3.4 Functional significance of the intermediate conformations
The current PRE results provide further evidence of disorder in the central and C-terminal regions of the OspA intermediates and similarities between the effects of pressure and temperature on the protein. In addition, a possible ensemble of partially disordered conformations was suggested from the MD simulation. These data indicate that the intermediates more readily expose the tick receptor-binding sites (i.e., residues 236–237 and 242–244)8 than does the basic folded conformation. Hence, the transition into the intermediatesin vivo may accelerate the binding of OspA to the tick receptor. Although the folded OspA protein and its C-terminal fragment have been included as vaccine candidates to prevent Borreliatransmission,17 the intermediates, in which the central and C-terminal domain are disordered, are unlikely to be recognized by antibodies targeted at the folded C-terminal portion, thereby reducing the effectiveness of the vaccine. The disordering of the C-terminal domain is also considered to be important when the protein is translocated through the outer membrane in the C-terminal-to-N-terminal direction. However, destabilization of the C-terminal domain by site-specific mutation induced protein degradation.16 Results from the present and previous studies indicate that a delicate interplay between localized conformational dynamics and thermodynamic stability may be required for OspA function.
Conclusion
Although structural characterization of protein intermediates remains challenging, the partial knowledge that we gathered here not only expands the current understanding on the molecular mechanism of protein function, but also facilitates novel drug design. The intermediate state of OspA consists of a mixture of conformations with different levels of partial disorder in the C-terminal region. The C-terminal region of the intermediate is partially disordered; it however retains weak specific contact with the N-terminal region, owing to a twist of the central β-sheet and increased flexibility of the polypeptide chain. These intermediates may escape antibody recognition. Information regarding localized conformational dynamics and thermodynamic stability might be useful for the rational design of effective OspA vaccines. Moreover, since PRE observation under high pressure is only limited in ubiquitin, the present is a confirmation of PRE under high pressure.24 A combination of PRE-assisted high-pressure NMR and MD simulation is, in general, useful for characterizing the dynamic structures of functionally essential protein intermediates.