% DMSO (control).

Plates were placed vertically in a growth chamber (16 h light/8 h dark light cycle, 100 µE s −1m −2 light intensity) for 10 days before pictures were taken, and root lengths were measured with ImageJ software. Hydrogen-deuterium exchange (HDX) mass spectrometry HDX of MYC3(5-242) in 20 mM Tris pH 8.0, 200 mM ammonium acetate, 1 mM EDTA, and 7% glycerol was performed at 4 °C using an automated system described previously. Briefly, protein was incubated in a D 2O buffer for a range of exchange times from 10 s to 1 h before quenching the deuterium exchange reaction with an acidic quench solution (pH 2.4) containing of 3 M Urea and 1% TFA. All mixing and digestions were carried out on a LEAP Technologies Twin HTS PAL liquid handling robot housed inside a temperature controlled fridge. Protein digestion was performed in-line with chromatography using an immobilized pepsin column. Mass spectra were acquired on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific).

Percent deuterium exchange values for peptide isotopic envelopes at each time point were calculated and processed using the Workbench Software. The unit of measure represented as a single value is percent deuterium incorporation (% D), which is determined by initially calculating the intensity weighted average (centroid) of all spectral data within defined m/z limits. The% D is then determined by comparing the result to defined minimum (0%) and maximum m/z values (100%) for each peptide. The minimum and maximum m/z values are determined using experimentally observed undeuterated and fully deuterated controls. The data representing each peptide are reduced to single value in the following manner: For each sample, the three individual time-point replicate% D (done in triplicate) at each time point are averaged. The mean of these values is then presented as a single value, representing the overall change in deuterium incorporation for the sample.

The first number in brackets is the representation of the propagation of error for the sample, which is determined by a root means squared approach using the standard deviations from each individual time point. The second number in brackets is the charge state of the detected peptide. Shows cumulative peptides fragments that were detected in MS/MS. Shorter fragments (4–10 residues) provide higher resolution information than longer peptides. Therefore, they supersede longer fragments ( 10 residues) and were used to manually overlay onto the atomic structure as in the case of.

No subtraction was employed. The peptide set used for structural overlay contained the shortest fragments from the complete data set (all peptides). Mapping of the JAZ9–MYC3 interface a and b, Yeast two-hybrid analysis of the interaction between LexA-JAZ9 and BD42(AD)-MYC3 constructs. Simplified diagrams of MYC3 ( a) and JAZ9 ( b) proteins are shown on top.

Blue yeast colonies indicate a positive interaction between two proteins. The experiment was repeated three times with same results. C, Sequence alignment of the Jas motif of the 12 A. Thaliana JAZ proteins.

The N-terminal five amino acids that are unwound in the crystal structure of the COI1-ligand-JAZ co-receptor complex are indicated by a red line on top of Jas JAZ1. Asterisks denote amino acids conserved in all of the sequences and colons denote similar amino acids. D, Interaction between purified His6Sumo-MYC3 N-terminal proteins and biotinylated Jas motif peptides by AlphaScreen luminescence proximity assay (n=3 technical replicates, error bars, s.d.).

indicate significant differences (p. Interactions of MYC2 and MYC4 with Jas motifs of JAZ8, JAZ9, and JAZ12 a, Yeast two-hybrid assays between MYC N-terminal proteins and full length JAZ9. The experiment was repeated three times with same results. B, AlphaScreen assay between His6Sumo-tagged MYC N-terminal proteins and biotinylated JAZ peptides (n=3 technical replicates, error bars, s.d.).indicate significant differences (p.

Surface accessibility and structural dynamics of MYC3(5-242) revealed by hydrogen deuterium exchange mass spectrometry (HDX) a, HDX heat map of MYC3(5-242). The color bar indicates% deuterium exchange. Three experimental repeats were performed for each HDX time point. B, HDX heat map overlaid onto the MYC3(5-242) apo structure. Peptides corresponding to the JID helix were not resolved (no HDX information, grey color), preventing a definitive assessment of the dynamics of the JID helix in solution. B-factor presentations of the four crystal structures The B-factor indicates the dynamic mobilities of different resolved parts within the structure.

The thicker the lines and the warmer the color, the higher is the mobility. Other than two linker regions (linker), the three helices that can occupy the JID helix have the highest B-factors in all four structures. The difficulties in crystallizing the MYC3(5-242)–Jas22 JAZ9 complex are therefore likely due to its high conformational flexibility due to the presence of all three dynamic helices as well as the unfolding of the α1’/α1 helix. Covalent fusion to MYC3(5-242) likely stabilizes the conformational flexibility of Jas22 JAZ9 and the complex. Note that the presence of the α1’/α1 helix does not interfere with the ability of MYC3 to bind the JAZ peptide (compare MYC3(5-242) and MYC3(44-238) in ). MYC3(44-238) in complex with Jas22 JAZ1 and Jas22 JAZ9 and phenotypes of the Arabidopsis med25 mutant a and b, MYC3(44-238) in complex with Jas22 JAZ1(aa 200-221; grey) overlaid with MYC3(44-238) in complex with Jas22 JAZ9(aa 218-239; pink). C, Arabidopsis med25 mutant ( pft1-2) plants are less susceptible to Pseudomonas syringae pv.

Tomato ( Pst) DC3000 than Arabidopsis wild-type (Col-0) plants. Disease symptoms (chlorotic lesions; upper panel) and bacterial population (lower panel) of Arabidopsis wild-type (Col-0) and med25 mutant ( pft1-2) plants 3 days after dip-inoculation with Pseudomonas syringae pv. Tomato ( Pst) DC3000 at 1×10 8 cfu/ml (n=4 biological replicates, error bars, s.e.m.). indicates a significant difference (p. A simplified diagram of the core components of the jasmonate signaling cascade (a) In the resting stage, JA response gene expression is restrained by a family of JAZ transcriptional repressors. JAZ repressors bind and inhibit the MYC family of transcription factors through (i) direct inhibition and (ii) recruiting TOPLESS (TPL) co-repressors either directly or through the NINJA adaptor. TPL in turn recruits histone deacetylases/methyltransferases (not shown) to repress gene expression through chromatin remodeling.

(b) In response to stress or developmental cues, plants synthesize JA-Ile, which serves as molecular glue to facilitate the formation of a co-receptor complex between JAZ and COI1. The formation of the COI1–JAZ co-receptor complex leads to ubiquitination and proteasome-dependent degradation of JAZ repressors. (C) JAZ-free MYCs interact with the MED25 subunit of the Mediator complex and recruit RNA polymerase II (not shown) to the promoters of JA responsive genes. Components examined in this study are colored.