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Involvement of hydrophobic amino acid residues in C7–C8 loop of Aspergillus oryzae hydrophobin RolA in hydrophobic interaction between RolA and a polyester a
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Takumi Tanaka , Hiroki Tanabe , Kenji Uehara , Toru Takahashi & Keietsu Abe a
Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan b
Laboratory of Enzymology, Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan c
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Microbial Genomics Laboratory, New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan Published online: 10 Jul 2014.
To cite this article: Takumi Tanaka, Hiroki Tanabe, Kenji Uehara, Toru Takahashi & Keietsu Abe (2014) Involvement of hydrophobic amino acid residues in C7–C8 loop of Aspergillus oryzae hydrophobin RolA in hydrophobic interaction between RolA and a polyester, Bioscience, Biotechnology, and Biochemistry, 78:10, 1693-1699, DOI: 10.1080/09168451.2014.932684 To link to this article: http://dx.doi.org/10.1080/09168451.2014.932684
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Bioscience, Biotechnology, and Biochemistry, 2014 Vol. 78, No. 10, 1693–1699
Involvement of hydrophobic amino acid residues in C7–C8 loop of Aspergillus oryzae hydrophobin RolA in hydrophobic interaction between RolA and a polyester Takumi Tanaka1, Hiroki Tanabe1, Kenji Uehara2, Toru Takahashi3 and Keietsu Abe1,3,* 1
Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan; 2Laboratory of Enzymology, Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan; 3Microbial Genomics Laboratory, New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan Received April 3, 2014; accepted April 28, 2014
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http://dx.doi.org/10.1080/09168451.2014.932684
Hydrophobins are amphipathic secretory proteins with eight conserved cysteine residues and are ubiquitous among filamentous fungi. The Cys3–Cys4 and Cys7–Cys8 loops of hydrophobins are thought to form hydrophobic segments involved in adsorption of hydrophobins on hydrophobic surfaces. When the fungus Aspergillus oryzae is grown in a liquid medium containing the polyester polybutylene succinate-co-adipate (PBSA), A. oryzae produces hydrophobin RolA, which attaches to PBSA. Here, we analyzed the kinetics of RolA adsorption on PBSA by using a PBSA pull-down assay and a quartz crystal microbalance (QCM) with PBSAcoated electrodes. We constructed RolA mutants in which hydrophobic amino acids in the two loops were replaced with serine, and we examined the kinetics of mutant adsorption on PBSA. QCM analysis revealed that mutants with replacements in the Cys7–Cys8 loop had lower affinity than wild-type RolA for PBSA, suggesting that this loop is involved in RolA adsorption on PBSA. Key words:
adsorption; Aspergillus oryzae; fungi; hydrophobic interaction; hydrophobin
Hydrophobins, which are small, amphipathic secretory proteins with eight conserved cysteine residues, are ubiquitous among filamentous fungi1–3) and play several important roles in fungal physiology. For example, they are involved in fungal adhesion to solid surfaces, in the formation of a protective surface coating on fugal cells, and in the reduction of water surface tension, and processes that support the growth of fungal aerial structures such as hyphae and conidiospores.4) When the fungus Aspergillus oryzae is grown under liquid culture conditions with the biodegradable aliphatic polyester polybutylene succinate-co-adipate (PBSA) as the sole carbon source, the fungus simultaneously produces both the biosurfactant protein hydrophobin RolA (GenBank *Corresponding author. Email: [email protected] © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
acc. no. AB094496)/HypA (GenBank acc. no. AB097447) and the cutinase CutL1, which hydrolyzes PBSA.5) The secreted RolA is adsorbed on the surface of PBSA, which has a chemical structure resembling that of the plant-wax polyester cutin,6) and efficiently recruits CutL1, resulting in condensation of CutL1 on the PBSA surface and stimulation of PBSA hydrolysis. This discovery of CutL1 recruitment by PBSA-bound RolA adds a previously unknown function to the above-described roles of hydrophobins. Recruitment of proteins by immobilized hydrophobins such as HGFI and HFBI has also been reported.7) The recruitment of hydrolytic enzymes by amphipathic proteins attached to solid surfaces could be applied to a large-scale degradation of hydrophobic solid materials at solid–liquid interfaces.5,8) Note that the soluble form of RolA does not interact with CutL1: that is, adsorption of the hydrophobin to a solid surface, such as PBSA, is essential for subsequent RolA-dependent CutL1 recruitment.5) Therefore, analysis of the kinetics of RolA adsorption on PBSA is important for understanding the hydrolysis of PBSA by CutL1 recruited by immobilized RolA. Hydrophobins possess conserved core β-barrel structures9) that divide the molecules into two segments: a hydrophilic segment and a hydrophobic segment that is predicted to be involved in adsorption of the proteins to hydrophobic solid surfaces.10,11) Involvement of the hydrophobic segment in self-assembly (biofilm formation) has been predicted by structural analyses and mutation analysis.9,10,12) However, direct involvement of the hydrophobic segments in hydrophobin adsorption to solid materials has not been confirmed, and the kinetics of hydrophobin adsorption on surfaces have not yet been investigated. Here, we used a pull-down assay with PBSA microparticles and a quartz crystal microbalance (QCM) with PBSA-coated electrodes to analyze the kinetics of adsorption of both wild-type RolA and RolA mutants in which hydrophobic amino acid residues in the hydrophobic segment (hydrophobic patches) were replaced with serine. On the basis of our
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results, we discuss the nature of the interaction between RolA and PBSA.
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Materials and methods Fungal strains, culture media, and growth conditions. A. oryzae niaD300 (niaD−).13) a niaD mutant derived from A. oryzae strain RIB40, was used as a recipient for transformation and protein expression experiments. These two strains were grown in YPG complete medium (1% yeast extract, 2% Polypepton™, and 2% glucose) or Czapek Dox minimal medium.14) Strains overexpressing RolA and the RolA mutants described below were grown in YPM liquid medium (1% yeast extract, 2% Polypepton™, and 2% maltose) to study the expression of, and to purify, RolA and its mutants. Shaken cultures of A. oryzae cells were generally grown at 30 °C on rotary shakers (160 rpm). Escherichia coli XL1-Blue (Stratagene, Tokyo, Japan) was used for plasmid construction, and E. coli DH5α (NIPPON GENE, Tokyo, Japan) was used for modification of constructed plasmids for creating RolA mutants. E. coli was grown at 37 °C in 2× Luria-Bertani medium (2% tryptone, 1% yeast extract, 2% NaCl). All basic molecular biology procedures were performed as described in the literature.15) Alignment analysis of amino acid sequences of hydrophobins. Amino acid sequences of the hydrophobins Neurospora crassa EAS (GenBank acc. no. EAA34064, class I),16) Trichoderma reesei HFBII (GenBank acc. no. P79073, class II),17) and A. oryzae RolA (GenBank acc. no. AB094496, class I)5) were obtained from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The alignment of the amino acid sequences of the three hydrophobins was generated with GENETYX sequence analysis software (ver. 10, GENETYX, Tokyo, Japan). Similarity scores among the amino acid sequences of the three hydrophobins were also calculated with GENETYX. Creation of RolA-overexpressing strain. An expression plasmid for RolA (pNG-eno-hyp) was constructed as described previously.5) A linear form of pNG-eno-hyp digested with MunI was introduced into A. oryzae niaD300 (niaD−) essentially according to the protoplast–PEG method.18) Candidate RolA-overexpressing strains were selected by colony PCR using the rolA and niaD primers as described previously.5,19) The resulting RolA-overexpressing strain was designated A. oryzae eno-hyp. Creation of strains overexpressing RolA mutants. Hydrophobic amino acid residues in hydrophobic patches of RolA (F70, V73, L77, L78, A79, L81, L82, L85, L86, A88, L95, L97, and L98 in the C3–C4 loop and L137, V138, L142, and P143 in the C7–C8 loop) were replaced with serine. The double mutant
RolA-L137S/L142S was also created. All of the RolA mutants were constructed with a QuickChange SiteDirected Mutagenesis Kit (Stratagene, Los Angeles, CA, USA). The template plasmid was pNG-eno-hyp, and the primers used are listed in Table S1. RolA mutant–overexpression plasmids were introduced into A. oryzae niaD300 according to the protoplast–PEG method.18) Candidate strains overexpressing RolA mutants were selected by colony PCR using the rolA and niaD primers, as described previously.5,19) The resulting strains overexpressing the mutants are designated A. oryzae eno-hyp mutants. Expression and purification of recombinant RolA and RolA mutants. Conidiospores of A. oryzae eno-hyp or A. oryzae eno-hyp mutants were inoculated into YPM liquid medium (1 × 106 conidia mL−1). After culture of the mutants for 20 h at 30 °C, the culture broth was filtered through Miracloth (Merck KGaA, Darmstadt, Germany) to separate mycelia. The filtrate (extracellular solution) was brought to 40% saturation by adding 90% ammonium sulfate, and the resultant suspension was centrifuged at 8,000g for 30 min to recover the supernatant. The supernatant was applied to a Phenyl Sepharose CL-4B column (3 × 18 cm, GE Healthcare Japan, Tokyo, Japan) equilibrated with 10 mM Tris–HCl buffer (pH 8.0) containing 40%-saturated ammonium sulfate. RolA was eluted with a 40–0% linear gradient of ammonium sulfate. After each fraction was checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; 3% stacking gel and 17.5% running gel), the fraction containing RolA was dialyzed against 5 mM Tris–HCl buffer (pH 9.0). The dialyzed solution containing RolA was subsequently applied to a Cellufine Q-500 column (3 × 12 cm, JNC Corp., Tokyo, Japan) equilibrated with the same Tris–HCl buffer. RolA was eluted with a 0–0.3 M linear gradient of NaCl. After the fractions were examined by SDS-PAGE, the fraction containing RolA was dialyzed against 10 mM sodium citrate buffer (pH 4.0). The dialyzed solution containing RolA was subsequently applied to an S-Sepharose FF column (2 × 18 cm, GE Healthcare Japan, Tokyo, Japan) equilibrated with the same sodium citrate buffer. RolA was eluted with a 0–0.3 M linear gradient of NaCl to obtain purified RolA. All the purification procedures described above were performed at 4 °C. To qualitatively determine the secreted RolA in the culture bloth, proteins separated by SDS-PAGE were blotted onto a Sequi-Blot PVDF Membrane (Bio-Rad, Tokyo, Japan) by using a Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad). A mouse polyclonal antibody against RolA purified from A. oryzae eno-hyp (T.K. Craft, Gunma, Japan),5) a horseradish phosphatase–conjugated rabbit polyclonal antibody against mouse IgG (Promega, Wisconsin, USA), and SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Yokohama, Japan) were used for Western blot analysis of RolA and RolA mutants. Chemiluminescence signals were detected with a ImageQuant LAS 4000mini lumino image analyzer (Fujifilm, Tokyo, Japan).
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Interaction of A. oryzae hydrophobin RolA with a polyester
Pull-down assay of RolA to PBSA microparticles. Conidiospores of A. oryzae niaD300, A. oryzae enohyp, or A. oryzae eno-hyp mutants were inoculated into YPM liquid medium (2 × 106 conidia mL−1). After shake-cultivation (160 rpm) for 20 h at 30 °C, the culture broth was filtered through Miracloth (Merck KGaA) to separate mycelia. Trichloroacetic acid solution (100% w/v, 0.5 mL) was added to a 1-mL aliquot of each culture broth, and the mixture was incubated at 4 °C for 1 h. After centrifugation (17,400g for 20 min), the precipitated protein was solubilized in 10 μL of SDS-sample buffer (Wako Pure Chemical Industries, Osaka, Japan) and then boiled. The boiled samples were subjected to SDS-PAGE. RolA and its mutants were stained with Coomassie Brilliant Blue R-250. After staining of the samples, gel images were captured with a GT-X 820 image scanner (Seiko Epson Corp., Nagano, Japan). The amounts of RolA and its mutants were quantified densitometrically with ImageJ software (ver. 1.44p; http://rsb.info.nih.gov/ij/). The concentration of RolA mutant in each culture broth was adjusted to the RolA concentration in the culture broth of A. oryzae niaD300 expressing wild-type RolA. The pH of each sample was adjusted to pH 6. PBSA microparticles (Showa Highpolymer Co., Tokyo, Japan) were added to each sample (final concentration of PBSA 0.1% w/v), and the mixture was incubated at 30 °C for 30 s, because the adsorption of wild-type RolA on PBSA was saturated within 30 s at 30 °C (Uehara et al., unpublished results). The microparticles were collected by centrifugation (17,400g for 20 min). SDS-sample buffer (10 μL) was added to the microparticles collected from each sample, the mixture was boiled, and the boiled sample was subjected to SDS-PAGE. RolA and RolA mutants adsorbed on the microparticles were stained with Coomassie Brilliant Blue R-250. The amounts of RolA and its mutants adsorbed on the microparticles were quantified densitometrically as described above. Circular dichroism measurements of RolA. Circular dichroism (CD) spectra of RolA were measured by using a method described previously.5) The spectra were recorded over the wavelength region from 190 to 250 nm on a J-725 CD spectrometer (JASCO International, Tokyo, Japan) by using a 1-mm quartz cuvette (GL Sciences, Tokyo, Japan). The temperature was kept at 25 °C, and the sample compartment was continuously flushed with N2 gas. Spectra are the averages of 10 scans, which were performed with a bandwidth of 1 nm, a step width of 1 nm, and 5-s averaging per point. The baseline of the CD spectrum was flat for 5 mM sodium phosphate buffer (pH 7.0), which was used as the RolA solvent. In order to eliminate self-assembled hydrophobin molecules, class I hydrophobins such as Schizophyllum commune SC3 and RolA are generally treated with 100% trifluoroacetic acid (TFA) and subsequent removal of TFA from the TFA-treated samples leads to refolding of hydrophobin molecules.5) Before CD analysis, purified RolA was condensed in a glass tube by lyophilization. Then the lyophilized RolA was treated with 100% TFA and dried with N2 gas for removal of TFA. Dried RolA
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was dissolved in 5 mM phosphate buffer (pH 7.0), and 250 μg/mL of RolA was placed in the cuvette. Analysis of RolA–PBSA interaction by means of a QCM method. The resonance frequency of a QCM electrode decreases linearly as the mass on the electrode increases at the nanogram level.20) A frequency change of 100 Hz corresponds to 3 ng of analyte protein bound to the QCM electrode. We modified the QCM method of Sato et al.21) for our QCM analyses of the RolA–PBSA interaction as follows. Preparation of electrodes: a PBSA film (thickness 15–20 μm, Showa Denko K.K., Tokyo, Japan) was dissolved in chloroform at a concentration of 0.05% w/v, and 3 μL of the dissolved PBSA was cast on a QCM electrode (Initium, Tokyo, Japan). The coated electrode was dried at room temperature for at least 20 min. We found that the amount of immobilized PBSA appropriate for obtaining fine detection sensitivity was 60–240 ng. QCM measurements were performed with an initial frequency of 27 MHz at 30 °C by using an Affinix QNμ system (Initium). The analysis chamber contained 500 μL of running buffer (10 mM GTA buffer [3.3 mM 3,3-dimethylglutaric acid, 3.3 mM Tris, and 3.3 mM 2-amino-2-methyl-1,3-propanediol], pH 7.0) in which a PBSA-coated electrode was immersed. Purified RolA or RolA mutants were dissolved in the running buffer, the solution was injected stepwise into the analysis chamber in which the PBSA-coated electrode was immersed, and QCM values were measured as a function of the concentration of RolA in the chamber (30– 700 nM). To evaluate the affinity of binding of RolA to PBSA, we analyzed the frequency changes upon the addition of RolA to the QCM analysis chamber by fitting to a Langmuir adsorption isotherm plot by using Aqua software (ver. 1.2, Initium) in accordance with the manufacturer’s instructions.22) The dissociation constant (KD) was calculated by the fitting based on the equation KD = [Analyte][Bmax/2], where Bmax is the maximum amount of an analyte bound to a ligand, and [Anlyte][Bmax/2] is the concentration of the analyte when the amount of the analyte bound to the ligand reaches Bmax/2.
Results Alignment analysis of A. oryzae RolA (class I), N. crassa EAS (class I), and T. reesei HFBII (class II) Hydrophobins are categorized as either class I or class II on the basis of their hydrophobicity and other physicochemical properties.3) Class I hydrophobins are generally more hydrophobic than class II hydrophobins and form polymer films made up of cylindrical rodlets.23,24) The outward-facing hydrophobic surfaces of the films show extremely low wettability,23,24) and the films are resistant to boiling in detergents or alkalis.2) Compared with class I hydrophobins, class II hydrophobins have lower hydropathy scores and are less robust, and they lack the rodlet morphology found in the polymer films of class I hydrophobins.25) Although the similarity of the amino acid sequences of N. crassa hydrophobin EAS (class I) and T. reesei hydrophobin HFBII (class II) is less than 30%, EAS and HFBII have
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9)
similar structural features. Based on their 3D structures, both of these hydrophobins are predicted to possess β-barrel core structures and to show a similar topology in which the central β-barrel core divides the hydrophobin molecule into two segments: a hydrophobic segment (hydrophobic patches) and a hydrophilic segment.9,10,26–28) Fig. 1 shows the alignment of the amino acid sequences of EAS, HFBII, and RolA and the homology among the three hydrophobins. Hydrophobins possess eight conserved cysteine residues (Fig. 1). The C3(Cys45)–C4(Cys71) loop and the C7(Cys87)–C8(Cys106) loop in EAS form hydrophobic patches and are thought to be involved in adsorption of EAS to hydrophobic surfaces.9) A. oryzae RolA possesses two amino acid loops similar to those of EAS: the C3(Cys61)–C4(Cys101) loop is rich in hydrophobic amino acid residues, and only half part of C7(Cys126)–C8(Cys144) loop is rich in hydrophobic residues (Fig. 1). Hydropathy analysis also suggests that the C3–C4 and C7–C8 loops of RolA comprise hydrophobic patches (data not shown). Pull-down Assay of RolA mutants to PBSA microparticles Because both RolA and EAS are predicted to have the C3–C4 and C7–C8 loops which are rich in hydrophobic amino acid residuess forming hydrophobic patches, we constructed a series of RolA mutants in which selected hydrophobic residues of the two loops were replaced with serine. To examine adsorption of the mutants on PBSA, we incubated crude culture broth
containing each mutant with PBSA microparticles and measured the amounts of the mutants adsorbed (Fig. 2). The amounts of RolA-L137S and RolAL142S mutants adsorbed on the PBSA microparticles were 60 and 40%, respectively, of the adsorbed amount of wild-type RolA. CD analysis of RolA mutants Because the pull-down assay revealed that L137 and L142 play important roles in RolA adsorption on PBSA, we examined whether single substitution or double substitution of these residues resulted in major changes in the structure of RolA. Because the peak wavelengths and absorbance of the CD spectra originating from the two single mutants (RolA-L137S, RolAL142S) and double mutant (RolA-L137S/L142S) were almost similar to those of the CD spectrum of wildtype RolA, the substitutions (L137S, L142S, and L137S/L142S) did not cause major structural changes in RolA (Fig. 3). Taken together with the results of the pull-down assay, these results indicate that the decrease in adsorption of the mutants on PBSA was attributable to a reduction in the hydrophobicity of the C7–C8 loop. QCM analysis of RolA adsorption on PBSA The pull-down assay and the CD spectra of the RolA mutants suggested that L137 and L142 were involved in the adsorption of RolA on PBSA. Therefore, we quantified the interaction between the mutants and
Fig. 1. Comparison of amino acid sequences of hydrophobins EAS, RolA, and HFBII. Notes: (A) Sequences of class I hydrophobins Aspergillus oryzae RolA and N. crassa EAS and class II hydrophobin T. reesei HFBII. The eight cysteine residues that form the conserved disulfide bonds are indicated in bold type. Selected hydrophobic amino acid residues of RolA for creating RolA mutants were highlighted by gray color. L137 and L142 of RolA were underlined. The alignment was generated with GENETYX (ver. 10). (B) Similarity scores of the amino acid sequences of RolA, EAS, and HFBII, calculated with GENETYX (ver. 10).
Interaction of A. oryzae hydrophobin RolA with a polyester
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Fig. 2. Pull-down Assay of RolA mutants to PBSA microparticles. Notes: Adsorption of RolA mutants in which a hydrophobic amino acid in the hydrophobic region was replaced with serine on PBSA microparticles was examined. PBSA microparticles were incubated with the culture supernatants of RolA mutant strains at 30 °C for 30 s. After the incubation, the RolA–PBSA complex was collected by centrifugation, and the proteins bound to the PBSA microparticles were extracted with SDS-PAGE loading buffer and subjected to SDS-PAGE. The data are means ± SD (n = 3).
Fig. 3. CD spectra of RolA in the soluble form. Notes: Wild-type RolA (solid line), RolA-L137S mutant (dotted line), RolA-L142S mutant (short-dashed line), and RolA-L137/L142S double mutant (long-dashed line). CD spectra are the averages of 10 scans collected by using a reference solution without the protein.
PBSA by using QCM analysis (Fig. 4 and Table 1). Although the KD value of RolA-L137S tended to be larger than that of wild-type RolA, the two KD values were statistically indistinguishable but the Bmax value of RolA-L137S was statistically smaller than that of wild-type RolA. The KD value of RolA-L142S was statistically larger than that of wild-type RolA; however, the Bmax value of RolA-L142S was not distinguishable from that of wild-type RolA. The KD value of RolAL137S/L142S was larger than that of wild-type RolA, and the Bmax value of RolA-L137S/L142S was smaller than that of wild-type RolA. But the KD value of RolA-L137S/L142S was statistically indistinguishable from those of RolA-L137S and RolA-L142S, and the Bmax value of RolA-L137S/L142S was statistically indistinguishable from that of RolA-L137S.
Discussion We previously reported that A. oryzae RolA attaches to PBSA surfaces and then recruits polyesterase CutL1 to the surfaces, and that condensation of CutL1 on the
Fig. 4. QCM analysis of interaction of RolA and its mutants with PBSA. Notes: To evaluate the affinity of binding of RolA and its mutants to PBSA, the frequency changes upon addition of RolA or its mutants to the QCM analysis chamber were analyzed by fitting to a Langmuir adsorption isotherm plot by using Aqua software (ver. 1.2, Initium). (A) wild-type RolA, (B) RolA-L137S mutant, (C) RolAL142S mutant, and (D) RolA-L137S/L142S double mutant. R2 is the coefficient of determination.
surfaces leads to stimulation of PBSA hydrolysis catalyzed by CutL1.5) The main driving force for the interaction between PBSA-bound RolA and CutL1 is predicted to be electrostatic interaction (unpublished results, Takahashi et al.). However, the mechanism and kinetics of the RolA–PBSA interaction remain unclear. Although it is known that the hydrophobic segments of hydrophobins are involved in their self-assembly (biofilm formation),11,12,29) whether the hydrophobic segments are directly involved in the adsorption of hydrophobins on solid surfaces remains unclear. Structural analysis of N. crassa class I hydrophobin EAS demonstrated that it contained the eight cysteine residues conserved among hydrophobins.9,10) The C3 (Cys45)–C4(Cys71) loop is rich in hydrophobic amino acid residues, and only half part of C7(Cys87)–C8 (Cys106) loop is rich in hydrophobic residues.9) Kwan et al. reported that the β-barrel core structure and the topology of N. crassa EAS are similar to those of class II hydrophobin HFBII from T. reesei, even though the
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Table 1. KD and Bmax values in the interaction of RolA and its mutants with PBSA. RolA
KD (nM) §
wild type
73.77 ± 13.49
L137S
139.75 ± 40.92
L142S L137S/L142S
303.17 ± 74.17 221.20 ± 53.54
Bmax (ng) § 6.28 ± 1.62 *
*
4.97 ± 1.11 *
6.11 ± 1.61 3.69 ± 0.92
* * * *
Dissociation constants (KD) were determined by fitting to a Langmuir adsorption isotherm plot; KD = [RolA][Bmax/2], where Bmax is the maximum amount of RolA bound to the PBSA-coated electrode, and RolA[Bmax/2] is the RolA concentration when the amount of RolA bound to the PBSA-coated electrode reaches Bmax/2. *p < 0.05 (n = 4).
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§
sequence homology of these two hydrophobins is not high.9) In both of these proteins, the region containing the C3–C4 loop, the C7–C8 loop, and the intersection of the two loops faces toward the air side (the hydrophobic side) at an air–water interface.9,10) These structural analyses, taken together with the results of biochemical studies of biofilm formation (self-assembly) of the two hydrophobins,11,12,29) suggest that the hydrophobic segments—that is, the C3–C4 and C7–C8 loops —are involved in self-assembly (biofilm formation). However, there is no direct evidence that the two loops are involved in hydrophobic interaction with solid surfaces. If the barrel topology of N. crassa EAS is shared by A. oryzae RolA, the parts of the C3–C4 and C7–C8 loops that contain hydrophobic residues (F70–L98 and L137–P143, respectively) of RolA are predicted to have the same orientation as the C3–C4 and C7–C8 loops of EAS. Therefore, to determine whether the C3– C4 and C7–C8 loops of RolA were directly involved in its interaction with PBSA, we constructed RolA mutants in which some of the hydrophobic amino acid residues in the loops were replaced with serine. Pulldown assay of the mutants with PBSA microparticles (Fig. 2) and QCM analysis revealed that the singlemutant RolA-L142S and double-mutant RolA-L137S/ L142S had statistically larger KD values and smaller Bmax values for PBSA than the wild-type protein (Fig. 4 and Table 1) and that the single-mutant RolA-L137S had a smaller Bmax value than that of wild-type protein. These results suggest that the L137S substitution in the C7–C8 loop affected the amount of protein adsorbed on PBSA at the steady-state adsorption and that the L142S substitution changed the affinity of the protein for PBSA. To compare the analysis time-length (60–240 min) and protein concentrations (30–700 nM) in the QCM analyses with those in the pull-down assay, the latter employed the shorter incubation time such as 30 s with the higher protein concentrations (2,000–12,000 nM). Therefore, because RolA-L137S showed the smaller Bmax value than wild-type and RolA-142S indicated larger KD value than wild-type, the lower levels of protein adsorption of the two mutants on PBSA microparticles than the adsorption level of wild-type RolA are attributable to both the kinetic properties of the two mutants and the conditions of the pull-down assay (Table 1, Fig. 2). The results of pull-down assay and QCM analysis indicate that the hydrophobic amino acid residues L137
and L142 are important for adsorption of RolA on PBSA. When the EAS topology is imposed on RolA, L137 and L142 are close to the interface region between the C3–C4 and C7–C8 loops. CD analysis of wild-type RolA and the three RolA mutants suggested that the mutations did not lead to any major structural changes. Consequently, the hydrophobicity of the region containing L137 and L142 must be important for the RolA–PBSA interaction. However, the two RolA mutants still indicated the low levels of adsorption on PBSA, so some other residues besides L137 and L142 must also be involved in the adsorption of RolA on PBSA. In conclusion, pull-down assays of RolA mutants with PBSA particles and QCM analysis of the mutants with PBSA-coated electrodes demonstrated that hydrophobic amino acid residues L137 and L142 in the C7–C8 loop of RolA are directly involved in adsorption of RolA on PBSA surfaces via hydrophobic interaction between RolA and PBSA. This is the first kinetic evidence that hydrophobic amino acid residues in the C7–C8 loop of hydrophobins are directly involved in their interaction with solid materials.
Supplemental material The supplemental material for this paper is available at http://dx.doi.org/10.1080/09168451.2014.932684.
Funding This work was partly supported by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science [20380175] (to K.A.); and Challenging Exploratory Research Grant [24658281] (to K.A.). K.A. also thanks the Research Institute of Innovative Technology for the Earth for financial support through the Joint Program to Promote Technological Development.
References [1] Wessels JGH, De Vries OMH, Asgeirsdottir SA, Schuren FHJ. Hydrophobin genes involved in formation of aerial hyphae and fruit bodies in Schizophyllum. Plant Cell. 1991;3:793–799. [2] De Vries OMH, Fekkes MP, Wösten HAB, Wessels JGH. Insoluble hydrophobin complexes in the walls of Schizophyllum commune and other filamentous fungi. Arch. Microbiol. 1993;159: 330–335. [3] Wessels JGH. Developmental regulation of fungal cell wall formation. Annu. Rev. Phytopathol. 1994;32:413–437. [4] Wösten HAB, van Wetter MA, Lugones LG, van der Mei HC, Busscher HJ, Wessels JGH. How a fungus escapes the water to grow into the air. Curr. Biol. 1999;9:85–88. [5] Takahashi T, Maeda H, Yoneda S, Ohtaki S, Yamagata Y, Hasegawa F, Gomi K, Nakajima T, Abe K. The fungal hydrophobin RolA recruits polyesterase and laterally moves on hydrophobic surfaces. Mol. Microbiol. 2005;57:1780–1796. [6] Fang X, Qiu F, Yan B, Wang H, Mort AJ, Stark RE. NMR studies of molecular structure in fruit cuticle polyesters. Phytochemistry. 2001;57:1035–1042. [7] Wang Z, Huang Y, Li S, Xu H, Linder MB, Qiao M. Hydrophilic modification of polystyrene with hydrophobin for time-resolved immunofluorometric assay. Biosens. Bioelectron. 2010;26: 1074–1079.
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Interaction of A. oryzae hydrophobin RolA with a polyester [8] Ohtaki S, Maeda H, Takahashi T, Yamagata Y, Hasegawa F, Gomi K, Nakajima T, Abe K. Novel hydrophobic surface binding protein, HsbA, produced by Aspergillus oryzae. Appl. Environ. Microbiol. 2006;72:2407–2413. [9] Kwan AH, Winefield RD, Sunde M, Matthews JM, Haverkamp RG, Templeton MD, Mackay JP. Structural basis for rodlet assembly in fungal hydrophobins. Proc. Nat. Acad. Sci. 2006;103: 3621–3626. [10] Kwan AH, Macindoe I, Vukašin PV, Morris VK, Kass I, Gupte R, Mark AE, Templeton MD, Mackay JP, Sunde M. The Cys3– Cys4 loop of the hydrophobin EAS is not required for rodlet formation and surface activity. J. Mol. Biol. 2008;382:708–720. [11] Gruné r MS, Szilvay GR, Berglin M, Lienemann M, Laaksonen P, Linder MB. Self-assembly of class II hydrophobins on polar surfaces. Langmuir. 2012;28:4293–4300. [12] Macindoe I, Kwan AH, Ren Q, Morris VK, Yang W, Mackay JP, Sunde M. Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS. Proc. Natl. Acad. Sci. USA. 2011;109:804–811. [13] Machida M. Progress of Aspergillus oryzae genomics. Adv. Appl. Microbiol. 2002;51:81–106. [14] Nakajima K, Kunihiro S, Sano M, Zhang Y, Eto S, Chang YC, Suzuki T, Jigami Y, Machida M. Comprehensive cloning and expression analysis of glycolytic genes from the filamentous fungus, Aspergillus oryzae. Curr. Genet. 2000;37:322–327. [15] Green MR, Sambrook J. Molecular cloning: a laboratory manual. 4th ed. New York: Cold Spring Harbor; 2012. [16] Galagan JE, Calvo SE, Borkovich KA, Selker EU, Read ND, Jaffe D, FitzHugh W, Ma LJ, Smirnov S, Purcell S, Rehman B, Elkins T, Engels R, Wang S, Nielsen CB, Butler J, Endrizzi M, Qui D, Ianakiev P, Bell-Pedersen D, Nelson MA, Werner-Washburne M, Selitrennikoff CP, Kinsey JA, Braun EL, Zelter A, Schulte U, Kothe GO, Jedd G, Mewes W, Staben C, Marcotte E, Greenberg D, Roy A, Foley K, Naylor J, Stange-Thomann N, Barrett R, Gnerre S, Kamal M, Kamvysselis M, Mauceli E, Bielke C, Rudd S, Frishman D, Krystofova S, Rasmussen C, Metzenberg RL, Perkins DD, Kroken S, Cogoni C, Macino G, Catcheside D, Li W, Pratt RJ, Osmani SA, DeSouza CP, Glass L, Orbach MJ, Berglund JA, Voelker R, Yarden O, Plamann M, Seiler S, Dunlap J, Radford A, Aramayo R, Natvig DO, Alex LA, Mannhaupt G, Ebbole DJ, Freitag M, Paulsen I, Sachs MS, Lander ES, Nusbaum C, Birren B. The genome sequence of the filamentous fungus Neurospora crassa. Nature. 2003;422:859–868. [17] Nakari-Setala T, Aro N, Ilmen M, Munoz G, Kalkkinen N, Penttila M. Differential expression of the vegetative and spore-bound hydrophobins of Trichoderma reesei cloning and
[18]
[19]
[20]
[21]
[22]
[23] [24]
[25]
[26]
[27]
[28]
[29]
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characterization of the hfb2 gene. Eur. J. Biochem. 1997;248: 415–423. Gomi K, Iimura Y, Hara S. Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans argB gene. Agric. Biol. Chem. 1987;51:2549–2555. Takahashi T, Mizutani O, Shiraishi Y, Yamada O. Development of an efficient gene-targeting system in Aspergillus luchuensis by deletion of the non-homologous end joining system. J. Biosci. Bioeng. 2011;112:529–534. Okahata Y, Niikura K, Sugiura Y, Sawada M, Morii T. Kinetic studies of sequence-specific binding of GCN4-bZIP peptides to DNA strands immobilized on a 27-MHz quartz-crystal microbalance. Biochemistry. 1998;37:5666–5672. Sato Y, Sagami I, Shimizu T. Identification of Caveolin-1-interacting sites in neuronal nitric-oxide synthase: molecular mechanism for inhibition of NO formation. J. Biol. Chem. 2004;279: 8827–8836. Hama H, Saito A, Takeda T, Tanuma A, Xie Y, Sato K, Kazama JJ, Gejyo F. Evidence indicating that renal tubular metabolism of leptin is mediated by megalin but not by the leptin receptors. Endocrinology. 2004;145:3935–3940. Beever RE, Dempsey G. Function of rodlets on the surface of fungal spores. Nature. 1978;272:608–610. Wösten HAB, De Vries OMH, Wessels JGH. Interfacial selfassembly of a fungal hydrophobin into a hydrophobic rodlet layer. Plant Cell. 1993;5:1567–1574. Paananen A, Vuorimaa E, Torkkeli M, Penttilä M, Kauranen M, Ikkala O, Lemmetyinen H, Serimaa R, Linder MB. Structural hierarchy in molecular films of two class II hydrophobins. Biochemistry. 2003;42:5253–5258. Hakanpää J, Parkkinen TAA, Hakulinen N, Linder MB, Rouvinen J. Crystallization and preliminary X-ray characterization of Trichoderma reesei hydrophobin HFBII. Acta Crystallogr., Sect D: Biol. Crystallogr. 2004;60:163–165. Hakanpää J, Paananen A, Askolin S, Nakari-Setala T, Parkkinen T, Penttila M, Linder MB, Rouvinen J. Atomic resolution structure of the HFBII hydrophobin, a self-assembling amphiphile. J. Biol. Chem. 2004;279:534–539. Hakanpää J, Linder MB, Popov A, Schmidt A, Rouvinen J. Hydrophobin HFBII in detail: ultrahigh-resolution structure at 0.75 Å. Acta Crystallogr., Sect D: Biol. Crystallogr. 2006;62: 356–367. Moldovan C, Thompson D. Molecular dynamics of the “hydrophobic patch” that immobilizes hydrophobin protein HFBII on silicon. J. Mol. Model. 2011;17:2227–2235.
Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Involvement of hydrophobic amino acid residues in C7–C8 loop of Aspergillus oryzae hydrophobin RolA in hydrophobic interaction between RolA and a polyester a
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Takumi Tanaka , Hiroki Tanabe , Kenji Uehara , Toru Takahashi & Keietsu Abe a
Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan b
Laboratory of Enzymology, Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan c
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Microbial Genomics Laboratory, New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan Published online: 10 Jul 2014.
To cite this article: Takumi Tanaka, Hiroki Tanabe, Kenji Uehara, Toru Takahashi & Keietsu Abe (2014) Involvement of hydrophobic amino acid residues in C7–C8 loop of Aspergillus oryzae hydrophobin RolA in hydrophobic interaction between RolA and a polyester, Bioscience, Biotechnology, and Biochemistry, 78:10, 1693-1699, DOI: 10.1080/09168451.2014.932684 To link to this article: http://dx.doi.org/10.1080/09168451.2014.932684
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Bioscience, Biotechnology, and Biochemistry, 2014 Vol. 78, No. 10, 1693–1699
Involvement of hydrophobic amino acid residues in C7–C8 loop of Aspergillus oryzae hydrophobin RolA in hydrophobic interaction between RolA and a polyester Takumi Tanaka1, Hiroki Tanabe1, Kenji Uehara2, Toru Takahashi3 and Keietsu Abe1,3,* 1
Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan; 2Laboratory of Enzymology, Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan; 3Microbial Genomics Laboratory, New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan Received April 3, 2014; accepted April 28, 2014
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http://dx.doi.org/10.1080/09168451.2014.932684
Hydrophobins are amphipathic secretory proteins with eight conserved cysteine residues and are ubiquitous among filamentous fungi. The Cys3–Cys4 and Cys7–Cys8 loops of hydrophobins are thought to form hydrophobic segments involved in adsorption of hydrophobins on hydrophobic surfaces. When the fungus Aspergillus oryzae is grown in a liquid medium containing the polyester polybutylene succinate-co-adipate (PBSA), A. oryzae produces hydrophobin RolA, which attaches to PBSA. Here, we analyzed the kinetics of RolA adsorption on PBSA by using a PBSA pull-down assay and a quartz crystal microbalance (QCM) with PBSAcoated electrodes. We constructed RolA mutants in which hydrophobic amino acids in the two loops were replaced with serine, and we examined the kinetics of mutant adsorption on PBSA. QCM analysis revealed that mutants with replacements in the Cys7–Cys8 loop had lower affinity than wild-type RolA for PBSA, suggesting that this loop is involved in RolA adsorption on PBSA. Key words:
adsorption; Aspergillus oryzae; fungi; hydrophobic interaction; hydrophobin
Hydrophobins, which are small, amphipathic secretory proteins with eight conserved cysteine residues, are ubiquitous among filamentous fungi1–3) and play several important roles in fungal physiology. For example, they are involved in fungal adhesion to solid surfaces, in the formation of a protective surface coating on fugal cells, and in the reduction of water surface tension, and processes that support the growth of fungal aerial structures such as hyphae and conidiospores.4) When the fungus Aspergillus oryzae is grown under liquid culture conditions with the biodegradable aliphatic polyester polybutylene succinate-co-adipate (PBSA) as the sole carbon source, the fungus simultaneously produces both the biosurfactant protein hydrophobin RolA (GenBank *Corresponding author. Email: [email protected] © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
acc. no. AB094496)/HypA (GenBank acc. no. AB097447) and the cutinase CutL1, which hydrolyzes PBSA.5) The secreted RolA is adsorbed on the surface of PBSA, which has a chemical structure resembling that of the plant-wax polyester cutin,6) and efficiently recruits CutL1, resulting in condensation of CutL1 on the PBSA surface and stimulation of PBSA hydrolysis. This discovery of CutL1 recruitment by PBSA-bound RolA adds a previously unknown function to the above-described roles of hydrophobins. Recruitment of proteins by immobilized hydrophobins such as HGFI and HFBI has also been reported.7) The recruitment of hydrolytic enzymes by amphipathic proteins attached to solid surfaces could be applied to a large-scale degradation of hydrophobic solid materials at solid–liquid interfaces.5,8) Note that the soluble form of RolA does not interact with CutL1: that is, adsorption of the hydrophobin to a solid surface, such as PBSA, is essential for subsequent RolA-dependent CutL1 recruitment.5) Therefore, analysis of the kinetics of RolA adsorption on PBSA is important for understanding the hydrolysis of PBSA by CutL1 recruited by immobilized RolA. Hydrophobins possess conserved core β-barrel structures9) that divide the molecules into two segments: a hydrophilic segment and a hydrophobic segment that is predicted to be involved in adsorption of the proteins to hydrophobic solid surfaces.10,11) Involvement of the hydrophobic segment in self-assembly (biofilm formation) has been predicted by structural analyses and mutation analysis.9,10,12) However, direct involvement of the hydrophobic segments in hydrophobin adsorption to solid materials has not been confirmed, and the kinetics of hydrophobin adsorption on surfaces have not yet been investigated. Here, we used a pull-down assay with PBSA microparticles and a quartz crystal microbalance (QCM) with PBSA-coated electrodes to analyze the kinetics of adsorption of both wild-type RolA and RolA mutants in which hydrophobic amino acid residues in the hydrophobic segment (hydrophobic patches) were replaced with serine. On the basis of our
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results, we discuss the nature of the interaction between RolA and PBSA.
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Materials and methods Fungal strains, culture media, and growth conditions. A. oryzae niaD300 (niaD−).13) a niaD mutant derived from A. oryzae strain RIB40, was used as a recipient for transformation and protein expression experiments. These two strains were grown in YPG complete medium (1% yeast extract, 2% Polypepton™, and 2% glucose) or Czapek Dox minimal medium.14) Strains overexpressing RolA and the RolA mutants described below were grown in YPM liquid medium (1% yeast extract, 2% Polypepton™, and 2% maltose) to study the expression of, and to purify, RolA and its mutants. Shaken cultures of A. oryzae cells were generally grown at 30 °C on rotary shakers (160 rpm). Escherichia coli XL1-Blue (Stratagene, Tokyo, Japan) was used for plasmid construction, and E. coli DH5α (NIPPON GENE, Tokyo, Japan) was used for modification of constructed plasmids for creating RolA mutants. E. coli was grown at 37 °C in 2× Luria-Bertani medium (2% tryptone, 1% yeast extract, 2% NaCl). All basic molecular biology procedures were performed as described in the literature.15) Alignment analysis of amino acid sequences of hydrophobins. Amino acid sequences of the hydrophobins Neurospora crassa EAS (GenBank acc. no. EAA34064, class I),16) Trichoderma reesei HFBII (GenBank acc. no. P79073, class II),17) and A. oryzae RolA (GenBank acc. no. AB094496, class I)5) were obtained from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The alignment of the amino acid sequences of the three hydrophobins was generated with GENETYX sequence analysis software (ver. 10, GENETYX, Tokyo, Japan). Similarity scores among the amino acid sequences of the three hydrophobins were also calculated with GENETYX. Creation of RolA-overexpressing strain. An expression plasmid for RolA (pNG-eno-hyp) was constructed as described previously.5) A linear form of pNG-eno-hyp digested with MunI was introduced into A. oryzae niaD300 (niaD−) essentially according to the protoplast–PEG method.18) Candidate RolA-overexpressing strains were selected by colony PCR using the rolA and niaD primers as described previously.5,19) The resulting RolA-overexpressing strain was designated A. oryzae eno-hyp. Creation of strains overexpressing RolA mutants. Hydrophobic amino acid residues in hydrophobic patches of RolA (F70, V73, L77, L78, A79, L81, L82, L85, L86, A88, L95, L97, and L98 in the C3–C4 loop and L137, V138, L142, and P143 in the C7–C8 loop) were replaced with serine. The double mutant
RolA-L137S/L142S was also created. All of the RolA mutants were constructed with a QuickChange SiteDirected Mutagenesis Kit (Stratagene, Los Angeles, CA, USA). The template plasmid was pNG-eno-hyp, and the primers used are listed in Table S1. RolA mutant–overexpression plasmids were introduced into A. oryzae niaD300 according to the protoplast–PEG method.18) Candidate strains overexpressing RolA mutants were selected by colony PCR using the rolA and niaD primers, as described previously.5,19) The resulting strains overexpressing the mutants are designated A. oryzae eno-hyp mutants. Expression and purification of recombinant RolA and RolA mutants. Conidiospores of A. oryzae eno-hyp or A. oryzae eno-hyp mutants were inoculated into YPM liquid medium (1 × 106 conidia mL−1). After culture of the mutants for 20 h at 30 °C, the culture broth was filtered through Miracloth (Merck KGaA, Darmstadt, Germany) to separate mycelia. The filtrate (extracellular solution) was brought to 40% saturation by adding 90% ammonium sulfate, and the resultant suspension was centrifuged at 8,000g for 30 min to recover the supernatant. The supernatant was applied to a Phenyl Sepharose CL-4B column (3 × 18 cm, GE Healthcare Japan, Tokyo, Japan) equilibrated with 10 mM Tris–HCl buffer (pH 8.0) containing 40%-saturated ammonium sulfate. RolA was eluted with a 40–0% linear gradient of ammonium sulfate. After each fraction was checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; 3% stacking gel and 17.5% running gel), the fraction containing RolA was dialyzed against 5 mM Tris–HCl buffer (pH 9.0). The dialyzed solution containing RolA was subsequently applied to a Cellufine Q-500 column (3 × 12 cm, JNC Corp., Tokyo, Japan) equilibrated with the same Tris–HCl buffer. RolA was eluted with a 0–0.3 M linear gradient of NaCl. After the fractions were examined by SDS-PAGE, the fraction containing RolA was dialyzed against 10 mM sodium citrate buffer (pH 4.0). The dialyzed solution containing RolA was subsequently applied to an S-Sepharose FF column (2 × 18 cm, GE Healthcare Japan, Tokyo, Japan) equilibrated with the same sodium citrate buffer. RolA was eluted with a 0–0.3 M linear gradient of NaCl to obtain purified RolA. All the purification procedures described above were performed at 4 °C. To qualitatively determine the secreted RolA in the culture bloth, proteins separated by SDS-PAGE were blotted onto a Sequi-Blot PVDF Membrane (Bio-Rad, Tokyo, Japan) by using a Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad). A mouse polyclonal antibody against RolA purified from A. oryzae eno-hyp (T.K. Craft, Gunma, Japan),5) a horseradish phosphatase–conjugated rabbit polyclonal antibody against mouse IgG (Promega, Wisconsin, USA), and SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Yokohama, Japan) were used for Western blot analysis of RolA and RolA mutants. Chemiluminescence signals were detected with a ImageQuant LAS 4000mini lumino image analyzer (Fujifilm, Tokyo, Japan).
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Interaction of A. oryzae hydrophobin RolA with a polyester
Pull-down assay of RolA to PBSA microparticles. Conidiospores of A. oryzae niaD300, A. oryzae enohyp, or A. oryzae eno-hyp mutants were inoculated into YPM liquid medium (2 × 106 conidia mL−1). After shake-cultivation (160 rpm) for 20 h at 30 °C, the culture broth was filtered through Miracloth (Merck KGaA) to separate mycelia. Trichloroacetic acid solution (100% w/v, 0.5 mL) was added to a 1-mL aliquot of each culture broth, and the mixture was incubated at 4 °C for 1 h. After centrifugation (17,400g for 20 min), the precipitated protein was solubilized in 10 μL of SDS-sample buffer (Wako Pure Chemical Industries, Osaka, Japan) and then boiled. The boiled samples were subjected to SDS-PAGE. RolA and its mutants were stained with Coomassie Brilliant Blue R-250. After staining of the samples, gel images were captured with a GT-X 820 image scanner (Seiko Epson Corp., Nagano, Japan). The amounts of RolA and its mutants were quantified densitometrically with ImageJ software (ver. 1.44p; http://rsb.info.nih.gov/ij/). The concentration of RolA mutant in each culture broth was adjusted to the RolA concentration in the culture broth of A. oryzae niaD300 expressing wild-type RolA. The pH of each sample was adjusted to pH 6. PBSA microparticles (Showa Highpolymer Co., Tokyo, Japan) were added to each sample (final concentration of PBSA 0.1% w/v), and the mixture was incubated at 30 °C for 30 s, because the adsorption of wild-type RolA on PBSA was saturated within 30 s at 30 °C (Uehara et al., unpublished results). The microparticles were collected by centrifugation (17,400g for 20 min). SDS-sample buffer (10 μL) was added to the microparticles collected from each sample, the mixture was boiled, and the boiled sample was subjected to SDS-PAGE. RolA and RolA mutants adsorbed on the microparticles were stained with Coomassie Brilliant Blue R-250. The amounts of RolA and its mutants adsorbed on the microparticles were quantified densitometrically as described above. Circular dichroism measurements of RolA. Circular dichroism (CD) spectra of RolA were measured by using a method described previously.5) The spectra were recorded over the wavelength region from 190 to 250 nm on a J-725 CD spectrometer (JASCO International, Tokyo, Japan) by using a 1-mm quartz cuvette (GL Sciences, Tokyo, Japan). The temperature was kept at 25 °C, and the sample compartment was continuously flushed with N2 gas. Spectra are the averages of 10 scans, which were performed with a bandwidth of 1 nm, a step width of 1 nm, and 5-s averaging per point. The baseline of the CD spectrum was flat for 5 mM sodium phosphate buffer (pH 7.0), which was used as the RolA solvent. In order to eliminate self-assembled hydrophobin molecules, class I hydrophobins such as Schizophyllum commune SC3 and RolA are generally treated with 100% trifluoroacetic acid (TFA) and subsequent removal of TFA from the TFA-treated samples leads to refolding of hydrophobin molecules.5) Before CD analysis, purified RolA was condensed in a glass tube by lyophilization. Then the lyophilized RolA was treated with 100% TFA and dried with N2 gas for removal of TFA. Dried RolA
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was dissolved in 5 mM phosphate buffer (pH 7.0), and 250 μg/mL of RolA was placed in the cuvette. Analysis of RolA–PBSA interaction by means of a QCM method. The resonance frequency of a QCM electrode decreases linearly as the mass on the electrode increases at the nanogram level.20) A frequency change of 100 Hz corresponds to 3 ng of analyte protein bound to the QCM electrode. We modified the QCM method of Sato et al.21) for our QCM analyses of the RolA–PBSA interaction as follows. Preparation of electrodes: a PBSA film (thickness 15–20 μm, Showa Denko K.K., Tokyo, Japan) was dissolved in chloroform at a concentration of 0.05% w/v, and 3 μL of the dissolved PBSA was cast on a QCM electrode (Initium, Tokyo, Japan). The coated electrode was dried at room temperature for at least 20 min. We found that the amount of immobilized PBSA appropriate for obtaining fine detection sensitivity was 60–240 ng. QCM measurements were performed with an initial frequency of 27 MHz at 30 °C by using an Affinix QNμ system (Initium). The analysis chamber contained 500 μL of running buffer (10 mM GTA buffer [3.3 mM 3,3-dimethylglutaric acid, 3.3 mM Tris, and 3.3 mM 2-amino-2-methyl-1,3-propanediol], pH 7.0) in which a PBSA-coated electrode was immersed. Purified RolA or RolA mutants were dissolved in the running buffer, the solution was injected stepwise into the analysis chamber in which the PBSA-coated electrode was immersed, and QCM values were measured as a function of the concentration of RolA in the chamber (30– 700 nM). To evaluate the affinity of binding of RolA to PBSA, we analyzed the frequency changes upon the addition of RolA to the QCM analysis chamber by fitting to a Langmuir adsorption isotherm plot by using Aqua software (ver. 1.2, Initium) in accordance with the manufacturer’s instructions.22) The dissociation constant (KD) was calculated by the fitting based on the equation KD = [Analyte][Bmax/2], where Bmax is the maximum amount of an analyte bound to a ligand, and [Anlyte][Bmax/2] is the concentration of the analyte when the amount of the analyte bound to the ligand reaches Bmax/2.
Results Alignment analysis of A. oryzae RolA (class I), N. crassa EAS (class I), and T. reesei HFBII (class II) Hydrophobins are categorized as either class I or class II on the basis of their hydrophobicity and other physicochemical properties.3) Class I hydrophobins are generally more hydrophobic than class II hydrophobins and form polymer films made up of cylindrical rodlets.23,24) The outward-facing hydrophobic surfaces of the films show extremely low wettability,23,24) and the films are resistant to boiling in detergents or alkalis.2) Compared with class I hydrophobins, class II hydrophobins have lower hydropathy scores and are less robust, and they lack the rodlet morphology found in the polymer films of class I hydrophobins.25) Although the similarity of the amino acid sequences of N. crassa hydrophobin EAS (class I) and T. reesei hydrophobin HFBII (class II) is less than 30%, EAS and HFBII have
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9)
similar structural features. Based on their 3D structures, both of these hydrophobins are predicted to possess β-barrel core structures and to show a similar topology in which the central β-barrel core divides the hydrophobin molecule into two segments: a hydrophobic segment (hydrophobic patches) and a hydrophilic segment.9,10,26–28) Fig. 1 shows the alignment of the amino acid sequences of EAS, HFBII, and RolA and the homology among the three hydrophobins. Hydrophobins possess eight conserved cysteine residues (Fig. 1). The C3(Cys45)–C4(Cys71) loop and the C7(Cys87)–C8(Cys106) loop in EAS form hydrophobic patches and are thought to be involved in adsorption of EAS to hydrophobic surfaces.9) A. oryzae RolA possesses two amino acid loops similar to those of EAS: the C3(Cys61)–C4(Cys101) loop is rich in hydrophobic amino acid residues, and only half part of C7(Cys126)–C8(Cys144) loop is rich in hydrophobic residues (Fig. 1). Hydropathy analysis also suggests that the C3–C4 and C7–C8 loops of RolA comprise hydrophobic patches (data not shown). Pull-down Assay of RolA mutants to PBSA microparticles Because both RolA and EAS are predicted to have the C3–C4 and C7–C8 loops which are rich in hydrophobic amino acid residuess forming hydrophobic patches, we constructed a series of RolA mutants in which selected hydrophobic residues of the two loops were replaced with serine. To examine adsorption of the mutants on PBSA, we incubated crude culture broth
containing each mutant with PBSA microparticles and measured the amounts of the mutants adsorbed (Fig. 2). The amounts of RolA-L137S and RolAL142S mutants adsorbed on the PBSA microparticles were 60 and 40%, respectively, of the adsorbed amount of wild-type RolA. CD analysis of RolA mutants Because the pull-down assay revealed that L137 and L142 play important roles in RolA adsorption on PBSA, we examined whether single substitution or double substitution of these residues resulted in major changes in the structure of RolA. Because the peak wavelengths and absorbance of the CD spectra originating from the two single mutants (RolA-L137S, RolAL142S) and double mutant (RolA-L137S/L142S) were almost similar to those of the CD spectrum of wildtype RolA, the substitutions (L137S, L142S, and L137S/L142S) did not cause major structural changes in RolA (Fig. 3). Taken together with the results of the pull-down assay, these results indicate that the decrease in adsorption of the mutants on PBSA was attributable to a reduction in the hydrophobicity of the C7–C8 loop. QCM analysis of RolA adsorption on PBSA The pull-down assay and the CD spectra of the RolA mutants suggested that L137 and L142 were involved in the adsorption of RolA on PBSA. Therefore, we quantified the interaction between the mutants and
Fig. 1. Comparison of amino acid sequences of hydrophobins EAS, RolA, and HFBII. Notes: (A) Sequences of class I hydrophobins Aspergillus oryzae RolA and N. crassa EAS and class II hydrophobin T. reesei HFBII. The eight cysteine residues that form the conserved disulfide bonds are indicated in bold type. Selected hydrophobic amino acid residues of RolA for creating RolA mutants were highlighted by gray color. L137 and L142 of RolA were underlined. The alignment was generated with GENETYX (ver. 10). (B) Similarity scores of the amino acid sequences of RolA, EAS, and HFBII, calculated with GENETYX (ver. 10).
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Fig. 2. Pull-down Assay of RolA mutants to PBSA microparticles. Notes: Adsorption of RolA mutants in which a hydrophobic amino acid in the hydrophobic region was replaced with serine on PBSA microparticles was examined. PBSA microparticles were incubated with the culture supernatants of RolA mutant strains at 30 °C for 30 s. After the incubation, the RolA–PBSA complex was collected by centrifugation, and the proteins bound to the PBSA microparticles were extracted with SDS-PAGE loading buffer and subjected to SDS-PAGE. The data are means ± SD (n = 3).
Fig. 3. CD spectra of RolA in the soluble form. Notes: Wild-type RolA (solid line), RolA-L137S mutant (dotted line), RolA-L142S mutant (short-dashed line), and RolA-L137/L142S double mutant (long-dashed line). CD spectra are the averages of 10 scans collected by using a reference solution without the protein.
PBSA by using QCM analysis (Fig. 4 and Table 1). Although the KD value of RolA-L137S tended to be larger than that of wild-type RolA, the two KD values were statistically indistinguishable but the Bmax value of RolA-L137S was statistically smaller than that of wild-type RolA. The KD value of RolA-L142S was statistically larger than that of wild-type RolA; however, the Bmax value of RolA-L142S was not distinguishable from that of wild-type RolA. The KD value of RolAL137S/L142S was larger than that of wild-type RolA, and the Bmax value of RolA-L137S/L142S was smaller than that of wild-type RolA. But the KD value of RolA-L137S/L142S was statistically indistinguishable from those of RolA-L137S and RolA-L142S, and the Bmax value of RolA-L137S/L142S was statistically indistinguishable from that of RolA-L137S.
Discussion We previously reported that A. oryzae RolA attaches to PBSA surfaces and then recruits polyesterase CutL1 to the surfaces, and that condensation of CutL1 on the
Fig. 4. QCM analysis of interaction of RolA and its mutants with PBSA. Notes: To evaluate the affinity of binding of RolA and its mutants to PBSA, the frequency changes upon addition of RolA or its mutants to the QCM analysis chamber were analyzed by fitting to a Langmuir adsorption isotherm plot by using Aqua software (ver. 1.2, Initium). (A) wild-type RolA, (B) RolA-L137S mutant, (C) RolAL142S mutant, and (D) RolA-L137S/L142S double mutant. R2 is the coefficient of determination.
surfaces leads to stimulation of PBSA hydrolysis catalyzed by CutL1.5) The main driving force for the interaction between PBSA-bound RolA and CutL1 is predicted to be electrostatic interaction (unpublished results, Takahashi et al.). However, the mechanism and kinetics of the RolA–PBSA interaction remain unclear. Although it is known that the hydrophobic segments of hydrophobins are involved in their self-assembly (biofilm formation),11,12,29) whether the hydrophobic segments are directly involved in the adsorption of hydrophobins on solid surfaces remains unclear. Structural analysis of N. crassa class I hydrophobin EAS demonstrated that it contained the eight cysteine residues conserved among hydrophobins.9,10) The C3 (Cys45)–C4(Cys71) loop is rich in hydrophobic amino acid residues, and only half part of C7(Cys87)–C8 (Cys106) loop is rich in hydrophobic residues.9) Kwan et al. reported that the β-barrel core structure and the topology of N. crassa EAS are similar to those of class II hydrophobin HFBII from T. reesei, even though the
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Table 1. KD and Bmax values in the interaction of RolA and its mutants with PBSA. RolA
KD (nM) §
wild type
73.77 ± 13.49
L137S
139.75 ± 40.92
L142S L137S/L142S
303.17 ± 74.17 221.20 ± 53.54
Bmax (ng) § 6.28 ± 1.62 *
*
4.97 ± 1.11 *
6.11 ± 1.61 3.69 ± 0.92
* * * *
Dissociation constants (KD) were determined by fitting to a Langmuir adsorption isotherm plot; KD = [RolA][Bmax/2], where Bmax is the maximum amount of RolA bound to the PBSA-coated electrode, and RolA[Bmax/2] is the RolA concentration when the amount of RolA bound to the PBSA-coated electrode reaches Bmax/2. *p < 0.05 (n = 4).
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§
sequence homology of these two hydrophobins is not high.9) In both of these proteins, the region containing the C3–C4 loop, the C7–C8 loop, and the intersection of the two loops faces toward the air side (the hydrophobic side) at an air–water interface.9,10) These structural analyses, taken together with the results of biochemical studies of biofilm formation (self-assembly) of the two hydrophobins,11,12,29) suggest that the hydrophobic segments—that is, the C3–C4 and C7–C8 loops —are involved in self-assembly (biofilm formation). However, there is no direct evidence that the two loops are involved in hydrophobic interaction with solid surfaces. If the barrel topology of N. crassa EAS is shared by A. oryzae RolA, the parts of the C3–C4 and C7–C8 loops that contain hydrophobic residues (F70–L98 and L137–P143, respectively) of RolA are predicted to have the same orientation as the C3–C4 and C7–C8 loops of EAS. Therefore, to determine whether the C3– C4 and C7–C8 loops of RolA were directly involved in its interaction with PBSA, we constructed RolA mutants in which some of the hydrophobic amino acid residues in the loops were replaced with serine. Pulldown assay of the mutants with PBSA microparticles (Fig. 2) and QCM analysis revealed that the singlemutant RolA-L142S and double-mutant RolA-L137S/ L142S had statistically larger KD values and smaller Bmax values for PBSA than the wild-type protein (Fig. 4 and Table 1) and that the single-mutant RolA-L137S had a smaller Bmax value than that of wild-type protein. These results suggest that the L137S substitution in the C7–C8 loop affected the amount of protein adsorbed on PBSA at the steady-state adsorption and that the L142S substitution changed the affinity of the protein for PBSA. To compare the analysis time-length (60–240 min) and protein concentrations (30–700 nM) in the QCM analyses with those in the pull-down assay, the latter employed the shorter incubation time such as 30 s with the higher protein concentrations (2,000–12,000 nM). Therefore, because RolA-L137S showed the smaller Bmax value than wild-type and RolA-142S indicated larger KD value than wild-type, the lower levels of protein adsorption of the two mutants on PBSA microparticles than the adsorption level of wild-type RolA are attributable to both the kinetic properties of the two mutants and the conditions of the pull-down assay (Table 1, Fig. 2). The results of pull-down assay and QCM analysis indicate that the hydrophobic amino acid residues L137
and L142 are important for adsorption of RolA on PBSA. When the EAS topology is imposed on RolA, L137 and L142 are close to the interface region between the C3–C4 and C7–C8 loops. CD analysis of wild-type RolA and the three RolA mutants suggested that the mutations did not lead to any major structural changes. Consequently, the hydrophobicity of the region containing L137 and L142 must be important for the RolA–PBSA interaction. However, the two RolA mutants still indicated the low levels of adsorption on PBSA, so some other residues besides L137 and L142 must also be involved in the adsorption of RolA on PBSA. In conclusion, pull-down assays of RolA mutants with PBSA particles and QCM analysis of the mutants with PBSA-coated electrodes demonstrated that hydrophobic amino acid residues L137 and L142 in the C7–C8 loop of RolA are directly involved in adsorption of RolA on PBSA surfaces via hydrophobic interaction between RolA and PBSA. This is the first kinetic evidence that hydrophobic amino acid residues in the C7–C8 loop of hydrophobins are directly involved in their interaction with solid materials.
Supplemental material The supplemental material for this paper is available at http://dx.doi.org/10.1080/09168451.2014.932684.
Funding This work was partly supported by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science [20380175] (to K.A.); and Challenging Exploratory Research Grant [24658281] (to K.A.). K.A. also thanks the Research Institute of Innovative Technology for the Earth for financial support through the Joint Program to Promote Technological Development.
References [1] Wessels JGH, De Vries OMH, Asgeirsdottir SA, Schuren FHJ. Hydrophobin genes involved in formation of aerial hyphae and fruit bodies in Schizophyllum. Plant Cell. 1991;3:793–799. [2] De Vries OMH, Fekkes MP, Wösten HAB, Wessels JGH. Insoluble hydrophobin complexes in the walls of Schizophyllum commune and other filamentous fungi. Arch. Microbiol. 1993;159: 330–335. [3] Wessels JGH. Developmental regulation of fungal cell wall formation. Annu. Rev. Phytopathol. 1994;32:413–437. [4] Wösten HAB, van Wetter MA, Lugones LG, van der Mei HC, Busscher HJ, Wessels JGH. How a fungus escapes the water to grow into the air. Curr. Biol. 1999;9:85–88. [5] Takahashi T, Maeda H, Yoneda S, Ohtaki S, Yamagata Y, Hasegawa F, Gomi K, Nakajima T, Abe K. The fungal hydrophobin RolA recruits polyesterase and laterally moves on hydrophobic surfaces. Mol. Microbiol. 2005;57:1780–1796. [6] Fang X, Qiu F, Yan B, Wang H, Mort AJ, Stark RE. NMR studies of molecular structure in fruit cuticle polyesters. Phytochemistry. 2001;57:1035–1042. [7] Wang Z, Huang Y, Li S, Xu H, Linder MB, Qiao M. Hydrophilic modification of polystyrene with hydrophobin for time-resolved immunofluorometric assay. Biosens. Bioelectron. 2010;26: 1074–1079.
Downloaded by [Lakehead University] at 02:14 14 March 2015
Interaction of A. oryzae hydrophobin RolA with a polyester [8] Ohtaki S, Maeda H, Takahashi T, Yamagata Y, Hasegawa F, Gomi K, Nakajima T, Abe K. Novel hydrophobic surface binding protein, HsbA, produced by Aspergillus oryzae. Appl. Environ. Microbiol. 2006;72:2407–2413. [9] Kwan AH, Winefield RD, Sunde M, Matthews JM, Haverkamp RG, Templeton MD, Mackay JP. Structural basis for rodlet assembly in fungal hydrophobins. Proc. Nat. Acad. Sci. 2006;103: 3621–3626. [10] Kwan AH, Macindoe I, Vukašin PV, Morris VK, Kass I, Gupte R, Mark AE, Templeton MD, Mackay JP, Sunde M. The Cys3– Cys4 loop of the hydrophobin EAS is not required for rodlet formation and surface activity. J. Mol. Biol. 2008;382:708–720. [11] Gruné r MS, Szilvay GR, Berglin M, Lienemann M, Laaksonen P, Linder MB. Self-assembly of class II hydrophobins on polar surfaces. Langmuir. 2012;28:4293–4300. [12] Macindoe I, Kwan AH, Ren Q, Morris VK, Yang W, Mackay JP, Sunde M. Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS. Proc. Natl. Acad. Sci. USA. 2011;109:804–811. [13] Machida M. Progress of Aspergillus oryzae genomics. Adv. Appl. Microbiol. 2002;51:81–106. [14] Nakajima K, Kunihiro S, Sano M, Zhang Y, Eto S, Chang YC, Suzuki T, Jigami Y, Machida M. Comprehensive cloning and expression analysis of glycolytic genes from the filamentous fungus, Aspergillus oryzae. Curr. Genet. 2000;37:322–327. [15] Green MR, Sambrook J. Molecular cloning: a laboratory manual. 4th ed. New York: Cold Spring Harbor; 2012. [16] Galagan JE, Calvo SE, Borkovich KA, Selker EU, Read ND, Jaffe D, FitzHugh W, Ma LJ, Smirnov S, Purcell S, Rehman B, Elkins T, Engels R, Wang S, Nielsen CB, Butler J, Endrizzi M, Qui D, Ianakiev P, Bell-Pedersen D, Nelson MA, Werner-Washburne M, Selitrennikoff CP, Kinsey JA, Braun EL, Zelter A, Schulte U, Kothe GO, Jedd G, Mewes W, Staben C, Marcotte E, Greenberg D, Roy A, Foley K, Naylor J, Stange-Thomann N, Barrett R, Gnerre S, Kamal M, Kamvysselis M, Mauceli E, Bielke C, Rudd S, Frishman D, Krystofova S, Rasmussen C, Metzenberg RL, Perkins DD, Kroken S, Cogoni C, Macino G, Catcheside D, Li W, Pratt RJ, Osmani SA, DeSouza CP, Glass L, Orbach MJ, Berglund JA, Voelker R, Yarden O, Plamann M, Seiler S, Dunlap J, Radford A, Aramayo R, Natvig DO, Alex LA, Mannhaupt G, Ebbole DJ, Freitag M, Paulsen I, Sachs MS, Lander ES, Nusbaum C, Birren B. The genome sequence of the filamentous fungus Neurospora crassa. Nature. 2003;422:859–868. [17] Nakari-Setala T, Aro N, Ilmen M, Munoz G, Kalkkinen N, Penttila M. Differential expression of the vegetative and spore-bound hydrophobins of Trichoderma reesei cloning and
[18]
[19]
[20]
[21]
[22]
[23] [24]
[25]
[26]
[27]
[28]
[29]
1699
characterization of the hfb2 gene. Eur. J. Biochem. 1997;248: 415–423. Gomi K, Iimura Y, Hara S. Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans argB gene. Agric. Biol. Chem. 1987;51:2549–2555. Takahashi T, Mizutani O, Shiraishi Y, Yamada O. Development of an efficient gene-targeting system in Aspergillus luchuensis by deletion of the non-homologous end joining system. J. Biosci. Bioeng. 2011;112:529–534. Okahata Y, Niikura K, Sugiura Y, Sawada M, Morii T. Kinetic studies of sequence-specific binding of GCN4-bZIP peptides to DNA strands immobilized on a 27-MHz quartz-crystal microbalance. Biochemistry. 1998;37:5666–5672. Sato Y, Sagami I, Shimizu T. Identification of Caveolin-1-interacting sites in neuronal nitric-oxide synthase: molecular mechanism for inhibition of NO formation. J. Biol. Chem. 2004;279: 8827–8836. Hama H, Saito A, Takeda T, Tanuma A, Xie Y, Sato K, Kazama JJ, Gejyo F. Evidence indicating that renal tubular metabolism of leptin is mediated by megalin but not by the leptin receptors. Endocrinology. 2004;145:3935–3940. Beever RE, Dempsey G. Function of rodlets on the surface of fungal spores. Nature. 1978;272:608–610. Wösten HAB, De Vries OMH, Wessels JGH. Interfacial selfassembly of a fungal hydrophobin into a hydrophobic rodlet layer. Plant Cell. 1993;5:1567–1574. Paananen A, Vuorimaa E, Torkkeli M, Penttilä M, Kauranen M, Ikkala O, Lemmetyinen H, Serimaa R, Linder MB. Structural hierarchy in molecular films of two class II hydrophobins. Biochemistry. 2003;42:5253–5258. Hakanpää J, Parkkinen TAA, Hakulinen N, Linder MB, Rouvinen J. Crystallization and preliminary X-ray characterization of Trichoderma reesei hydrophobin HFBII. Acta Crystallogr., Sect D: Biol. Crystallogr. 2004;60:163–165. Hakanpää J, Paananen A, Askolin S, Nakari-Setala T, Parkkinen T, Penttila M, Linder MB, Rouvinen J. Atomic resolution structure of the HFBII hydrophobin, a self-assembling amphiphile. J. Biol. Chem. 2004;279:534–539. Hakanpää J, Linder MB, Popov A, Schmidt A, Rouvinen J. Hydrophobin HFBII in detail: ultrahigh-resolution structure at 0.75 Å. Acta Crystallogr., Sect D: Biol. Crystallogr. 2006;62: 356–367. Moldovan C, Thompson D. Molecular dynamics of the “hydrophobic patch” that immobilizes hydrophobin protein HFBII on silicon. J. Mol. Model. 2011;17:2227–2235.
