All NMR experiments were acquired at 298 K on Bruker Avance 500 or 800 MHz spectrometers, both equipped with a TCI HCN-z cryoprobe. The following spectra were used to complete the sequence-specific backbone assignment (recorded at 500-MHz 1H Larmor frequency): 2D [1H,15N] HSQC, 3D HNCACB, 3D CBCA(CO)NH, 3D HNCA, 3D (H)CC(CO)NH, and 3D HBHA(CO)NH. Together with these spectra, 3D HC(C)H–total correlation spectroscopy (TOCSY) (Tm = 11.3 ms) was used for the assignment of aliphatic side-chain 1H and 13C resonances. Aromatic side chains were assigned using 2D [1H,1H] NOE spectroscopy (NOESY) (Tm = 70 ms), 2D [1H,1H] TOCSY (Tm = 60 ms), and 2D [1H,1H] correlation spectroscopy (COSY) spectra of GMCBM21 in 20 mM sodium phosphate (pH 6.5), 50 mM NaCl, 10 mM DTT, and 100% D2O. A 2D 15N[1H]-NOE (heteronuclear NOE) experiment was recorded at 500 MHz 1H Larmor frequency with a saturation delay of 5 s and evaluated using the Dynamics Center 2.0 software (Bruker).

All spectra were processed using Topspin 2.1/3.0/3.1 (Bruker, Billerica, MA), and chemical shift assignments were achieved using Cara ( NMR spectra of GMCBM21 were acquired using either 15N- or 15N,13C-labeled protein at a final concentration of 0.8 mM in 20 mM sodium phosphate (pH 6.5), 50 mM NaCl, 10 mM DTT, and 90% H2O/10% D2O. The interaction of GMCBM21 with carbohydrates was tested by NMR titration experiments using α-CD (Thermo Fisher Scientific) and β-CD (Acros Organics).

For GMCBM21, only Asn224, Asn228, and two cloning artifacts (His2 and Met3) have no sequence-specific backbone assignment. The high-quality spectral data also enabled a 98% completeness of the side-chain assignment, except Pro110 Cδ/Qδ resonances and all of the side-chain resonances for Gly1, His2, Phe192, Phe220, and Tyr106. Aromatic side-chain assignment was more challenging owing to the large number of aromatic residues in GMCBM21—a total of 16—a characteristic feature of SBDs.

The interaction between GM64–237 and PP1α7–330 was studied by direct comparison of 2D [1H,15N] TROSY spectra of free and PP1α7–330-bound (2H,15N)-labeled GM64–237. The final concentration used was 0.1 mM GM64–237:PP1α7–330 complex in 20 mM bis-tris (pH 6.8), 150 mM NaCl, 0.5 mM TCEP, and 90% H2O/10% D2O. The spectra were processed using Topspin 4.0.3 (Bruker, Billerica, MA) and analyzed using Sparky. The NMR spectra were acquired on a Bruker Avance NEO 800 MHz 1H Larmor frequency NMR spectrometer equipped with a TCI-active HCN-cooled z-gradient cryoprobe at 298 K.

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