Revealing Mechanisms of Pinholin Activation through Thermodynamic Integration and Mutational Analysis

Pinholin is a protein employed by bacteriophages, consisting of two transmembrane domains (TMDs) that drive cell membrane rupture. This is achieved by inducing the formation of small membrane pores to disrupt proton gradients, thus activating larger holin proteins. The mechanism of pinholin activation is well characterized and involves a conformational rearrangement in which TMD1 leaves the membrane to become exposed to the solvent. Then, TMD2 can oligomerize with neighboring pinholin proteins and form the membrane pore.

While the mechanism has been explored thoroughly, the thermodynamic contributions of each amino acid to the activation process are however unknown. Experimentally, many mutations have been characterized in terms of conformational shifts and the tendency of TMD1 to translocate between the membrane and the solvent. Spin label distance measurements from electron paramagnetic resonance have shown that mutations in A17 and S16 amino acids can cause early activation. From these experiments, a general structure emerges, but the exact molecular-level coordinates remain unknown. To generate a more detailed atomistic structure, molecular dynamics (MD) simulations have been conducted to explore conformational ensembles over timescales up to hundreds of nanoseconds. Molecular models were validated by comparing structure and dynamics to experimental data, showing close agreement between the two. However, the timescales of conformational rearrangement involving the translocation of TMD1 are too long for standard MD simulations to probe. To compensate for slow kinetics, an alternative thermodynamic model has been employed to compare the relative free energies of translocation for various experimentally characterized mutants. Thermodynamic integration in the form of alchemical mutations provides an alternate route to characterize the thermodynamic cycle of pinholin activation. Thus, through complementary experimental and computational mutational analysis, the thermodynamic contributions of specific amino acids to pinholin rearrangement can be probed relatively quickly. Within this thermodynamic integration framework, we have tested the extent to which (a) hydrophobic interactions between TMD1, TMD2, and the membrane and (b) hydrogen bond interactions between TMD1 and TMD2 affect the conformational stability of pinholin’s active and inactive states. We foresee future applications of this methodology to other systems with inconveniently long time scales.