Molecular Microbiology (2015) 96(1), 14–27 ■

doi:10.1111/mmi.12915 First published online 11 February 2015

Ionic interaction of positive amino acid residues of fungal hydrophobin RolA with acidic amino acid residues of cutinase CutL1 Toru Takahashi,1‡ Takumi Tanaka,2‡ Yusei Tsushima,2‡ Kimihide Muragaki,2‡ Kenji Uehara,3 Shunsuke Takeuchi,3 Hiroshi Maeda,1,4 Youhei Yamagata,1,4 Mayumi Nakayama,1,2 Akira Yoshimi1,2 and Keietsu Abe1,2* 1 Microbial Genomics Laboratory, New Industry Creation Hatchery Center, 2Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science and 3Laboratory of Enzymology, Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi 981-8555, Japan. 4 Department of Applied Molecular Biology and Biochemistry, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan.

Summary Hydrophobins are amphipathic proteins secreted by filamentous fungi. When the industrial fungus Aspergillus oryzae is grown in a liquid medium containing the polyester polybutylene succinate co-adipate (PBSA), it produces RolA, a hydrophobin, and CutL1, a PBSA-degrading cutinase. Secreted RolA attaches to the surface of the PBSA particles and recruits CutL1, which then condenses on the particles and stimulates the hydrolysis of PBSA. Here, we identified amino acid residues that are required for the RolA–CutL1 interaction by using site-directed mutagenesis. We quantitatively analyzed kinetic profiles of the interactions between RolA variants and CutL1 variants by using a quartz crystal microbalance (QCM). The QCM analyses revealed that Asp142, Asp171 and Glu31, located on the hydrophilic molecular surface of CutL1, and His32 and Lys34, located in the N-terminus of RolA, play crucial roles in the RolA–CutL1 interaction via ionic interactions. RolA immobilized on a QCM electrode

Accepted 17 December, 2014. *For correspondence. E-mail [email protected] biochem.tohoku.ac.jp; Tel. (+81) 22717 8779; Fax (+81) 22717 8780. ‡ These authors contributed equally to this work.

© 2015 John Wiley & Sons Ltd

strongly interacted with CutL1 (KD = 6.5 nM); however, RolA with CutL1 variants, or RolA variants with CutL1, showed markedly larger KD values, particularly in the interaction between the double variant RolA-H32S/ K34S and the triple variant CutL1-E31S/D142S/D171S (KD = 78.0 nM). We discuss a molecular prototype model of hydrophobin-based enzyme recruitment at the solid–water interface.

Introduction In nature, numerous species of filamentous fungi are involved in the enzymatic hydrolysis of bio-polymers such as polysaccharides, proteins and polyesters, and thus play important roles in material recycling in the global ecosystem (Ichishima, 2000; Maeda et al., 2005; Chandra et al., 2009). Such filamentous fungi include pathogenic fungi against plants and animals, and industrial molds used for fermentation (Wang and St Leger, 2005; Machida et al., 2008; Nishimura et al., 2009). For over 1,000 years, Aspergillus oryzae and A. sojae have been used in solid-state fermentation, which utilizes processes that mimic events in the pathogenesis of plants by plant-pathogenic fungi, to produce a wide range of polymer-degrading enzymes. Efficient enzymatic hydrolysis of solid grains is considered the most important process for the fungal industry, and enzymatic degradation is a crucial process for fungal pathogens as they enter hosts through outer layers of protectant polymers (Purdy and Kolattukudy, 1975; Sweigard et al., 1992). We previously discovered that A. oryzae produces the polyesterase/cutinase CutL1 and that CutL1 hydrolyzes the biodegradable plastic polybutylene succinate co-adipate (PBSA) (Maeda et al., 2005). PBSA is an aliphatic polyester, and its chemical structure resembles that of cutin, a plant-wax polyester (Fang et al., 2001). Cutinases are also produced by plant pathogens such as Fusarium solani f. pisi and Magnaporthe grisea (Purdy and Kolattukudy, 1975; Sweigard et al., 1992). CutL1 of A. oryzae can degrade not only PBSA but also polylactic acid and polycaprolactone (Maeda et al., 2005; Liu et al., 2009). When we studied PBSA hydrolysis catalyzed by the CutL1 of A. oryzae, we discovered that during growth under liquid culture conditions with PBSA as the

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Ionic interaction between hydrophobin and cutinase 15 CutL1 recruited by RolA-PFA350 CutL1 recruited by RolA-PFA350 (+NaCl)

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Fig. 1. Quantification of the amount of CutL1 recruited by the RolA–PFA350 complex at various pHs. Black bars: pH profile of the amount of CutL1 bound to the RolA–PFA350 complex in the absence of NaCl (pH 3–10). White bars: pH profile of the amount of CutL1 bound to the RolA–PFA350 complex in the presence of 250 mM NaCl (pH 3–10). Data are from three independent experiments and are presented as the mean ± standard deviation.

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sole carbon source, A. oryzae simultaneously produced both CutL1 and the bio-surfactant hydrophobin RolA (Takahashi et al., 2005). The secreted RolA adsorbed to the hydrophobic PBSA surface and efficiently recruited CutL1, resulting in CutL1 condensing on the PBSA surface and stimulating PBSA hydrolysis (Takahashi et al., 2005). Hydrophobins are small, secreted amphipathic proteins that contain eight conserved cysteine residues and are ubiquitous among filamentous fungi (Wessels et al., 1991; de Vries et al., 1993; Wessels, 1994). Hydrophobins play several important roles in fungal physiology, for example, in fungal adhesion to hydrophobic surfaces, in the formation of a protective surface coating and in the reduction of water surface tension; these roles support the growth of fungal aerial structures such as hyphae and conidiospores (Wösten et al., 1999). Hydrophobins are classified into two categories (class I and class II) according to the hydropathy pattern of the hydrophobin amino acid sequence and the solubility of their assembled films (Wösten, 2001). RolA has been shown to contain the signature eight cysteine residues that are conserved in class I hydrophobins and also to have a hydropathy profile similar to those of known class I hydrophobins (Takahashi et al., 2005). Furthermore, a comparative analysis of the amino acid sequences of A. oryzae RolA and A. nidulans RodA (class I) has demonstrated that A. oryzae RolA is an ortholog of A. nidulans RodA (Tanaka et al., 2014). In addition, RolA has been shown to strongly adsorb to hydrophobic materials such as PBSA and Teflon and be difficult to remove, even by treatment with sodium dodecyl sulfate (SDS) or ethanol, which is similar to the properties of known class I hydrophobins such as Schizophyllum commune SC3 (Takahashi et al., 2005). As the recruitment of hydrolytic enzymes by amphipathic proteins attached to solid surfaces is a novel mechanism for the degradation of hydrophobic solid materials at the solid– liquid interface (Takahashi et al., 2005; Ohtaki et al., 2006), the RolA–CutL1 interaction is considered a prototype molecular interaction model; however, the molecular mechanism involved in the RolA–CutL1 interaction remains unclear. © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 14–27

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Here, we used site-directed mutagenesis and a quartz crystal microbalance (QCM) to identify which amino acid residues are involved in the RolA–CutL1 interaction. Our study provides insights into a novel molecular mechanism for the interaction of scaffold biosurfactant proteins with enzymes.

Results Involvement of ionic bonds in the RolA–CutL1 interaction As protein–protein interactions generally depend on several types of forces such as ionic interaction, hydrophobic interaction, hydrogen bonds and van der Waals forces, we first examined whether ionic bonds are involved in the RolA–CutL1 interaction. Prior to the CutL1 pull-down assay with RolA-PFA350, we confirmed that PFA350 was fully coated with RolA under our RolA-coating conditions by comparing the adsorption of BSA to non-RolA-coated PFA350 and RolA-coated PFA350. BSA was used as the control protein because it does not interact with RolA bound to solid surfaces (Takahashi et al., 2005). PFA350 adsorbed BSA (0.1–2 nmol/m2 PFA350) at pH 3, 5 and 7 but scarcely did so at pH 10 (Fig. S1). RolA-coated PFA350 scarcely adsorbed BSA at pH 3, 7 and 10 and adsorbed only a small amount of BSA (0.2 nmol/m2 PFA350) at pH 5. The low levels of BSA adsorption to RolA-PFA350 confirmed that PFA350 was fully coated with RolA under our RolA-coating conditions. Next, we measured the amount of CutL1 recruited by RolA bound to PFA350 by using a pull-down assay at pH 3–10, which showed that the amount of CutL1 recruited to RolA was decreased between pH 8 and pH 10 and saturated between pH 4 and pH 7 (Fig. 1). At pH 3, the amount of CutL1 recruited was markedly decreased regardless of the presence of NaCl. Between pH 4 and 7, the amount of CutL1 recruited was decreased in the presence of NaCl. In the absence of NaCl, at pH 8, 9 and 10, the amount of CutL1 recruited to the RolA–PFA350 complex was approximately one-third of that recruited at pH 4–7 and

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The pH dependence of the RolA–CutL1 interaction (Fig. 1) implied the involvement of the single histidine residues of RolA and CutL1. As the three-dimensional (3D) structure of CutL1 (Liu et al., 2009) shows that His194 is located inside the protein as part of the catalytic domain, we hypothesized that His32 located in the N-terminus of RolA was involved in the ionic interaction between RolA and CutL1. We constructed an RolA variant in which His32 was substituted with serine (RolA-H32S) and examined the interaction of RolA-H32S with CutL1. To further confirm the involvement of His32 in the RolA–CutL1 interaction, wildtype RolA was treated with diethylpyrocarbonate (DEPC), which at pH 6 preferentially modifies histidine residues (Miles, 1977). CutL1 recruitment by PFA350 coated with the DEPC-treated RolA was then examined with a pulldown assay. The amount of CutL1 recruited by RolAH32S–coated PFA350 was decreased to 60% ± 12% (mean ± standard deviation) of that recruited by wild-type RolA-coated PFA350 (Fig. 2). Furthermore, DEPC-treated RolA-coated PFA350 only adsorbed 44% ± 8% of the amount of CutL1 recruited by wild-type RolA-coated PFA350 (Fig. 2). These results show that His32 of RolA is important for the RolA–CutL1 interaction. As RolA-H32S still recruited some CutL1, other amino acid residues besides His32 were assumed to be involved in the RolA– CutL1 interaction. Because DEPC-treated RolA recruited less CutL1 than did RolA-H32S, and because DEPC modifies not only histidine residues but also the ε-amino group of lysine residues (Miles, 1977; Kawai et al., 2005), we hypothesized that Lys34 located behind His32 is also involved in the ionic interaction between RolA and CutL1. We constructed a Lys variant of RolA in which Lys34 was substituted with serine (RolA-K34S), and the amount of CutL1 recruited by RolA-K34S was 63% ± 11% of that recruited by wild-type RolA (Fig. 2). As His32 and Lys34 were indicated to play important roles in the RolA–CutL1 interaction, we constructed a

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almost the same as that recruited with NaCl (Fig. 1). These results suggest that ionic interactions are involved in the RolA–CutL1 interaction at pH 4–7. As both BSA and CutL1 were adsorbed to non-RolA-coated PFA350 in the presence or absence of 250 mM NaCl at pH 3–10 (Figs S1 and S2A), and our RolA-coating conditions produced fully RolA-coated PFA350 (Fig. S1), the amount of CutL1 recruited by RolA-coated PFA350 was considered to quantitatively indicate the molecular interaction between CutL1 and PFA350-bound RolA (Figs 1 and S2B).

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Fig. 2. Quantification of the amount of CutL1 recruited by the DEPC-treated RolA–PFA350 complex or RolA variant–PFA350 complex. The amounts of CutL1 bound to the DEPC-treated RolA–PFA350 complex (DEPC–RolA), RolA single variant–PFA350 complex (RolA-H32S or RolA-K34S) and RolA double variant–PFA350 complex (RolA-H32S/K34S) were quantified at pH 5. Data are from three independent experiments and are presented as the mean ± standard deviation. Statistical significance was evaluated with Student’s t-test (*P < 0.01, **P < 0.05).

RolA double mutant in which both His32 and Lys34 were substituted with serine (RolA-H32S/K34S). The amount of CutL1 recruited by RolA-H32S/K34S was 46% ± 10% of that recruited by wild-type RolA and similar to that recruited by DEPC-treated RolA but 10–15% lower than that recruited by RolA single mutants (RolA-H32S and RolA-K34S) (Fig. 2). Because the circular dichroism (CD) spectra of both the soluble- and PFA350-attached forms of the RolA variants were very similar to the spectra of the respective forms of wild-type RolA, RolA variants RolA-H32S, RolA-K34S and RolA-H32S/K34S can be considered structurally similar to wild-type RolA (Fig. S3). Furthermore, the amounts of the RolA variants (RolA-H32S, RolA-K34S and RolA-H32S/ K34S) bound to non-RolA-coated PFA350 were statistically indistinguishable from that of wild-type RolA (Fig. S4). Thus, the physicochemical properties of the RolA variants with regard to adsorption on PFA350 were scarcely changed by the mutagenesis.

Negatively charged amino acid residues in CutL1 are involved in the RolA–CutL1 interaction As the positively charged residues His32 and Lys34 in the N-terminus of RolA are likely involved in the RolA–CutL1 interaction through ionic interactions, we hypothesized that negatively charged amino acid residues in CutL1 must also be involved. We treated CutL1 with a carboxyl group– modifying reagent, N-cyclohexyl-N′-(4-dimethylaminoalpha-naphthyl) carbodiimide (NCD-4) and examined whether NCD-4-treated CutL1 was recruited by RolA. The © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 14–27

Ionic interaction between hydrophobin and cutinase 17

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Fig. 3. Quantification of the amount of NCD4-treated CutL1 recruited by the RolA–PFA350 complex at various pHs. Black bars: pH profile of the amount of wild-type CutL1 bound to the RolA–PFA350 complex. Gray bars: pH profile of the amount of wild-type CutL1 bound to the RolA–PFA350 complex in the presence of 250 mM NaCl. White bars: pH profile of the amount of NCD4-treated CutL1 bound to the RolA–PFA350 complex. Data are from three independent experiments and are presented as the mean ± standard deviation.

amount of NCD-4-treated CutL1 recruited by wild-type RolA was 50–70% of the amount of wild-type CutL1 recruited but was similar to the amount of wild-type CutL1 recruited in the presence of NaCl (Fig. 3), suggesting that negatively charged amino acid residues in CutL1 are involved in the RolA–CutL1 interaction.

Asp142, Asp171 and Glu31 of CutL1 are involved in the RolA–CutL1 interaction

CutL1 (mol/mol of RolA)

As wild-type RolA recruited only about half the amount of NCD-4-treated CutL1 compared with wild-type CutL1, we looked for negatively charged amino acid residues in CutL1 that could be involved in the RolA–CutL1 interaction. A. oryzae CutC (GenBank Accession number: AB733178.1), which is a homolog of CutL1, also hydrolyzes PBSA, and QCM analysis demonstrated that CutC also interacts with RolA that is bound to solid surfaces

(Fig. S5). Alignment analysis of the amino acid sequences of these two cutinase homologs revealed nine conserved negatively charged amino acid residues that were candidates for involvement in RolA interactions. A 3D structure of CutL1 based on crystallography (Protein Data Bank ID code: 3GBS) predicted that six of these residues are located on the molecular surface of CutL1 (Liu et al., 2009). We therefore performed competition assays of the RolA–CutL1 interaction with synthetic peptides containing the six candidates in partial amino acid sequences of CutL1 (Fig. S6A). CutL1 peptide III, which contained Asp142 and Asp145, and CutL1 peptide IV, which contained Asp171, inhibited the RolA–CutL1 interaction (Fig. S6B). Subsequently, we generated six CutL1 variants in which one of the six negatively charged residues was substituted with serine and examined recruitment of the six variants by wild-type RolA–PFA350 complex. The pull-down assays revealed that the recruited amounts of CutL1-E31S, CutL1-D142S and CutL1-D171S variants were significantly decreased compared with wild-type CutL1 (P < 0.05, Fig. 4). The amounts of two single variants (CutL1-E31S and CutL1-D142S), three double variants (CutL1-E31S/D142S, CutL1-D142S/D171S and CutL1-E31S/D171S) and a triple variant (CutL1-E31S/ D142S/D171S) recruited to the wild-type RolA–PFA350 complex were lower than that of the single variant CutL1D171S (P < 0.01, Fig. 4). The CutL1 single variants, double variants and triple variant were all adsorbed to non-RolA-coated PFA350 (Fig. S7A); however, BSA (negative control) was scarcely adsorbed to RolA-coated PFA350. Thus, the level of adsorption of wild-type CutL1 or of each CutL1 variant to RolA-coated PFA350 quantitatively indicated a specific interaction between CutL1 derivatives and RolA (Fig. 4, Figs S1 and S7B and C), suggesting that Asp142, Asp171 and Glu31 of CutL1 play important roles in the RolA–CutL1 interaction. We next confirmed whether the conformations of the CutL1 variants were the same as that of the wild-type by

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Fig. 4. Quantification of the amount of CutL1 variants recruited by the RolA–PFA350 complex at pH 5. CutL1-E31S, CutL1-E109S, CutL1-D142S, CutL1-D145S, CutL1-D171S and CutL1-D203S are CutL1 single variants. CutL1-E31S/D142S, CutL1-E31S/D171S and CutL1 D142S/D171S are CutL1 double variants. CutL1-E31S/D142S/D171S is a CutL1 triple variant. Data are from three independent experiments and are presented as the mean ± standard deviation. Statistical significance was evaluated with Student’s t-test (*P < 0.01).

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using CD spectroscopic analysis. The CD spectra of the CutL1 variants were very similar to that of the wild-type (Fig. S8), suggesting that the conformation of each variant was very similar to that of the wild-type.

QCM analysis of the interaction between RolA variants and wild-type CutL1

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variants. A. KD values of the interaction between wild-type RolA or RolA variants and wild-type CutL1 in the absence or presence of 250 mM NaCl at pH 6. B. KD values of the interaction between wild-type RolA and CutL1 variants in the absence or presence of 250 mM NaCl at pH 6. C. KD values of the interaction between wild-type RolA or RolA-H32/K34 and CutL1-E31S/D142S/D171S in the absence or presence of 250 mM NaCl at pH 6. Data are from three to eight independent experiments and are presented as the mean ± standard deviation. Statistical significance was evaluated with Student’s t-test (*P < 0.01, **P < 0.05).

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As His32 and Lys34 in the N-terminus of RolA were shown to be involved in the RolA–CutL1 interaction by the pulldown assay of RolA variants, we next quantitatively analyzed the interaction of the RolA variants and CutL1 by using a QCM. Purified wild-type RolA, RolA-H32S, RolAK34S and RolA-H32S/K34S were immobilized on QCM sensor electrodes that were then immersed in analysis chambers filled with analysis buffer solution. Wild-type CutL1 was then added to the chamber in a stepwise manner in concentrations ranging from 0 to 900 nM. The KD values of the wild-type RolA, RolA-H32S and RolA-K34S were 6.5 nM, 35.8 nM and 44.8 nM respectively (Fig. 5A). The KD value (6.5 nM) of the interaction between wild-type RolA and wild-type CutL1 suggests that the RolA–CutL1 interaction is a strong protein–protein interaction similar to that of antigen–antibody interactions (Kontermann and Duebel, 2010). The KD values of the RolA single variants were six- to sevenfold higher than that of wild-type RolA. The KD value of the double variant was 46.1 nM, which was similar to the KD values of the RolA single variants (Fig. 5A). These results further confirmed the involvement of His32 and Lys34 of RolA in the ionic interaction with CutL1. The KD value of the interaction between RolA-K34S and CutL1 was larger than that of the interaction between RolA-H32S and CutL1, suggesting that Lys34 plays an important role in the RolA–CutL1 interaction. When a running buffer containing 250 mM NaCl was used for the QCM analysis, the KD value of the interaction between wild-type RolA and wild-type CutL1 (concentration range: 0–2000 nM) was 874.0 nM (Fig. 5A). According to the observed KD values of the RolA variants vs. wild-type CutL1, the affinity originating from the ionic interaction between the two molecules ranges between KD values of 6.5 and 874.0 nM (Fig. 5A).

vs. CutL1-E31S/D142S/D171S

© 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 14–27

Ionic interaction between hydrophobin and cutinase 19

QCM analysis of the interaction between CutL1 variants and wild-type RolA As the Asp142, Asp171 and Glu31 of CutL1 were shown to be involved in the RolA–CutL1 interaction by the pulldown assay of CutL1 variants (Fig. 4), we next quantitatively analyzed the interaction of wild-type CutL1 or its variants with wild-type RolA by using a QCM. Wild-type RolA was attached to QCM electrodes, and wild-type CutL1 or one of its variants was added to the analysis chamber in a stepwise manner at concentrations ranging from 0 to 900 nM. The KD values of the wild-type CutL1, CutL1-E31S, CutL1-D142S or CutL1-D171S with wildtype RolA were 6.5 nM, 17.2 nM, 20.4 nM and 14.5 nM respectively (Fig. 5B). The KD values of the CutL1 single variants were two- to threefold higher than that of wildtype CutL1. The KD values for the interaction between the CutL1 double variants and wild-type RolA were 60.5 nM for CutL1-E31S/D142S, 31.6 nM for CutL1-D171S/E31S and 37.9 nM for CutL1-D142S/D171S (Fig. 5B). The KD values of the CutL1 double variants were two- to fourfold higher than those of the CutL1 single variants. CutL1D142S had the largest KD value among the three single CutL1 variants, and the double variants containing the D142S substitution also had larger KD values than did the double variant without D142S, suggesting a prominent role for D142S in the RolA–CutL1 interaction. The KD value for the interaction between the triple variant CutL1E31S/D142S/D171S and wild-type RolA was 75.5 nM, which was 1.2- to 2.4-fold higher than those of the CutL1 double variants; thus, it is likely that the three acidic residues of CutL1 additively contribute to the interaction with RolA (Fig. 5B). When the running buffer contained 250 mM NaCl, the KD value for CutL1-E31S/D142S/ D171S and wild-type RolA was 783.7 nM, which was almost the same as the KD value between wild-type RolA and wild-type CutL1 in the presence of NaCl (Fig. 5A). Overall, the QCM analyses revealed that Glu31, Asp142 and Asp171 of CutL1 cooperatively play an important role in the ionic interaction with RolA; however, as NaCl (250 mM) in the running buffer increased the KD value of wild-type RolA for CutL1-E31S/D142S/D171S by 10-fold compared with the value without NaCl (KD = 75.5 nM), other amino acid residues must be involved in the ionic interaction with RolA.

QCM analysis of the interaction between RolA-H32S/K34S and CutL1-E31S/D142S/D171S To quantitatively examine the contribution of the two positively charged residues of RolA (His32 and Lys34) and the three negatively charged residues of CutL1 (Glu31, Asp142 and Asp171) to the ionic interaction between immobilized RolA and CutL1, we performed QCM analysis © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 14–27

of the interaction between the RolA-H32S/K34S double variant and the CutL1-E31S/D142S/D171S triple variant in the presence or absence of 250 mM NaCl. RolA-K32S/ H34S was immobilized on a QCM electrode, and CutL1E31S/D142S/S171S was added to the analysis chamber in a stepwise manner in concentrations ranging from 0 to 900 nM without NaCl and from 0 to 4000 nM with 250 mM NaCl. The KD value for the interaction between RolA-H32S/ K34S and CutL1-E31S/D142S/D171S was 78.0 nM (Fig. 5C). When the running buffer contained 250 mM NaCl, the KD value was 812.7 nM (Fig. 5C). These results suggest that some ionic interaction remains between the RolA double variant and the CutL1 triple variant. Moreover, because the KD value between the RolA double variant and the CutL1 triple variant (78.0 nM; Fig. 5C) was approximately the same as that between wild-type RolA and the CutL1 triple variant (75.5 nM; Fig. 5B), the results suggest that His32 and Lys34 of RolA directly interact with Glu31, Asp142 and Asp171 of CutL1. QCM analysis of the interaction between A. nidulans RodA and A. oryzae CutL1 As described above, CutL1 recruitment by RolA immobilized on a solid surface is mediated via ionic interaction, we next examined whether other hydrophobins besides RolA are able to recruit CutL1. A. oryzae RolA is an ortholog of the class I hydrophobin A. nidulans RodA, so we compared the amino acid sequences of RodA orthologs derived from aspergilli and Penicillium chrysogenum (Fig. S9). Multiple alignment of the amino acid sequences of RodA orthologs showed that most RodA orthologs have several positively charged residues (including Lys, Arg and His) in their N-terminal region (upstream of the first cysteine residue) (Fig. S9A and B). Moreover, multiple alignment of those of CutL1 orthologs derived from aspergilli and P. chrysogenum showed conserved acidic residues, including residues corresponding to Glu31, Asp142 and Asp171 of A. oryzae CutL1 (Fig. S10A and B). The conservation of N-terminal positively charged residues in RodA orthologs and acidic residues on the molecular surface of CutL1 orthologs implies a broad distribution of interactions of RodA orthologs with CutL1 orthologs among aspergilli and other fungal species. We purified A. nidulans RodA expressed by A. oryzae and examined whether RodA immobilized on QCM electrodes could recruit A. oryzae CutL1 in the presence or absence of 250 mM NaCl. We found that A. nidulans RodA immobilized on QCM electrodes recruited A. oryzae CutL1 (Fig. 6). The KD values for the RodA–CutL1 interaction were KD = 242.7 ± 88.0 nM without NaCl and KD = 983.7 ± 156.2 nM in the presence of NaCl, suggesting that RodA recruits CutL1 via an ionic interaction.

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the mechanism of CutL1 recruitment via RolA that is attached to a solid surface is a novel function of RolA and is thus considered a prototype model of hydrophobindependent protein recruitment (Takahashi et al., 2005). As not only RolA, but also HsbA, another surface-active protein produced by A. oryzae, is able to recruit CutL1 to the surface of PBSA (Ohtaki et al., 2006), the mechanism may play an important role in polymer degradation or infection by fungi in nature. In the present study, we identified the amino acid residues required for the RolA–CutL1 interaction and determined the kinetic properties of the amino acid residues in the recruitment reaction by using QCM analysis. QCM analysis revealed that two positively charged residues (His32 and Lys34) in the N-terminus of RolA and three negatively charged residues (Asp142, Asp171 and Glu31) on the hydrophilic molecular surface of CutL1 are critically involved in RolA-dependent CutL1 recruitment via an ionic interaction (Fig. 7).

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Curve A: KD = 242.7 ± 88.0 nM

250 ΔFrequency (Hz)

30

200 150 Curve B: KD = 983.7 ± 156.2 nM

100 50 0 0

500

1000 1500 2000 2500 3000 3500 CutL1 concentration (nM)

Fig. 6. QCM analysis of the interaction between RodA and CutL1. A. Interaction of wild-type CutL1 with RodA immobilized on a QCM electrode in the absence of 250 mM NaCl at pH 6. Arrows indicate final concentrations of CutL1 in the analysis chamber. B. Interaction of wild-type CutL1 with RodA immobilized on a QCM electrode in the presence of 250 mM NaCl at pH 6. Arrows indicate final concentrations of CutL1 in the analysis chamber. C. Graph used to calculate the kinetic parameters for the interaction between immobilized RodA and CutL1; KD values were determined by fitting to a Langmuir adsorption isotherm. Interaction between RodA and wild-type CutL1 in the absence (curve A) and presence (curve B) of 250 mM NaCl at pH 6.

Discussion Previously, we reported that the A. oryzae hydrophobin RolA attaches to the surface of PBSA particles where it efficiently recruits CutL1, which then promotes the hydrolysis of PBSA. Soluble RolA does not interact with CutL1, so

The amount of wild-type CutL1 recruited by PFA350-bound wild-type RolA was pH dependent and decreased in the presence of NaCl (Fig. 1), suggesting that ionic interaction was the major force for the RolA–CutL1 interaction. In the presence of NaCl, one-half of the level of CutL1 recruitment in the absence of NaCl remained (Fig. 3), implying the involvement also of hydrophobic interactions, hydrogen bonds and van der Waals forces. Corvis et al. previously reported that S. commune SC3 bound to a glassycarbon electrode interacts with glucose oxidase and horseradish peroxidase and that the complex can be used as an enzyme electrode (Corvis et al., 2005). Wang et al. also reported that the class I hydrophobin HGFI and class II hydrophobin HFBI of Trichoderma reesei attached to the surface of hydrophobic materials bind BSA, monoclonal IgG, avidin and glucose oxidase via charged amino acid residues located on the hydrophobin molecular surface (Wang et al., 2010). As the binding of these proteins to HGFI and HFBI was pH dependent, Wang et al. considered ionic interactions to be involved in the interactions (Wang et al., 2010). His32, Lys34 and other charged amino acid residues of RolA are involved in its interaction with CutL1 The pull-down assay and QCM analysis of RolA-H32S, RolA-K34S and RolA-H32S/K34S revealed that both His32 and Lys34 in the N-terminal region of RolA contribute to the RolA–CutL1 interaction (Figs 2 and 5A). The KD value of RolA-H32S/K34S and CutL1 was only slightly larger than that of RolA-H32S and was similar to that of RolA-K34S (Fig. 5A). RolA-H32S/K34S still showed a KD © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 14–27

Ionic interaction between hydrophobin and cutinase 21

A

Y-

CutL1 E31-

D142-

X+

D171KD = 46.1 nM

KD = 6.5 nM + K34+ H32

+ K34+ H32

YX+

RolA

Y-

RolA

X+

B

Y-

CutL1 E31-

D142-

KD = 75.5 nM

X+

D171-

KD = 78.0 nM

+ K34+ H32

+ K34+ H32

YX+

RolA

YX+

RolA

Fig. 7. Schematic model of the interaction between RolA and CutL1 on a hydrophobic surface. A. RolA adsorbed onto a hydrophobic surface interacts with CutL1. QCM analysis revealed that the interaction between wild-type RolA and wild-type CutL1 has a KD value of 6.5 nM. RolA-H32S/K34S showed a KD value of 46.1 nM. However, because the interactions between wild-type RolA or RolA variants and wild-type CutL1 or CutL1 variants have KD values around 800 nM in the presence of NaCl (see Fig. 5C), RolA-H32S/K34S is predicted to have some undetermined positively charged (X+) and/or negatively charged (Y−) amino acid residue(s) that are also involved in the interaction with wild-type CutL1. B. As the interaction between CutL1-E31S/D142S/D171S and wild-type RolA had a KD value of 75.5 nM, and the interaction between CutL1-E31S/D142S/D171S and RolA-H32S/K34S had a KD value of 78.0 nM, it is likely that His32 and Lys34 located in the N-terminus of RolA directly interact with Asp142, Asp171 and Glu31 located on the hydrophilic molecular surface of CutL1. Moreover, CutL1-E31S/D142S/D171S is predicted to have some undetermined positively charged (X+) and/or negatively charged (Y−) amino acid residue(s) that are involved in the interaction with wild-type RolA.

© 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 14–27

value of 46.1 nM with wild-type CutL1 (Fig. 5A), but wildtype RolA showed a KD value of 874.0 nM with wild-type CutL1 in the presence of 250 mM NaCl (Fig. 5A), implying that some remaining charged resides in RolA-H32S/K34S are involved in the interaction with CutL1 (Fig. 7A). Since RolA-H32S/K34S showed KD values of 46.1 nM and 78.0 nM with wild-type CutL1 and CutL1-E31S/D142S/ D171S, respectively (Fig. 5A and C), one or more of the three acidic residues in CutL1 must be interacting with positively charged residues other than His32 and Lys34 in RolA (Fig. 7A, right; 7B, right). The 3D structures of the class I hydrophobin EAS of Neurospora crassa and the class II hydrophobins HFBI and HFBII have been determined with triple-resonance Nuclear Magnetic Resonance (NMR) and X-ray crystallography respectively (Hakanpää et al., 2004; 2006; Kwan et al., 2006). The predicted 3D structures of HFBI and HFBII are compact and globular and consist of a core β-barrel comprising two adjoining β-hairpins (Hakanpää et al., 2006). Like HFBI and HFBII, the core structure of EAS is centered on two interlocking β-hairpins that form a four-stranded β-barrel (Sunde et al., 2008). As charged amino acid residues are present in the β-barrel structure, EAS is thought to interact with the water surface via charged amino acid residues localized at the side of its β-barrel structure at the water–air interface (Kwan et al., 2006). We previously reported from a CD analysis that the structure of the class I hydrophobin RolA attached to PFA350 particles differs from that of the soluble form (Takahashi et al., 2005). Because only the attached form of RolA, not the soluble form, can recruit CutL1 (Takahashi et al., 2005), the two positively charged amino acid residues (His32 and Lys34) involved in the RolA–CutL1 interaction in the N-terminus of RolA appear to be displayed to the hydrophilic aqueous side after RolA is attached to hydrophobic solid surfaces. Scholtmeijer et al. constructed a hydrophobin S. commune SC3 derivative in which fibronectin binding domain (Arg-Gly-Asp; RGD) was inserted downstream of the N-terminal signal sequence, and the growth of fibroblasts was stimulated on the surface of Teflon coated with the SC3 derivative (Janssen et al., 2002; Scholtmeijer et al., 2002). Another SC3 derivative in which the 25 N-terminal amino acid residues, including the glycosylation site, were deleted and RGD residues were inserted also showed a stimulatory effect on the growth of fibroblasts on Teflon coated with the SC3 derivative (Janssen et al., 2004). Janssen et al. also reported that glycosylation of the SC3 N-terminus results in the Nterminus becoming less exposed when SC3 adopts the β-sheet state and that the N-terminus is important for the growth of fibroblasts on SC3-coated Teflon (Janssen et al., 2004). These results suggest that the N-terminus of SC3 can be modified to improve its function. The results of these previous studies together with those of the present study

22 T. Takahashi et al. ■

indicate that the hydrophobin N-terminal region faces the hydrophilic side of the interface between water and a hydrophobic solid and that it is able to interact with other hydrophilic macromolecules. Asp142, Asp171 and Glu31 in CutL1 are involved in the interaction with RolA Although it is possible that the carboxyl groups of CutL1 were only partially modified with NCD-4 in the pull-down experiment shown in Figure 3, the pull-down assay and QCM analysis with CutL1 variants against wild-type RolA demonstrated that three negatively charged amino acid residues (Asp142, Asp171 and Glu31) predicted to be located at the molecular surface of CutL1 play important roles in the interaction (Figs 3, 4 and 5B). In the pull-down assay under acidic pH conditions (below pH 4), protonation of the carboxyl groups of acidic amino acid residues might weaken the ionic interaction between CutL1 and RolA (Fig. 1). In the presence of 250 mM NaCl, the triple variant showed a KD value of 783.7 nM for the interaction with wild-type RolA, suggesting that some charged residues of CutL1 other than the three acidic residues substituted are involved in the ionic interaction between CutL1 and RolA (Fig. 7B). His32 and Lys34 of RolA directly interact with Asp142, Asp171 and Glu31 of CutL1 Because the KD values for the interaction of wild-type RolA or RolA-H32S/K34S with CutL1-E31S/D142S/D171S were 75.5 nM and 78.0 nM, respectively, and were statistically indistinguishable (Fig. 5C), the two positively charged residues (His32 and Lys34) of Ro1A are likely to interact directly with the three acidic residues (Glu31, Asp142 and Asp171) of CutL1 (Fig. 7B). The KD value of the interaction between RolA-H32S/K34S and CutL1E31S/D142S/D171S was 78.0 nM in the absence of NaCl but that in the presence of 250 mM NaCl was 812.7 nM (Fig. 5C). The KD value between wild-type RolA and wildtype CutL1 in the presence of 250 mM NaCl was 874.0 nM (Fig. 5A). The latter two KD values were statistically indistinguishable, implying that the ionic interaction ranges between KD values of approximately 6.5–800 nM and that some charged amino acid residues other than the two positively charged residues (His32 and Lys34) of RolA and three acidic residues (Glu31, Asp142 and Asp171) of CutL1 are involved in the remaining ionic interaction between RolA-H32S/K34S and Cutl1-E31S/D142S/ D171S (Fig. 7B). The remaining amino acid residues involved in the ionic interaction will be determined in future studies. The recruitment of A. oryzae CutL1 by A. nidulans RodA immobilized on a QCM electrode (Fig. 6) and the results of

the alignment analyses of RodA orthologs (Fig. S9) and CutL1 orthologs (Fig. S10) suggest the possibility that ionic interaction between the two molecules is common among aspergilli and other filamentous fungi. SWISS-MODEL (http://swissmodel.expasy.org/) homology modeling of RolA and RodA, using the class I hydrophobin A. nidulans DewA (Protein Data Bank ID code: 2SLH) as the template (data not shown), revealed that RolA and RodA both possess the β-barrel core structure typically found in hydrophobins (Hakanpää et al., 2004; 2006; Kwan et al., 2006; Morris et al., 2012) and also that an α-helical structure exists just upstream of the first cysteine in the N-terminal region of RolA (Thr44–Lys51) and RodA (Thr49–Ala53). Moreover, the N-terminal region upstream of the α-helix of the two hydrophobins was predicted to be a random structure. Both RolA and RodA contain three positively charged residues in their N-terminal random structures (Fig. S9A); however, the three positively charged residues of RodA (His23, Lys35 and Lys41) are widely dispersed, whereas those of RolA (His32, Lys34 and Lys41) are clustered together. Therefore, the clustered residues of RolA are more likely to contribute to ionic interactions than are those of RodA, which may explain the large difference between the KD values for the RolA–CutL1 (Fig. 5) and RodA–CutL1 (Fig. 6) interactions. Because the combination of A. oryzae CutL1 with A. nidulans RodA is heterologous and not an authentic combination, QCM analysis with the authentic combination of A. nidulans CutL1 (and its variants) with A. nidulans RodA (and its variants), or with N-terminal chimeric variants between RolA and RodA, is necessary to further confirm which amino acid residues are involved in the interaction and whether the different distributions of the positively charged N-terminal residues in the two hydrophobins explains the large difference in the KD values for the RolA–CutL1 and RodA–CutL1 interactions. Based on the phylogenetic analysis of hydrophobins by Degani et al. (2013), we performed a phylogenetic analysis of RodA orthologs and several other class I hydrophobins derived from ascomycetes and basidiomycetes (Fig. S9C). Alignment analysis of the N-terminal regions of class I hydrophobins upstream of the first cysteine revealed that the N-terminal regions of ascomycetes class I hydrophobins contain several positively charged residues that are clustered in some of the hydrophobins including RodA orthologs. The N-terminal regions of basidiomycetes class I hydrophobins tend to be shorter and generally contain fewer unclustered positively charged residues than those of ascomycetes class I hydrophobins; however, there are some exceptions, for instance, the class I hydrophobin Hydpt1 of Pisolithus tinctorius belonging to basidiomycetes possesses a cluster of positively charged residues in its N-terminal region upstream of the first cysteine. Therefore, the clustered positively charged residues found in the N-terminal regions of ascomycetes/ © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 14–27

Ionic interaction between hydrophobin and cutinase 23

basidiomycetes class I hydrophobins may contribute to ionic interactions with other proteins such as cutinases. In addition, phylogenetic analysis of cutinases derived from ascomycetes and basidiomycetes revealed that most filamentous fungi possess several cutinases, and some filamentous fungi, including Neurospora crassa, also contain several carbohydrate esterase five family acetylxylan esterases (AXEs; underlined in Fig. S10C) that possess amino acid sequences with high similarities to those of cutinases. The cutinases and AXEs form three groups as follows: (i) ascomycetes cutinases, including all aspergilli cutinases, (ii) cutinases derived from other ascomycetes and basidiomycetes, and (iii) AXEs and cutinases showing high similarities to AXEs (Fig. S10C). Acidic amino acid residues corresponding to CutL1 Glu31, Asp142 and Asp171 were highly conserved in the group (i) ascomycetes cutinases, which included all aspergilli cutinases. However, those acidic amino acid residues were only partly conserved in groups (ii) and (iii). The exceptions SCHCODRAFT_104032 and SCHCODRAFT_109319 of S. commune in group (iii) possess conserved acidic amino acid residues corresponding to CutL1 Glu31, Asp142 and Asp171. Therefore, in the cutinases and AXEs, the conserved acidic amino acid residues corresponding to CutL1 Glu31, Asp142 and Asp171 may also contribute to ionic interactions with other proteins such as hydrophobins. There have been several previous studies of the interactions of hydrophobins with other macromolecules. Coexistence of the S. commune class I hydrophobin SC3 and the Trichoderma reesei class II hydrophobins HFBI and HFBII did not affect the self-assembly of SC3; however, it did reduce the precipitation of SC3, suggesting the formation of mixed patches of SC3 and HFBI (or HFBII) via interaction of SC3 with HFBI (or HFBII) (Askolin et al., 2006). Scholtmeijer et al. reported that schizophyllan (β(1– 3),(1–6)-glucan) promotes amyloid formation of SC3 at the water–air interface or the interface between water and a hydrophobic solid such as Teflon (Scholtmeijer et al., 2009). Heparan sulfate has also been shown to induce the β-sheet state of SC3 (Scholtmeijer et al., 2009). Taking the results of these previous studies with those of the present study, it is likely that hydrophobins have not only a surfaceactive function but also a function as scaffolds in the interactions with other macromolecules at various interfaces. In conclusion, we identified amino acid residues involved in the RolA–CutL1 interaction in both molecules. QCM analysis with RolA and CutL1 variants clearly demonstrated that the RolA–CutL1 interaction depends on the ionic interaction between two positively charged residues (His32 and Lys34) in the N-terminus of RolA and three negatively charged residues (Asp142, Asp171 and Glu31) that are probably located on the molecular surface of CutL1 (Fig. 7). © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 14–27

Experimental procedures Strains, media and growth conditions A. oryzae niaD300, a niaD mutant derived from A. oryzae RIB40 was used as the recipient for the transformation and expression of wild-type CutC and CutL1 variants. A. oryzae gla-cut is a wild-type CutL1-expressing strain created in our previous study (Takahashi et al., 2005). A. oryzae NSlDtApEnBdIVdV2 (Yoon et al., 2009) was used as the recipient for the transformation and expression of wild-type RolA, RolA variants and RodA. Wild-type CutL1–, CutL1 variant–, wildtype RolA–, RolA variant– and RodA–overexpressing strains were grown in YPM liquid medium (1% yeast extract, 2% polypeptone, 2% maltose). Escherichia coli DH5α (Invitrogen, Tokyo, Japan) was used for plasmid construction. E. coli was grown at 37°C in 2 × lysogeny broth (2% tryptone, 1% yeast extract, 2% NaCl). All basic molecular biology procedures were carried out as described by Green and Sambrook (Green and Sambrook, 2012).

Creation of RolA-overexpressing strains Creation of a wild-type RolA expression strain was performed as described previously (Takahashi et al., 2005; 2011). A. oryzae NSlD-tApEnBdIVdV2 was used as the recipient. In pNEN142-based plasmids, expression of the inserted ORFs was induced by using maltose as described previously (Takahashi et al., 2005).

Creation of RolA variant– and CutL1 variant–overexpressing strains All of the RolA and CutL1 variants were constructed by using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The template plasmids and primers used are shown in Table S1. RolA variant– and CutL1 variant– overexpression plasmids were introduced into A. oryzae NSlD-tApEnBdIVdV2 or A. oryzae niaD300 according to the protoplast–PEG method (Gomi et al., 1987). Candidate RolA variant– or CutL1 variant–overexpressing strains were selected by colony polymerase chain reaction (PCR) using the rolA and niaD primers for the rolA gene and the cutL1 and niaD primers for the cutL1 gene (Takahashi et al., 2005). The primer sequences were described previously (Takahashi et al., 2005).

Creation of a CutC-overexpressing strain A wild-type CutC-expression plasmid, pNG-CutC, was constructed as follows: the cutC gene was amplified by PCR by using cutCF (5′-CTATCCCATAGGTCGACAAGATGGTG-3′) and cutCR primers (5′-GCCAACGTTACTCTAGAAGAGT ACCTC-3′), and A. oryzae RIB40 genomic DNA as the template. The PCR product was digested with SalI and XbaI and ligated into the SalI/XbaI site of the pNGA142 vector (Hata et al., 1992). A linear form of pNG-CutC digested with BamHI was introduced into A. oryzae niaD300 according to the protoplast–PEG method (Gomi et al., 1987). Candidate CutC-overexpressing strains were selected by means of colony PCR using the cutCF and niaD primers as described

24 T. Takahashi et al. ■

previously (Takahashi et al., 2005). The created CutCoverexpressing strain was designated as A. oryzae CutC. In pNGA142-based plasmids, expression of the inserted ORFs was induced by using maltose as described previously (Hata et al., 1992).

Creation of RodA-overexpressing strains A RodA expression plasmid, pNGA142-rodA, was constructed as follows: the rodA gene was amplified by PCR by using the rodAF1 (5′-GCTATATTCACCACTAGTTCAATTC CTC-3′), rodAF2 (5′-CACCCATATGGCATGAAGTTCTC-3′), rodAR primers (5′-CCCTAAGAAAGGCTTCTAGAGACTGT CGT-3′) and A. nidulans ABPU1 genomic DNA as the template. The PCR product was digested with NdeI and XbaI and ligated into the NdeI/XbaI site of the pNGA142 vector (Hata et al., 1992). A linear form of pNGA142-rodA digested with MunI was introduced into A. oryzae NSlD-tApEnBdIVdV2 according to the protoplast–PEG method (Gomi et al., 1987). Candidate RodA–overexpressing strains were selected by colony PCR using the rodAF2 and rodAR primers as described previously (Takahashi et al., 2005; 2011).

Purification of RolA and CutL1 Purification of wild-type RolA, RolA variants, wild-type CutL1 and CutL1 variants was performed as described previously (Takahashi et al., 2005).

Purification of CutC Conidiospores of A. oryzae CutinaseC (CutC) were inoculated into YPM liquid medium (1 × 106 spores/ml). After cultivation for 24 h at 30°C, the culture broth was filtered through Miracloth (Calbiochem, La Jolla, CA, USA) to remove mycelia. Ammonium sulfate was added to the filtrate (extracellular solution) (15% saturation), and the resultant suspension was centrifuged at 8,000 g for 30 min. The supernatant was recovered and applied to a Phenyl Sepharose CL-4B column (3 × 18 cm, GE Healthcare Bio-Sciences Co., NJ, USA) equilibrated with 10 mM Tris-HCl buffer (pH 8.0) containing 15% saturated ammonium sulfate. CutC was eluted with a 15–0% linear gradient of ammonium sulfate. The CutCcontaining fraction was identified by measurement of esterase activity according to the method described by Maeda et al. (Maeda et al., 2005). The fraction containing CutC was dialyzed against 10 mM 2-morpholinoethanesulfonic acid (MES)-NaOH buffer (pH 5.4). The dialyzed solution containing CutC was then applied to a HiTrap SP column (GE Healthcare Bio-Sciences Co.) equilibrated with the same MES-NaOH buffer (pH 5.4). CutC was eluted with a 0–0.1 M linear gradient of NaCl to obtain purified CutC.

Purification of RodA Purification of RodA was performed by following the method used for RolA purification described previously (Takahashi et al., 2005). The N-terminal amino acid sequence of the purified RodA was determined to be identical to the predicted N-terminus of RodA (i.e., LPPAH) (Fig. S9).

Quantification of the amount of CutL1 recruited by the RolA–PFA350 complex We used perfluoroalkoxy fluorocarbon (Teflon PFA350 suspension; average diameter, 200 nm; specific surface area, 8.95 m2 ml−1) to quantify the amount of CutL1 binding to RolA adsorbed on a hydrophobic surface (de Vocht et al., 1998; Wang et al., 2002; Takahashi et al., 2005). The PFA350 suspension was kindly donated by Du Pont-Mitsui Fluorochemicals (Shizuoka, Japan). Because RolA adsorption to PFA350 (total surface area, 2,780 mm2) was saturated with the addition of over 3.0 μg of purified RolA (data not shown), purified RolA (5 μg) was dissolved in 100 μl of 5 mM MES-NaOH buffer (pH 5.0) containing PFA350 (total surface area, 2,780 mm2). The suspension was incubated at 30°C for 10 min in a glass tube, and the RolA–PFA350 complex was collected by centrifugation (6,300 g for 10 sec at 4°C). The RolA–PFA350 complex was then washed three times with 100 μl of 5 mM MES-NaOH buffer (pH 5.0). Purified CutL1 (5 μg) was dissolved in 350 μl of 10 mM GTA broad buffer [3,3-dimethylglutaric acid, tris (hydroxymethyl) aminomethane, 2-amino-2-methyl-1,3-propanediol] or 0.5 M NaCl containing 10 mM GTA broad buffer adjusted to between pH 3 and 10. CutL1 solution was added to the RolA–PFA350 complex, and the mixture was incubated at 30°C for 1 h in a glass tube. CutL1 bound to RolA–PFA350 complex was harvested by centrifugation and washed three times with 10 mM GTA broad buffer (pH 3–10). SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (Wako, Osaka, Japan) was added to the CutL1–RolA–Teflon complex, boiled, and the boiled sample was subjected to SDS-PAGE. CutL1 was stained with Coomassie Brilliant Blue R-250 (Sigma-Aldrich corp., St. Louis, Missouri, US). After staining, the gel image was captured with a GT-X 820 image scanner (Seiko Epson Corp., Nagano, Japan). Quantification of the amount of CutL1 bound to the RolA–PFA350 complex was analyzed densitometrically with the National Institutes of Health’s (NIH) ImageJ software (http://rsb.info.nih.gov/nih-image/about.html). To quantify the amount of CutL1 or CutL1 variants bound to RolA or RolA variants, 10 mM GTA broad buffer (pH 5.0) was used as both the dissolving buffer and the wash buffer. Other experimental methods were performed as described above.

DEPC modification of RolA Diethylpyrocarbonate treatment of RolA was performed as described by Hsiao et al. (Hsiao et al., 2004) with minor modifications. DEPC was dissolved as stock in absolute alcohol. DEPC modification was performed in 100 mM MES buffer (pH 6.0) containing 1 mg ml−1 purified RolA and 2 mM DEPC, and the mixture was incubated at 37°C for 30 min. After incubation, the reaction mixture was dialyzed against purified water. Because the N-carbethoxylated imidazole in modified histidine residues has an absorbance maximum at 242 nm and RolA contains a single histidine residue, we used spectrophotometry to confirm the DEPC modification.

NCD-4 modification of CutL1 N-cyclohexyl-N′-(4-dimethylamino-alpha-naphthyl) carbodiimide (NCD-4) treatment of CutL1 was performed as © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 14–27

Ionic interaction between hydrophobin and cutinase 25

described by Wang et al. (Wang et al., 1998) with minor modifications. NCD-4 was dissolved as stock in absolute alcohol. NCD-4 modification was performed in 50 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 6.2) containing 1 mg ml−1 purified CutL1 and 100 mM NCD-4, and the mixture was incubated at 4°C for 12 h. After incubation, the reaction mixture was dialyzed against 10 mM Tris-HCl buffer (pH 8.0). We confirmed the NCD-4 modification of CutL1 fluorospectrophotometrically with excitation at 334 nm and emission at 420 nm.

Competitive inhibition assay using CutL1 synthetic peptides Preparation of the RolA–PFA350 complex was performed as described above. The RolA–PFA350 complex (total surface area of PFA350, 2,780 mm2), CutL1 synthetic peptide (2.24 nmol) and purified CutL1 (22.4 nmol) were incubated in 350 μl of 5 mM MES-NaOH buffer (pH 5.0) at 37°C for 1 h. Quantification of the amount of CutL1 bound to the RolA– PFA350 complex was performed as described above. The sequences of the CutL1 synthetic peptides are shown in Fig. S6A.

Analysis of dissociation constants between CutL1 and RolA, CutC and RolA, and CutL1 and RodA by means of a quartz crystal microbalance QCM analysis was performed as described previously (Takahashi et al., 2005) with minor modifications. Preparation of electrodes: wild-type RolA, a RolA variant, or RodA (100 μg ml−1, dissolved in purified water) were immobilized on a QCM electrode (Initium, Tokyo, Japan) at room temperature for 30 min in a plastic Petri dish. To determine whether RolA was adsorbed on the QCM electrode, an anti-RolA polyclonal antibody was injected into the QCM analyzer, and the signal output was monitored. QCM analysis: QCM measurements were performed with a 27-MHz QCM (Affinix Q, Affinix QNμ; Initium Co, Kanagawa, Japan) at 30°C. The analysis chamber contained 8 ml (for Affinix Q) or 0.5 ml (for Affinix QNμ) of running buffer (10 mM sodium phosphate buffer, pH 6.0) or 250 mM NaCl containing running buffer, in which an electrode immobilized with RolA or RolA variant or RodA was immersed. Wild-type CutL1, CutL1 variants and CutC were dialyzed against the running buffer. Dialyzed CutL1 or CutC (stock solution, 1 mg ml−1) was injected stepwise into the analysis chamber. To evaluate the binding affinity of CutL1 or CutC to RolA or RodA, the frequency changes upon addition of CutL1 or CutC to the QCM analysis chamber in a stepwise manner in concentrations ranging from 0 to 900 nM without NaCl and from 0 to 4000 nM with 250 mM NaCl were analyzed by fitting to a Langmuir adsorption isotherm by using AQUA software version 1.2 (Initium) in accordance with the manufacturer’s instructions (Hama et al., 2004; Takahashi et al., 2005). The dissociation constant (KD) was calculated by fitting to the equation KD = [Analyte (CutL1 or its variant)][Bmax/2], where Bmax is the maximum amount of analyte bound to the ligand (RolA–PFA350 complex), and [Analyte][Bmax/2] is the concentration of analyte when the amount of analyte bound to the ligand reaches Bmax/2. © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 14–27

Quantification of the amount of RolA variants bound to PFA350 Purified RolA variants (5 μg) were dissolved in 100 μl of 5 mM MES-NaOH buffer (pH 5.0) containing PFA350, as described above (average diameter, 200 nm; total surface area, 2,780 mm2). Suspensions were incubated at 30°C for 10 min in a glass tube, and the RolA–PFA350 complexes were then collected by centrifugation (6,300 g for 10 sec at 4°C). RolA–PFA350 complexes were washed three times with 100 μl of 5 mM MES-NaOH buffer (pH 5.0). RolA (or RolA variant) was then extracted from the complex with 100% TFA, and the TFA fraction was dried with N2 gas, and the RolA (or RolA variant) was collected. The RolA (or RolA variant) was then dissolved in 5 mM phosphate buffer (pH 7.0). SDS-PAGE sample buffer (Wako, Osaka, Japan) was added to the TFA-extracted RolA (or RolA variant), boiled, and the boiled samples were subjected to SDS-PAGE. RolA variants were stained with Coomassie Brilliant Blue R-250. After staining, the gel image was captured by means of a GT-X 820 image scanner (Seiko Epson Corp.). The amount of RolA variants bound to PFA350 was quantified densitometrically with NIH’s ImageJ software.

Quantification of the amount of BSA bound to PFA350 with or without RolA coating BSA (5 μg) was dissolved in 100 μl of 10 mM GTA buffer (pH 3, 5, 7 or 10) containing PFA350 or RolA-PFA350 (total surface area, 2,780 mm2). The suspension was incubated at 30°C for 10 min in a glass tube, and the BSA–PFA350 (or BSA–RolA–PFA350) complex was collected by centrifugation (6,300g for 10 sec at 4°C). The complex was then washed three times with 100 μl of 10 mM GTA buffer (pH 3, 5, 7 or 10). BSA was then extracted from the complex with SDSPAGE sample buffer, boiled, and subjected to SDS-PAGE. The amount of extracted BSA was quantified by means of imaging analysis as described above. CutL1 recruitment was quantified as described above.

Circular dichroism spectra of CutL1 variants Circular dichroism spectra of CutL1 variants were obtained with minor modification of the methods described previously (Takahashi et al., 2005). CutL1 variants were dissolved in 1 mM sodium citrate buffer (pH 5.0) (protein concentration, 100 μg/ml). The CD spectra were recorded over the 190 (or 195) to 250 nm wavelength region on a J-720 circular dichroism spectrometer (JASCO International, Tokyo, Japan), using a 1-mm quartz cuvette (TOSO, Tokyo, Japan). The temperature was kept at 20°C, and the sample compartment was continuously flushed with N2 gas. Spectra shown are averages of 10 scans, using a bandwidth of 1 nm and a step width of 1 nm.

CD spectra of RolA variants CD spectra of RolA variants were obtained with minor modification of the methods described previously (Takahashi

26 T. Takahashi et al. ■

et al., 2005). RolA variants were dissolved in 1 mM sodium citrate buffer pH 5 (protein concentration, 40 μg/ml). Analysis conditions were as described above.

Acknowledgements This work was partly supported by KAKENHI (Japan Society for the Promotion of Science), a Grant-in-Aid for Scientific Research (B) (grant no. 20380175 to K.A.), and Challenging Exploratory Research (grant no. 24658281 to K.A.). K.A. also would like to thank RITE (Research Institute of Innovative Technology for the Earth) for their financial support through the Joint Program to Promote Technological Development. We thank Du Pont-Mitsui Fluorochemicals for the gift of the Teflon PFA350 suspension. We also thank Katsuhiko Kitamoto for providing the Aspergillus oryzae NSlD-tApEnBdIVdV2. We thank Hideaki Koike, Tomonori Fujioka, Osamu Mizutani, Kentaro Furukawa, Kei Nanatani, Keiko Orui, Yoonkyung Kim, Daiki Sato, Megumi Nagayama, Yuki Terauchi, Naoki Abe and Natsumi Okada for their helpful discussions and technical assistance.

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Ionic interaction of positive amino acid residues of fungal hydrophobin RolA with acidic amino acid residues of cutinase CutL1.

Hydrophobins are amphipathic proteins secreted by filamentous fungi. When the industrial fungus Aspergillus oryzae is grown in a liquid medium contain...
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