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.