Formation of the single-layer β-sheet of Borrelia burgdorferi OspA in the absence of the C-terminal capping globular domain

Borrelia outer surface protein A (OspA) contains a unique single-layer β-sheet that connects N and C-terminal globular domains. This single-layer β-sheet segment (β-strands 8–10) is highly stable in solution, although it is exposed to the solvent on both faces of the sheet and thus it does not contain a hydrophobic core. Here, we tested whether interactions with the C-terminal domain are essential for the formation of the single-layer β-sheet. We characterized the solution structure, dynamics and stability of an OspA fragment corresponding to β-strands 1–12 (termed OspA[27–163]), which lacks a majority of the C-terminal globular domain. Analyses of NMR chemical shifts and backbone nuclear Overhauser effect (NOE) connectivities showed that OspA[27–163] is folded except the 12th and final β-strand. 1H-15N heteronuclear NOE measurements and amide H-2H exchange revealed that the single-layer β-sheet in this fragment is more flexible than the corresponding region in full-length OspA. Thermal-denaturation experiments using differential scanning calorimetry and NMR spectroscopy revealed that the N-terminal globular domain in the fragment has a conformational stability similar to that of the same region in the full-length protein, and that the single-layer β-sheet region also has a modest thermal stability. These results demonstrate that the unique single-layer β-sheet retains its conformation in the absence of its interactions with the C-terminal domain. This fragment is significantly smaller than the full-length OspA, and thus it is expected to facilitate studies of the folding mechanism of this unusual β-sheet structure.