Proteins and their binding partners with complementary shapes form complexes. Fisher was onto something when he introduced the “key and lock mechanism” in 1896. For the first time the shape of molecules was considered crucial for molecular recognition. Since then there have been various attempts to improve upon this theory in order incorporate the structural diversity of proteins and their binding partners.
Koshland proposed the “induced fit” mechanism in 1956, which states that interacting partners undergo local conformational changes upon complex formation to strengthen binding. An additional mechanism “conformational selection” was introduced by Monod, Wyman and Changeux who argued that the conformational change occurred before binding driven by the inherent conformational heterogeneity of proteins. Given a protein that fluctuates between two states A and B and a substrate C that only interacts with one of these states, the chance of complex formation depends on the probability of our protein being in state A or B. Furthermore, one could imagine a scenario where a protein has multiple binding partners, each binding to a different conformational state. This means that some proteins exists in an equilibrium of different structural states, which determines the prevalence of interactions with different binding partners.
Based on this observation Michielssens et al. used various in-silico methods to manipulate the populations of binding-competent states of ubiquitin in order to change its protein binding behaviour. Ubiquitin is known to take on two equally visited states along the “pincer mode” (the collective motion describing the first PCA-eigenvector); closed and open.
Different binding partners prefer either the closed, open or both states. By introducing point mutations in the core of ubiquitin, away from the binding interface, Michielssens et al. managed to shift the conformational equilibrium between open and closed states, thereby changing binding specificity.
Point mutations were selected according to the following criteria:
⁃ residues must be located in the hydrophobic core
⁃ binding interface must be unchanged by the mutation
⁃ only hydrophobic residues may be introduced (as well as serine/threonine)
⁃ glycine and tryptophan were excluded because of their size
Fast growth thermal integration (FGTI), a method based on molecular dynamics, was used to calculate the relative de-/stabilisation of the open/closed state caused by each mutation. Interestingly, most mutations that caused stabilisation of the open states were concentrated on one residues, Isoleucine 36 (Slide 7).
For the 15 most significant mutations a complete free energy profile was calculated using Umbrella sampling.
To further validate that they correctly categorised their mutants into stabilising the open or closed state, six X-ray structure of ubiquitin in complex with a binding partner that prefers either the open or closed state were simulated with each of their mutations. Figure 5 shows the change in binding free energy that is caused by the mutation in compatible, neutral and incompatible complexes (compatible may refer to an “open favouring mutation” (blue) in an open complex (blue) and vice versa).
In their last step a selection of open and closed mutations was introduced into an open complex and the change in binding free energy was compared between experiment (NMR) and their simulations. For this example, their mutants behaved as expected and an increase in binding free energy was observed when the closed mutations were introduced into the open complex while only subtle changes were seen when the “compatible” closed mutations were introduced.
The authors suggest that in the future this computational protocol may be a corner stone to designing allosteric switches. However, given that this approach requires pre-existing knowledge and is tailored to proteins with well defined conformational states it may take some time until we discover its full potential.