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Supplemental Information.
Structure of Decorin Binding Protein B from Borrelia burgdorferi and Its Interactions with Glycosaminoglycans
Wei Feng and Xu Wang
Figures S1. 1H-15N HSQC overlays (upper panel) of WT DBPB titrated with DS dp10 and fitting curve (lower panel) of residue K184. Contours are color-coded with increasing concentrations of GAG fragments (0, 0.2, 0.6, 1.0, 1.4, 1.8 and 2.2 mM). The sample contains 400 uL of 100 uM protein in 50 mM NaH2PO4 and 150 mM NaCl buffer (pH 6.5). K184 is displayed with an arrow indicating migration direction.
Figures S2. DS-induced DBPB conformational exchange characterized by CPMG-based relaxation dispersion experiments. (A) Residue specific Rex values derived from the experiment. Rex is taken as the difference between R2,eff values at field strengths of 10 and 210. (B) Changes in R2,eff of residues G55 and T66 as field strength increases.
Figure S3. DBPB titrations with heparin dp6. (A) 1H-15N HSQC overlays of WT B31 DBPB with increasing concentrations of heparin dp6. Signals with large migrations are labeled with their residue numbers and arrows to indicate migration directions. (B) Fitting curves of DBPB residues S72 and K185 when titrated with heparin dp6.
Figure S4. Backbone dynamics of DBPB in the presence and absence of heparin dp10. Order parameters of backbone amide nitrogen atoms for WT B31 DBPB with (red) or without (black) 10 molar equivalents of heparin dp10.
Figures S5. 1H-15N HSQC overlays (A) K65S/K69S DBPB. (B) R78S/K81S DBPB. (C) K81S/K169S DBPB. (D) 184SSS187 DBPB. (E) DBPB21-183. Contours are color-coded with increasing concentrations of GAG fragments (0, 0.2, 0.6, 1.0, 1.4, 1.8 and 2.2 mM). The sample contains 400 uL of 100 uM protein in 50 mM NaH2PO4 and 150 mM NaCl buffer (pH 6.5). Residues S72 and K185 were used in the Kd calculations.
Figures S5. 1H-15N HSQC overlays (A) K65S/K69S DBPB. (B) R78S/K81S DBPB. (C) K81S/K169S DBPB. (D) 184SSS187 DBPB. (E) DBPB21-183. Contours are color-coded with increasing concentrations of GAG fragments (0, 0.2, 0.6, 1.0, 1.4, 1.8 and 2.2 mM). The sample contains 400 uL of 100 uM protein in 50 mM NaH2PO4 and 150 mM NaCl buffer (pH 6.5). Residues S72 and K185 were used in the Kd calculations.
Figures S5. 1H-15N HSQC overlays (A) K65S/K69S DBPB. (B) R78S/K81S DBPB. (C) K81S/K169S DBPB. (D) 184SSS187 DBPB. (E) DBPB21-183. Contours are color-coded with increasing concentrations of GAG fragments (0, 0.2, 0.6, 1.0, 1.4, 1.8 and 2.2 mM). The sample contains 400 uL of 100 uM protein in 50 mM NaH2PO4 and 150 mM NaCl buffer (pH 6.5). Residues S72 and K185 were used in the Kd calculations.
Figure S6. Kd binding curves of DBPBs. Kd fittings were obtained for NMR-monitored titrations using xcrvfit (http://www.bionmr.ualberta.ca/bds/software/xcrvfit/).
Figure S7. (A) Results of heparin and DS ELISA comparing the GAG affinities of B31 DBPA and DBPB. The two proteins have equal affinities for native heparin and DS. (B) Heparin dp6 GMSA of B31 DBPA and DBPB. Both proteins’ affinities for size defined heparin are also similar.
