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Cited by 8Biofuel production and bioconversionAdvanced Cellulose Research StudiesPolysaccharides and Plant Cell Walls

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SANS investigation of fungal loosenins reveals substrate-dependent impacts of protein action on the inter-microfibril arrangement of cellulosic substrates

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Dahiya, Deepika, Péter-Szabó, Zsuzsanna, Senanayake, Manjula, Pingali, Sai Venkatesh, Leite, Wellington C., Byrnes, James, Buchko, Garry W., Sivan, Pramod, Vilaplana, Francisco, Master, Emma R., O’Neill, Hugh·2025·Biotechnology for Biofuels and Bioproducts
DOI 10.1186/s13068-025-02618-5PMC PMC11869483

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Abstract

BACKGROUND: Microbial expansin-related proteins include fungal loosenins, which have been previously shown to disrupt cellulose networks and enhance the enzymatic conversion of cellulosic substrates. Despite showing beneficial impacts to cellulose processing, detailed characterization of cellulosic materials after loosenin treatment is lacking. In this study, small-angle neutron scattering (SANS) was used to investigate the effects of three recombinantly produced loosenins that originate from Phanerochaete carnosa, PcaLOOL7, PcaLOOL9, and PcaLOOL12, on the organization of holocellulose preparations from Eucalyptus and Spruce wood samples. RESULTS: Whereas the SANS analysis of Spruce holocellulose revealed an increase in interfibril spacing of neighboring cellulose microfibrils following treatment with PcaLOOL12 and to a lesser extent PcaLOOL7, the analysis of Eucalyptus holocellulose revealed a reduction in packing number following treatment with PcaLOOL12 and to a lesser extent PcaLOOL9. Parallel SEC-SAXS characterization of PcaLOOL7, PcaLOOL9, and PcaLOOL12 indicated the proteins likely function as monomers; moreover, all appear to retain a flexible disordered N-terminus and folded C-terminal region. The comparatively high impact of PcaLOOL12 motivated its NMR structural characterization, revealing a double-psi b-barrel (DPBB) domain surrounded by three alpha-helices - the largest nestled against the DPBB core and the other two part of loops extending from the core. CONCLUSIONS: The SANS analysis of PcaLOOL action on holocellulose samples confirms their ability to disrupt cellulose fiber networks and suggests a progression from reducing microfibril packing to increasing interfibril distance. The most impactful PcaLOOL, PcaLOOL12, was previously observed to be the most highly expressed loosenin in P. carnosa. Its structural characterization herein reveals its stabilization through two disulfide linkages, and an extended N-terminal region distal to a negatively charged and surface accessible polysaccharide binding groove.

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Figures

Fig. 1 Solution NMR structure of PcaLOOL12 (9CE9). A The primary amino acid sequence of PcaLOOL12 with the β-strand and α-helix elements of secondary structure colored blue and yellow, respectively. Disulfide bonds are observed between C26–C50 and C73–C76 with the position of these four cysteine residues identified with red residue numbers above the sequence. The P. carnosa genome contains 12 loosenin genes divided into two subgroups based on the position of an insertion relative to the predicted polysaccharide binding groove (PcaLOOL12 belongs in subgroup B) [15]. The red box is the characteristic 12–18 residue insertion observed in subgroup B loosenins and the asterisk after G44 indicates the position of the characteristic 8–9 residue insertion observed in subgroup A loosenins. The rectangles below the sequence denote conserved residues in the P. carnosa loosenin family predicted to form a polysaccharide binding groove, with the blue rectangles denoting residues associated with expansin activity (EXLX1) in this groove. The first two N-terminal residues are “scars” left over after cleavage of the thioredoxin moiety of the original construct. The six β-strands form a double-psi β-barrel (DPBB) structural fold. B A cartoon superposition of the ensemble of 20 solution structures calculated for PcaLOOL12. C A cartoon representation of a single structure in the ensemble rotated 180 º in the y-axis to better illustrate the location of the two disulfide bonds (side chains colored red) and the three β-strands on the back side of the protein. D A topology diagram illustrating the relative orientation of the elements of secondary structure with the α-helices colored yellow and the β-strands colored blue (psi-1) or purple (psi-2). The β-strands and α-helices are drawn to scale but not the connecting loops and turns. E Isolation of the two psi-motifs in the DPBB structural fold. F A view similar to (C) of the solvent-accessible surface of PcaLOOL12 with the residues predicted to form a polysaccharide binding groove colored cyan or blue (A). The three labeled blue residues are associated with activity in expansins. G A view identical to (F) highlighting the electrostatic potentials at the solvent-accessible surface of PcaLOOL12 (–5 to + 5 kT/e) with the negative and positive regions colored red and blue, respectively
Fig. 1 Solution NMR structure of PcaLOOL12 (9CE9). A The primary amino acid sequence of PcaLOOL12 with the β-strand and α-helix elements of secondary structure colored blue and yellow, respectively. Disulfide bonds are observed between C26–C50 and C73–C76 with the position of these four cysteine residues identified with red residue numbers above the sequence. The P. carnosa genome contains 12 loosenin genes divided into two subgroups based on the position of an insertion relative to the predicted polysaccharide binding groove (PcaLOOL12 belongs in subgroup B) [15]. The red box is the characteristic 12–18 residue insertion observed in subgroup B loosenins and the asterisk after G44 indicates the position of the characteristic 8–9 residue insertion observed in subgroup A loosenins. The rectangles below the sequence denote conserved residues in the P. carnosa loosenin family predicted to form a polysaccharide binding groove, with the blue rectangles denoting residues associated with expansin activity (EXLX1) in this groove. The first two N-terminal residues are “scars” left over after cleavage of the thioredoxin moiety of the original construct. The six β-strands form a double-psi β-barrel (DPBB) structural fold. B A cartoon superposition of the ensemble of 20 solution structures calculated for PcaLOOL12. C A cartoon representation of a single structure in the ensemble rotated 180 º in the y-axis to better illustrate the location of the two disulfide bonds (side chains colored red) and the three β-strands on the back side of the protein. D A topology diagram illustrating the relative orientation of the elements of secondary structure with the α-helices colored yellow and the β-strands colored blue (psi-1) or purple (psi-2). The β-strands and α-helices are drawn to scale but not the connecting loops and turns. E Isolation of the two psi-motifs in the DPBB structural fold. F A view similar to (C) of the solvent-accessible surface of PcaLOOL12 with the residues predicted to form a polysaccharide binding groove colored cyan or blue (A). The three labeled blue residues are associated with activity in expansins. G A view identical to (F) highlighting the electrostatic potentials at the solvent-accessible surface of PcaLOOL12 (–5 to + 5 kT/e) with the negative and positive regions colored red and blue, respectivelyCC BY-NC-ND · PubMed Central
Fig. 2 Structural analysis of PcaLOOLs. Panels A–C Experimental SAXS profiles of PcaLOOL12 (pink), PcaLOOL7 (orange), and PcaLOOL9 (green) are shown as open hexagons. Theoretical SAXS profiles from AlphaFold2 and Normal Mode Analysis are shown as dashed light and solid dark blue lines, respectively. Error-normalized residual plots are shown below the SAXS profiles. Panels D–F Normal Mode Analysis models of PcaLOOL12 (pink), PcaLOOL7 (orange), and PcaLOOL9 (green)
Fig. 2 Structural analysis of PcaLOOLs. Panels A–C Experimental SAXS profiles of PcaLOOL12 (pink), PcaLOOL7 (orange), and PcaLOOL9 (green) are shown as open hexagons. Theoretical SAXS profiles from AlphaFold2 and Normal Mode Analysis are shown as dashed light and solid dark blue lines, respectively. Error-normalized residual plots are shown below the SAXS profiles. Panels D–F Normal Mode Analysis models of PcaLOOL12 (pink), PcaLOOL7 (orange), and PcaLOOL9 (green)CC BY-NC-ND · PubMed Central
Fig. 3 Relative monosaccharide and lignin composition of untreated wood (Eucalyptus and Spruce) and holocellulose samples. Values represent the relative weight of the corresponding monosaccharide from acid hydrolysis and the measured lignin after the acetyl bromide dissolution in the total weight of the biomass sample. 4O-MeGlcA (4-O-methyl-D-glucuronic acid); GalA (galacturonic acid); GlcA (glucuronic acid)
Fig. 3 Relative monosaccharide and lignin composition of untreated wood (Eucalyptus and Spruce) and holocellulose samples. Values represent the relative weight of the corresponding monosaccharide from acid hydrolysis and the measured lignin after the acetyl bromide dissolution in the total weight of the biomass sample. 4O-MeGlcA (4-O-methyl-D-glucuronic acid); GalA (galacturonic acid); GlcA (glucuronic acid)CC BY-NC-ND · PubMed Central
Fig. 4 Two-dimensional SANS detector images of native Eucalyptus (A) and Spruce (B), and holocellulose from Eucalyptus (C) and Spruce (D) in 20 mM sodium acetate buffer, pH 5.5 in 100% D2O. Each panel shows the main (left) and wing (right) detector images from the SANS instrument. Lines in panel A represent the equatorial and meridional sectors that were used for data analysis and interpretation
Fig. 4 Two-dimensional SANS detector images of native Eucalyptus (A) and Spruce (B), and holocellulose from Eucalyptus (C) and Spruce (D) in 20 mM sodium acetate buffer, pH 5.5 in 100% D2O. Each panel shows the main (left) and wing (right) detector images from the SANS instrument. Lines in panel A represent the equatorial and meridional sectors that were used for data analysis and interpretationCC BY-NC-ND · PubMed Central
Fig. 5 One-dimensional equatorial SANS profiles of Eucalyptus (A) and Spruce (B). The color labeling schemes for the left and right panels are identical. The holocellulose samples were incubated for 24 h with the PcaLOOL proteins before SANS measurements. Native wood sample (grey); holocellulose control (blue); PcaLOO12 (pink); PcaLOOL7 (orange); and PcaLOOL9 (green). The PcaLOOL12, PcaLOOL7, and PcaLOOL9 curves were multiplied by multiples of 4, and native wood sample was multiplied by a factor of 0.16 for data visualization purposes. The solid black lines are the fits using the Unified Fit approach as described in the Materials and Methods section
Fig. 5 One-dimensional equatorial SANS profiles of Eucalyptus (A) and Spruce (B). The color labeling schemes for the left and right panels are identical. The holocellulose samples were incubated for 24 h with the PcaLOOL proteins before SANS measurements. Native wood sample (grey); holocellulose control (blue); PcaLOO12 (pink); PcaLOOL7 (orange); and PcaLOOL9 (green). The PcaLOOL12, PcaLOOL7, and PcaLOOL9 curves were multiplied by multiples of 4, and native wood sample was multiplied by a factor of 0.16 for data visualization purposes. The solid black lines are the fits using the Unified Fit approach as described in the Materials and Methods sectionCC BY-NC-ND · PubMed Central
Fig. 6 One-dimensional meridional SANS profiles of Eucalyptus (A) and Spruce (B). The color labeling schemes for the left and right panels are identical. The holocellulose samples were incubated for 24 h with the PcaLOOL proteins before SANS measurements. Native wood sample (grey); holocellulose control (blue); PcaLOO12 (pink); PcaLOOL7 (orange); and PcaLOOL9 (green). The PcaLOOL12, PcaLOOL7, and PcaLOOL9 curves were multiplied by multiples of 4, and native wood sample was multiplied by a factor of 0.16 for data visualization purposes. The solid black lines are the fits using the Unified Fit approach as described in the Materials and Methods section
Fig. 6 One-dimensional meridional SANS profiles of Eucalyptus (A) and Spruce (B). The color labeling schemes for the left and right panels are identical. The holocellulose samples were incubated for 24 h with the PcaLOOL proteins before SANS measurements. Native wood sample (grey); holocellulose control (blue); PcaLOO12 (pink); PcaLOOL7 (orange); and PcaLOOL9 (green). The PcaLOOL12, PcaLOOL7, and PcaLOOL9 curves were multiplied by multiples of 4, and native wood sample was multiplied by a factor of 0.16 for data visualization purposes. The solid black lines are the fits using the Unified Fit approach as described in the Materials and Methods sectionCC BY-NC-ND · PubMed Central
Fig. 7 Inter-microfibril distance (A) and packing parameter (B) obtained from fitting the Eucalyptus (open squares) and the Spruce (open hexagons) data for holocellulose (Holocel’), and PcaLOOL12, PcaLOOL7, and PcaLOOL9-treated samples
Fig. 7 Inter-microfibril distance (A) and packing parameter (B) obtained from fitting the Eucalyptus (open squares) and the Spruce (open hexagons) data for holocellulose (Holocel’), and PcaLOOL12, PcaLOOL7, and PcaLOOL9-treated samplesCC BY-NC-ND · PubMed Central

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Metadata

Authors
Dahiya, D., Péter-Szabó, Z., Senanayake, M., Pingali, S. V., Leite, W. C., Byrnes, J., Buchko, G. W., Sivan, P., Vilaplana, F., Master, E. R., & O’Neill, H.
Affiliations
  • Aalto University (FI)
  • KTH Royal Institute of Technology (SE)
  • Oak Ridge National Laboratory (US)
  • Brookhaven National Laboratory (US)
  • Pacific Northwest National Laboratory (US)
  • Wallenberg Wood Science Center (SE)
  • University of Toronto (CA)
Corresponding author
  • Master, Emma R.
  • O’Neill, Hugh
ORCID
Venue
Biotechnology for Biofuels and Bioproducts
Publisher
Springer Science and Business Media LLC
Published
2025-02-28
Volume
18
Issue
1
Pages
27-27
ISSN
2731-3654 (ISSN-L)
DOI
10.1186/s13068-025-02618-5
PMC
PMC11869483
OpenAlex
W4408066007
Cited by
8
References
2
Citations by year
2026: 4 · 2025: 4
FWCI
1.95
Type
journal article
Language
en
Open access
Licence
Locations
  • Biotechnology for Biofuels and Bioproducts (publishedVersion, cc-by-nc-nd)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Aaltodoc (Aalto University) (acceptedVersion, other-oa)
  • PubMed Central (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
Funders
  • Horizon 2020 Framework Programme (964764)
  • Office of Science (FICUS -60859)
  • Center for Structural Molecular Biology, USA (FWP ERKP291)
  • Genomic Science Program, USA (FWP ERKP752)
  • Biological and Environmental Research (KP1605010, DE-AC05-76RL01830)
  • National Institute of General Medical Sciences (P30GM133893)
  • National Institutes of Health (S10 OD012331)
Topics
  • Physical Sciences › Engineering › Biomedical Engineering › Biofuel production and bioconversion
  • Physical Sciences › Materials Science › Biomaterials › Advanced Cellulose Research Studies
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Polysaccharides and Plant Cell Walls
Keywords
holocellulose, eucalyptus wood, spruce wood, protein structure, disulfide bonds

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