Interactions of a fungal lytic polysaccharide monooxygenase with β-glucan substrates and cellobiose dehydrogenase

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds using molecular oxygen and an external electron donor. We have used NMR and isothermal titration calorimetry (ITC) to study the interactions of a broad-specificity fungal LPMO, NcLPMO9C, with various substrates and with cellobiose dehydrogenase (CDH), a known natural supplier of electrons. The NMR studies revealed interactions with cellohexaose that center around the copper site. NMR studies with xyloglucans, i.e., branched β-glucans, showed an extended binding surface compared with cellohexaose, whereas ITC experiments showed slightly higher affinity and a different thermodynamic signature of binding. The ITC data also showed that although the copper ion alone hardly contributes to affinity, substrate binding is enhanced for metal-loaded enzymes that are supplied with cyanide, a mimic of O2 (-) Studies with CDH and its isolated heme b cytochrome domain unambiguously showed that the cytochrome domain of CDH interacts with the copper site of the LPMO and that substrate binding precludes interaction with CDH. Apart from providing insights into enzyme-substrate interactions in LPMOs, the present observations shed new light on possible mechanisms for electron supply during LPMO action.

Keywords: LPMO; cellobiose dehydrogenase; cellulose; lytic polysaccharide monooxygenase; xyloglucan.

Fig. S1.

Secondary structure of NcLPMO9C in solution, as derived from secondary chemical shifts. Secondary chemical shifts allow estimation of dihedral angles, which in turn can be used to predict secondary structure propensities, which are indicated by the blue bars. The pink bars indicate secondary structure assignments based on the crystal structure, and comparison shows that there is excellent agreement between the secondary structure elements observed in solution and those seen in the X-ray crystallographic structure (PDB ID: 4D7U) (26). Such high similarity is not unexpected and has previously been shown to be valid for another LPMO, CBP21, for which both the NMR (PDB ID: 2LHS) (9) and the X-ray (PDB ID: 2BEM) (52) structures are available.

Fig. S2.

The ¹⁵N relaxation data and ¹⁵N-{¹H} heteronuclear NOEs for apo-NcLPMO9C. (A) Longitudinal relaxation time/transverse relaxation time (T₁/T₂). The T₁:T₂ ratio is a direct measure of the correlation time for overall rotational tumbling of the protein. The average T₁:T₂ ratio for NcLPMO9C was calculated to be 13.8 ± 0.7 (SD), which, assuming a spherical particle, corresponds to an overall rotational correlation time, τ_c, of 11.8 ± 0.5 ns (SD) (44), indicating a tightly packed structure. The Northeast Structural Genomics Consortium has a database with NMR-determined correlation times for globular proteins with different molecular weights. The protein WR73 (which is a 21.9-kDa globular protein) has a longer τ_c = 13.0 ns. The database is available online at www.nmr2.buffalo.edu/nesg.wiki/NMR_determined_Rotational_correlation_time (accessed March 22, 2016). (B) Steady-state ¹⁵N-{¹H} NOEs measured for the backbone amide nitrogen atoms.

Fig. 1.

Interaction of apo-NcLPMO9C with substrates. (A) Overlay of an area of interest from the ¹⁵N-HSQC spectrum for NcLPMO9C (black) in the presence of 4.5 mM GlcNAc₆ (labeled as NAG₆; red) or increasing concentrations of Glc₆ (from lighter to darker blue). The H^N/N chemical shift of Val42 is not affected by the interaction, and therefore, the peak is shown as a reference. (B–D) Compound change in chemical shifts larger than 12 Hz (Fig. S3) upon substrate binding mapped on the structure of NcLPMO9C. The backbone of NcLPMO9C (shown in cartoon and surface representation) is colored according to the compound change in chemical shift (¹⁵N-HSQC) upon adding 2.6 mM Glc₆ (B), 1.3 mM XG₁₄ (C), or 4.2 µM polyXG (D) using the indicated coloring scheme (gray coloring represents no change). The NcLPMO9C structure is shown by a side view (Left) and a top view (Right). The side chains of residues His1, Ala80, His83, and His155 are shown in stick representation. In addition, the side chain of Tyr204 is shown in green. The positions of the L3 loop and the β8-strand are marked on the structures. The LC loop spans the stretch from Gly177 (marked with a red triangle) to the C terminus (marked with a red arrow). The N-terminal amino group (His1) is not observed in ¹⁵N-HSQC spectra because of its fast exchange. The ¹³C-aromatic HSQC spectra showed clear changes in chemical shift for this residue, with all three substrates, with the strongest effects (a vanished signal) being observed with XG₁₄ and polyXG. Based on these observations, for illustrative purposes, His-1 is colored purple (B) or red (C and D) in the figures.

Fig. S3.

Compound change in chemical shift (¹⁵N-HSQC) upon adding (A) 2.6 mM Glc₆, (B) 1.3 mM XG₁₄, or (C) 4.2 µM polyXG to apo-NcLPMO9C. No change in the chemical shifts was observed upon adding 4.5 mM GlcNAc₆. For His1, clear effects on the side chain chemical shifts were observed in a ¹³C-aromatic HSQC spectrum.

Fig. S4.

Thermograms (Upper) and binding isotherms with theoretical fits (Lower) for the binding of 4.0 mM XG14 to 15 μM of NcLPMO9C apo (Left), in the presence of Cu²⁺ (Middle), and in the presence of Cu²⁺/CN⁻ (Right) at t = 25 °C.

Fig. 2.

Interaction model of Glc₆ and NcLPMO9C produced by HADDOCK (39). The backbone is shown as a cartoon and surface, and the side chains of residues known from the NMR experiments to be strongly affected by substrate binding (His1, His64, Ala80, His83, and His155) are shown as sticks. In addition, the picture shows the side chain of a selected surface residue (Tyr204) that shows a high degree of sequence conservation and that may be involved in substrate binding, possibly without an effect of substrate binding on the compound change in chemical shift (¹⁵N-HSQC). In the shown complex, the scissile glycosidic bond is located at 5 Å from the copper atom (orange sphere). The L3 loop (containing His64) and the long LC loop (containing Tyr204) are displayed in blue and magenta, respectively. The sugar residues are numbered by subsite, where the sugar that is closest to His1 is sugar +1, in accordance with recent crystallographic data (28) (Discussion). The HADDOCK energies were (with SD) as follows: van der Waals energy = −39.6 ± 2.7 kcal⋅mol⁻¹, electrostatic energy = −27.8 ± 7.7 kcal⋅mol⁻¹, and desolvation energy = −8.5 ± 3.2 kcal⋅mol⁻¹.

Fig. 3.

Interaction of apo-NcLPMO9C with CDH and CYT. (A) Compound change in chemical shifts (¹⁵N-HSQC) for each amino acid in NcLPMO9C upon addition of CYT (rhombi) or CDH (crosses). (B) Compound change in chemical shifts larger than 12 Hz mapped on the NcLPMO9C structure. The backbone of NcLPMO9C (shown in cartoon and surface representation) is colored using the indicated coloring scheme. The side chains of residues His1, Ala80, His83, and His155 are shown in stick representation. The ¹³C-aromatic HSQC spectra showed clear changes in chemical shift for His1, and this residue was treated as described in the legend of Fig. 1. (C) Overlay of an area of interest from the ¹⁵N-HSQC spectrum for 0.10 mM apo-NcLPMO9C in the absence of an interaction partner (black) and in the presence of 0.12 mM CDH (red), 2.6 mM Glc₆ (cyan), or both (blue).