Structural mechanisms underlying sequence-dependent variations in GAG affinities of decorin binding protein A, a Borrelia burgdorferi adhesin.

Decorin-binding protein A (DBPA) is an important surface adhesin of the bacterium Borrelia burgdorferi, the causative agent of Lyme disease. DBPA facilitates the bacteria's colonization of human tissue by adhering to glycosaminoglycan (GAG), a sulfated polysaccharide. Interestingly, DBPA sequence variation among different strains of Borrelia spirochetes is high, resulting in significant differences in their GAG affinities. However, the structural mechanisms contributing to these differences are unknown. We determined the solution structures of DBPAs from strain N40 of B. burgdorferi and strain PBr of Borrelia garinii, two DBPA variants whose GAG affinities deviate significantly from strain B31, the best characterized version of DBPA. Our structures revealed that significant differences exist between PBr DBPA and B31/N40 DBPAs. In particular, the C-terminus of PBr DBPA, unlike C-termini from B31 and N40 DBPAs, is positioned away from the GAG-binding pocket and the linker between helices one and two of PBr DBPA is highly structured and retracted from the GAG-binding pocket. The repositioning of the C-terminus allowed the formation of an extra GAG-binding epitope in PBr DBPA and the retracted linker gave GAG ligands more access to the GAG-binding epitopes than other DBPAs. Characterization of GAG ligands' interactions with wild-type (WT) PBr and mutants confirmed the importance of the second major GAG-binding epitope and established the fact that the two epitopes are independent of one another and the new epitope is as important to GAG binding as the traditional epitope.

Flexible Linker Modulates Glycosaminoglycan Affinity of Decorin Binding Protein A.

Decorin binding protein A (DBPA) is a glycosaminoglycan (GAG)-binding adhesin found on the surface of the bacterium Borrelia burgdorferi (B. burgdorferi), the causative agent of Lyme disease. DBPA facilitates bacterial adherence to extracellular matrices of human tissues and is crucial during the early stage of the infection process. Interestingly, DBPA from different strains (B31, N40, and PBr) show significant differences in GAG affinities, but the structural basis for the differences is not clear. In this study, we show that GAG affinity of N40 DBPA is modulated in part by flexible segments that control access to the GAG binding site, such that shortening of the linker leads to higher GAG affinity when analyzed using ELISA, gel mobility shift assay, solution NMR, and isothermal titration calorimetry. Our observation that GAG affinity differences among different B. burgdorferi strains can be attributed to a flexible linker domain regulating access to the GAG-binding domain is novel. It also provides a rare example of how neutral amino acids and dynamic segments in GAG binding proteins can have a large influence on GAG affinity and provides insights into why the number of basic amino acids in the GAG-binding site may not be the only factor determining GAG affinity of proteins.

The novel heparin-binding motif in decorin-binding protein A from strain B31 of Borrelia burgdorferi explains the higher binding affinity.

Decorin-binding protein A (DBPA), a glycosaminoglycan (GAG)-binding lipoprotein found in Borrelia burgdorferi, is crucial to the transmission of Lyme disease in its earliest stages. Because of its role in the initial transmission of the disease, DBPA is an ideal target for vaccine development. DBPA sequences from different strains also contain considerable heterogeneity, leading to differing affinities for GAGs and proteoglycans among different DBPA sequences. Through biophysical and structural analysis of DBPA from strain B31, we have discovered a novel and important GAG-binding epitope in B31 DBPA. Removal of the epitope greatly attenuated its affinity for DBPA and may explain the differential GAG affinities seen in DBPAs from other strains of B. burgdorferi. Paramagnetic perturbation of the protein with TEMPO-labeled heparin fragments showed bound GAGs are located close to the linker region containing the BXBB motif that plays a significant role in determining the specific affinity and orientation of binding of GAG to DBPA. Thermodynamic contributions of the new motif to GAG binding were also characterized by both nuclear magnetic resonance and isothermal titration calorimetry and compared with those of other DBPA residues previously known to be involved in GAG interactions. These analyses showed the motif is as important as other known binding epitopes. The discovery of the motif offers a possible structural explanation for the previously observed differences in GAG affinities of DBPA variants from different Borrelia strains and improves our understanding of DBPA-GAG interactions.

Interactions of the Chemokine CCL5/RANTES with Medium-Sized Chondroitin Sulfate Ligands.

Interactions of the chemokine CCL5 (RANTES) with glycosaminoglycans (GAGs) are crucial to the CCL5-mediated inflammation process. However, structural information on interactions between CCL5 and longer GAG fragments is lacking. In this study, the interactions between oligosaccharides derived from chondroitin sulfate and a dimeric variant of CCL5 were investigated using solution nuclear magnetic resonance. The data indicate that, in addition to the BBXB motif in the 40s loop, GAGs also contact residues in the N loop in a manner similar to interactions between chemokine and the receptor N terminus, leading to possible stabilization of the dimer. Using 2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl-tagged hexasaccharides, the binding orientation of the hexasaccharides was shown to be highly dependent on the sulfation pattern of the N-acetyl galactosamine groups. Finally, a model of the CCL5 dimer complexed to chondroitin sulfate hexasaccharides was constructed using paramagnetic relaxation enhancement and intra- and intermolecular nuclear Overhauser effect constraints.