Commentary and Perspective
Updating view of membrane transport proteins by simulation studies
2023 Volume 20 Issue 4 Article ID: e200041
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2023 Volume 20 Issue 4 Article ID: e200041
Membrane transport proteins play essential roles in many physiological functions, such as maintenance of concentration gradients, nerve conductions, and synthesis of ATP. In principle, conformational changes at the molecular level should be involved in these functions, and observation of such molecular motions at the molecular level is necessary to fully understand their mechanisms. Although such observation by experiments is still difficult, it is possible with the use of computer simulations. Especially, recent advances in high-performance computers have enabled us to perform long time simulations, which paved a way to relate molecular motions to biological functions of membrane transport proteins.
At the 61st Annual Meeting of the Biophysical Society of Japan in Nagoya, a symposium organized by Takashi Sumikama and Kei-ichi Okazaki is held to present recent advances in this field. The invited presentations are given by 4 speakers from Japan and 1 speaker from Australia. This symposium covers (1) ion conduction and selectivity mechanism through the ion channels (Takashi Sumikama, Ben Corry), (2) those through the ion pumps (Junichi Ono, Chigusa Kobayashi), (3) alternating-access conformational dynamics of transporters (Kei-ichi Okazaki). Summaries of invited presentations are described below.
K+ and Na+ channels are the central molecules that generate action potentials or nerve impulses in neurons. Most textbooks describe rapid and selective ion permeation through the K+ channels at the molecular level as follows: K+ ions snugly fit into the K+ channels. It results in a high affinity, leading to high selectivity over Na+ ions. For rapid permeation, this high affinity must be cut by the ion-ion repulsion between the ions in the channel and the next ion entering the channel (the knock-on mechanism). However, recent results by Sumikama et al. obtained by molecular dynamics (MD) simulations were opposite to these explanations [1–3]. It was found that the knock-on mechanism is not always necessary for ion permeation [2]. This implies that the affinity and thus the selectivity is not high. In fact, selectivity calculated by MD simulation was approximately 40 [3]. Experimental evidence to support this scenario is also shown.
Sumikama also presents a novel gating mechanism of a Na+ channel [4]. It has been believed that in the voltage-gated cation channels, structural changes in voltage sensor domains (VSDs) lead to structural changes in pore domains directly associated to VSDs, which opens and closes the gate of the channel. Here, by combining the results of high-speed atomic force microscopy, MD simulations, and a theoretical estimation using Monte Carlo simulation, it is shown that it is possible to form networks of the Na+ channels when the gate closes. This network has to be cooperatively disconnected to open the channel. Thus, it offers a plausible explanation for rapid onset of action potentials at the molecular level, which has not been addressed since the discovery of rapid onset by Hodgkin and Huxley [5].
The ability to discriminate between ions is essential to the function of many biological channels [6]. Indeed, selective transport mechanisms exist for a huge variety of ions including Na+, K+, Cl–, Ca2+, and nitrates. Without this, cells would not be able to maintain electrochemical gradients or control their electrical excitability [7]. Many factors have been suggested to dictate selectivity in biological channels, each of which can be important in different situations. In this talk Corry goes back to basics, examining the essential conditions that must be met and the simple physical principles that can be used to discriminate between ions. He gives examples where each factor may be important from his own research and that of others, including in simplified models and in simulations of sodium, potassium and calcium channels.
By tracing the origins of ion discrimination in sequentially more complex cases it is possible to see some of the different physical mechanisms that can be employed. In narrow pores, ions have to be dehydrated before they can enter. When the pore itself has limited interaction with the ion, such as in pores with hydrophobic walls, the selectivity is largely dictated by the different dehydration energies [8]. When the pore can offer specific interactions with the passing ion, a wealth of additional factors can contribute. These include the intrinsic electrostatic properties of the pore, the number of ligands that coordinate permeating ions, the size of a rigid pore [9], the vibrational motion of the ligands [10], the interactions between multiple ions and the kinetics of the transport process [11].
Microbial rhodopsins, one of the photoreceptive membrane proteins, have diverse biological functions such as light-driven ion pumps and light-gated ion channels, despite the similarity in the structures and reaction mechanisms; for example, the isomerization of retinal chromophore from all-trans/15-anti to 13-cis/15-anti forms as the initial trigger of the photocycle, resulting in the functionally relevant conformational changes and reactions followed by the thermal isomerization of retinal to the initial resting state. A recent experimental study based on X-ray free electron laser (XFEL) for nonlabens marinus rhodopsin-3 (NM-R3), a light-driven chloride-pumping rhodopsin, reported the unusual 13-cis/15-syn form instead of the 13-cis/15-anti form of retinal in the intermediate state [12], highlighting the importance of theoretical analyses based on MD simulations for the reaction mechanisms at spatiotemporal scales that are experimentally inaccessible. So far, large-scale quantum MD and metadynamics (MetaD) simulations, in which all the atoms were treated quantum-mechanically, have revealed the functional mechanism of proton transfers in bacteriorhodopsin, one of the most representative rhodopsins [13,14]. In the present study, quantum MD and MetaD simulations for NM-R3 were carried out with a focus on the thermal isomerization reactions of retinal in both the resting and intermediate states. In this presentation, free energy analyses of the reaction mechanism in NM-R3 are discussed in detail.
Sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) transports two Ca2+ across the membrane against large concentration gradient by utilizing ATP hydrolysis. In the step from the E1P state to the E2P state, X-ray structures of the E1P and E2P states have been revealed [15–18] and biochemical experiments [19] have suggested the existence of intermediate states. Recently, Cryo-EM [20,21] and FRET [22] have also shown the structural changes in the step. In this study, Kobayashi et al. have performed MD simulations of SERCA1a [23] and 2b [24] to understand a mechanism. The simulations show a series of structural changes starting from the cytoplasmic domains, and gradually occurring in the TM helices, and then in the Ca2+ binding site.
Transporter proteins change their conformation during substrate transport across the membrane. The conformational dynamics are key to understanding the function of transporter proteins. First, Okazaki et al. focus on oxalate transporter (OxlT), an oxalate:formate antiporter from a gut bacterium [25]. They explored the missing inward-open conformation of OxlT by leveraging accelerated MD simulations and AlphaFold2. They revealed the inward-open conformation and possible state-shifting mutation favoring the inward-open state. Second, they focus on the membrane domain Vo of the V/A-ATPase. By changing protonation state of the conserved glutamate and applying torque during MD simulation, they identified a coupling mechanism between c-ring rotation and proton transport.
