3.1 Discovery of novel mutation in exon 9 of PIK3CA
Initial screening via PCR/HRM was performed on DNA extracted from 40 tumor samples and the HRM positive samples were subsequently investigated by Sanger sequencing for mutations on exon 9. Four tumors displayed an altered melting pattern in PCR/HRM of which three were confirmed to harbor at least one of the E542K, E545K or L551Q mutations. E542K and E545K variants were previously reported as pathogenic mutations in My Cancer Genome (21), cosmic (20) and TCGA (22), however, L551Q mutation did not exist in any of the cancer databases. Interestingly, the sample with L551Q variant had a pathogenic mutation, E542K, in hotspot region of exon 9 (Table 1). This prompted us to investigate the pathological effect of E542K, E545K, and the newly detected L551Q by further in silico studies.
3.2 p110α L551Q dynamics displays a carcinogenic activity
The impact of L551Q was first predicted to be disease causing using various variant annotation tools that is described in Table 2 in details. We then used molecular dynamics simulation to study the structural effects of all mutations we found in p110α helical domain. Although the p110α-p85α is a large complex, the p110α kinase and p85α nSH2 domains are the main functional domains of the complex, with the p110α helical domain mediating their interactions (Figure 1). We restricted our simulation systems to these three domains that have the central role in PI3Kα activity to investigate how the kinase activity of p110α is regulated by p85α.
p110α helical domain directly interacts with the nSH2 domain of p85α but the function of this interaction has not been fully uncovered. Thus, first we compared the dynamics cross-correlation map (DCCM) of simulated systems to find out to what extent the motions of helical domain are correlated with nSH2 and kinase domains, and if helical domain mutations could affect this correlation (Figure 2). In WT, all of helical, kinase and nSH2 domains have a high intradomain correlation score. In addition, helical domain motions are highly correlated with nSH2 motions. N-terminal region of kinase domain is highly anti-correlated with helical and nSH2 domains whereas C-terminal region (encompassing the enzyme catalytic site) is highly correlated with helical and nSH2.
All of the mutant systems show a huge reduction in both intra and interdomain correlations (Figure 2). Surprisingly, the helical domain mutations have a greater impact on the kinase domain correlations. The pattern of DCCM from all mutants, including L551Q, are similar, in which helical domain and nSH2 show intradomain correlations, but their motions are no longer correlated with those of kinase domain, specifically in E545K. Additionally, the intradomain motions of kinase domain are disturbed, in particular in the C-terminal region.
To better visualize how helical domain mutations affect the overall motions of the p110α-p85α complex, we rendered dynamics of all systems along their first principal mode of motion (PC1) (Figure 2, tube structures). In WT, all of helical domain, kinase domain C-terminal region, and nSH2 domain residues move as an integrated unit. However, the N-terminal region of kinase domain is less dynamic and moves to an opposite direction, as shown as an anti-correlated moiety in the WT DCCM.
PC1 shows that helical domain mutations, including L551Q, have slowed down the dynamics of p110α-p85α complex, to the extent that kinase domain is almost static and does not respond to helical and nSH2 domains dynamics. In other words, there is no integrated motions of domains in the mutant complexes unlike what is seen in the WT. The helical and nSH2 domains move slowly towards the C-terminal region of kinase domain in E542K and E545K. Inversely, the helical domain slowly moves towards the N-terminal region of kinase domain in L551Q. The nSH2 domain residues in L551Q show erratic motions along their PC1.
Taken together, DCCM and PC analyses show that helical domain mutations extremely affect dynamics of p110α-p85α complex. In WT, motions of kinase domain are completely regulated by coordinated motions of helical and nSH2 domains. However, mutants have disrupted this dynamic regulation, turning kinase domain into a detached part which no longer moves with helical and nSH2 domains. Indeed, dynamic behavior of L551Q is compatible with that of E542K and E545K mutants. Thus, these results strongly suggest that the novel p110α L551Q mutation could harbor carcinogenic features similar to previously known mutations.
3.3 nSH2 allosterically regulates p110α catalytic activity through helical domain
We then asked how a small change in helical domain can affect dynamics of kinase domain. To answer that, we utilized community network analysis to investigate the pathways that connect motions of kinase to helical and nSH2 domains. Edge betweenness measurements revealed that E542 mediates the communication between nSH2 and helical domains in WT. Then D538 in the helical domain, in vicinity of E542, interacts with N996 in the kinase domain, and directly spreads the nSH2 regulatory signal throughout the kinase domain (Figure 3A). So, these three residues (E542, D538, and N996) have a central role in conducting the regulatory signal from nSH2 domain to kinase domain.
As D538 is spatially oriented in the middle of the E542 and N996, changing the repulsive force of E542-D538 to an attractive one by E542K mutation influences the orientation of D538 relative to N996, and consequently disrupts the interaction between helical and kinase domains. Therefore, E542K mutant cannot conduct the nSH2 regulatory signal to kinase domain, but instead, diverts the signal to the helical domain, leading to the detachment of kinase domain from helical and nSH2 domains. On the other hand, E545K and L551Q mutations change helical-nSH2 and helical domain internal interaction respectively, both resulting in an impaired interaction of D538-N996.
For further confirmation of these results, we tried to find suboptimal pathways from nSH2 domain to three kinase domain residues including: K776 in the p-loop which identifies p110α substrate specificity, H917 in the catalytic loop which accommodate ATP and catalysis the enzymatic reaction, and K941 in the activation loop, which makes the initial interactions with substrates. In WT, signals from nSH2 domain allosterically regulate catalytic activity by two main pathways: one passes through kinase domain H12, and the other through helical domain (data not shown). In all mutants, these allosteric pathways become significantly longer (Figure 3B), because the nSH2 regulatory signal has been trapped in the helical domain.
Collectively, communication network analysis shows that the helical domain mutations disrupt the connection between helical and kinase domains, resulting in detachment of kinase domain from rest of the complex. Consequently, nSH2 signal is trapped in the helical domain since it cannot find its way towards kinase domain which makes it inaccessible to nSH2 domain in all three mutants.
3.4 P110α substrate affinity is different in each mutant
Some of the known mutants of p110α has been proved to result in resistance to anti-p110α drugs (26, 27). Thus, it is clinically important to find out whether breast cancer patients with the novel carcinogenic p110α L551Q mutation may be drug resistance. To this end, we investigated the flexibility and shape of the p110α substrate binding pocket (SBP) using pairwise root-mean-square deviation (RMSD) and radius of gyration (Rg) calculations, respectively. RMSD distribution plot (Figure 4) shows that the flexibility of the SBP has been slightly reduced in all mutants, specifically in E545K compared to WT. For an enzyme with various substrates, a more rigid SBP could result in higher affinity for a specific substrate. To further inspect this hypothesis about p110α catalytic site, we compared distributions of Rg of p110α SBP in all simulated systems (Figure 4B). Rg variation shows the major difference between E542K and E545K mutants, suggesting that the shape of apo-enzyme SBP differs among mutants.
Accordingly, geometric analysis suggests that all helical domain mutations result in a slightly rigid p110α SBP with different shapes. This proposes that a specific therapeutic compound may not have the same affinity for all p110α mutants. Further biophysical experiments are warranted to test this hypothesis.