Novel US-CpHMD Protocol to Study the Protonation-Dependent Mechanism of the ATP/ADP Carrier

We have designed a protocol combining
constant-pH molecular dynamics
(CpHMD) simulations with an umbrella sampling (US) scheme (US-CpHMD)
to study the mechanism of ADP/ATP transport (import and export) by
their inner mitochondrial membrane carrier protein [ADP/ATP carrier
(AAC)]. The US scheme helped overcome the limitations of sampling
the slow kinetics involved in these substrates’ transport,
while CpHMD simulations provided an unprecedented realism by correctly
capturing the associated protonation changes. The import of anionic
substrates along the mitochondrial membrane has a strong energetic
disadvantage due to a smaller substrate concentration and an unfavorable
membrane potential. These limitations may have created an evolutionary
pressure on AAC to develop specific features benefiting the import
of ADP. In our work, the potential of mean force profiles showed a
clear selectivity in the import of ADP compared to ATP, while in the
export, no selectivity was observed. We also observed that AAC sequestered
both substrates at longer distances in the import compared to the
export process. Furthermore, only in the import process do we observe
transient protonation of both substrates when going through the AAC
cavity, which is an important advantage to counteract the unfavorable
mitochondrial membrane potential. Finally, we observed a substrate-induced
disruption of the matrix salt-bridge network, which can promote the
conformational transition (from the C- to M-state) required to complete
the import process. This work unraveled several important structural
features where the complex electrostatic interactions were pivotal
to interpreting the protein function and illustrated the potential
of applying the US-CpHMD protocol to other transport processes involving
membrane proteins.