If you are familiar with the reader, writer & eraser concepts or you are passionate about epigenetics and arginines, this recent publication might be of interest to you. The study addresses the transcription factor E2F-1, which plays a crucial role in the control of cell cycle and is linked with cancer. Like Yin-yang, it has opposing functional roles: to promote cell-cycle progression and to induce apoptosis. The results demonstrate that the biological outcome of E2F-1 activity is affected by arginine methylation marks. While asymmetric arginine methylation causes apoptosis, the symmetrical methylation results in proliferation. This reader-writer interplay determined by the two types of marks governs the function of E2F-1 and potentially the fate of the cell.
In this study we selected point mutations resulting in Pim-1 variants that are expressed in cancer tissues and reported in SNP databases, such as FastSNP and COSMIC. These Pim-1 variants have been comprehensively characterized to investigate the effect of single amino acid substitution on Pim-1 thermal and thermodynamic stability and structure in solution. Our results indicate that the effects of the mutation observed in cancer tissues cause local changes of tertiary structure, but do not affect binding to type I kinase inhibitors.
This work has been pioneered by researches at the Department of Biochemical Sciences “A. Rossi Fanelli”, Sapienza University of Rome and served as an inspiration for one of my thesis chapters.
Last week I was presenting my DPhil work. In one of my projects I address the reasons for inhibitor selectivity in PIM protein kinase family. PIM kinases play key roles in signalling pathways and have been identified as oncogenes long time ago. Slightly unusual for protein kinases ATP-binding sites and cancer roles have prompted the investigation of potential PIM-selective inhibitors for anticancer therapy. Due to overlapping functions of the three PIM isoforms, efficacious inhibitors should bind to all three isozymes. However, most reported inhibitors show considerable selectivity for PIM1 and PIM3 over PIM2 and the mechanisms leading to this selectivity remain unclear.
To establish the sequence determinants of inhibitor selectivity we investigated the phylogenetic relationships of PIM kinases and their structural conformations upon ligand binding (Figure 1). Together with my OPIG supervisor Charlotte Deane we predicted a set of candidates for site-directed mutagenesis as illustrated in Figure 2. The mutants were designed to convert PIM1 residues into analogous PIM2 residues at the same positions.
I then moved to the wetlab to test the hypotheses experimentally. Under guidance of Oleg Fedorov, I screened the SGC library of kinase inhibitors using differential scanning fluorimetry (DSF). After comparing melting temperature shift values across the PIM kinases and mutants, a set of potent inhibitors with different chemical scaffolds have been selected for quantitative binding analysis. I worked with Peter Drueker’s team at Novartis on PIMs enzymology, where I measured activities, Km values for ATP and IC50s using mobility shift assay. For my final set of measurements I performed isothermal titration calorimetry (ITC) experiments back at the SGC and determined binding constants and enthalpic/entropic contributions to the total free energy of ligand binding.
The data are yet to be published, I only briefly state the results here. The hinge mutant E124L demonstrated reduced thermal stability probably due to removal of E124-R122 salt bridge. The P-loop mutants had intermediate Km ATP values between PIM1 and PIM2, indicating that those residues could be responsible for stronger ATP binding in PIM2. As shown in Figure 2, the residues are located at the tip of the P-loop and might have involvement in the P-loop movement. Importantly, three mutants have shown reduced affinity to inhibitors validating my initial hypotheses.
Ideally having PIM1 and PIM2 co-crystal structures with the same inhibitors would allow direct comparison of the binding modes. So far I was able to solve apo-PIM2 structure in addition to the single PIM2 pdb, which will be deposited shortly.
I will update you soon about on my second project which involves more mutants, type II inhibitors, equilibrium shifts and speculations about conformational transitions. Keep visiting us!
Molecular recognition is the mechanism by which two or more molecules come together to form a specific complex. But how do molecules recognise and interact with each other?
In the TIBS Opinion article by Ruth Nussinov group, an extended conformational selection model is described. This model includes the classical lock-and-key, induced fit, conformational selection mechanisms and their combination.
The general concept of equilibrium shift of the ensemble was proposed nearly 15 years ago, or perharps earlier. The basic idea is that proteins in solution pre-exist in a number of conformational substates, including those with binding sites complementary to a ligand. The distribution of the substates can be visualised as free energy landscape (see figure above), which helps in understanding the dynamic nature of the conformational equilibrium.
This equilibrium is not static, it is sensitive to the environment and many other factors. An equilibrium shift can be achieved by (a) sequence modifications of special protein regions termed protein segments, (b) post-translational modifications of a protein, (c) ligand binding, etc.
So why are these concepts discussed and published again?
While the theory is straight-forward, proving conformational selection is hard and it is even harder to quantify it computationally. Experimental techniques such Nuclear Magnetic Resonance (NMR), single molecule studies (e.g. protein yoga), targeted mutagenesis and its effect on the energy landscape, plus molecular dynamics (MD) simulations have been helping to conceptualise conformational transitions. Meanwhile, there is still a long way to go before a full understanding of atomic scale pathways is achieved.