2.4 | Alternative conformations of three kinases,
T1195-T1197
An interesting set of ensemble targets was provided by Charalampos
Kalodimos. These are the principal and some low population alternative
conformations for three kinase domains (Src, BRAF, and P38a) under
particular conditions, obtained using similar methods to those described
in (21). The low population states may represent conformations that are
highly populated under other conditions and/or are important in the
functions of these kinases, or may be non-functional conformations that
nevertheless contain potential new sites for drug binding. Structures of
three newly determined conformations that were shared with CASP are
substantially different from the existing PDB entries with GDT_TS to
corresponding X-ray structures in the range of 64-76.
Participants were asked to include models for all ensemble members (two
or three per target) within the set of five submissions allowed for each
target. Comparison of submissions with the target structures shows that
all conformations represented by a structure in the PDB have highly
accurate submissions by at least some groups (highest GDT_TS typically
over 95, in some case 100). There is less agreement for the newly
derived NMR structures (GDT_TS in the 70s for T1195 and T1196, and
mid-80s for T1197). Note that some reference structures that were used
for assessment contain mutations with respect to the sequences released
to participants. These mutations were introduced to boost population
levels, but they did not substantially affect conformation.
Kinases have been extensively characterized in terms of local structural
and functional motifs (22), providing a useful basis for evaluation.
Figure 3 shows the motif regions for CASP targets. All ensembles have
differences among their members for three functional regions: the N-lobe
β-sheet, for some kinases involved in activity regulation through SH2
binding; the N-lobe αC helix, usually characterized as having an ‘in’ or
’out’ position, with the ‘in’ position allowing formation of a key salt
bridge; and the activation loop, often involved in regulating activity.
Each of the three targets has an additional region of conformational
difference included in the analysis: one of the two N to C lobe
connecting loop for T1195 and the second one for T1196; and a C-lobe
helix for T1197. Supplementary Table 1 lists the motif regions and
results for each kinase.
As noted above, for all three kinases, at least some CASP models are
very similar to the PDB-derived experimental structures, and
consequently for those structures the local motifs are all accurately
reproduced. For the new, NMR-derived structures, the results are more
variable, but for all motifs the closest predicted conformations have a
smaller deviation from the NMR structure than the corresponding X-ray
structures do. Also, at least one group exploring various MSAs with the
AlphaFold2 machine included all the ensemble members (two or three,
depending on the target). At the individual motif level, this is an
impressive result. However, if we examine the likely reason for this
success, perhaps performance is less impressive. The number of
successful groups for a particular NMR-determined motif is often low,
and no single group stands out as performing well against the complete
set of motifs across all targets. Nevertheless, these results again
demonstrate that current methods do produce relevant alternative
conformations, at least at the motif level.