Corrections

PERSPECTIVECorrection for “Integrating the invisible fabric of nature intofisheries management,” by Joseph Travis, Felicia C. Coleman,Peter J. Auster, Philippe M. Cury, James A. Estes, Jose Orensanz,Charles H. Peterson, Mary E. Power, Robert S. Steneck, andJ. Timothy Wootton, which appeared in issue 2, January 14,2014, of Proc Natl Acad Sci USA (111:581–584; first publishedDecember 23, 2013; 10.1073/pnas.1305853111).The authors note that on page 582, left column, first paragraph,

line 3, “10 metric tons (MT)” should instead appear as “10 millionmetric tons (MT).”

www.pnas.org/cgi/doi/10.1073/pnas.1402460111

BIOPHYSICS AND COMPUTATIONAL BIOLOGYCorrection for “Dynamics transitions at the outer vestibule ofthe KcsA potassium channel during gating,” by H. Raghuraman,Shahidul M. Islam, Soumi Mukherjee, Benoit Roux, and EduardoPerozo, which appeared in issue 5, February 4, 2014, of ProcNatl Acad Sci USA (111:1831–1836; first published January 15,2014; 10.1073/pnas.1314875111).The authors note that, due to a PNAS error, the author con-

tributions footnote appeared incorrectly. The corrected authorcontributions footnote appears below.

Author contributions: H.R. and E.P. designed research; H.R. performed research; H.R. andS.M. performed fluorescence measurements; S.M.I. and B.R. contributed the computa-tional analysis; H.R., S.M.I., S.M., B.R., and E.P. analyzed data; and H.R. and E.P. wrotethe paper.

www.pnas.org/cgi/doi/10.1073/pnas.1402205111

MEDICAL SCIENCESCorrection for “Blocking CD40-TRAF6 signaling is a therapeu-tic target in obesity-associated insulin resistance,” by AntoniosChatzigeorgiou, Tom Seijkens, Barbara Zarzycka, David Engel,Marjorie Poggi, Susan van den Berg, Sjoerd van den Berg, OliverSoehnlein, Holger Winkels, Linda Beckers, Dirk Lievens, AnnDriessen, Pascal Kusters, Erik Biessen, Ruben Garcia-Martin,Anne Klotzsche-von Ameln, Marion Gijbels, Randolph Noelle,Louis Boon, Tilman Hackeng, Klaus Schulte, Aimin Xu, GertVriend, Sander Nabuurs, Kyoung-Jin Chung, Ko Willems van Dijk,Patrick C. N. Rensen, Norbert Gerdes, Menno de Winther,Norman L. Block, Andrew V. Schally, Christian Weber, Stefan R.Bornstein, Gerry Nicolaes, Triantafyllos Chavakis, and EstherLutgens, which appeared in issue 7, February 18, 2014, of ProcNatl Acad Sci USA (111:2686–2691; first published February 3,2014; 10.1073/pnas.1400419111).The authors note that the author name Klaus Schulte should

instead appear as Klaus-Martin Schulte. The corrected authorline appears below. The online version has been corrected.

Antonios Chatzigeorgiou, Tom Seijkens, BarbaraZarzycka, David Engel, Marjorie Poggi, Susanvan den Berg, Sjoerd van den Berg, Oliver Soehnlein,Holger Winkels, Linda Beckers, Dirk Lievens, AnnDriessen, Pascal Kusters, Erik Biessen, RubenGarcia-Martin, Anne Klotzsche-von Ameln, MarionGijbels, Randolph Noelle, Louis Boon, Tilman Hackeng,Klaus-Martin Schulte, Aimin Xu, Gert Vriend, SanderNabuurs, Kyoung-Jin Chung, Ko Willems van Dijk,Patrick C. N. Rensen, Norbert Gerdes, Menno de Winther,Norman L. Block, Andrew V. Schally, Christian Weber,Stefan R. Bornstein, Gerry Nicolaes, TriantafyllosChavakis, and Esther Lutgens

www.pnas.org/cgi/doi/10.1073/pnas.1403231111

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PHYSIOLOGYCorrection for “Reversible DNA methylation regulates seasonalphotoperiodic time measurement,” by Tyler J. Stevenson andBrian J. Prendergast, which appeared in issue 41, October 8, 2013,of Proc Natl Acad Sci USA (110:16651–16656; first publishedSeptember 25, 2013; 10.1073/pnas.1310643110).The authors note the following: “Recent high throughput se-

quencing has indicated that an upstream region of the dio3promoter sequence in our paper was the result of a PCR fusionerror. The reverse primer located –140bp upstream of the startcodon was not specific to Siberian hamsters. As a result, the tran-scription factor binding site analysis and sodium bisulfite-treatedDNA sequence analyses in the original publication were incorrect.To correct this error, we have resequenced the dio3 proximalpromoter and conducted replications of the transcription factorbinding site analyses and sequencing of sodium bisulfite-treatedDNA, using primer sequences with confirmed specificity to Siberianhamster DNA. The corrected dio3 promoter sequence exhibitedgreater homology with mice and human dio3 promoter (revised

Fig. S1), a greater number of CpG sites and a higher CpG fre-quency (revised Fig. S2) than previously reported. Analysis ofsodium bisulfite-treated DNA in the acute LD-SD (Fig. 1F) andphotorefractory experiments (Fig. 3E) yielded results consistentwith the originally-published report: dio3 promoter DNA meth-ylation was reduced in SD (revised Fig. 1F) and increased in SD-R(revised Fig. 3E). Revised transcription factor binding site analyseshave also been performed (revised Table S1). See corrected TableS2 for primers used on sodium bisulfite treated DNA. In addition,the reverse primers for the sequencing and MSRE PCR reactions(Table S2) were originally listed in the incorrect (3′-5′) orientation;the correct orientation (5′-3′) now appears in the revised version ofTable S2.“We thank Drs. Hugues Dardente and David Hazlerigg for

their assistance in identifying these errors.”As a result of this error, Figs. 1 and 3 and their legends appeared

incorrectly. The figures and their corrected legends appear below.These errors do not affect the main conclusions of the article.

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Fig. 1. Short photoperiods inhibit reproduction and activate hypothalamic mRNA expression via epigenetic mechanisms. Acute transfer from LD to SD pho-toperiods caused gonadal regression (A), increased hypothalamic dio3 mRNA expression (B), and decreased hypothalamic dnmt1 and dnmt3b mRNA expression(C) after 8 wk. (D) Immunocytochemical localization of DNMT3b (DNMT3b-ir) in the hamster mediobasal hypothalamus (MBH). DNMT3b-ir was evidentthroughout the MBH and in the ependymal cell (EC) layer along the third ventricle (III). (E) Transfer from LD to SD reduced DNA methylation in the dio3 proximalpromoter, as measured using an MSRE assay. (F) Proportion of LD and SD hamsters in which no unmethylated DNA was detected at each of 17 CpG sites in thedio3 proximal promoter, as assessed by direct sequencing of sodium bisulfite-treated DNA. The abscissa (not to scale) depicts the 17 CpG sites from –249 to thestart codon of the dio3 proximal promoter. Averaging across the entire promoter region that was sequenced, evidence of unmethylation was evident on 15% ofCpG sites in LD (i.e., on 85% of CpG sites examined in LD hamsters, no detectable C-to-T bisulfite conversion occurred), whereas in SD, evidence of unmethylatedDNA was present on 42% of CpG sites (χ2 = 18.4, P < 0.002). All data in panels A–E are mean ± SEM. *P < 0.05, ***P < 0.005 vs. LD value.

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Fig. 3. Neuroendocrine refractoriness to SD reverses patterns of DNA methylation induced by acute SD exposure. (A) Acute (10 wk, SD) exposure to SDinduced gonadal regression, whereas prolonged exposure (42 wk, SD-R) triggered neuroendocrine refractoriness and gonadal recrudescence. Refractorinessin SD-R hamsters was characterized by a complete reversal of hypothalamic dio3 and dnmt3b mRNA expression (B and C) and by remethylation of DNA in thedio3 proximal promoter (D). (E) Proportion of LD, SD, and SD-R hamsters in which no unmethylated DNA was detected in each of 17 CpG sites in the dio3proximal promoter, as assessed by direct sequencing of sodium bisulfite DNA from the whole hypothalamus. The abscissa (not to scale) depicts the 17 CpGsites from –249 to the start codon of the dio3 proximal promoter. Averaging across the promoter region that was sequenced, evidence of unmethylation wasevident on 25% of CpG sites in LD hamsters, whereas in SD hamsters, evidence of unmethylation was present on 39% of CpG sites (χ2 = 5.08, P < 0.03). In SD-Rhamsters, methylation patterns returned to LD-like values, and evidence of unmethylation was detected on 29% of CpG sites examined (χ2 = 0.46, P > 0.40 vs.LD). Five of 17 sites (sites 16, 13, 12, 2, and 1) exhibited reversals in the pattern of methylation in SD-R hamsters. All data in panels A–D are mean ± SEM. *P <0.05; ***P < 0.005 vs. LD value.

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Dynamics transitions at the outer vestibule of the KcsApotassium channel during gatingH. Raghuraman, Shahidul M. Islam, Soumi Mukherjee, Benoit Roux, and Eduardo Perozo1

Department of Biochemistry and Molecular Biology, Center for Integrative Sciences, The University of Chicago, Chicago, IL 60637

Edited by Richard W. Aldrich, The University of Texas at Austin, Austin, TX, and approved December 13, 2013 (received for review September 9, 2013)

In K+ channels, the selectivity filter, pore helix, and outer vestibuleplay a crucial role in gating mechanisms. The outer vestibule is animportant structurally extended region of KcsA in which toxins,blockers, and metal ions bind and modulate the gating behavior ofK+ channels. Despite its functional significance, the gating-relatedstructural dynamics at the outer vestibule are not well understood.Under steady-state conditions, inactivating WT and noninactivat-ing E71A KcsA stabilize the nonconductive and conductive filterconformations upon opening the activation gate. Site-directedfluorescence polarization of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled outer vestibule residues shows that the outer vestibule ofopen/conductive conformation is highly dynamic compared withthe motional restriction experienced by the outer vestibule duringinactivation gating. A wavelength-selective fluorescence approachshows a change in hydration dynamics in inactivated and noninac-tivated conformations, and supports a possible role of restricted/boundwater molecules in C-type inactivation gating. Using a uniquerestrained ensemble simulation method, along with distance mea-surements by EPR, we show that, on average, the outer vestibuleundergoes a modest backbone conformational change during itstransition to various functional states, although the structural dy-namics of the outer vestibule are significantly altered during activa-tion and inactivation gating. Taken together, our results support therole of a hydrogen bond network behind the selectivity filter, side-chain conformational dynamics, and water molecules in the gatingmechanisms of K+ channels.

ion channel | EPR spectroscopy | REES | pulsed EPR

The functional behavior of K+ channels is defined by a seriesof structural rearrangements associated with the processes of

activation and inactivation gating (1–6). In response to a pro-longed stimulus and in the absence of an N-terminal inactivatingparticle, most K+ channels become nonconductive through a pro-cess known as C-type inactivation (7). This C-type inactivation iscrucial in controlling the firing patterns in excitable cells and isfundamental in determining the length and frequency of the car-diac action potential (8). C-type inactivation is inhibited by highextracellular K+ (9, 10), and the blocker tetraethylammonium(TEA) (11) can also be slowed down in the presence of permeantions with a long residence time in the selectivity filter (Rb+, Cs+,and NH4+) (10).The prokaryotic pH-gated K+ channel KcsA shares most of

the mechanistic properties of C-type inactivation in voltage-dependent K+ channels (5, 6, 12–16). Recent crystal structuresof open/inactivated KcsA reveal that there is a remarkable corre-lation between the degree of opening at the activation gate and theconformation and ion occupancy of the selectivity filter (5). InKcsA, the selectivity filter is stabilized by a hydrogen bond net-work, with key interactions between residues Glu71, Asp80, andTrp67 and a bound water molecule (17). Disrupting this hydrogenbond network favors the conductive conformation of the selectivityfilter (12, 13, 15).Early electrophysiological experiments have suggested that the

outer vestibule (around T449 residue in Shaker and Y82 residuein KcsA) undergoes significant conformational rearrangementduring C-type inactivation gating (16, 18, 19). However, com-parison of the WT KcsA crystal structure, where the filter is in its

conductive conformation, with either the structure obtained withlow K+ (collapsed filter) (17) or the crystal structure of open-inactivated KcsA with maximum opening (inactivated filter) (5)does not show major conformational changes in the outer ves-tibule that would explain these results (Fig. 1A). We have sug-gested that this apparent discrepancy can be understood if wetake into consideration the potential differences in the dynamicbehavior of the outer vestibule changes as the K+ channelundergoes its gating cycle (16).We have probed the gating-induced structural dynamics at the

outer vestibule of KcsA using site-directed fluorescence and site-directed spin labeling and pulsed EPR approaches in combina-tion with a recently developed computational method, restrainedensemble (RE) simulations. RE simulation was used to constrainthe outer vestibule using experimentally derived distance histo-grams in different functional states (closed, open/inactivated,and open/conductive) and to monitor the extent of backboneconformational changes during gating. To this end, we tookadvantage of our ability to stabilize both the open/conductive(E71A mutant) and the open/inactivated (WT) conformationsof KcsA upon opening the activation gate under steady-stateconditions (Fig. 1B).Our data show that the outer vestibule in the open/conductive

conformation is highly dynamic. In addition, the red edge exci-tation shift (REES) points to a change in hydration dynamicsbetween conductive and nonconductive outer vestibule con-formations, suggesting a role of restricted water molecules inC-type inactivation gating. We suggest that, on average, thebackbone conformation of the outer vestibule does not changesignificantly between different functional states but that localdynamics change significantly, underlining the importance of thehydrogen bond network behind the selectivity filter and the mi-croscopic observables (e.g., dynamics of hydration) in K+ channelgating and C-type inactivation.

Significance

C-type inactivation gating in K+ channels plays an importantrole in controlling the firing patterns of excitable cells and isfundamental in determining the length and frequency of thecardiac action potential. At a molecular level, toxins, blockers,and metal ions bind to the outer vestibule and modulate thefunctional behavior of K+ channels. Using KcsA, we show thatthe shuttling between the inactivated and conductive states ofK+ channels is accompanied by changes in local outer vestibuledynamics, in the absence of large conformational changes. Thealtered structural and hydration dynamics of the outer vesti-bule appear to be likely and important modulators of selec-tivity filter gating transitions in K+ channels.

Author contributions: H.R. and E.P. designed research; H.R. performed research; H.R. andS.M. performed fluorescence measurements; S.M.I. and B.R. contributed new reagents/analytic tools; H.R., S.M.I., S.M., B.R., and E.P. analyzed data; and H.R. and E.P. wrote thepaper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: eperozo@uchicago.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1314875111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1314875111 PNAS | February 4, 2014 | vol. 111 | no. 5 | 1831–1836

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ResultsChanges in Outer Vestibule Conformational Dynamics. Cysteinemutants corresponding to KcsA outer vestibule residues weregenerated at positions 51–60 and 81–86 in either WT (inacti-vating) or E71A (noninactivating) background. Of these, resi-dues 51, 53, 55, 59, and 83 did not express well or were notbiochemically stable, and therefore were not investigated. Fig.1C shows the gating-induced changes in EPR spectra of spin-labeled mutants of WT and E71A KcsA reconstituted in mem-branes. As expected from the small structural differences seen inthe crystal structures (5, 19) (Fig. 1A), there was little or nochange in the overall EPR spectral line shape (Fig. 1C, Left),suggesting the outer vestibule is structurally stable upon innergate opening and subsequent inactivation. However, when in-activation is abrogated via the E71A mutation, an increase inoverall spectral mobility can be seen in a few positions. In par-ticular, the noninactivating Y82-SL/E71A construct shows a con-siderable reduction in spin-spin coupling (moving away from thepermeation path) and a large increase in probe mobility (Fig. 1C,

Right). A direct interpretation of this result would suggest theexistence of regions of high dynamics in the outer vestibule as thechannel populates the conductive state (18). However, the factthat these changes are not fully reversible (Fig. S1) might suggestadditional local effects. In general, EPR is sensitive to nanosec-ond-to-microsecond time scale motions, whereas fluorescencepolarization is sensitive to picosecond-to-nanosecond motions(20). Because the individual subunit chain that forms the outervestibule in KcsA is in an unusually extended conformation, it ispossible that some of the changes in local dynamics are notproperly monitored in the EPR time scale. We therefore focusedon site-directed fluorescence approaches using the environment-sensitive fluorophore NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) (21)to investigate the nature of the outer vestibule dynamics in open/inactivated and open/conductive KcsA further.Fig. 2 shows the steady-state polarization of KcsA NBD ana-

logs, and its overall changes, under conditions that promote ei-ther the inactivated (WT) or conductive (E71A mutant) of theselectivity filter. The polarization values generally indicate thatthe NBD rotational mobility is representative of motionally re-stricted environments in the outer vestibule of closed KcsA.However, triggering the opening of the inner bundle gate gen-erates a series of changes in outer vestibule dynamics dependingon whether the selectivity filter populates the conductive or theinactivated conformation. In open/inactivated KcsA (WT, lowpH), the polarization values are significantly higher for most ofthe residues (Gly53, Ala54, Gln58, Leu81, and Val84) than forthose in the closed state, indicating that the residues in the outervestibule are more motionally restricted once inactivated (Fig. 2,Left). When the same experiment is repeated on the non-inactivating E71A KcsA background, some of the outer vestibuleresidues undergo increased rotational motion (Arg52 and Gly53in the outer loop and Leu81, Tyr82, and Leu86 in the inner loopabove the selectivity filter), reflecting an increased conforma-tional flexibility in the open/conductive conformation. It shouldbe noted that we do not find significant differences in the po-larization values in the closed states of WT and E71A KcsA formost of the outer vestibule residues (Fig. S2). However, signifi-cant differences in the polarization values do arise upon openingthe lower gate of the channel.Mapping the differences in polarization between the inactivated

and conductive conformations illustrates the differential dynamicsin the outer vestibule of KcsA during selectivity filter gating (Fig.2, Bottom). Both EPR (Fig. 1C, Right) and fluorescence polari-zation experiments demonstrate that the mobility of Tyr82 issimilar during inactivation gating but is significantly increased inY82-SL/E71A and Y82-NBD/E71A constructs. These differentialdynamics might be responsible for our recent observation (16) thatCd2+ bridges promote the rate of inactivation of Y82C KcsA buthave no effect on the noninactivating E71A background. Thegating-induced changes in dynamics observed for the E71A/Y82-NBD construct are fully reversible, with macroscopic currentscharacteristic of E71A KcsA (Fig. S1). These results could beattributed to the perturbed hydrogen bond network between thepore helix and the selectivity filter, and we propose that thehigher dynamics of the outer vestibule are a characteristic fea-ture of the open/conductive conformation of K+ channels.

Changes in Hydration Dynamics During Gating. REES representsa unique and powerful approach that can be used to directlymonitor the environment-induced restriction and dynamicsaround a fluorophore in a complex biological system (see refs. 22and 23 for reviews and SI Materials and Methods for details).Because REES provides information on the relative rates ofwater relaxation dynamics (24, 25), it is sensitive to changes inlocal hydration dynamics.Fig. 3 shows the magnitude of REES for NBD-labeled

mutants in both closed and open states under steady-state con-ditions, along with ΔREES (closed open) mapped in structures,for WT and E71A KcsA. In general, the outer vestibule of KcsAexhibits significant REES, an indication of a motionally restricted

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Fig. 1. Comparison of outer vestibule conformation in KcsA structures withconductive and collapsed/inactivated filters. (A) High-K+ KcsA structure[Protein Data Bank (PDB) ID code 1K4C; yellow] is compared with a low-K+

KcsA structure (PDB ID code 1K4D; blue) in the closed state (Left) and open/inactivated conformation (PDB ID code 3F5W; green) (Right). The outervestibule residues are depicted as red spheres, and relevant residues arelabeled. (B) Schematic representation of typical macroscopic currents elicitedby pH-jump experiments in WT (inactivating) and E71A (noninactivating)KcsA channels at a depolarizing membrane potential is shown. Conditionsthat stabilize the closed, open/inactivated, and open/conductive con-formations at the steady state are indicated with a black circle. (C) Effect ofopening the lower gate on the mobility of spin-labeled outer vestibuleresidues in palmitoyloleoylphosphatidyl choline/palmitoyloleoylphosphatidylglycerol (POPC/POPG) (3:1, moles/moles) reconstituted WT (Left) and non-inactivating mutant E71A (Right) backgrounds for the closed (pH 7, red) andopen (pH 4, black) states of KcsA, as determined by continuous wave (CW)EPR. The spectra shown are amplitude-normalized. Details are provided in SIMaterials and Methods.

1832 | www.pnas.org/cgi/doi/10.1073/pnas.1314875111 Raghuraman et al.

environment. Furthermore, the magnitude of REES (0–6 nm)suggests that there is a considerable difference in the relaxationof solvent molecules (dynamics of hydration) in different posi-tions of the outer vestibule of KcsA. It has been shown thata change of a few nanometers in REES is significant, and theobserved changes in the magnitude of REES (0–6 nm) in ourcase fall within the range of expected REES changes (∼5 nm)associated with changes from fully restricted to freely relaxingenvironments (24, 25). It should be mentioned that REES doesnot generally provide a quantitative estimate of the water re-laxation dynamics because the magnitude of REES is not alwayslinear and is dependent on the given system under investigation(22). In WT KcsA, the solvent relaxation rate is considerablyslower around most of the residues during inactivation gating(residues Arg52, Gly53, Ala54, Gln58, and Ile60 in the outerloop and Leu81, Tyr82, and Val84 in the inner loop). This sug-gests that in the outer vestibule of KcsA, the dynamics of hy-dration become slower during inactivation gating. In contrast,solvent relaxation is considerably faster in the outer vestibule ofchannels that remain conductive and do not undergo inactivation(Fig. 3, Right). It is interesting to note that these gating-relatedchanges in hydration dynamics are not solely due to changes inthe polarity of the local microenvironment, because the outer

vestibule residues experience a relatively nonpolar environment inboth these states upon gating (Fig. S3). Mapping of ΔREES high-lights the changes in hydration dynamics between the open/inacti-vated and open/conductive outer vestibules of KcsA. Residuessurrounding the central pore offer a considerable motional re-striction to solvent relaxation dynamics during inactivation gating.These results clearly show that not only is the outer vestibule ofopen, conductive conformation highly dynamic (Fig. 2) but thatthe microenvironment associated with the outer vestibule displaysfaster relaxation. In addition, these results imply a role of bound(restricted) water molecules in inactivation gating (26), and thebound/restricted water could possibly stabilize the inactivatedfilter long enough to affect K+ ion permeation (26, 27).

Conformational Changes at the Outer Vestibule of KcsA. The resultsof the mobility and hydration dynamics suggest that the outervestibule of KcsA displays different dynamic behavior in theopen/inactivated and open/conductive states. This is in agree-ment with recently reported metal ion binding experiments (16).To probe the changes in structural dynamics further, we mea-sured distances between the symmetrical diagonal subunits ofKcsA under conditions that stabilize the conductive and inacti-vated filter conformations. Using tandem dimer (TD) constructs(28), long distances (∼>20 Å) were measured using doubleelectron-electron resonance (DEER) spectroscopy (29), whereasshort distances (∼<20 Å) were monitored using continuous waveEPR dipolar broadening experiments.Outer vestibule distances and distance histograms from inac-

tivating (WT) and noninactivating (E71A) KcsA are shown inFigs. S4 and S5. The analysis of conformational changes in theouter vestibule in different functional states of KcsA is compli-cated due to broad and heterogeneous distance distributions.Nevertheless, mapping the distance differences between closedand open states from the predominant distance peak (Fig. S6)suggests that residues surrounding the central pore tend to comecloser to each other in the open/inactivated state, in line withearlier metal ion binding experiments (16). However, this con-stricting transition does not seem to be present in the open/noninactivating (conductive) conformation (Fig. S6), as reportedrecently (30).Although there is a considerable change in intersubunit dis-

tances as KcsA transitions between the inactivated and conduc-tive conformations, it is difficult to interpret these changes at thebackbone level due to broad and heterogeneous distance dis-tributions. We have used a recently developed molecular dy-namics simulation technique, the RE method (31, 32), to evaluatethe degree of overall motion in the outer vestibule using the ex-perimentally derived distance histograms (Figs. S7 and S8) obtainedfrom DEER distance determinations. In the RE method, interspindistance distribution histograms calculated from multiple spin labelcopies in a molecular dynamics simulation are forced to matchthose obtained from experimentally derived distance histograms viaa global ensemble-based energy restraint. The closed/conductive[Protein Data Bank (PDB) ID code 1K4C], open/inactivated (PDBID code 3F5W), and “flipped” E71A (PDB ID code 2ATK) con-formations of KcsA were used as templates in all RE simulations. Arepresentative KcsA structure with multiple copies of spin labelsattached to positions 57 and 86 in the outer vestibule is shown inFig. 4A. The final models for the outer vestibule, obtained using theREmethod and several refinement cycles, for all three structures ofKcsA are shown in Fig. 4B, along with the respective changes inrmsd (for residues 51–88) as a function of refinement cycles (Fig.4C). Our calculations suggest that there are no major static con-formational changes in the outer vestibule of different functionalstates of KcsA compared with the starting template structures (Fig.4B). The change in rmsd for the backbone atoms is modest and isin the range of ∼0.4–1.0 Å (Fig. 4C and Table S1). It is interestingto note that even for the flipped E71A starting structure, whichdiffers significantly from other starting conformations, changesin rmsd do not exceed ∼1 Å. These results suggest that all struc-tures are physiologically relevant and the modest conformational

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Fig. 2. Outer vestibule of the open/noninactivating conformation is highlydynamic. (Top) Steady-state polarization of NBD fluorescence measured forNBD-labeled outer vestibule residues of full-length WT (Left) and E71A(Right) KcsA reconstituted in POPC/POPG (3:1, moles/moles) liposomes.Measurements at pH 7.5 and pH 4 correspond to the closed and open statesof the channel, respectively. The excitation wavelength used was 480 nm;emission was monitored at 535 nm in all cases. The difference in polarizationvalues (ΔP) represent mean ± SE of three independent measurements. TheΔPs (Middle) are shown and were mapped on the crystal structure of KcsA(1K4C) to highlight the changes in dynamics between the different func-tional states of KcsA (Bottom) upon gating. Details are provided in SIMaterials and Methods.

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change in the outer vestibule seen in several crystal structures ofKcsA might not be a Fab-mediated conformation. Further, thisclearly suggests that the side-chain conformational dynamics atthe KcsA outer vestibule might be responsible for the functionaldifferences observed during inactivation and noninactivationgating (12, 16).

DiscussionThere is now ample evidence suggesting that in K+ channels, theselectivity filter and its support structures (the pore helix and theouter vestibule) play a key role in gating, both at the slow (C-typeinactivation) and fast (single-channel flicker) time scales (3, 5,12, 13, 15, 16, 18, 19, 33–36). Indeed, strong experimental evi-dence points to the hydrogen bond network behind the selectivityfilter as a driver of the inactivation process (12, 13, 15).The outer vestibule of KcsA is an important unstructured re-

gion in which toxins (37), blockers (35, 38), and metal ions (16)bind and modulate the gating behavior of K+ channels. Severalresults suggest a dynamic structural rearrangement in the outervestibule during C-type inactivation gating in KcsA and ShakerK+ channels (16, 18, 19). However, despite its functional im-portance, the outer vestibule does not show appreciable con-formational changes in several crystal structures of KcsA trappedin various functional states (5, 17). Because the high-resolution

KcsA crystal structures were obtained using Fab that binds at theouter mouth of the pore and slows down the rate of inactivation(12), it is reasonable to speculate that Fab binding limits thefunctionally relevant conformational changes. We have lookedinto this issue by monitoring the structural dynamics of theouter vestibule in KcsA under conditions that stabilize differentfunctional conformations.Our results show that the conformational dynamics of the

outer vestibule change according to the functional state of thechannel. During inactivation gating, the mobility of the outervestibule is restricted, whereas the outer vestibule is highly dy-namic in the open/conductive conformation of KcsA (Fig. 2).This is consistent with earlier findings on the Shaker K+ channel,which indicate that the C-type–inactivated state is more orderedthan the noninactivated state (39). In particular, the spectro-scopic probes (nitroxide spin label and NBD fluorophore) at-tached to Y82C KcsA show a significant increase in dynamics asthe lower gate opens and the selectivity filter remains in theconductive conformation (E71A mutant). Further, unlike in theinactivated conformation (16), the average distance obtainedbetween spin labels in the TD Y82C KcsA changes from 12 Å inthe closed state to ∼17 Å in the noninactivating E71A mutant,indicating that the selectivity filter appears to be more plastic inthe open/conductive conformation (Fig. S5). This is supportiveof the alternate conformations of the Y82 side chain in the outervestibule, as seen in the flipped structure of E71A (12). In thecrystal structures of WT KcsA and flipped E71A KcsA, thedifference in the distance between the Cα-Cα and OH-OH atomsof Tyr82 residues in diagonally symmetrical subunits increases by∼0.8 Å and ∼4.3 Å, respectively. The excursion between twoorientations of the Tyr82 side chain might therefore be re-sponsible for the ∼50-fold lower extracellular TEA affinity ob-served in E71A KcsA (38) and for the inability of cadmium ions

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Fig. 3. Solvent relaxation dynamics probed by the REES. REES of NBD-la-beled outer vestibule residues of full-length WT (Left) and E71A (Right) KcsAreconstituted in POPC/POPG (3:1, moles/moles) liposomes. Measurements atpH 7.5 and pH 4 correspond to the closed and open states of the channel.(Top) Change in the emission maximum (in nanometers) as a function ofchanging the excitation wavelength from 465 to 515 nm is shown as theREES. The error is typically ±0.5 nm of the reported values. The difference inREES values (ΔREES) (Middle) are shown and were mapped on the crystalstructure of KcsA to highlight the changes in solvent relaxation dynamicsbetween different functional states of KcsA (Bottom) upon gating. Detailsare provided in SI Materials and Methods.

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Fig. 4. “All-atom” RE simulations. (A) Side view (Left) and top view (Right)of the WT KcsA tetramer (PDB ID code 1K4C) with multiple copies of the spinlabel (shown as sticks) attached in silico to positions 57 and 86. The potas-sium ion (K+) is shown as magenta spheres. (B) Backbone conformationscorresponding to residues 51–88 representative of closed (red), open/inac-tivated (blue), and conductive (flipped E71A, green) conformations derivedafter RE simulations using the respective experimentally derived histogramsfor the closed, open/inactivated, and open/conductive states of KcsA. (C)Change in rmsd as a function of refinement cycle index during RE simu-lations for closed (red), open/inactivated (blue), and conductive (flippedE71A, green) states of KcsA with respect to the template structures used.Details are provided in SI Materials and Methods.

1834 | www.pnas.org/cgi/doi/10.1073/pnas.1314875111 Raghuraman et al.

to form metal bridges between adjacent subunits in the open/conductive conformation of Y82C KcsA (16). Based on theseresults, it is tempting to suggest that the Y82 side-chain con-formation observed in the flipped E71A structure, possibly dueto increased outer vestibule dynamics, could be a characteristicfeature in the open/conductive conformation of KcsA, especiallywhen the strength of the interaction between the pore helix andthe outer mouth is disrupted.Hydration and dynamics play an important role in lipid–pro-

tein interactions (21) and ion channel selectivity (40) becauseprotein fluctuations, slow solvation, and the dynamics of hydratingwater are all intrinsically related (41). It has been shown thatheterogeneity in the dynamics of water interactions is coupled todifferential time scales of hydrogen bond formation and breaking(42). Using a wavelength-selective fluorescence approach (REES),we showed that entry to the inactivated state is associated with thepresence of restricted/bound water molecules in the outer vestibuleof KcsA (Fig. 3). These restricted/bound water molecules mightpotentially act as hydrogen bonding ligands with the residues as-sociated with the selectivity filter, affecting filter dynamics and ki-netics of entry and exit from the inactivated state (26, 27).It is interesting to note that three crystallographic water mole-

cules are present in the nonconducting filter conformation (1K4D)behind the selectivity filter, whereas only one buried ordered watermolecule is observed in the crystal structure of the conductive filter(1K4C). Very recently, using a long molecular dynamics simulation(∼17 μs), it has been shown that the recovery from C-type in-activation is directly controlled by these buried ordered watermolecules (26), thus making these restricted/bound water mole-cules a vital part of the protein structure when the selectivity filteris trapped in the nonconductive conformation. Additional evi-dence of the presence of bound water molecules comes from

a recent NMR study (27), which shows that the carbonyl group ofV76 seems to form hydrogen bonds with the water molecule in theselectivity filter only in the inactivated state. In contrast, we ob-served a relatively fast solvent relaxation in the open/conductivestate, suggesting that the water molecules in the outer vestibule andbehind the selectivity filter might be rapidly exchanging with free/bulk water. These increased dynamics of hydration are consistentwith the dynamic properties of the open/conductive outer vestibule,as seen in the E71A mutant (Fig. 2). The nature of differentialhydration dynamics in the outer vestibule of different functionalstates of KcsA support a role for water molecules in selectivityfilter gating of K+ channels, and could be relevant in un-derstanding the interplay between ions, the selectivity filter, andthe resulting distinct gating fluctuations (26, 36, 43).RE simulations, along with distance measurements, support

modest backbone conformational changes in the outer vestibuleof KcsA in the open/conductive and open/inactivated states. Thisreiterates the role of side-chain conformational dynamics inmodulating the gating mechanisms in K+ channels. Importantly,such conformational changes in the side chains of several keyresidues, such as Trp67, Asp80, and Tyr82, are observed in theflipped E71A structure (12). The outward “flipping” of theAsp80 side chain relative to its position in WT KcsA has beenobserved in flipped E71A [both high K+ (12) and low K+ (33)]and other Glu71 mutants that exhibit substantial loss of C-typeinactivation (36), as well as in the Kir 3.1 channel (44).We propose a model (Fig. 5) that shows key differences as-

sociated with the outer vestibule in open/inactivated and open/conductive conformations of KcsA and their likely consequencesin gating mechanisms. The outer vestibule is highly dynamic inchannels that do not undergo inactivation, which is accompaniedby increased hydration dynamics. In contrast, the outer vestibuleof the inactivated state is motionally restricted, and the re-stricted/bound water molecules might play a crucial role inmodulating the extent of inactivation. This might involve bindingof water molecules to selectivity filter residues in the inactivatedstate, most notably the carbonyl group of V76 when it flips awayfrom the conduction pathway (5, 26, 27, 36). Depending on therelative water relaxation dynamics, the exchange between thebound and free water might therefore be affected, which, in turn,would affect the organization and occupancy of water molecule(s)behind the selectivity filter. The conformational change of theouter vestibule, on average, is modest when the channel transitsbetween different functional states, and the constriction of thepore is possible only in the inactivated state depending on thepresence of external ligands, such as metal ions (16). In the open/conductive conformation, the concerted changes due to increaseddynamics and flipping of Asp80 and Tyr82 might not allow theouter mouth to constrict the pore, thereby eliminating inac-tivation by preventing the selectivity filter from collapsing. Overall,this model highlights the importance of the hydrogen bond net-work behind the selectivity filter, side-chain conformational dy-namics, and microscopic observables (e.g., water molecules) in thegating mechanisms of K+ channels.

Materials and MethodsWe have used pQE32-KcsA construct in all cases. Tandem dimer constructs ofKcsA were used for distance measurements. Detailed information regardingcloning, mutagenesis, channel biochemistry, generation of tandem KcsAconstructs, liposome patch-clamp, fluorescence and EPR spectroscopy, andDEER and RE simulations can be found in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank F. Bezanilla (The University of Chicago) forproviding access to the Photon Technology Instruments steady-state spectro-fluorometer. This work was supported in part by National Institutes ofHealth Grant R01-GM57846 (to E.P.).

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H+

H+

H+

H+

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free waterrestricted/bound water

+K ion

V76

Fig. 5. Gating-induced structural dynamics at the outer vestibule of KcsA.Schematic representations of the events associated with the outer vestibulein open/inactivated (Left) and open/conductive (Right) conformations ofKcsA are shown. The diagonally symmetrical subunits are shown for clarity.(Right) Outer vestibule is highly dynamic in open/conductive conformations,as depicted by the wiggly nature of the outer vestibule. Further, upongating, the hydration dynamics are faster in the open/conductive confor-mation, which is indicative of the increased ratio of bulk/free (blue spheres)vs. restricted/bound (purple spheres) water molecules. (Left) In contrast, theopening of the activation gate imposes motional restriction on the outervestibule in channels that undergo inactivation. This is accompanied by in-creased restricted/bound water molecules that might play an important rolein further stabilizing the inactivated/collapsed (26, 27) filter by possiblyinteracting with selectivity filter residues (V76 is shown as an example). Theexchange rate of water molecules in the selectivity filter and the outervestibule is shown as curved arrows. (Left) Tendency of the side chains tocome closer during inactivation gating is depicted as yellow arrows. Detailsare provided in Discussion.

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