TN16 is one of the most promising inhibitors of α, β dimer of tubulin that occupies the cavity in the β-subunit located at the dimeric interface, known as the colchicine binding site. The experimentally determined structure of the complex (Protein Data Bank entry 3HKD) presents the conformation and position of the ligand based on the "best fit", keeping the controversy of other significant binding modes open for further investigation. Computation has already revealed that TN16 experiences fluctuations within the binding pocket, but the insight from that previous report was limited by the shorter windows of sampling and by the approximations on the surrounding environment by implicit solvation. This article reports that in most of the cases straightforward MMGBSA calculations of binding energy revealed a gradual loss of stabilization that was inconsistent with the structural observations, and thus, it indicated the lack of consideration of stabilizing factors with appropriate weightage. Consideration of the structurally packed water molecules in the space between the ligand and receptor successfully eliminated such discrepancies between the structure and stability, serving as the "litmus test" of the importance of explicit consideration of such structurally packed water in the calculations. Such consideration has further evidenced a quasi-degenerate character of the different binding modes of TN16 that has rationalized the observed intrinsic fluctuations of TN16 within the pocket, which is likely to be the most critical insight into its entropy-dominated binding. Quantum mechanical calculations have revealed a relay of electron density from TN16 to the protein via a water molecule in a concerted manner.

The dynamic instability of the microtubule originates from the conformational switching of its building block, that is, the α, β-tubulin dimer. Ligands occupying the interface of the α-β dimer bias the switch toward the disintegration of the microtubule, which in turn controls the cell division. A little loop of tubulin is structurally encoded as a biophysical "gear" that works by changing its structural packing. The consequence of such change propagates to the quaternary level to alter the global dynamics and is reflected as a swapping between the relative contributions of dominating internal modes. Simulation shows that there is an appreciable separation between the conformational space accessed by the liganded and unliganded systems; the clusters of conformations differ in their intrinsic tendencies to "bend" and "twist". The correlation between the altered breathing modes and conformational space rationally hypothesizes a mechanism of straight-bent interconversion of the system. In this mechanism, a ligand is understood to bias the state of the "gear" that detours the conformational equilibrium away from its native preference. Thus, a fundamental biophysical insight into the mechanism of the conformational switching of tubulin is presented as a multiscale process that also shows promise to yield newer concept of ligand design.

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The basic framework of understanding the mechanisms of protein functions is achieved from the knowledge of their structures which can model the molecular recognition. Recent advancement in the structural biology has revealed that in spite of the availability of the structural data, it is nontrivial to predict the mechanism of the molecular recognition which progresses via situation-dependent structural adaptation. The mutual selectivity of protein-protein and protein-ligand interactions often depends on the modulations of conformations empowered by their inherent flexibility, which in turn regulates the function. The mechanism of a protein's function, which used to be explained by the ideas of 'lock and key' has evolved today as the concept of 'induced fit' as well as the 'population shift' models. It is felt that the 'dynamics' is an essential feature to take into account for understanding the mechanism of protein's function. The design principles of therapeutic molecules suffer from the problems of plasticity of the receptors whose binding conformations are accurately not predictable from the prior knowledge of a template structure. On the other hand, flexibility of the receptors provides the opportunity to improve the binding affinity of a ligand by suitable substitution that will maximize the binding by modulating the receptors surface. In this paper, we discuss with example how the protein's flexibility is correlated with its functions in various systems, revealing the importance of its understanding and for making applications. We also highlight the methodological challenges to investigate it computationally and to account for the flexible nature of the molecules in drug design.

The α,β-tubulin is the building block of microtubules, which is associated with and dissociated from the microtubular architecture complying with the dynamic instability of the microtubules. This dynamic instability has a direct relation with the spindle formation by the microtubules and cell division kinetics. E7010 is one of the promising ligands of an α,β-tubulin protein that binds at the core of this protein and can diminish the protein’s ability to fit to a growing microtubule, thus frustrating cell division. Although X-ray crystallography has reported a specific binding conformation of E7010 in PDB, molecular dynamics (MD) simulations have revealed two other conformational states of the ligand capable of binding to tubulin with stabilities close to that state reported in PDB. To rationalize this quasidegeneracy of ligand binding modes, MD simulations have further revealed that the understanding of the mechanism of E7010-tubulin binding remains incomplete unless the role of water molecules to bridge this interaction is taken into consideration, a very critical insight that was not visible from the PDB structure. Further, these water molecules differ from the standard examples of “bridging” waters which generally exist as isolated water molecules between the receptor and the ligand. In the present case, the water molecules sandwiched between ligand and protein, sequestered from the bulk solvent, integrate with each other by an H-bonds network forming a group, which appear as microclusters of water. The structural packing with the ligand binding pocket and the bridging interactions between protein and ligand take place through such clusters. The presence of this microcluster of water is not just cosmetic, instead they have a crucial impact on the ligand binding thermodynamics. Only with the explicit consideration of these water clusters in the binding energy calculations (MMGBSA) is the stability of the native mode of ligand binding reported in PDB rationalized. At the same time, two other binding modes are elucidated to be quasi-degenerate with the native state and that indicates the further possibility in gaining more entropic stabilization of the complex. The role of such “bridging” water clusters to enhance the protein–ligand interaction will be insightful for designing the next generation prospective compounds in the field of cancer therapeutics.