Dissecting the Molecular Mechanism of Nucleotide-Dependent Activation of the KtrAB K+ Transporter

RESEARCH ARTICLE Dissecting the Molecular Mechanism of Nucleotide-Dependent Activation of the KtrAB K + Transporter Andras Szollosi 1¤a , Ricardo S. Vieira-Pires 1¤b , Celso M. Teixeira-Duarte 1,2 , Rita Rocha 1,2 , João H. Morais-Cabral 1,2 * 1IBMC, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal, 2Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal ¤a Current address: Department of Medical Biochemistry, Semmelweis University, Budapest H-1094, Hungary ¤b Current address: Center for Neuroscience and Cell Biology (CNBC/UC), University of Coimbra, Portugal *[email protected] Abstract KtrAB belongs to the Trk/Ktr/HKT superfamily of monovalent cation (K + and Na + ) transport proteins that closely resemble K + channels. These proteins underlie a plethora of cellular functions that are crucial for environmental adaptation in plants, fungi, archaea, and bacteria. The activation mechanism of the Trk/Ktr/HKT proteins remains unknown. It has been shown that ATP stimulates the activity of KtrAB while ADP does not. Here, we present X-ray structural information on the KtrAB complex with bound ADP. A comparison with the KtrABATP structure reveals conformational changes in the ring and in the membrane protein. In combination with a biochemical and functional analysis, we uncover how ligand-dependent changes in the KtrA ring are propagated to the KtrB membrane protein and conclude that, despite their structural similarity, the activation mechanism of KtrAB is markedly different from the activation mechanism of K + channels. Author Summary Animals have organs that regulate the balance of water and ions in the fluids bathing their cells. In contrast, the cells of plants, bacteria, and fungi have little or no control over those fluids and, thus, they have to cope with changes in the local environment. These cells have therefore evolved specific molecular systems that are crucial for environmental adaptation. We study the molecular properties of the membrane protein KtrAB—a member of the Trk/Ktr/HKT superfamily of transport proteins that shuffle K + and Na + ions across the plasma membrane, closely resemble K + channels, and underlie environmental adaptation of cells of plants, fungi, bacteria, and archaea. KtrAB is formed by the KtrB membrane protein and the KtrA cytosolic ring protein. KtrA binds to both ADP and ATP, resulting in a low-activity ADP-bound state and a high-activity ATP-bound state, respectively. We determined a low resolution structure of a low-activity form of the transport protein. A PLOS Biology | DOI:10.1371/journal.pbio.1002356 January 15, 2016 1/21 OPEN ACCESS Citation: Szollosi A, Vieira-Pires RS, Teixeira-Duarte CM, Rocha R, Morais-Cabral JH (2016) Dissecting the Molecular Mechanism of Nucleotide-Dependent Activation of the KtrAB K + Transporter. PLoS Biol 14 (1): e1002356. doi:10.1371/journal.pbio.1002356 Academic Editor: Raimund Dutzler, University of Zurich, SWITZERLAND Received: June 4, 2015 Accepted: December 10, 2015 Published: January 15, 2016 Copyright: © 2016 Szollosi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Coordinate and diffraction data files are available from the PDB database (accession number 5BUT). Numerical data for Figures 1a-b; 3a-b, 4b-e, 5c-d; S10a-c, and S11ad are provided in Supplemental Material (S1 Data) Funding: AS was supported by FEBS (Long term fellowship). This work was financially supported by national funds through FCT—Fundação para a Ciência e a Tecnologia/MEC—Ministério da Educação e Ciência and when applicable co-funded by FEDER funds within the Partnership Agreement PT2020 related with the research unit number 4293 and by FEDER funds through the Operational comparison of this structure with the structure of ATP-bound KtrAB reveals changes in both the KtrA ring and the KtrB membrane protein. We uncover how changes in the KtrA ring are propagated to KtrB and conclude that, despite their structural similarity, the activation mechanism of KtrAB is markedly different from the activation mechanism of K + channels. Introduction KtrAB belongs to the Trk/Ktr/HKT superfamily of monovalent cation (K + and Na + ) transport proteins that are found ubiquitously in nonanimal cells [1–3]. The Trk/Ktr/HKT superfamily comprises uniporters (K + or Na + ) and symporters (K + /Na + or K + /H + ) and underlies a plethora of cellular functions in plants, fungi, archaea, and bacteria such as K + and Na + uptake, regulation of cellular electrical activity, turgor compensation, osmotic adjustment (thereby contributing to resistance to drought and salinity), intraand intercellular ion transport, motor cellular functions, and adjustment of membrane potential [2,4]. The structure of the KtrAB complex from the bacterium Bacillus subtilis was recently determined [5]. This protein complex plays an important role in the osmotic adaptation mechanism of this bacterium [6]. It is composed by a homodimeric KtrB membrane protein (GenBank: KIX81591.1) assembled with the KtrA octameric ring (GenBank: KIX81590.1). KtrB is responsible for ion permeation; each KtrB subunit has the architecture of a potassium channel pore domain [1,5,7](S1A–S1D Fig), consisting of four M1-P-M2 (transmembrane helix 1-pore helix and looptransmembrane helix 2) structural repeats (D1 to D4), which embrace an ion-permeable pore. KtrA binds ATP and adenosine diphosphate (ADP) and is responsible for regulation of KtrAB activity [5,8]. This cytosolic protein is a regulate conductance of K + (RCK) domain [5,9–11]that assembles as an octameric ring, closely resembling the RCK gating rings of the MthK [12,13], BK [14–16], and GsuK [17] potassium channels (S1E–S1F Fig). The architecture of the KtrAB complex is also present in the TrkHA complex, another member of the Trk/Ktr/HKT superfamily [18]. Importantly, KtrB displays structural features that are not present in K + channels. In particular, repeat D3 of KtrB has an insertion of ~10 residues that form an intramembrane loop pointing into the cytosolic pore. This intramembrane loop is also found in other members of the Trk/Ktr/HKT superfamily [1,7,19], and together with a highly conserved arginine residue (R417 in KtrB from B.subtilis), it obstructs the ion pathway of KtrAB and TrkHA [5,18,20]. The intramembrane loop and the conserved arginine are thought to be a central feature of the activation mechanism of KtrAB and TrkHA. In KtrB from Vibrio alginolyticus, cell-based studies showed that truncations introduced in the intramembrane loop enhance the maximum uptake velocity for K + [21]; and an electron paramagnetic resonance study showed a K + -dependent relative motion of the intramembrane loop [22]. In TrkH, mutation of the conserved arginine to alanine enhanced K + flux in a liposome-based assay [20] and electrophysiological recordings with the TrkHA complex showed that truncation of the intramembrane loop changes the response to the ligand and increases the open probability in the absence of ligand [18]. These results, together with the structures of KtrAB and TrkHA, have led to the proposal that the intramembrane loop and the conserved arginine function as a pore gate. It has been shown that some orthologs of KtrAB are regulated by cyclic-diAMP [23–25], and additionally, in B.subtilis, the KtrAB operon responds to cyclic-diAMP [26]. Importantly, it is generally accepted that activation of KtrAB and TrkHA involves ATP binding to the KtrA and TrkA gating rings, respectively. Structures of isolated KtrA and TrkA rings show liganddependent conformational changes. In TrkHA, the open probability increases with ATP and Conformational Changes in KtrAB PLOS Biology | DOI:10.1371/journal.pbio.1002356 January 15, 2016 2/21 Competitiveness Program–COMPETE and by National Funds through FCT–Fundação para a Ciência e a Tecnologia under the project FCOMP-010124-FEDER-028115 (PTDC/BBB-BEP/2017/2012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: ADP, adenosine diphosphate; cpm., counts per minute; DEN, Deformable Elastic Network; DTNB, 5,5’-dithio-bis(2-nitrobenzoic acid); KtrA ΔC , KtrA protein with C-terminal domain truncated; LLG, log-of-likelihood; M1D2, M1 transmembrane helix in repeat D2; RCK, regulate conductance of K + ; SEM, standard error of the mean; TNB, thio-nitrobenzoate. decreases with ADP [18]. In KtrAB, ion flux is stimulated by ATP and not by ADP; the ADPbound KtrAB is in a low-activity state, displaying basal activity that is also present in KtrB alone [5,8]. Not much more is understood about the molecular mechanism of activation of these transporters; in particular, the conformational changes induced by ATP and ADP in the KtrAB or TrkHA complexes have not been described. We present a structure of KtrAB in the low-activity, ADP-bound state and reveal conformational changes in the gating ring and the membrane protein relative to the high-activity ATPbound KtrAB. We also perform a functional and biochemical characterization of the mechanism of activation. Based on these results, we propose an activation mechanism of the KtrAB complex by ATP. Results The Structure of the KtrA ΔC B Complex To gain structural insights into the mechanism of activation of the KtrAB K + transporter, we crystallized the ADP-bound KtrAB complex from B.subtilis but could only measure diffraction data to ~8 Å from these crystals. As an alternative, we turned to the KtrA ΔC B complex, which is formed by wild-type KtrB and a C-terminal domain truncated form of KtrA (KtrA ΔC ). This KtrA form lacks residues 145 to 222 that correspond to the whole C-terminal domain (S1E Fig). Ligand-binding studies show that KtrA ΔC from B.subtilis is able to bind ATP and ADP and crystal structures have shown that it forms an octameric ring [11]. Also, it has been reported that versions of KtrA ΔC B from V.alginolyticus are inactive [8]. We verified by sizeexclusion chromatography that the complex between KtrB and KtrA ΔC is formed both in the presence of ADP and ATP (S2A Fig). We also measured KtrA ΔC B-mediated 86 Rb + flux using a liposome-based assay (Fig 1A) and compared it with an equivalent characterization of wild type KtrAB (Fig 1B). As demonstrated before [5], ATP stimulates wild type KtrAB flux relative to ADP and to KtrB alone. Despite a larger background signal (a detailed explanation for this observation can be found in Material and Methods), it is clear that after 1 h, KtrA ΔC B-mediated 86 Rb + uptake levels are identical for KtrA ΔC B-ATP and KtrA ΔC B-ADP and similar to uptake levels of KtrB alone. Thus, the stimulatory effect of ATP is abolished in KtrA ΔC B, leading us to conclude that this complex is trapped in a low-activity state. We crystallized KtrA ΔC B bound to ADP, collected diffraction data to 6 Å (S2 Table), and solved the structure (Protein Data Bank accession number 5BUT) by molecular replacement using the KtrB dimer structure from KtrAB and one of the octameric ring structures previously determined for KtrA ΔC [11]. As in the KtrAB-ATP and TrkHA structures, the asymmetric unit contains two membrane–protein dimers associated with opposing faces of an octameric RCK ring [5,18](S4 Fig). The calculated electron-density map was then 4-fold and 8-fold averaged in the molecular envelopes corresponding to KtrB and KtrA, respectively. The resulting 6Å electron-density map is of very high quality (Fig 1C), clearly showing the position of all transmembrane and pore helices in KtrB as well as the position of different secondary-structure elements in the KtrA subunits. The final KtrA ΔC B-ADP model (Fig 1D and S4 Fig) was generated by manual adjustment of the M1 helix in the KtrB D2 repeat to better fit the map (more details below) and Deformable Elastic Network (DEN) refinement (S2 Table). The final model fits very well in the averaged electron-density map (Fig 1C). Importantly, we could establish that KtrA ΔC B-ADP and KtrAB-ADP are structurally similar and distinct from KtrAB-ATP. Using as search models a collection of different KtrA ΔC ring structures (S5 Fig) and two KtrB structures (from KtrAB-ATP and KtrA ΔC B-ADP; Fig 1D and 1E), we performed molecular replacement searches against an 8 Å diffraction dataset collected from wild-type KtrAB-ADP crystals. The six different RCK ring structures together with the Conformational Changes in KtrAB PLOS Biology | DOI:10.1371/journal.pbio.1002356 January 15, 2016 3/21 two different KtrB structures cover a range of potential conformations of the KtrAB complex and allowed us to get a low resolution model of the KtrAB-ADP structure. The procedure involved sequential searches with the membrane proteins and with the gating rings; for many of these pairs, we were able to obtain molecular replacement solutions that display the expected packing between the membrane protein dimer and the gating ring. The values for the log-oflikelihood (LLG) function (a measure of how well the structural model agrees with the data) are shown in S3 Table; they clearly show that the pair formed by the KtrB homodimer from the KtrA ΔC B-ADP together with the KtrA ΔC ring also from the KtrA ΔC B-ADP (model 7) has the highest LLG value (LLG = 515). A detailed analysis of the significance and sensitivity of LLG values is presented with S3 Table. Overall, these LLG results (S3 Table) show that the best fit for the KtrAB-ADP diffraction data is obtained with the protein components of the KtrA ΔC B-ADP structure, strongly indicating that KtrA ΔC B-ADP is structurally similar to KtrAB-ADP. In contrast, the pair formed by the KtrB homodimer from KtrAB-ATP and KtrA-ATP (without the C-terminal domain) has a LLG = 223 and incorrect packing, indicating that the ATP-bound complex is structurally distinct from KtrAB-ADP. We also determined an averaged density map for the KtrAB-ADP structure using the starting phases calculated from the KtrA ΔC B model after rigid body Fig 1. Structure of KtrA ΔC B. 86 Rb + uptake into proteoliposomes with a) KtrA ΔC B (with ATP or ADP) and KtrB alone (with ATP), or with b) wild type KtrAB (with ATP or ADP) and KtrB alone (with ATP). Control liposomes were formed in the presence of KtrA ΔC or KtrA alone; liposome float-up experiments are shown in S3 Fig; KtrA ΔC increases liposome leakiness as reflected in the increased control signal (see raw data in S1 Table). Mean ±standard error of the mean (SEM) values calculated from 5–8 assays from 3 separate liposome preparations. counts per minute (cpm). c) Averaged electron-density map of KtrA ΔC B in mesh with superposed Cαtrace of refined KtrA ΔC B structure. Side views of d) KtrA ΔC B-ADP and e) wild type KtrAB-ATP. KtrB dimer is shown at the top and colored dark-grey and wheat, with D1-D2 domain of KtrA ΔC B in magenta. RCK rings are shown in light grey and red with N-terminal domain as cartoon and C-terminal domain in KtrAB-ATP as thin Cαtrace; K + ions as green spheres; tip and lateral contacts are labeled; putative membrane limits are indicated by horizontal lines. Numerical values of data displayed in panels a and b are included in S1 Data. doi:10.1371/journal.pbio.1002356.g001 Conformational Changes in KtrAB PLOS Biology | DOI:10.1371/journal.pbio.1002356 January 15, 2016 4/21 refinement (S6 Fig). Map quality is relatively poor due to the low resolution of the data and limited averaging power, and caution must be exerted during interpretation. In any case, the density supports our conclusion that the KtrA ΔC B-ADP and KtrAB-ADP structures are similar, with density that matches the conformation of the KtrA ΔC ring and new density that appears to correspond to the C-terminal domains of full-length KtrA. The overall organization of KtrA ΔC B-ADP and KtrAB-ATP, including the relative disposition of the KtrB homodimer subunits, is similar (Fig 1D–1E). Importantly, the KtrA ΔC ring is asymmetrically expanded along the diagonal defined by the tip contacts (Fig 2A), revealing that in the KtrAB complex the ligand-dependent conformational change of the RCK ring occurs along the tip contacts. As a consequence of this change, the KtrB-KtrA interface contact sites are affected differently. In the lateral contacts, the spatial relationship between the KtrB C termini and two KtrA subunits is almost unaltered. The Cα-Cαdistance separating F71 (or L66) in the two lateral contact KtrA subunits is almost unchanged: ~66 and ~65 Å for F71, ~48 and ~49 Å for L66, in KtrAB-ATP and KtrA ΔC B-ADP respectively. In the tip contact KtrA subunits, the Cα-Cαdistance F71 distance increases from ~68 Å (KtrAB-ATP) to ~87 Å (KtrA ΔCB-ADP), and for L66, from ~50 to ~76 Å. This expansion also includes a KtrA movement away from the membrane protein (Fig 2B and 2C); the distance separating a reference spatial Fig 2. Comparison of KtrA ΔC B-ADP and KtrAB-ATP. a) Face view of RCK rings from KtrA ΔC B-ADP (cyan) and KtrAB-ATP (red) superposed through KtrB homodimers; lateral contacts indicated with KtrB C terminus in purple coil. After superposition of KtrA ΔC B-ADP and KtrAB-ATP, a D1-D2 domain from KtrAB-ATP (colored magenta and wheat) is shown together with a KtrA subunit from b) KtrAB-ATP (red and grey) and c) KtrA ΔC B-ADP (cyan). Q115, V126, and V130 are shown in dark blue dot representation; Cαatoms of L66, F71, and L108 shown as spheres. d) and e) Averaged density (mesh) of M1D2 helix in two different KtrB subunits. KtrAB-ATP M1D2 helices are shown in red (on the left), and KtrA ΔC B-ADP M1D2 helices are in cyan (on the right). Arrow indicates shift applied to cytosolic end of helix to bring into density. f) Overall view of superposed KtrB subunits from KtrA ΔC B-ADP (cyan) and KtrAB-ATP (red). Star indicates moved M1D2 helix; I127 Cαin the two structures is shown as yellow sphere; K + ion as green sphere. g) Averaged density (mesh) of D1-D2 domain region together with refined KtrA ΔC B-ADP model. Lack of density for M3D1 and D1-D2 helices is apparent. doi:10.1371/journal.pbio.1002356.g002 Conformational Changes in KtrAB PLOS Biology | DOI:10.1371/journal.pbio.1002356 January 15, 2016 5/21 position (the Cαatom of KtrB-L108 in KtrAB-ATP) from the Cαof KtrA-F71 increases from ~9 Å in KtrAB-ATP to ~21 Å in KtrA ΔC B-ADP. Although the overall conformations of the KtrA ΔC ring and isolated KtrA-ADP ring [5] resemble each other, with one diagonal of the ring longer than the other, they are different structures (S7A Fig). This is clearly seen in the Cα-Cαdistances between F71 residues in opposing ring subunits. In the isolated KtrA-ADP structure, these distances are ~68 and ~82 Å, while in KtrA ΔC from KtrA ΔC B-ADP the distances are ~66 and ~87 Å. The intradimer arrangement in KtrA ΔC B-ADP is also different from the one seen in the isolated full-length KtrA-ADP structure (S7B Fig); the angle between the KtrA dimer subunits in KtrA ΔC B-ADP has changed by ~15° relative to KtrA-ADP. In any case, the angle difference between KtrA ΔC and KtrA in KtrAB-ATP is even larger, being close to 30° (S7C Fig). Importantly, the averaged electron-density map also reveals structural changes in the KtrB protein. As in the KtrA ring, all apparent changes are centered on the KtrB tip contact region, and no alterations are detected around the lateral contact. The position of the cytosolic end of the M1 transmembrane helix in repeat D2 (M1D2) in the averaged density is clearly different from the one seen in the KtrAB-ATP structure (Fig 2D and 2E and S8 Fig); the need for a positional adjustment of the helix is also detectable in a difference map (S9 Fig). The M1D2 helix was adjusted with an outward movement of the cytosolic end so that the Cαatom of I127 is shifted by ~3–4Å(Fig 2D–2F). The better fit of the final model to the averaged electron-density map (Fig 2D–2E and S8 Fig) is supported by an increase in the map correlation coefficient calculated for the main chain and Cβatoms of the cytosolic half of the M1D2 helices (residues 128 to 140) in the 4 KtrB asymmetric unit copies: 0.50 in KtrAB-ATP and 0.71 in KtrA ΔCB-ADP. As a result of this conformational change, the M1D2 helix in KtrA ΔC B-ADP has been straightened while in KtrAB-ATP it is bent towards the cytosolic pore. In addition, while all other KtrB helices are visible in the averaged electron-density map (including the pore helices and the M3 helices of repeats D2, D3, and D4), there is no density for the two helices just before M1D2, M3D1, and the D1-D2 loop helix (Fig 2G); in KtrAB-ATP, these two helices, together with M1D2, form a domain-like structure (the D1-D2 domain) that functions as a KtrB foot on the tip contact (Fig 1D and 1E). The lack of density for the M3D1 and D1-D2 helices in the 6Å resolution KtrA ΔC B-ADP averaged map probably results from either unwinding of the helices, as a manifestation of increased local disorder, or from a breakdown of the KtrB “homodimer”symmetry in this region. Remodeling of the Tip-Contact Region during Activation To explore in more detail the ligand-dependent remodeling of the D1-D2 domain in KtrB, we evaluated the ligand-dependent accessibility of cysteine residues introduced in the D1-D2 domain. We engineered single-cysteine mutations in a cysteineless KtrB (Fig 2B and 2C): Q115C is a semiburied residue on the D1-D2 loop; V126C and V130C are on the M1D2 helix and are semi- (V126C) or fully buried (V130C). These mutants were assembled with cysteineless KtrA (KtrA C0 ), and we confirmed that the properties of the mutant complexes are similar to wild type KtrAB (S10 Fig): by size-exclusion chromatography, we verified that the complexes are assembled in the presence of ATP and ADP and, using the 86 Rb + flux assay, we verified that the mechanism of ligand activation (stimulation by ATP relative to ADP) is not markedly altered. Using a fast injection system, we mixed the complex with ~20-fold molar excess DTNB (5,5’-dithio-bis(2-nitrobenzoic acid)) in the presence of ATP or ADP and followed the reaction time course. DTNB, or Ellman’s reagent, reacts rapidly with reduced thiols in tissues and proteins [27–29] and irreversibly in our experimental conditions, generating stoichiometric amounts of the yellow thio-nitrobenzoate (TNB), which absorbs at 412 nm. Conformational Changes in KtrAB PLOS Biology | DOI:10.1371/journal.pbio.1002356 January 15, 2016 6/21 The DTNB modification time courses for Q115C (Fig 3A) clearly show that the reaction is much faster (~65 times faster) in the presence of ADP than in the presence of ATP; the modification halftimes are ~0.2 sec and ~13 sec with ADP and ATP, respectively (Table 1). With the KtrAB V126C mutant, the reactions are remarkably fast in the presence of either ligand, and we cannot detect a difference in reactivity (Table 1,S10B Fig), but the modification halftime for KtrAB V130C (~10 s) is also shorter for ADP than with ATP (~21 s) (Fig 3B and Table 1). Faster reaction time courses for ADP relative to ATP for two different cysteine positions in the D1-D2 domain are consistent with an increase in cysteine accessibility to DTNB in the ADPbound state and support the proposal that the domain has undergone a structural change. This biochemical analysis together with our structural comparison establishes that during ligand activation the RCK ring conformational change is associated with remodeling of the tip contact interface and D1-D2 domain. The KtrB Cytosolic Pore Becomes Narrower during Activation A central feature of the activation mechanism of many K + channels is the opening of a cytoplasmic gate and increased access to the cytosolic pore. Since Trk/Ktr/HKT proteins are Fig 3. Remodeling of the tip contact. Time course of DTNB modification reaction for a) KtrB Q115C and b) KtrB V130C in complex with KtrA C0 bound to ADP (gray) or ATP (black). Representatives of four separate modification reactions are shown, normalized to maximum (last recorded) value. For each mutant, the final absorption values between ADP and ATP varied by less than 30%, showing that differences in initial cysteine oxidation levels are small. The initial fast jump observed in the time course is due to the time resolution of our system (100 ms). Numerical values are included in S1 Data. doi:10.1371/journal.pbio.1002356.g003 Table 1. DTNB modification reaction halftimes for mutant cysteines. ADP (s) ATP (s) Cysteines introduced in the D1-D2 domain Q115C 0.23 ±0.01 12.75 ±0.48 V126C 0.21 ±0.01 0.21 ±0.02 V130C 10.48 ±0.09 20.95 ±0.39 Cysteines introduced in the cytosolic pore N119C 0.24 ±0.01 0.55 ±0.01 P121C 0.23 ±0.02 0.91 ±0.05 F443C 0.48 ±0.01 63.73 ±0.43 T444C 52.43±4.02 386.55 ±106.99 * SEM values are determined from 3–4 separate modification reactions. *Halftime for T444C with ATP is underestimated since the reaction is very slow and does not reach its end point. doi:10.1371/journal.pbio.1002356.t001 Conformational Changes in KtrAB PLOS Biology | DOI:10.1371/journal.pbio.1002356 January 15, 2016 7/21 structurally similar to K + channels, we asked whether the same happens in KtrAB during activation. To assess the ligand-dependent conformational changes in the pore of KtrB, we made use of the DTNB assay (introduced above) and probed ligand-induced changes in accessibility of cysteine residues introduced on the wall of the cytosolic pore of KtrB. In the KtrAB-ATP structure, N119C is positioned at the mouth of the pore, while P121C, F443C, and T444C are positioned deep in the pore (Fig 4A). These mutants showed no alteration in their ability to assemble with KtrA and retained the ATP stimulation effect, although reduced for F443C (S11 Fig). The DTNB modification halftimes measured for the four cysteine mutants were consistently shorter with ADP than with ATP (Fig 4B–4E,Table 1), showing that all cysteine thiol groups are less reactive in the ATP-bound state. This trend across four different positions strongly indicates a reduction in DTNB accessibility. These results, together with the ligand-dependent repositioning of the M1D2 helix towards the cytosolic pore in KtrAB-ATP (Fig 2F), suggest that the cytosolic pore of KtrAB becomes narrower upon ATP activation. Functional and Biochemical Characterization of the Intramembrane Gate The intramembrane loop and the conserved arginine are thought to function as a gate in KtrAB and TrkHA. We analyzed the functional impact of mutations both in the loop and in Fig 4. Narrowing of cytosolic pore. a) View of cytosolic pore wall residues (in yellow stick) that were mutated to cysteines. KtrB C-terminus shown as orange coil; D1-D2 domain from KtrAB-ATP shown in magenta and wheat; K + ion as green sphere. Time course of DTNB modification reaction for b) KtrB N119C ,c) KtrB P121C ,d) KtrB F443C and e) KtrB T444C in complex with KtrA C0 bound to ADP (gray) or ATP (black). Representatives of four separate modification reactions are shown. Normalized to maximum (last recorded) value. The initial fast jump in the time course (very noticeable in ATP time courses of F443C and T444C) is due to the time resolution of our system (100 ms). For N119C, P121C, and F443C, the final absorption values between ADP and ATP varied by less than 30%, showing that differences in cysteine oxidation are small; this value could not be determined for T444C with ATP due to its very slow reactivity. Numerical values of data displayed in panels b–e are included in S1 Data. doi:10.1371/journal.pbio.1002356.g004 Conformational Changes in KtrAB PLOS Biology | DOI:10.1371/journal.pbio.1002356 January 15, 2016 8/21 the arginine (R417). We truncated the intramembrane loop (KtrB Δloop —truncation of residues G306 to A311), mutated two residues in the loop (KtrB G306S and KtrB S309D ), and mutated the conserved arginine to a lysine (KtrB R417K ). Strikingly, the intramembrane loop seems to be very sensitive to alterations since both the truncation (Fig 5A) and the two single point mutations (Fig 5B) have a destabilizing effect on the interaction between KtrB and KtrA. Hanelt and colleagues [21] described the same effect for truncations of the intramembrane loop in V.alginolyticus KtrB but did not observe destabilization with a mutation equivalent to G306S. In contrast, KtrB R417K assembles with KtrA (S2B Fig) in the presence of ADP or ATP. Flux assays with liposome-reconstituted KtrB Δloop and KtrB R417K show that the uptake in KtrB Δloop is faster than in the KtrB wild type protein (single exponential time constants [τ]are ~5 min for KtrB Δloop and ~11 min for wild type KtrB) as expected if an obstacle to ion flow has been removed (Fig 5C). On the other hand, the rate of uptake in KtrB R417K alone (τ~22 min) is slow. More interestingly, the uptake rate of KtrAB R417K -ATP (τ~6 min) is as fast as in KtrB Δloop (Fig 5D) while KtrAB R417K -ADP (τ~22 min) is comparable to wild type KtrAB (τ~19 and ~17 min, for KtrAB-ATP and KtrAB-ADP respectively). This suggests that the conservative substitution in the KtrAB R417K complex is functionally similar to the removal of the intramembrane loop from the cytosolic pore of KtrB. Note however that this effect is ligand-dependent since it only occurs when KtrB R417K is associated with KtrA with bound ATP and not with ADP. Overall, changes in the intramembrane loop or in R417 modify the ion permeation properties in a way that is consistent with a role in the intramembrane gate. Discussion The structures of the isolated RCK rings from the KtrAB and TrkHA ion transporters led to the proposal that ligand-induced conformational changes in the RCK rings (KtrA or TrkA) are at the basis of the activation mechanisms of the Ktr and Trk ion transporters [5,8,18]. In KtrAB in particular, it was shown that the isolated KtrA ring expands asymmetrically upon exchange of ATP for ADP. However, those studies did not show how the asymmetric expansion/contraction occurs within the complex. In fact, no conformational changes had been demonstrated in the KtrAB and TrkHA complexes. Fig 5. Properties of intramembrane gate mutants. Size-exclusion profiles of intramembrane loop a) truncation and b) G306S and S309D single mutants assembled with KtrA in the presence of ATP and ADP. Inverted arrow heads indicate elution volumes of KtrAB complex (closed symbol) and of separate components (open symbol). SDS-PAGE of fractions are shown in S12 Fig;c) Time course of 86 Rb + uptake for KtrB wt and mutants (KtrB Δloop , KtrB R417K )in the absence of KtrA. d) Time course of 86 Rb + uptake for KtrB R417K alone and KtrAB R417K with ADP or ATP. Liposome float-up experiments are shown in S13 Fig. Numerical values of data displayed in panels c–d are included in S1 Data. doi:10.1371/journal.pbio.1002356.g005 Conformational Changes in KtrAB PLOS Biology | DOI:10.1371/journal.pbio.1002356 January 15, 2016 9/21 2HMU); f) KtrA ΔC from KtrA ΔC B structure (model 6 and 7—PDB code 5BUT). F71-Cαatoms at each of the subunits are shown as black spheres. Distances between F71 in pairs of opposite subunits are indicated for each ring in Angstroms. (TIF) S6 Fig. Averaged map of KtrAB-ADP. Averaged map calculated with KtrAB-ADP 8 Å diffraction data and using starting phases calculated from the model 7 molecular replacement solution (S3 Table). Superposed on the map is the KtrB model from KtrA ΔC B and the fulllength KtrA-ADP structure previously determined. Two KtrB dimers are indicated with density covering one of the dimers. Density covering the N-terminal domain and C-terminal domain (not included in the initial phasing) of two KtrA subunits is also indicated. (TIF) S7 Fig. Comparison of RCK rings in KtrA ΔC B and KtrA-ADP. a) Face view of superposed KtrA-ADP (black Cαtrace) and KtrA ΔC (cyan). F71-Cαatoms are shown as cyan and black spheres. Superposition (through one of the N-terminal domains) of KtrA ΔC ring dimer (cyan) with dimer from b) KtrA-ADP (black) or c) KtrA-ATP (red) from KtrAB-ATP structure. Cterminal domains are not shown. Angle values referring to the structural changes between RCK ring dimers, and quoted in the main text, were measured between axes of the last α-helix in the N-terminal domain, as indicated by cyan, black, and red lines. (TIF) S8 Fig. Movement of M1D2 helix. Averaged density (mesh) of M1D2 helix in two different KtrB subunits. Together with Fig 2D and 2E, these two panels show the averaged density for all M1D2 helices of the 4 KtrB subunits in the asymmetric unit of KtrA ΔC B. KtrAB-ATP M1D2 helices are shown in red (on the left) and KtrA ΔC B-ADP M1D2 helices are in cyan (on the right). Arrow indicates shift applied to cytosolic end of helix to bring it into density. (TIF) S9 Fig. KtrA ΔC B Difference map. Fo-Fc difference map calculated with structure factors from KtrA ΔC B dataset and structure factors and phases from molecular replacement solution after rigid body refinement. Red mesh indicates negative density at 3 sigma contour level; green mesh indicates positive density at 3 sigma contour level. Initial model is shown superposed with density. The largest red mesh peaks correspond to the cytosolic end of M1D2 helix and part of the M1D3 helix indicating the model needs adjustments in these regions. (TIF) S10 Fig. Control experiments for cysteine residues introduced in D1-D2 domain. a) Q115C, b) V126C, and d) V130C. Size exclusion chromatography profiles (left) and 86 Rb + uptake (center) values at 0 and 60 min of KtrAB complexes formed by KtrA C0 and KtrB mutants with ATP and ADP. For V126C, the time course of modification by DTNB is also shown (right); for the other mutants, the equivalent data is presented in the main text. Closed arrowhead indicates elution volume of KtrAB complex; open arrowhead indicates elution volume of individual components. All mutants form a complex with KtrA C0 in the presence of ATP or ADP, and retain the functional stimulation by ATP relative to ADP. SDS-PAGE of size-exclusion fractions are shown as insets. Inset in a) 1KtrA+KtrB Q115C with ADP eluting at closed arrow; 2KtrA+KtrB Q115C with ADP eluting at open arrow; 3KtrA+KtrB Q115C with ATP eluting at closed arrow; 4KtrA+KtrB Q115C with ATP eluting at open arrow; Inset in b) 1KtrA+KtrB V126C with ADP eluting at closed arrow; 2KtrA+KtrB V126C with ADP eluting at open arrow; 3sample buffer; 4KtrA+KtrB V126C with ATP eluting at closed arrow; 5KtrA+- KtrB V126C with ATP eluting at open arrow. Inset in c) 1pure KtrBV130C; 2KtrA+KtrB V130C Conformational Changes in KtrAB PLOS Biology | DOI:10.1371/journal.pbio.1002356 January 15, 2016 16 / 21 with ADP eluting at closed arrow; 3KtrA+KtrB V130C with ADP eluting at open arrow; 4KtrA+KtrB V130C with ATP eluting at closed arrow; 5KtrA+KtrB V130C with ATP eluting at open arrow. Lower horizontal arrow indicates KtrA; horizontal arrow with M indicates KtrB monomer. Numerical values are included in S1 Data. (TIF) S11 Fig. Control experiments for cytosolic pore cysteines. a) N119C, b) P121C, d) F443C and e) T444C. Size exclusion chromatography profiles (left) and 86 Rb + uptake (right) values at 0 and 60 min of KtrAB complexes formed by KtrA C0 and KtrB mutants with ATP and ADP. Closed arrowhead indicates elution volume of KtrAB complex; open arrowhead indicates elution volume of individual components. All mutants form a complex with KtrA C0 in the presence of ATP or ADP and retain the functional stimulation by ATP relative to ADP, although reduced in F443C. SDS-PAGE of size-exclusion fractions are shown as insets. Inset in a) 1KtrA+KtrB N119C with ADP eluting at closed arrow; 2KtrA+KtrB N119C with ADP eluting at open arrow; 3KtrA+KtrB N119C with ATP eluting at closed arrow; 4KtrA+KtrB N119C with ATP eluting at open arrow; Inset in b) 1pure KtrB P121C . 2KtrA+KtrB P121C with ADP eluting at closed arrow; 3KtrA+KtrB P121C with ADP eluting at open arrow; 4KtrA+KtrB P121C with ATP eluting at closed arrow; 5KtrA+KtrB P121C with ATP eluting at open arrow. Inset in c) 1pure KtrB F443C . 2KtrA+KtrB F443C with ADP eluting at closed arrow; 3KtrA+KtrB F443C with ADP eluting at open arrow; 4KtrA+KtrB F443C with ATP eluting at closed arrow; 5KtrA+- KtrB F443C with ATP eluting at open arrow. Inset in d) 1pure KtrA; 2pure KtrA; 3pure KtrB T444C ; 4KtrA+KtrB T444C with ADP eluting at closed arrow; 5KtrA+KtrB T444C with ATP eluting at closed arrow. Lower horizontal arrow indicates KtrA; horizontal arrow with M indicates KtrB monomer. Numerical values are included in S1 Data. (TIF) S12 Fig. SDS-PAGE of size-exclusion fractions of KtrB Δloop , KtrB G306S , and KtrB S309D .a) KtrB Δloop . Lanes 1 to 3 correspond to 1 ml fractions from chromatography of KtrA+KtrB Δloop with ADP (Fig 5A) collected around open arrow peak. Lanes 4 to 8 correspond to 1 ml fractions from chromatography of KtrA+KtrB Δloop with ATP (Fig 5A), starting at closed arrow and covering the open arrow peak. b) KtrB G306S . 1pure KtrA; 2KtrB G306S before size-exclusion; 3KtrA+ KtrB G306S with ADP at open arrow; 4KtrA+ KtrB G306S with ATP at open arrow. c) KtrB S309D . 1pure KtrA; 2KtrB S309D before size-exclusion; 3KtrA+ KtrB S309D with ADP at open arrow; 4KtrA+ KtrB S309D with ATP at open arrow. Lower horizontal arrow indicates KtrA; horizontal arrow with M indicates KtrB monomer. (TIF) S13 Fig. Western blots of fractions collected from float-up experiments. Westerns were probed with anti-KtrB antibody. Fractions 1 to 5 correspond to 40 μl fractions collected from low to high sucrose concentration with liposomes floating to the low density fractions. KtrB float-up distribution for a) wild type KtrB and KtrB Δloop reconstituted liposomes; b) wild type KtrB and KtrB R417K reconstituted liposomes or c) KtrAB R417K -ATP and -ADP reconstituted liposomes. The membrane protein is detected in the low sucrose concentration fractions, showing that it is associated with the liposomes. Horizontal arrows with M or D indicate KtrB monomer or dimer, respectively. (TIF) S1 Table. Averaged absolute 86 Rb + uptake levels (counts per minute) of functional assays depicted in Fig 1.Tables show the averaged raw data (before valinomycin normalization) collected for the KtrA ΔC B and KtrAB functional assays plotted in Fig 1A and 1B, respectively. The increased background values seen in the KtrA ΔC B functional assay are explained by the much Conformational Changes in KtrAB PLOS Biology | DOI:10.1371/journal.pbio.1002356 January 15, 2016 17 / 21 lower valinomycin values for the Control time course in KtrA ΔC B (~26,000 cpm) relative to the same values in KtrAB (~54,000 cpm). The valinomcyin values are obtained at the end of the time course and are used for normalization of the Rb + uptake, as a consequence normalized values for the KtrA ΔC B control are larger than for the KtrAB control. It is important to realize that in all biochemical and functional assays, we use an excess of RCK ring to favor the formation of the KtrAB complex, which has 1 dimer and 1 ring and minimize the formation of the complex with 1 RCK ring and 2 dimers of KtrB (see S4 Fig). In these circumstances, control liposomes were formed in the presence of KtrA ΔC and contain a large amount of free ring; in contrast, in liposomes reconstituted with KtrA ΔC B, a large fraction of the ring is involved in the formation of the complex. We do not know why KtrA ΔC appears to increase leakiness while full-length KtrA does not. The normalized data plotted in Fig 1 are slightly different from normalized values calculated with data shown in S1 Table. For Fig 1, we first normalized each individual time course using the corresponding valinomycin value and then calculated the average for each timepoint. In S1 Table, the values for a particular time point or valinomycin addition are the average of different sample preparations, reconstitutions, and time courses prior to normalization. (DOCX) S2 Table. Diffraction data and refinement statistics. Rmsd: root-mean-square deviation; values in parenthesis correspond to highest resolution bin. (DOCX) S3 Table. Log of likelihood values for molecular replacement analysis. - Incorrect packing between KtrB and the octameric ring. Molecular replacement functions for the wild-type KtrAB-ADP 8 Å diffraction dataset were calculated with PHASER and search models composed by KtrB homodimers from either the KtrAB-ATP or KtrA ΔC B-ADP structures and KtrA octameric rings adopting different conformations (after removing their C-terminal domains). The procedure involved sequential searches with the membrane protein and the gating ring; for many of these pairs, we were able to obtain molecular replacement solutions that display the expected packing between the membrane protein dimer and the gating ring. The values for the LLG function are a measure of how well the structural model agrees with the data. The pair formed by the KtrB homodimer and KtrA ΔC ring both from KtrA ΔC B-ADP has the highest LLG value (LLG = 515) and therefore appears to fit the data better than the other models. To evaluate the sensitivity of LLG parameter to small improvements in the KtrB model (in particular, the capacity of LLG to distinguish the goodness of fit between search models 6 and 7), we performed a series of tests. We first distorted the KtrB search model from KtrA ΔC B-ADP with a 10° tilt (see below explanation for this tilt) of the cytosolic halves of the M1D1 (residues 15 to 29) or M1D3 (residues 227–241) helices. This conformational change has not been observed in any of the existing structures, and so with this tilting the new KtrB search models are distorted (worsened) relative to KtrA ΔC B-ADP and KtrAB-ATP. Molecular replacement searches were performed with PHASER using the distorted KtrB dimers together with KtrA ΔC against the KtrAB-ADP 8 Å data. If the LLG parameter calculated in the search is sensitive to a distortion affecting 15 residues, then its values should be lower than 515, the value found for the final refined model of KtrA ΔC B-ADP; LLG for the model distorted at M1D1 was 506, and at M1D3 it was 503. For both cases, the packing of the different components was correct. This demonstrates that LLG is sensitive even to relatively small distortions of the search model. It also shows that even small changes in LLG (in these cases changes of less than 10%) indicate a model that is a worse or better fit to the data. We then altered KtrB of KtrAB-ATP. We changed the cytosolic half (residues128 to 140) of the M1D2 helix in so that it coincides with the helices of the KtrB search model in KtrA ΔC B-ADP. We called this new model KtrB improved . Conformational Changes in KtrAB PLOS Biology | DOI:10.1371/journal.pbio.1002356 January 15, 2016 18 / 21 We performed the inverse operation on the KtrB search model of KtrA ΔC B-ADP so that it resembles KtrAB-ATP and called it KtrB worse . These changes corresponded to a 10–11° tilt of the cytosolic ends of the M1D2 helices around a pivot point, residue 140. Once again, if LLG is sensitive to these changes, then molecular replacement searches using KtrB improved (together with KtrA ΔC ) against the KtrAB-ADP 8 Å data should show an increase in LLG relative to the unmodified model, while KtrB worse should result in a decrease in LLG relative to the unmodified model. KtrB improved LLG went up from 473 to 496 while for KtrB worse LLG went down from 515 to 496. Note also that the LLG value for KtrB improved is still lower than 515, the value obtained with the components of KtrA ΔC B; this shows that besides the difference in the M1D2 helices, there are many other small adjustments that occurred during refinement of KtrA ΔCB-ADP. These small adjustments improved the model and made it even more like KtrAB-ADP so that the LLG value is the highest. Overall, these experiments demonstrate how sensitive LLG in Phaser is to the goodness of fit of a search model to a crystal structure. Moreover, they demonstrate that the difference between 473 and 515 shown in the two bottom searches listed on S3 Table is significant, supporting our conclusion that the conformation of both the membrane protein and the RCK ring in full-length KtrAB-ADP is similar to the conformations observed in our KtrA ΔC B-ADP structure. (DOCX) Acknowledgments We are grateful for access to Proxima2 and Proxima1 at SOLEIL and to ID14-4/ID-29 at ESRF (through the Portuguese BAG), and thank the respective support staff. We thank Carol Harley for critical reading of the manuscript. Author Contributions Conceived and designed the experiments: AS JHMC. Performed the experiments: AS RSVP CMTD RR JHMC. Analyzed the data: AS RSVP CMTD RR JHMC. Wrote the paper: AS JHMC. References 1. Durell SR, Hao Y, Nakamura T, Bakker EP, Guy HR. Evolutionary relationship between K(+) channels and symporters. Biophys J. 1999 Aug; 77(2):775–88. PMID: 10423425 2. Corratge-Faillie C, Jabnoune M, Zimmermann S, Very AA, Fizames C, Sentenac H. Potassium and sodium transport in non-animal cells: the Trk/Ktr/HKT transporter family. Cell Mol Life Sci. 2010 Aug; 67 (15):2511–32. doi: 10.1007/s00018-010-0317-7 PMID: 20333436 3. Rhoads DB, Waters FB, Epstein W. Cation transport in Escherichia coli. VIII. Potassium transport mutants. J Gen Physiol. 1976 Mar; 67(3):325–41. PMID: 4578 4. Diskowski M, Mikusevic V, Stock C, Hanelt I. Functional diversity of the superfamily of K+ transporters to meet various requirements. Biol Chem. 2015 Apr 2. 5. Vieira-Pires RS, Szollosi A, Morais-Cabral JH. The structure of the KtrAB potassium transporter. Nature. 2013 Apr 18; 496(7445):323–8. doi: 10.1038/nature12055 PMID: 23598340 6. Holtmann G, Bakker EP, Uozumi N, Bremer E. KtrAB and KtrCD: two K+ uptake systems in Bacillus subtilis and their role in adaptation to hypertonicity. J Bacteriol. 2003 Feb; 185(4):1289–98. PMID: 12562800 7. Durell SR, Guy HR. Structural models of the KtrB, TrkH, and Trk1,2 symporters based on the structure of the KcsA K(+) channel. Biophys J. 1999 Aug; 77(2):789–807. PMID: 10423426 8. Kroning N, Willenborg M, Tholema N, Hanelt I, Schmid R, Bakker EP. ATP binding to the KTN/RCK subunit KtrA from the K+ -uptake system KtrAB of Vibrio alginolyticus: its role in the formation of the KtrAB complex and its requirement in vivo. J Biol Chem. 2007 May 11; 282(19):14018–27. PMID: 17344221 Conformational Changes in KtrAB PLOS Biology | DOI:10.1371/journal.pbio.1002356 January 15, 2016 19 / 21 9. Jiang Y, Pico A, Cadene M, Chait BT, MacKinnon R. Structure of the RCK domain from the E. coli K+ channel and demonstration of its presence in the human BK channel. Neuron. 2001 Mar; 29(3):593– 601. PMID: 11301020 10. Roosild TP, Miller S, Booth IR, Choe S. A mechanism of regulating transmembrane potassium flux through a ligand-mediated conformational switch. Cell. 2002 Jun 14; 109(6):781–91. PMID: 12086676 11. Albright RA, Ibar JL, Kim CU, Gruner SM, Morais-Cabral JH. The RCK domain of the KtrAB K+ transporter: multiple conformations of an octameric ring. Cell. 2006 Sep 22; 126(6):1147–59. PMID: 16990138 12. Jiang Y, Lee A, Chen J, Cadene M, Chait BT, MacKinnon R. The open pore conformation of potassium channels. Nature. 2002 May 30; 417(6888):523–6. PMID: 12037560 13. Jiang Y, Lee A, Chen J, Cadene M, Chait BT, MacKinnon R. Crystal structure and mechanism of a calcium-gated potassium channel. Nature. 2002 May 30; 417(6888):515–22. PMID: 12037559 14. Yuan P, Leonetti MD, Pico AR, Hsiung Y, MacKinnon R. Structure of the human BK channel Ca2+-activation apparatus at 3.0 A resolution. Science. 2010 Jul 9; 329(5988):182–6. doi: 10.1126/science. 1190414 PMID: 20508092 15. Yuan P, Leonetti MD, Hsiung Y, MacKinnon R. Open structure of the Ca2+ gating ring in the high-conductance Ca2+-activated K+ channel. Nature. 2012 Jan 5; 481(7379):94–7. 16. Wu Y, Yang Y, Ye S, Jiang Y. Structure of the gating ring from the human large-conductance Ca(2 +)-gated K(+) channel. Nature. 2010 Jul 15; 466(7304):393–7. doi: 10.1038/nature09252 PMID: 20574420 17. Kong C, Zeng W, Ye S, Chen L, Sauer DB, Lam Y, et al. Distinct gating mechanisms revealed by the structures of a multi-ligand gated K(+) channel. Elife. 2012; 1:e00184. doi: 10.7554/eLife.00184 PMID: 23240087 18. Cao Y, Pan Y, Huang H, Jin X, Levin EJ, Kloss B, et al. Gating of the TrkH ion channel by its associated RCK protein TrkA. Nature. 2013 Apr 18; 496(7445):317–22. doi: 10.1038/nature12056 PMID: 23598339 19. Durell SR, Bakker EP, Guy HR. Does the KdpA subunit from the high affinity K(+)-translocating P-type KDP-ATPase have a structure similar to that of K(+) channels? Biophys J. 2000 Jan; 78(1):188–99. PMID: 10620285 20. Cao Y, Jin X, Huang H, Derebe MG, Levin EJ, Kabaleeswaran V, et al. Crystal structure of a potassium ion transporter, TrkH. Nature. 2011 Mar 17; 471(7338):336–40. doi: 10.1038/nature09731 PMID: 21317882 21. Hanelt I, Lochte S, Sundermann L, Elbers K, Vor der Bruggen M, Bakker EP. Gain of function mutations in membrane region M2C2 of KtrB open a gate controlling K+ transport by the KtrAB system from Vibrio alginolyticus. J Biol Chem. 2010 Apr 2; 285(14):10318–27. doi: 10.1074/jbc.M109.089870 PMID: 20097755 22. Hanelt I, Wunnicke D, Muller-Trimbusch M, Vor der Bruggen M, Kraus I, Bakker EP, et al. Membrane region M2C2 in subunit KtrB of the K+ uptake system KtrAB from Vibrio alginolyticus forms a flexible gate controlling K+ flux: an electron paramagnetic resonance study. J Biol Chem. 2010 Sep 3; 285 (36):28210–9. doi: 10.1074/jbc.M110.139311 PMID: 20573964 23. Bai Y, Yang J, Zarrella TM, Zhang Y, Metzger DW, Bai G. Cyclic di-AMP impairs potassium uptake mediated by a cyclic di-AMP binding protein in Streptococcus pneumoniae. J Bacteriol. 2014 Feb; 196 (3):614–23. doi: 10.1128/JB.01041-13 PMID: 24272783 24. Kim H, Youn SJ, Kim SO, Ko J, Lee JO, Choi BS. Structural Studies of Potassium Transport Protein KtrA Regulator of Conductance of K+ (RCK) C Domain in Complex with Cyclic Diadenosine Monophosphate (c-di-AMP). J Biol Chem. 2015 Jun 26; 290(26):16393–402. doi: 10.1074/jbc.M115.641340 PMID: 25957408 25. Corrigan RM, Campeotto I, Jeganathan T, Roelofs KG, Lee VT, Grundling A. Systematic identification of conserved bacterial c-di-AMP receptor proteins. Proc Natl Acad Sci U S A. 2013 May 28; 110 (22):9084–9. doi: 10.1073/pnas.1300595110 PMID: 23671116 26. Nelson JW, Sudarsan N, Furukawa K, Weinberg Z, Wang JX, Breaker RR. Riboswitches in eubacteria sense the second messenger c-di-AMP. Nat Chem Biol. 2013 Dec; 9(12):834–9. doi: 10.1038/ nchembio.1363 PMID: 24141192 27. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959 May; 82(1):70–7. PMID: 13650640 28. Czerski L, Sanders CR. Thiol modification of diacylglycerol kinase: dependence upon site membrane disposition and reagent hydrophobicity. FEBS Lett. 2000 Apr 28; 472(2–3):225–9. PMID: 10788616 29. Vopel T, Kunzelmann S, Herrmann C. Nucleotide dependent cysteine reactivity of hGBP1 uncovers a domain movement during GTP hydrolysis. FEBS Lett. 2009 Jun 18; 583(12):1923–7. doi: 10.1016/j. febslet.2009.05.027 PMID: 19463820 Conformational Changes in KtrAB PLOS Biology | DOI:10.1371/journal.pbio.1002356 January 15, 2016 20 / 21 30. Kato N, Akai M, Zulkifli L, Matsuda N, Kato Y, Goshima S, et al. Role of positively charged amino acids in the M2D transmembrane helix of Ktr/Trk/HKT type cation transporters. Channels (Austin). 2007 MayJun; 1(3):161–71. 31. Zhang J, Xue F, Liu Y, Yang H, Wang X. The structural mechanism of the Cys-loop receptor desensitization. Mol Neurobiol. 2013 Aug; 48(1):97–108. doi: 10.1007/s12035-013-8420-z PMID: 23397136 32. Lester HA, Dibas MI, Dahan DS, Leite JF, Dougherty DA. Cys-loop receptors: new twists and turns. Trends Neurosci. 2004 Jun; 27(6):329–36. PMID: 15165737 33. Meyerson JR, Kumar J, Chittori S, Rao P, Pierson J, Bartesaghi A, et al. Structural mechanism of glutamate receptor activation and desensitization. Nature. 2014 Oct 16; 514(7522):328–34. doi: 10.1038/ nature13603 PMID: 25119039 34. Chaudhry C, Weston MC, Schuck P, Rosenmund C, Mayer ML. Stability of ligand-binding domain dimer assembly controls kainate receptor desensitization. EMBO J. 2009 May 20; 28(10):1518–30. doi: 10.1038/emboj.2009.86 PMID: 19339989 35. Ye S, Li Y, Chen L, Jiang Y. Crystal structures of a ligand-free MthK gating ring: insights into the ligand gating mechanism of K+ channels. Cell. 2006 Sep 22; 126(6):1161–73. PMID: 16990139 36. Li W, Aldrich RW. State-dependent block of BK channels by synthesized shaker ball peptides. J Gen Physiol. 2006 Oct; 128(4):423–41. PMID: 16966472 37. Liu Y, Holmgren M, Jurman ME, Yellen G. Gated access to the pore of a voltage-dependent K+ channel. Neuron. 1997 Jul; 19(1):175–84. PMID: 9247273 38. Holmgren M, Smith PL, Yellen G. Trapping of organic blockers by closing of voltage-dependent K+ channels: evidence for a trap door mechanism of activation gating. J Gen Physiol. 1997 May; 109 (5):527–35. PMID: 9154902 39. Rothberg BS, Shin KS, Phale PS, Yellen G. Voltage-controlled gating at the intracellular entrance to a hyperpolarization-activated cation channel. J Gen Physiol. 2002 Jan; 119(1):83–91. PMID: 11773240 40. Uysal S, Cuello LG, Cortes DM, Koide S, Kossiakoff AA, Perozo E. Mechanism of activation gating in the full-length KcsA K+ channel. Proc Natl Acad Sci U S A. 2011 Jul 19; 108(29):11896–9. doi: 10. 1073/pnas.1105112108 PMID: 21730186 41. Kabsch W. Xds. Acta Crystallogr D Biol Crystallogr. 2010 Feb; 66(Pt 2):125–32. doi: 10.1107/ S0907444909047337 PMID: 20124692 42. Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr. 2011 Apr; 67(Pt 4):235–42. doi: 10. 1107/S0907444910045749 PMID: 21460441 43. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr. 2010 Feb; 66(Pt 2):213–21. doi: 10.1107/S0907444909052925 PMID: 20124702 44. Brunger AT, Adams PD, Fromme P, Fromme R, Levitt M, Schroder GF. Improving the accuracy of macromolecular structure refinement at 7 A resolution. Structure. 2012 Jun 6; 20(6):957–66. doi: 10.1016/j. str.2012.04.020 PMID: 22681901 Conformational Changes in KtrAB PLOS Biology | DOI:10.1371/journal.pbio.1002356 January 15, 2016 21 / 21