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Function-Related Dynamics in Multi-Spanning Helical Membrane Proteins Revealed by Solution NMR

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Function-Related Dynamics in Multi-Spanning Helical Membrane Proteins Revealed by Solution NMR membranes Review Function-Related Dynamics in Multi-Spanning Helical Membrane Proteins Revealed by Solution NMR Koh Takeuchi 1,* , Yutaka Kofuku 2, Shunsuke Imai 3, Takumi Ueda 2, Yuji Tokunaga 1, Yuki Toyama 2 , Yutaro Shiraishi 3 and Ichio Shimada 2,3,* 1 Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology, Aomi, Koto, Tokyo 135-0064, Japan; [email protected] 2 Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo, Tokyo 113-0033, Japan; [email protected] (Y.K.); [email protected] (T.U.); [email protected] (Y.T.) 3 Center for Biosystems Dynamics Research, RIKEN, Suehiro, Tsurumi, Yokohama 230-0045, Japan; [email protected] (S.I.); [email protected] (Y.S.) * Correspondence: [email protected] (K.T.); [email protected] (I.S.) Abstract: A primary biological function of multi-spanning membrane proteins is to transfer informa- tion and/or materials through a membrane by changing their conformations. Therefore, particular dynamics of the membrane proteins are tightly associated with their function. The semi-atomic resolution dynamics information revealed by NMR is able to discriminate function-related dynamics from random fluctuations. This review will discuss several studies in which quantitative dynamics information by solution NMR has contributed to revealing the structural basis of the function of multi-spanning membrane proteins, such as ion channels, GPCRs, and transporters. Citation: Takeuchi, K.; Kofuku, Y.; Keywords: membrane protein; NMR; ion channel; GPCR; transporter; dynamics Imai, S.; Ueda, T.; Tokunaga, Y.; Toyama, Y.; Shiraishi, Y.; Shimada, I. Function-Related Dynamics in Multi-Spanning Helical Membrane 1. Introduction Proteins Revealed by Solution NMR. Membranes 2021, 11, 604. Multi-spanning helical membrane proteins, such as GPCRs, ion channels, and trans- https://doi.org/10.3390/ porters, play a critical role in transferring information and materials through a membrane. membranes11080604 Since the function and malfunction of multi-spanning helical membrane proteins are critical in many biological processes and their associated diseases, understanding the functional Academic Editor: Izuru Kawamura mechanism of these proteins is of great interest from biological, medical, and pharmaceuti- cal aspects. The recent advances in X-ray crystallography and cryo-electron microscopy Received: 14 July 2021 (Cryo-EM) unveiled a significant number of structures of membrane proteins with bio- Accepted: 5 August 2021 logical, medical, and pharmacological importance. The number of published membrane Published: 9 August 2021 protein structures is currently over 4000 and rapidly increases after the establishment of single-particle analysis of Cryo-EM. Publisher’s Note: MDPI stays neutral The atomic-resolution structures of membrane proteins have contributed to vari- with regard to jurisdictional claims in ous research fields as a structural basis for understanding their functions. One of the published maps and institutional affil- early landmark examples is the determination of potassium ion channel structure by iations. R. Mackinnon [1]. The structure of the KcsA potassium ion channel revealed the selec- tive ion conductance mechanism of the channel by the formation of square antiprism coordination of carbonyl oxygens around each K+ binding site as if to mimic the waters of hydration [1,2](Figure1A ). The following crystal structure of a calcium-dependent Copyright: © 2021 by the authors. potassium channel MthK in the opened state unveiled the structural difference between Licensee MDPI, Basel, Switzerland. the closed and opened potassium channels [3,4]. Under the closed conditions, the inner This article is an open access article helix adopts straight conformation to form a bundle, closing the ion-conducting path in the distributed under the terms and intracellular side (Figure1A). In contrast, the inner helices are in the bent configuration in conditions of the Creative Commons the open conditions, which sprays out the intracellular gate, allowing the ion conductance Attribution (CC BY) license (https:// (Figure1B). In addition, the structures of ion channels with different regulation mecha- creativecommons.org/licenses/by/ nisms have been solved to unveil how ion conductance is regulated by various inputs such 4.0/). Membranes 2021, 11, 604. https://doi.org/10.3390/membranes11080604 https://www.mdpi.com/journal/membranes Membranes 2021, 11, 604 2 of 16 Membranes 2021, 11, x FOR PEER REVIEW 3 of 17 as pH, ligands, temperature, membrane voltage, ion concentrations, mechanosensation, [15].and The protein–protein activation-coupled interactions, inactivation etc. [5 is–12 present]. The recentin almost image all K processing+ channels technique and many in otherCryo-EM ion channels; even allows thus, the it reflects classification the common of open structural and closed behavior conformations of ion channels. of ion channels in the mixture to quantify the increased fraction of open conformation with the exposure to the inducers [13]. FigureFigure 1. 1. FunctionalFunctional dynamics dynamics of of an an ion ion channel. channel. (A (A) Crystal) Crystal structure structure of of KcsA KcsA in in the the closed closed conformation conformation at at neutral neutral pH pH (PDB(PDB ID: ID: 1BL8). 1BL8). ( (BB)) Crystal structurestructure ofof pore pore domain domain of of MthK MthK in thein the open open conformation conformation (PDB (PDB ID: 1LNQ). ID: 1LNQ). (C) Single-channel (C) Single- channelrecording recording of KcsA of inKcsA acidic in conditionacidic condition [14]. ( D[14].) A ( macroscopicD) A macroscopic current current of KcsA of KcsA after pHafter jump. pH jump. (E) Close-up (E) Close-up view view of the ofTrp the indole Trp indole region region of the of1H– the15 1NH– TROSY15N TROSY HSQC HSQC spectra spectra of H αof-partially Hα-partially protonated/ protonated/2H/152H/N-labeled15N-labeledD125–160 Δ125–160 KcsA. KcsA. The Theresonances resonances from from Trp-68 Trp-68 and and Trp-87 Trp-87 overlap overlap with with each each other. othe Assignmentsr. Assignments were were established established by site-specific by site-specific mutagenesis. mutagen- The esis.dashed The dashed lines connect lines connect corresponding corresponding signals. signals. (F) The conformational(F) The conformational state of HBCstate andof HBC SF in and the SF corresponding in the corresponding condition conditionis shown is in shown panel in (D panel). (G) Methyl-TROSY(D). (G) Methyl-TROSY spectra ofspectra WT (Left), of WT the (Left), E71A the (Middle), E71A (Middle), and Y82A and (Right) Y82A mutants(Right) mutants at pH 3.2 at pH 3.2 and 25 °C. The numbers in parentheses are the relative intensities of the two signals of V76 γ. The figure was and 25 ◦C. The numbers in parentheses are the relative intensities of the two signals of V76 γ. The figure was reproduced reproduced from [14,16,17] with permission. from [14–16] with permission. WhileThe dynamicthe open behavior probability of membrane of KcsA in proteins the inactivated is of importance condition as theyis known are required to be as to muchchange as 10% their [14], structure our NMR in response study revealed to various that stimuli intracellular and transfer HBC theis fully information open even in andin 1 15 theout inactivated of the membrane condition to [16]. exert In their a series functions. of H N-TROSY For example, HSQC the spectra single-channel measured recording at var- iousshows pH values, that ion significant channels chemical in the open shift conditions changes were are notdetected continuously between permeatingpH 3.9 and 5.2, ions. reflectingThey are a instead conformational under the re equilibriumarrangement between associated open, with closed, the andgating inactive of the conformations, ion channel. Sincereflected the 2D in theNMR stochastic measurement profile oftakes the hours, ion permeabilization the acidic condition through spectra a channel are those (Figure that1C). experienceThe open the dwelling activation-coupled times are mostly inactivati in theon. order The of pH-dependent milliseconds, whichchemical correspond shift changes to the wereexchange primarily rate observed 1000 s−1. in Since the residues the open near probability the intracellular (i.e., the populationHBC. Especially, of open the state chem- per icalunit shifts time) Trp-26 determined and Trp-113 from the are open/closed distinct at acidic dwell and time neutral and the pH, ion indicating conductivity that in the the intracellular gate changes to a completely different structure, presumably in open confor- Membranes 2021, 11, 604 3 of 16 open state defines the total ion flux, the activity of the ion channel can be described from the stochastic dynamic behavior of the proteins and is beyond the description by static snapshot structures. NMR is an optimal spectroscopic technique to reveal such dynamics of proteins. NMR is able to define the function-associated dynamics in a site-specific manner and quantify the population of each conformation under an equilibrium, along with the rate of inter- conversion. For this purpose, various NMR measurements and stable-isotope labeling strategies are available. The quantitative analysis of the NMR relaxation rate can detect a high-energy functional state that populates less than one percent in an equilibrium. Due to its versatility to the condition, NMR can monitor the changes in structure
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