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.