Class I Hydrophobin Vmh2 Adopts Atypical Mechanisms to Self

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CLASS I HYDROPHOBIN VMH2 ADOPTS ATYPICAL MECHANISMS TO SELF-ASSEMBLE INTO FUNCTIONAL AMYLOID FIBRILS Alfredo Maria Gravagnuolo, Sara Longobardi, Alessandra Luchini, Marie-Sousai Appavou, Luca De Stefano, Eugenio Notomista, Luigi Paduano, and Paola Giardina Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01632 • Publication Date (Web): 01 Feb 2016 Downloaded from http://pubs.acs.org on February 1, 2016

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Biomacromolecules

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CLASS I HYDROPHOBIN VMH2 ADOPTS

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ATYPICAL MECHANISMS TO SELF-

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ASSEMBLE INTO FUNCTIONAL AMYLOID

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FIBRILS

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Alfredo Maria Gravagnuolo1, Sara Longobardi1, Alessandra Luchini1, Marie-Sousai Appavou4,

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Luca De Stefano2, Eugenio Notomista3, Luigi Paduano1, Paola Giardina1* 1

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Department of Chemical Sciences, University of Naples ‘Federico II’, Via Cintia 4, 80126

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Naples, Italy 2

Unit of Naples, Institute for Microelectronics and Microsystems, National Council of Research,

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Via Pietro Castellino 111, 80131 Naples, Italy 3

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Department of Biology, University of Naples ‘Federico II’, Via Cintia 4, 80126 Naples, Italy

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Jülich Centre for Neutron Science JCNS, Forschungszentrum Jülich GmbH, Outstation at MLZ,

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Lichtenbergstraße 1, 85747 Garching, Germany

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KEYWORDS

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Fungi, amyloid fibrils, cryo-TEM, Dynamic Light Scattering, Pleurotus ostreatus, protein self-

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assembly

ACS Paragon Plus Environment

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ABSTRACT

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Hydrophobins are fungal proteins whose functions are mainly based on their capability to self-

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assemble into amphiphilic films at hydrophobic-hydrophilic interfaces (HHI). It is widely

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accepted that Class I hydrophobins form amyloid-like structures, named rodlets, which are

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hundreds of nanometers long, packed into ordered lateral assemblies and do not exhibit an

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overall helical structure. We studied the self-assembly of the Class I hydrophobin Vmh2 from

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Pleurotus ostreatus in aqueous solutions by dynamic light scattering (DLS), thioflavin T (ThT)

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fluorescence assay, circular dichroism (CD), cryogenic trasmission electron microscopy (Cryo-

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TEM) and TEM. Vmh2 does not form fibrillar aggregates at HHI. It exhibits spherical and

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fibrillar assemblies whose ratio depends on the protein concentration, when freshly solubilized at

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pH ≥ 7. Moreover it spontaneously self-assembles into isolated, micrometer long, and twisted

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amyloid fibrils, observed for the first time in fungal hydrophobins. This process is promoted by

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acidic pH, temperature, and Ca2+ ions. A model of self-assembly into amyloid-like structures has

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been proposed.


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INTRODUCTION

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Protein self-assembly is a complex phenomenon intensively studied over past few decades, due

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to its implications in the living systems.1,2 This spontaneous process is gaining interest of

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researchers, due to the wide spectrum of potential applications of protein layers in nano-

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biotechnological field, specifically in the design and production of novel advanced materials.3,4

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Understanding the mechanism of self-assembly is crucial to control the process and to exploit the

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potential of these systems, moreover it might shed light on amyloid diseases and their

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treatments.5–7

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Hydrophobins are fungal proteins whose function is determined by the ability to self-assemble

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into amphiphilic films at a hydrophobic-hydrophilic interface (HHI).8,9 They play a key role in

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the growth and morphogenesis of the majority of filamentous fungi.10 Hydrophobins and their

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encoding genes have been identified in both ascomycete and basidiomycete phyla that represent

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most species in the fungal kingdom, and more recently in bacteria.11 The presence of

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hydrophobins is not associated with a specific microbial lifestyle, since they are produced by

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saprophytic, pathogenic, and symbiotic fungi. Due to their ability to coat both hydrophobic and

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hydrophilic solid surfaces and reverse their hydropathy, their main biological functions are to

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allow fungi to escape an aqueous environment and to facilitate dispersal of the spores by their

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hydrophobization.10

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Generally, hydrophobins show extensive differences in their sequence, however all of the

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known three dimensional (3D) structures exhibit a similar β-barrel structure interrupted by some

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disordered regions and share eight conserved cysteine residues that form four disulfide bridges.12

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Based on the spacing of the cysteine residues and the properties of the layer formed by

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hydrophobins, they have been split in two classes. Class I hydrophobins assemble into a protein


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layer that can only be dissociated using pure trifluoroacetic acid (TFA) or formic acid. In

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contrast, assemblages of class II hydrophobins can be dissociated in 60% ethanol, or 2% sodium

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dodecyl sulfate (SDS).9

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Fibrillar structures formed by Class I hydrophobins, called rodlets, share many structural

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analogies with amyloid fibrils: they bind the amyloid specific dyes, i.e. Congo red and

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Thioflavin T (ThT)13 and exhibit the typical X-ray diffraction pattern.14 Amyloid fibrils show a

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common structural motif, the cross-β structure, and have historically been associated with

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pathologies, such as Alzheimer’s and Parkinson diseases. However rodlets formed by class I

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hydrophobins provide biologically functional molecules, defined functional amyloids. Other

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proteins from bacteria, insect, fish, spider, and from humans also belong to the functional

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amyloid group.15

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Based on the literature review, there is a scope of research in understanding the mechanism of

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rodlet formation and the role of HHI in the assembly process. The extensively studied class I

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hydrophobin, SC3 from Schizophyllum commune spontaneously self-assembles via an α-helical

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intermediate state into a stable β-sheet end configuration at a water-air interface.16,17 It has been

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also demonstrated that SC3 adopts the amyloid state at the water-Teflon interface by heating the

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sample in the presence of detergent or at high protein concentration and prolonged incubation.

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The presence of some polysaccharides, such as schizophyllan, also promotes SC3 amyloid

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formation.18

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Morris et al. studied the behavior of the class I hydrophobins EAS and DewA.19 They reported

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the key role of HHI in rodlet formation related not only to a local protein concentration increase,

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but also to specific conformational changes that expose amyloidogenic regions and result in


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cross-β rodlet structure formation. The assembly rate of these hydrophobins, induced by solution

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agitation reduced, when the percentage of non-polar solvents increased.

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In our research group, the most abundant hydrophobin produced by the edible basidiomycete

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fungus Pleurotus ostreatus, named Vmh2, is widely studied.20,21 Peñas et al.22 firstly observed

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Vmh2 gene expression in vegetative mycelia in all the tested nutritional conditions. Vmh2 was

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detected throughout the culture time, both associated to the cell wall and secreted into the

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medium. It belongs to the Class I hydrophobins and can form rodlets, as shown by atomic force

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microscopy.23,24 Fluorescence analysis in the presence of ThT and Fourier transform infrared

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spectroscopy confirm the cross-β structure of Vmh2 aggregates that can be only disassembled in

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strong acids.21,25

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In a previous paper we have reported that Vmh2 is quite insoluble in water, while its solubility

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increases in less polar solvents (i.e., ethanol, ≥ 40% v/v).21 The presence of alcohol in an

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aqueous environment can be required to mask the large exposed hydrophobic areas of

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hydrophobin molecules, thus, reducing protein-protein interactions. In these solvents the protein

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adopts mainly a stable α-helix conformation. A decreased Vmh2 solubility was observed by

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increasing the solvent polarity, even if no conformational transition was observed. On the other

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hand, Vmh2 conformational change occurs by increasing the pH of the alcoholic solution (pH ≥

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6), and a self-assembled β-sheet rich state is formed. Vmh2 self-assembling is also induced in the

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presence of Ca2+. Analogously the recombinant class I hydrophobin H* protein A shows

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propensity to self-assemble in the latter condition.26

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In this study, we evaluate the self-assembly characteristics of the Class I hydrophobin Vmh2

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under an array of experimental conditions in an attempt to define the key factors controlling the

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process. Since we now assess that Vmh2 can be solubilized in water at pH ≥ 7, its behavior can


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be compared to that of the other Class I hydrophobins in the same conditions. Findings indicate

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that Vmh2 conversion into the β-sheet rich, assembled form does not occur at HHI, while it can

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self-assemble in true amyloid fibrils in other conditions. A model is proposed which can explain

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the mechanism of self-assembling of the hydrophobin Vmh2.

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Biomacromolecules

MATERIALS and METHODS

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Vmh2 extraction from P. ostreatus mycelia.

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White-rot fungus, P. ostreatus (Jacq.: Fr.) Kummer (type: Florida; ATCC No. MYA-2306)

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was maintained at 4 °C through periodic transfer on potato dextrose agar (Difco) plates in the

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presence of 0.5% yeast extract. Mycelia were inoculated in 1 L flasks containing 500 mL of

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potato-dextrose broth (24 g/L) supplemented with 0.5% yeast extract, grown at 28 °C in shaken

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mode (150 rpm). After 10 days of fungal growth, mycelia were separated by filtration through

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gauze, treated twice with 2% SDS in a boiling water bath for 10 min, washed three times with

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water and once with 60% ethanol to completely remove the detergent. The residue was freeze-

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dried, grinded and treated with 100% TFA in a water bath sonicator (Elmasonic S30, Elma) for

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30 min, and centrifuged (10 min at 3200 g). The supernatant was dried, dissolved in 60% ethanol

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and centrifuged (20 min at 3200 g) obtaining a raw extract solution. The ethanol was removed

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from the raw extract under vacuum at 40 °C using rotavapor and the material was freeze-dried,

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then lipids were extracted in a mixture of water-methanol-chloroform 2:2:1 v/v (5 min in bath

16

sonicator). After centrifugation, proteins appeared as a solid aggregate at the interface between

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the water-methanol and the chloroform phases. They were recovered by removal of liquid

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phases. The aggregated protein was dried, treated with TFA for 30 min in bath sonicator, re-

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dried, dissolved in 60% ethanol and centrifuged (90 min at 12000 g). The supernatant was dried,

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treated with TFA as above-described and re-dissolved in the appropriate solution. All the

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analyses were performed using samples just after TFA treatment and solubilization, unless

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specifically indicated.

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Protein Concentration Determination.

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Protein concentration was evaluated using the PIERCE 660 nm or BCA Protein Assay kit

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using ovalbumin as standard.

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Spectroscopy Techniques.

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Far-UV Circular Dichroism (CD) spectra were recorded on a Jasco J715 spectropolarimeter

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equipped with a Peltier thermostatic cell holder in a quartz cell (0.1 cm light path) from 190 to

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250 nm. The temperature was kept at 20 °C and the sample compartment was continuously

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flushed with nitrogen gas. The final spectra were obtained by averaging three scans, using a

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bandwidth of 1 nm, a step width of 0.5 nm, and a 4 s averaging per point.

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Fluorescence spectra were recorded at 25 °C with a HORIBA Scientific Fluoromax-4

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spectrofluorometers. Slits were set to 3 and 6 nm spectral bandpass in excitation and emission

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monochromators, respectively. ThT (Sigma, 30 µM final concentration) was added, the samples

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were excited at 435 nm and emission was monitored from 460 to 600 nm.

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Dynamic Light Scattering (DLS)

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DLS measurements were performed with a home-made instrument composed by a Photocor

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compact goniometer, a SMD 6000 Laser Quantum 50 mW light source operating at 532.5 nm, a

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photomultiplier (PMT-120-OP/B) and a correlator (Flex02-01D) from Correlator.com.27 The

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measurements were performed at (25.00 ± 0.05) °C with temperature controlled through the use

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of a thermostat bath. All experiments were performed at the scattering angle of 90° (θ), the value

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of the scattering vector q = 4 π n/λ sin (θ/2) were calculated assuming the refractive index of the

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solution n = 1.33. The scattered intensity correlation function was analyzed using a


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regularization algorithm (Precision Deconvolve 32).28 The measured diffusion coefficient was

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taken as the z-average diffusion coefficient of the obtained distributions.29

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For spheres diffusing in a continuum medium at infinite dilution, the diffusion coefficient,

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D ≡ D ∞ is dependent on the radius of the sphere,30 called hydrodynamic radius (RH), through

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the Stokes–Einstein equation:

RH =

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kT 6πη D

∞

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where k is the Boltzmann constant, T is the absolute temperature and η is the medium viscosity

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corresponding to 0.89 cP. For not spherical particles, RH represents the radius of equivalent

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spherical aggregates. In this hypothesis, Stokes–Einstein equation can be reasonably used to

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estimate the averaged RH of the aggregates.31 Herein RH was estimated from the average value of at least three measurements of the diffusion coefficients of the aggregates for each analyzed sample.

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Homology modeling of Vmh2

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“DeepView Project Mode” option of the Swiss-Model homology-modeling server was used to

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prepare the homology model of Vmh2.32,33 The sequences of the hydrophobins Vmh2, DewA

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and

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(http://www.ebi.ac.uk/Tools/msa/clustalw2/).34 The structures of hydrophobins DewA (pdb code:

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2LSH) and EAS (pdb code: 2FMC) were superimposed using the DeepView/Swiss-PdbViewer

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software,33 hence the sequence of Vmh2 was manually aligned to the structural alignment of the

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two known structures using the ClustalW2 alignment as reference and finally, the “DeepView

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project” was uploaded to the Swiss-Model server. Structures and Vmh2 model were analyzed

EAS

were

aligned

using

the

ClustalW2

server


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using PyMol (“The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC”

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http://www.pymol.org/).

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Lysine derivatization

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Acetylation of amines of Vmh2 was performed by a transesterification reaction using

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trifluoroacetic anhydride. 60 µg of Vmh2, after the TFA treatment, were dried in a stream of

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nitrogen and dissolved in 300 µl mix of trifluoroacetic anhydride/acetic acid 2:1 vol:vol, treated

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in bath sonicator for 2 min and one hour at room temperature. The sample was again dried in a

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stream of nitrogen, dissolved in methanol, dried once more to remove volatile impurities, TFA

10

treated and dissolved in 60% ethanol solution or 50 mM sodium phosphate, pH 7 at 200 µg mL-1.

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Electron Microscopy

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Samples were prepared for transmission electron microscopy (TEM) analysis by incubating a 5

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µl drop for 10 minutes on a standard carbon-coated copper grids (100 mesh) covered with a

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Formvar film. Then the drop was removed and the grid was air-dried. Images were acquired

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using a FEI Tecnai 12 transmission electron microscope (FEI Company, Hillsboro, Oregon,

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USA) equipped with a Veleta CCD digital camera (Olympus Soft Imaging Solutions GmbH,

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Münster, Germany) and operating at 120 kV, at magnifications of 21000X, 30000X and 68000X.

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Cryo-TEM measurements were performed at the Heinz Meier Leibnitz Source, Garching

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Forschungszentrum facilities. A Multi A 300 mesh copper grid coated with holey carbon film

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(Quantifoil Micro Tools GmbH) was dipped into the solution and then placed in the chamber of a

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cryo-plunge (EMGP Leica GmbH) maintained at 20 °C and 80% relative humidity. The excess

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liquid was removed with filter paper. This sample was cryo-fixed by rapid immersion with the


10

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cryo-plunge into liquid ethane between -170 and -180 °C. The specimen was inserted into a cryo-

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transfer holder (HTTC 914, Gatan, Munich, Germany) and transferred to a JEM 2200 FS

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EFTEM instrument (JEOL, Tokyo, Japan). Examinations were carried out at temperatures

4

around -180°C. The transmission electron microscope was operated at an acceleration voltage of

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200 kV. Zero-loss filtered images were taken under reduced dose conditions ( 6 µm) (Figure 4D), and cryo-TEM analysis (Figure 4E,F) revealed

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thick and twisted fibrils in addition to spherical aggregates. Longer fibrils were observed by

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TEM analysis of samples stored for two months (Figure 5)

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Figure 5. TEM image of a 200 µg mL-1 Vmh2 sample dissolved in 50 mM Na phosphate buffer, pH 7, and stored for two months at room temperature.

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When Vmh2 was incubated at 60 °C, conformational changes, ThT fluorescence intensity

6

increase, and protein precipitation occurred just after 30 minutes (Figure SI-5). Conversely,

7

samples stored at 4 °C did not show any variations in 7 days.

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Effects of CaCl2 addition

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It has been reported by Longobardi et al.21 that Vmh2, dissolved in 60% ethanol, rapidly self-

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assembles after the addition of CaCl2. Herein, the formation of a precipitate some minutes after

12

addition of CaCl2 was observed to the naked eye also when Vmh2 was dissolved in aqueous

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buffer (50 mM Tris HCl, pH 7 or 8), alongside the occurrence of conformational changes and

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ThT fluorescence intensity increase (Figure SI-6). DLS measurements could not be performed in

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this case because of the precipitation of large particles in the measurement cell.

16 17

Effect of HHI

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Class I hydrophobins are known to self-assemble forming amyloid-like structures by

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increasing HHI,9 e.g. by agitation of the solution. Vmh2 samples were analyzed after vortexing,


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changing several conditions e.g. vortexing time, sample concentration and pH. In any case no

2

significant change was detected by protein concentration assay (after centrifugation), CD and

3

DLS. However, formation of foam stable for at least three days after vortexing, was observed

4

(Figure SI-7). This foam stabilizing property was lost after Vmh2 aggregation, either at high

5

temperature, or in the presence of calcium ions or at low pH, suggesting that the assembled form

6

of Vmh2 has no tendency to reach HHI.

7 8

Effect of solvent polarity

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As aforementioned, Vmh2 shows an opposite behavior in aqueous solution (soluble at

10

neutral/basic pH, self-assembled at acid pH) and in low polar solvents (soluble in 60% ethanol

11

solution at acidic pH, self-assembled at neutral/basic pH). Notably the protein is stable for more

12

than one year in the low polar solution at low pH. These findings suggest the occurrence of

13

different mechanisms of stabilization/aggregation in solvents at diverse polarity.

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In order to prove the effect of solvent polarity, ethanol was added to Vmh2 dissolved in

15

aqueous solution at pH 7÷8 (initial Vmh2 concentration 100 µg mL-1 dissolved in 10 mM NH3

16

after TFA treatment), reaching 60% of ethanol. CD analysis of this sample and slight higher ThT

17

fluorescence intensity indicated the increase in β-sheet content and β-sheet stacking (Figure SI-

18

8). TEM analysis exhibited the presence of oligomers, clusters of fibrils, and very large

19

aggregates never detected in aqueous solutions of Vmh2 (Figure 6A,B).

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Figure 6. A-B, TEM images of 200 µg mL-1 Vmh2 samples dissolved at pH 7÷8 using 10mM

3

NH3, upon ethanol addition up to 60%, diluted 1:8 before the image acquisition. C, TEM image

4

of Vmh2 in 60% ethanol self-assembled upon ammonia addition (pH 7÷8).

5 6

To perform further comparative study, Vmh2 samples in 60% ethanol self-assembled by

7

ammonia addition (final pH 7÷8) were analyzed by TEM (Figure 6C). Again very large

8

aggregates together with short fibrils were observed. Likewise, rapid formation of large

9

aggregates (RH > 1 µm) was also shown by DLS analysis in this condition (data not shown).

10 11

These results demonstrated that self-assembly in low polar solvent at neutral/basic pH occurs independent of the starting conditions in which the protein is solubilized.

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All the reported results allow us to conclude that, the hydrophobin Vmh2:

14

i) is soluble in aqueous buffers at pH ≥ 7; ii) when freshly dissolved, forms both spherical and

15

fibrillar assemblies, whose relative amounts depends on protein concentration; iii) spontaneously

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self-assembles into amyloid fibrils and the process is promoted by temperature increase, pH < 6,

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or in the presence of Ca2+ ions; iv) forms both large aggregates and amyloid fibrils in 60%


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ethanol at pH>7; v) only in its soluble form acts as a surfactant (foam stabilizer) and its

2

exposition to HHI (i.e. by vortexing) does not induce amyloid formation.

3

As shown in Figure 7, the protein adopts the same final conformation, characterized by higher

4

contribution of β structure, irrespective of the specific condition promoting fibril formation. This

5

conformation is reached even when Vmh2 self-assembles in 60% ethanol at neutral/basic pH.

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7 8

Figure 7. CD spectra of (a) Vmh2 (200µg mL-1) in Na phosphate buffer pH 7, (b) after 5 days at

9

30 °C, (c) after 30 min at 60 °C, (d) in the presence of 10 mM CaCl2 (in this case Tris HCl buffer

10

pH 7.0 was used to avoid precipitation of calcium phosphate), (e) at pH 3.2 (intensity of this

11

spectrum normalized with respect to spectrum b), (f) after addition of ethanol up to 60% v/v (in

12

this case 10 mM NH3 was used to avoid precipitation of salts).

13 14

Inference from Vmh2 3D structure model

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In order to shed light on the mechanism of self-assembling of Vmh2 the 3D structure of the

16

protein was modeled using the structure of hydrophobin DewA (pdb code: 2LSH) as template


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(for details see Supporting information “Modeling of the Vmh2 3D structure” and Figure SI-9 ).

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The structural alignment (Figure 8A) of Vmh2 with DewA and EAS, the only two class I

3

hydrophobins of known structure, highlights the significant differences in the sequence and

4

structure of these three proteins.

5


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Figure 8. A, Structural alignment of DewA (pdb code: 2LSH), EAS (pdb code: 2FMC) and

3

Vmh2 (UniProt Accession Number Q8WZI2; chain 25-111). The letters H and S at the bottom of


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the alignment indicate the position of α-helices and β-strands, respectively, in the two known

2

structures. Residues are colored according to their properties (red, acidic; blue, basic; magenta,

3

hydrophilic; green, hydrophobic, gray, glycine and proline; yellow, cysteine). The region of EAS

4

involved in the formation of rodlets is underlined. The position of loops L1-3 is indicated at the

5

top of the alignment. B-E, Comparison between the DewA structure (pdb code: 2LSH; panels B

6

and D) and the homology model of Vmh2 (panels C and E). In B and C the structures are shown

7

as ribbons to highlight position and length of loops L1 (yellow), L2 (green) and L3 (cyan). In

8

panel B the first seven residues are not shown for clarity. In panel C side chains of Asp2, Asp22,

9

Asp40, Ser81 and Lys19 are shown as sticks. Hydrogen bonds are shown as magenta dotted

10

lines. Panels D and E show the solvent accessible surface colored according residues properties

11

(colors as panel A). Green arrows in panels C and E indicate the large hydrophobic patch created

12

by the packing of L1 and L3.

13 14

Beyond the different length of the loops connecting the conserved cysteines, the three proteins

15

show significant differences in the number and distribution of hydrophobic and charged residues

16

(Figure 8). For example the loop L1 which is connecting cysteines 3 and 4 in Vmh2 and DewA,

17

is considerably longer than in EAS. The first part of loop L1 of both Vmh2 and DewA is very

18

hydrophilic, the central region is rich in hydrophobic residues in both proteins, whereas the C-

19

terminal end is very hydrophobic in Vmh2 and hydrophilic in DewA. It is worth noting that

20

Vmh2 shows an aspartate (Asp40) in this region which is not present in DewA. The loop L2,

21

connecting cysteines 4 and 5, is much longer in DewA than in EAS and Vmh2. On the contrary

22

the loop L3 which is connecting cysteines 7 and 8, is much longer in EAS than in DewA, and in

23

Vmh2 it has an intermediate length.


27

Biomacromolecules

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Page 28 of 40

1

Very interestingly Vmh2 model shows a large hydrophobic exposed area (Figure 8E),

2

corresponding to 27% of the total area (2117 Å2 out of 7811 Å2), which is not present in DewA

3

(Figure 8D). This hydrophobic patch is created by the packing of the hydrophobic sequences in

4

L1 and L3.

5

Vmh2 contains much less charged residues than the other two hydrophobins. Indeed only three

6

aspartates

and

one

lysine

are

present

in

Vmh2,

whereas

DewA

contains

ten

7

aspartates/glutamates, six lysines and a histidine. Moreover, EAS contains five aspartates and

8

three lysines (Figure 8A). Except Asp2 that, in our model, is solvent exposed and not involved in

9

stabilizing contacts, the other charged residues are located in crucial positions at both ends of

10

loop L1: Lys19 and Asp22 form a ionic pair stabilizing the first short helix of L1, whereas Asp40

11

forms a hydrogen bond with the OH group of Ser81 thus anchoring the turn located at the C-end

12

of L1 to body of the protein (Figure 8C).

13

In order to validate the proposed model, amino groups of the protein (the key side chain of

14

Lys19 and the N-term of the sequence) were modified by acetylation using trifluoroacetic

15

anhydride. The acetylated Vmh2 became insoluble in the aqueous buffer at pH 7, thus supporting

16

the hypothesis that the formation of the ionic couple between Lys19 and Asp22 is crucial to

17

stabilize the aqueous form of the protein. On the other hand, the modification of Lys19 did not

18

affect the protein solubility in 60% ethanol, as demonstrated by the protein concentration assay,

19

but only its structure, as shown by an evident change of the CD spectrum intensity (Figure SI-

20

10).

21


28

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1

Biomacromolecules

DISCUSSION

2

Proteins can use different mechanisms to control their own assembly into fibrils, such as the

3

controlled release of an amyloidogenic region from the folded protein, the inclusion of

4

chaperoning domain, or the adjustment of fibrillation propensity according to pH.38 It is widely

5

accepted that Class I hydrophobins assemble into amyloid-like structures, the so-called rodlets,

6

when exposed to HHI. A plausible mechanism has been recently described for the Neurospora

7

crassa hydrophobin EAS, whose NMR structure is known.2,14 According to this model, contact

8

with an air/water interface should induce the formation of an amphiphilic secondary structure

9

from a disordered loop. A region of EAS has been identified that drives the intermolecular

10

association and the formation of the cross-β structure.

11 12

The main issue of this work is to explore the self-assembly of Class I hydrophobins showing C D that it is not regulated by a unique, common mechanism, but different mechanisms can trigger

13

and control functional amyloid formation within this class of proteins. Exposition to HHI does

14

not induce formation of amyloid-like structures in the case of the Class I hydrophobin Vmh2

15

from P. ostreatus in any of the conditions tested, whereas the phenomenon spontaneously occurs

16

and it is controlled by other parameters (i.e. pH, protein concentration, temperature, presence of

17

Ca2+, solvent polarity). Among the known Class I hydrophobins, SC3 from Schizophyllum

18

commune shows the highest sequence similarity to Vmh2,20 however SC3 is much more

19

hydrophilic, being a glycosylated protein.39 It is generally reported that SC3, upon assembly at

20

water-air interface, proceeds via an α-helical intermediate state to a stable amyloid-like β-sheet

21

state. On the other hand at water and hydrophobic solid interface, SC3 does not spontaneously

22

form amyloid-like fibrils, but is arrested in the α-helical intermediate state, unless transition to

23

the β-sheet state is induced by particular conditions (heating in the presence of detergent, high


29

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 40

1

protein concentration, presence of cell-wall polysaccharides).18 Hence, differently from EAS,40

2

SC3 requires specific conditions to form amyloid-like fibrils at HHI. Recently, Zykwinska et al.

3

reported that SC3 rodlets are formed in aqueous solution, tuned by pH and ionic strength.41 The

4

Class I hydrophobin ABH1 from Agaricus bisporus also shows unusual self-assembly properties.

5

As reported by Paslay et al., ABH1 undergoes a direct transition to the β-sheet state at elevated

6

temperature.42 These examples demonstrate that, although the amphiphilic characteristics of

7

Class I hydrophobins determine their tendency to migrate towards HHI, this migration is not the

8

necessary and sufficient condition to form amyloid-like structures.

9

Vmh2 shows a very high hydrophobic index (grand average of hydropathy, GRAVY

10

+0.829),43 and just four charged residues (one Lys and three Asp) are present in the sequence of

11

the mature protein (isoelectric point about 4). It is conceivable that the protein is soluble in low

12

polar solvent (40÷60% ethanol) at low pH where Asp residues are protonated and it has low net

13

charge. In this condition the protein assumes a α-helical conformation, but it does not show a

14

tendency to reach the air-liquid interface.21 It has been reported that the interface properties (e.g.

15

the surface tension of the solution) are of critical importance for the rodlet formation of both

16

EAS and DewA.40 Indeed it was not observed rodlet formation in the case of DewA in 20%

17

ethanol. Since we now assess that Vmh2 is soluble in water at pH ≥ 7, we can compare its

18

behavior to that of the other Class I hydrophobins in aqueous solutions. Our results clearly

19

indicate that formation of amyloid-like structures is not induced by exposition to HHI.

20

This result is in agreement with our previous study in which atomic force spectroscopy (AFM)

21

images of the Langmuir-Blodgett films displayed a layer of Vmh2 monomers at water-air

22

interface. Actually, rodlets were only observed after repeated compression-expansion cycles 23,24.


30

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Biomacromolecules

1

When aqueous solutions of Vmh2 freshly dissolved at neutral pH were analyzed, two

2

populations were detected by DLS, whose smallest RH (10÷20 nm, which is larger than that one

3

expected from a single molecule, ≈1.5nm) was ascribed to small oligomers. Furthermore, on the

4

basis of the DLS analysis and ThT assay, the large size population (RH 50÷80 nm) is rich in

5

amyloid-like structures and its amount greatly increases as a function of protein concentration.

6

TEM imaging of these assemblies showed small and large oligomers as well as short (1-2 µm)

7

amyloid fibrils, much more abundant at the highest concentration tested, 0.4 mg mL-1. However,

8

the size of the fibrils observed in these conditions was more than one order of magnitude higher

9

than that of the previously mentioned rodlets of Vmh2, shown by AFM.23,24 The presence of

10

fibrillar structures of Vmh2 in the aqueous solutions immediately upon dissolution, could be due

11

to its hydrophobic exposed area, which is much larger than that of other hydrophobins,

12

determining its higher propensity to self-assemble in water.

13

The results discussed above were obtained using proteins freshly dissolved after TFA

14

depolymerization treatment. However few

days after protein

dissolution, extensive

15

conformational changes were detected by CD with a concomitant increase of size and amount of

16

the amyloid aggregates, as revealed by DLS and by ThT assay. As expected, the kinetics of the

17

self-assembly process depends on temperature, the higher the temperature, the faster the

18

aggregate formation. Moreover the protein rapidly undergoes conformational changes by

19

decreasing the pH or in the presence of Ca2+. Upon decreasing pH or depending on incubation

20

time, TEM and cryo-TEM imaging showed the presence of isolated, several micrometers long,

21

and twisted fibrils. It is generally reported that rodlets formed by hydrophobin proteins are

22

hundreds of nanometers long, packed into ordered lateral assemblies and do not exhibit an


31

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 40

1

overall helical structure.2,44–47 Thus, to the best of our knowledge, this is the first time that fibrils

2

appearing as true amyloids, formed by hydrophobins have been observed.

3

Sunde and co-workers have proposed a molecular mechanism for the formation of EAS

4

amyloid-like fibrils. They have demonstrated by a mutational and deletion analysis that loop L3

5

of EAS is able to trigger aggregation when transferred to a non-amyloidogenic hydrophobin.2

6

According to this mechanism, regions Asp64-Thr68 and Ser71-Ile75 of loop L3 would form the

7

stems of a β-hairpin responsible for the aggregation process. More recently the same research

8

group has analyzed the structure of DewA19 and observed that the loop L3 in this protein is too

9

short and does not contain amyloidogenic sequences, so they have proposed that the long loop

10

L2 could drive the self-assembly process of DewA. However, at the moment, this hypothesis has

11

not been verified.

12

The 3D structure of Vmh2 has not been determined before, however a possible mechanism of

13

formation of amyloid-like structures is discussed on the basis of the homology model herein

14

proposed. Even if the loop L3 of Vmh2 shows a region very similar in sequence (residues 71-78)

15

to the second stem of the β-hairpin, which is responsible for the self-assembly process in EAS

16

(Figure 8A), this loop seems too short to be the sole or the main responsible for the aggregation,

17

as in the case of DewA. Moreover the loop L3 does not contain charged residues that could

18

explain the pH and calcium dependence of the process.

19

According to the mechanism of self-assembly of Vmh2 that we propose, protonation of Asp22

20

and Asp40 would induce a destabilization of the loop L1 thus triggering a conformational change

21

that in turn would determine the aggregation of the protein. Moreover, calcium ions, whose

22

affinity for aspartate is well known, could trigger a similar conformational switch. Both loops L1

23

and L3 might participate to the process, in fact, the two loops are closely packed in the model


32

Page 33 of 40

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Biomacromolecules

1

(Figure 8C) and a conformational change of the loop L1 could expose the loop L3 to the solvent

2

thus making it available to intermolecular contacts. Notably, both loops contain very

3

hydrophobic stretches that could contribute to hydrophobic interactions (green arrows in Figure

4

8C,E). The exposition of loops L1 and L3 to the solvent may also occur spontaneously and the

5

process can be accelerated by the increase of temperature, thus triggering the conformational

6

switch and the formation of amyloid structures.

7

It is worth noting that the presence of ethanol at 60% remarkably stabilizes the protein at low

8

pH. In this condition Vmh2 shows a higher content of α-helical structure than in aqueous buffers

9

at pH ≥ 7, moreover neither conformational change nor aggregation is detected in more than one

10

year. Actually, self-assembly in the presence of ethanol occurs at neutral/basic pH. TEM and

11

DLS analysis of these assemblies showed rapid formation of very large amorphous aggregates,

12

never seen in the aqueous solutions of Vmh2, together with some fibrils. On this basis, a

13

completely different mechanism of solubilization/aggregation has to be supposed, moreover the

14

model of 3D structure is not valid in 60% ethanol solution. The increase of pH of the solution

15

should convert the Asp residues into their unprotonated form, thus forming new charges on the

16

protein surface. Albeit this should enhance the electrostatic repulsions of protein molecules, it is

17

known that unfavorable interactions between charged solutes and low dielectric organic solvents,

18

such as ethanol, can cause reduction of protein solubility, aggregation and precipitation.48,49

19

Large aggregates formed by the Class I hydrophobin SC3 have previously been observed by

20

Stroud et al.45 slowly appearing in 120 hours in undisturbed aqueous solutions. These large

21

aggregates showed lower β-sheet stacking with respect to amyloid-like fibrils, as indicated by

22

ThT fluorescence assays, analogously to those ones formed by Vmh2 in 60% ethanol. However,

23

also in this case, aggregation of Vmh2 occurs in a condition different from that of another


33

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Page 34 of 40

hydrophobin.

2 3

CONCLUSIONS

4

The conversion of Vmh2 into the β-sheet rich forms occurs in conditions which are different

5

from those of other Class I hydrophobins. Indeed exposition of Vmh2 to water-air HHI does not

6

induce fibril formation, whereas the process spontaneously occurs over time, and is promoted by

7

high protein concentration, pH < 6, temperature increase, or in presence of Ca2+ ions, resulting

8

into the same final conformation. This conversion leads to the formation of isolated, long and

9

twisted amyloid fibrils, so far never observed in the other Class I hydrophobins. On the basis of

10

the 3D structure model of the protein, a self-assembling mechanism can be inferred. Protonation

11

of two aspartates, as well as interaction with Ca2+ ions or increase of temperature would induce

12

the destabilization of a long loop thus triggering the conformational change that would determine

13

the formation of amyloid fibrils. Some of the distinctive properties of the hydrophobin Vmh2,

14

such as the higher hydrophobic character, specific conditions leading to the self-assembly and

15

special structures formed, allow us to suppose that it should play a peculiar functional role in the

16

life cycle of P. ostreatus. Further studies will be carried out to elucidate the natural function of

17

Vmh2.

18 19

ASSOCIATED CONTENT

20

Supporting Information

21

This supporting information is available free of charge on the ACS publications website

22

http://pubs.acs.org.


34

Page 35 of 40

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Biomacromolecules

1

Optimization of TFA treatment of Vmh2; modeling of the 3D structure; additional CD spectra,

2

ThT fluorescence assays, DLS analysis and TEM images of Vmh2 samples in different

3

conditions, e.g. concentration, temperature, time, upon Ca2+ or ethanol addition; pictures of

4

Vmh2 samples after agitation; CD spectra of Lys acetylated Vmh2.

5 6 7 8

AUTHOR INFORMATION

9

Corresponding Author

10

*Paola Giardina. Department of Chemical Sciences, University of Naples ‘Federico II’, Via

11

Cintia 4, 80126 Naples, Italy - Email: [email protected] - Tel: +39 081 674319 - Fax +39 081

12

674 310

13

Author Contributions

14

The manuscript was written through contributions of all authors. All authors have given

15

approval to the final version of the manuscript.Funding Sources

16

This work was supported by grant from the Ministero dell’Università e della Ricerca

17

Scientifica -Industrial Research Project “Integrated agro-industrial chains with high energy

18

efficiency for the development of eco-compatible processes of energy and biochemicals

19

production from renewable sources and for the land valorization (EnerbioChem)”

20

PON01_01966, funded in the frame of Operative National Programme Research and

21

Competitiveness 2007–2013 D. D. Prot. n. 01/Ric. 18.1.2010.

22 23

ACKNOWLEDGEMENTS


35

Biomacromolecules

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Page 36 of 40

1

The authors thank Dr. Marinella Pirozzi of the Bioimaging Facility, Institute of Protein

2

Biochemistry (CNR), Naples, for help with TEM experiments and Dr. Jasneet Kaur, of the

3

Department of Physics, University of Naples, Federico II, for editorial assistance.

4 5 6


36

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1

Biomacromolecules

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Biomacromolecules

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Class I Hydrophobin Vmh2 Adopts Atypical Mechanisms to Self

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