© 2013 Nature America, Inc. All rights reserved. hydrophilic pore for water permeation. Specificity for involves water Specificity for pore permeation. water hydrophilic a form to barrel, a in as another transmem one against six pack that of helices brane consists monomer forms Each water. monomer for pore each own its which in tetramers ways are different AQPs in Structurally, regulated being and characteristics meability per substrate unique possessing isoform each manner, with specific across molecules water membranes. of Humansflux differentiallythe expressfacilitate 13 that AQP isoformslife, of in a tissue-kingdoms Ca intracellular in changes to response dynamic a with proteins target its of function the modulate rapidly transducer can that signal efficient an as acts CaM way,low-Ca this under In conditions. interaction constitutive a for allowing thus state, Ca its in targets cellular its of many bind to able is CaM addition, In substrates. of set diverse a to binding for allows ibility supports CaM’s Ca change conformational This proteins. target recognizes that pocket Ca upon binding rearrangements domain linker Ca C-terminal Ca cytoplasmic to response in by CaM modulated Correspondence should Correspondence be addressed to J.E.H. ( Irvine, Irvine, California, USA. 1 nels family of Ca neuronal voltage-gatedand ion channels cardiac the as such channels, and transporters as act that teins pro Many membrane of proteins. of hundreds function the regulate Ca in several messenger ondary Ca a is CaM the allosterically function. aquaporin-0 membrane membrane correspondingly Calmodulin J Douglas Tobias Steve L Reichow Ca Allosteric mechanism of water-channel gating by nature structural & molecular biology molecular & structural nature Received 4 April; accepted 5 June; published online 28 July 2013; Howard Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA. Aquaporins (AQPs) are a family of water channels, found in all all in found channels, water of family a are (AQPs) Aquaporins and N- of composed protein bilobed (16.7-kDa) small a is CaM

8– fully 1 2+ 0 , , gap junctions

assembled

Molecular –calmodulin

2+ proteins channels

2+ (CaM)

(AQP0, -release channels -release 16– closing 2+ -binding protein that functions as a ubiquitous sec ubiquitous a as functions that protein -binding -binding domains connected by an internal flexible flexible internal an by connected domains -binding 1

9 modulates 2+ . The N- and C-terminal lobes undergo structural structural undergo lobes . The N- and C-terminal

1 -dependent -dependent recognition mechanism, and its flex is

3

, channel,

has

4

, , James E Hall

dynamics 11–

and Bos Bos taurus the , , Daniel M Clemens a

universal 1

remained 3 3

Department Department of Chemistry, University of California, Irvine, Irvine, California, USA. cytoplasmic and water the aquaporin channels transporters.

their

5– into 2+

and 7 2+ signaling pathways to dynamically pathways to dynamically signaling ) , transient receptor potential , chan receptor transient potential

regulatory in to expose a to hydrophobic binding expose

1– function.

how elusive.

functional 4 complex , , the receptorryanodine (RyR) 2

& Tamir Gonen [email protected] gate

However, CaM

In

of This

protein regulates

2

with AQP0. this 2+ ,

4 mutation advance online publication online advance , , J FreitesAlfredo

2+ levels key

) ) or T.G. (

understanding fluctuations.

study, CaM,

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that 2+ Our 20

do multimeric -free (apo) (apo) -free , 2

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using [email protected] 1 1 communicates

. 14 mechanistic ,

1 determined 5

are

protein EM 2 2+ 2

------

reveal . of

to channels

the ous structures of CaM bound to fragments of membrane proteins proteins membrane of fragments to bound CaM of structures ous numer Instead, CaM. with complex in channel protein membrane full-length any for models structural no currently are there biology, understood. poorly is occurs permeation this by which mechanism water of inhibition the is AQP0 to binding stoichiometry simultaneously by fashion to helices two result in AQP0 an 2:1 C-terminal (AQP0/CaM) overall noncanonical a in interacts Ca AQP0, of case the However, in helix. amphipathic single Ca (refs. AQP0 of domain C-terminal cytoplasmic Ca a through junctions channel by modulated directly is CaM permeability whose and lens eye malian regulation. channel for crucial often are domains these and domains, C-terminal and N- cytoplasmic their in greatly AQPs vary of different structure sequences primary the architecture, pore and transmembrane this share AQPs all Although vestibule. protons of passage the prevent to used site proton-exclusion the form to dues resi asparagine of pair a contribute (NPA)motifs Asn-Pro-Ala two molecules larger to but barrier is a for through to water steric pass is enough large which Å, ~3 of diameter a to pore water the constricts CSI vestibule. lular at extracel the located I site (CSI)) (constriction filter selectivity the 3

elucidate , , Karin L Németh-Cahalan

is

Despite the importance of CaM regulation in membrane protein protein membrane in regulation CaM of importance the Despite mam the in expressed exclusively is that channel a water AQP0 is

2+ model

how structural

important –CaM recognizes and binds its targets by wrapping around a around wrapping by targets its binds and recognizes –CaM the 25 the 0 2 , 2 Department Department of Physiology and Biophysics, University of California,

27– 6

CaM by pseudoatomic ). . . AQP0 a serves dual in function the lens by as acting a water presence 31–

provides 3 23

0 facilitating how 3 or as an adhesive protein mediating cell-cell adhesive adhesive cell-cell mediating protein adhesive an as or , 3 2 2+

3 . Regulation of water permeability by CaM is achieved achieved is by CaM permeability of water . Regulation 4 binding mechanisms 5 . The pore then expands to form the cytoplasmic cytoplasmic the form to expands then pore The .

-dependent interaction between Ca between interaction -dependent for . The functional consequence of Ca of consequence functional The .

this

of

2 controlling new 3

. Further down the pore, roughly at its center, its at roughly pore, the down Further .

signaling calcium

inhibits 4

cooperativity These These authors contributed equally to this work.

structure insight,

driving

to

AQP0 protein the

only 2 its

of , , Matthias Heyden

activity

CaM

molecular full-length

water between possible

modulates

regulation

of permeability

hundreds

26

in adjacent targets

26 mammalian s e l c i t r a

, the 34 ,

2+ 2+ 3 water-channel 5

, although the the although , –CaM binding binding –CaM , –CaM and the the and –CaM of 3

context 5

). Typically, ). and full-length

3 of subunits.

, by 2+

–CaM –CaM

of



- - - -

© 2013 Nature America, Inc. All rights reserved. aie Q0 n rcmiat a wr prfe t homoge to purified were CaM recombinant and AQP0 Native Pseudoatomic RESULTS channels. membrane other of functions the modulates allosterically CaM how into cues offers and AQPs regulating in ity Our channel. into the role of insights new provides cooperativ model mechanistic full-length any to bound CaM of knowledge, our to model, structural first the provides work This and studies. mutagenesis permeability by experimentally this tested we turn, in and ity, AQP0 modulates permeabil water-channel CaM dynamically which by mechanism the dissect to us allowed model structural Our CaM. with complex in AQP0 full-length the of structure pseudoatomic a eling, molecular dynamics and mod functional mutation studies to structural develop EM, used we AQP0, of permeability water the late function. channel to allow preliminary understanding channel, of how CaM dynamically modulates full-length a to bound CaM of model structural a yield to required are techniques hybrid that clear became it systems, ous and the lack of of from information difficulties Because these numer obtained. been has complex complete the of CaM regulation, and of no pseudoatomic model understanding mechanistic a prevented has channel RyR the for information tural previously however, reported an absence of high-resolution struc been have CaM with complex in RyR the of reconstructions Cryo-EM analysis. structural to refractory domains flexible by mediated and transient with CaM can be and with, associations their work to difficult often are channels length CaM with complex ~20 small residue of peptides or domains of the channel in typically are these and exist, tetramer. AQP0 the of helices C-terminal cytoplasmic the to bound B) and (A molecules CaM two displaying complex ( mesh). (gray reconstruction 3D the into (blue) CaM and yellow) and (orange AQP0 of ( complex. AQP0–CaM the of reconstruction 3D ( complex. AQP0–CaM the of averages projection symmetrized Inset, particles. AQP0–CaM stained negatively of ( kDa. ~13 at band a diffuse as migrating CaM free 3, lane (AQP0) 2:1 the and cross-link AQP0–CaM 1:1 the monomer, AQP0 the to corresponding kDa, 65 and 39 26, at migrating bands protein three into SDS in dissociated complex, cross-linked CaM AQP0– purified 2, lane material; starting total 1, Lane purification. chromatography exclusion size- the from fractions showing SDS-PAGE ( units. milliabsorbance mAU, chromatography. size-exclusion by CaM free excess from complex cross-linked AQP0–CaM ( EM. by determined complex AQP0–CaM the of 1 Figure s e l c i t r a  as calcium, of presence the in formed was complex the and neity, e a f

) Pseudoatomic model of the AQP0–CaM AQP0–CaM the of model ) Pseudoatomic ) Fitting of the crystallographic structures structures crystallographic the of ) Fitting the of purification showing ) Chromatogram To gain a mechanistic understanding of how CaM is used to modu

Purification and pseudoatomic model model pseudoatomic and Purification 2 –CaM cross-link, respectively; respectively; cross-link, –CaM

model c ) Electron micrograph micrograph ) Electron 36– d ) Different views of the the of views ) Different 3

9 of . This is because full- . is This because

the b ) Silver-stained ) Silver-stained

AQP0–calmodulin

40

, 4 1 -

; f e d a

complex UV280 (mAU) 10 15 20 0 5 (extracellular view) 0 Calmodulin (A) Top view advance online publication online advance 5 Aquaporin-0 tetramer 10 65 65 Time (min) Å Å ------

15 90 90 ° ° AQP0–CaM FREALIGN tilted- methods the conical random by from set data pair reconstruction (3D) three-dimensional initial an ( views side as interpreted features, bilobed of square-shaped structures ( ( grid EM the on distributed evenly were and size in nm ~7 particles CaM complex, the AQP0–CaM complexes appeared as homogeneous ( respectively cross-link, (AQP0) 2:1 and the cross-link monomer, AQP0–CaM 1:1 the weights of ~26 kDa, ~39 kDa and ~65 kDa corresponding to molecular the apparent AQP0 with species three identified which SDS-PAGE, ( molecules CaM two with complex in micelle detergent a in comprised tetramer AQP0 an and complex AQP0–CaM consist assembled time fully the retention with a ent with species monodispersed a as eluted ( chromatography by CaM size-exclusion effectively excess from complex and cross-linked AQP0–CaM (EDC), separated carbodiimide (3-dimethylaminopropyl) 1-ethyl-3- reagent cross-linking zero-length the using studies, EM described previously Fig. Fig. 1 Calmodulin (B) 20 Upon negative staining and EM visualization of purified AQP0– purified of visualization EM and staining negative Upon 40 Side view c 25 ). Projection averages revealed predominant views consisting consisting predominant views averages revealed ). Projection Å CaM Extracellular Cytoplasm 4 Fig. 1 Fig. 40 30 3 to produce a final ~25-Å reconstruction ( reconstruction ~25-Å final a produce to Å b

MW (kDa) 100 130 10 15 25 35 40 55 70 a 75

). We assessed the peak fraction by denaturing denaturing by fraction peak the assessed We ). 90 2 1 35 40 Å nature structural & molecular biology molecular & structural nature Total 3 5 90 ° 90 Å Å . We stabilized the AQP0–CaM complex for for complex AQP0–CaM the stabilized We . Complex ° 3 Fig. 1 Fig. ° Free CaM CaM AQP0 (AQP0)–CaM (AQP0) Fig. Fig. 1 Side view b and and 2 –CaM Fig. 1a Fig. c ) ) and views containing distinctive Supplementary Fig. 1 Fig. Supplementary c 1 00 nm 35 40 , 4 b 2 Å Å 90 90 and further refined with with refined further and ). The purified complex complex purified The ). Fig. 1 Fig. ° ° (cytoplasmic view) Bottom view c ). We calculated calculated We ). Fig. 1 Fig. ). 2 90 d –CaM –CaM ). Å -

© 2013 Nature America, Inc. All rights reserved. an initial high-affinity event with an association constant ( constant association an with event high-affinity initial an ( to model binding a two-state well fit that binding of heats large produced CaM, with mixed when AQP0 3 Fig. Supplementary ( (ITC) calorimetry titration isothermal using CaM, to binding on to effects site thermodynamic the individual characterized and each alanine mutated we recognition, CaM in involved pockets binding C-lobe and Ca N- in hydrophobic the by be could recognized that residues hydrophobic conserved highly several with (AQP0 domain CaM-binding AQP0 The Calmodulin the to compared map. when experimental 0.95 of map cross-correlation a calculated yielded a Å and 25 at clashes, steric no displayed model final the ( monomers adjacent of helices directly beneath two of the AQP0 subunits and bound to the C-terminal complex the contains two AQP0–CaM Ca 2 Fig. and Supplementary Methods (Online 223–226) (residues domains linker short by α 227–241) by using the antiparallel ptGAD We the C-terminal modeled cytoplasmic of the lobes 40 well within × ( 35 Å in the EM reconstruction plex complex (ptGAD)–CaM decarboxylase glutamate plant the to belong These Ca of structures ( Bank Data Protein the in Of fashion. the antiparallel >200 CaM deposited complexes currently an in together come to helices C-terminal neighboring two for was in mode binding this the AQP0 that arrangement facilitated tetramer simultaneously helices AQP0 C-terminal ( fitting initial the minimize computationally Chimera Weused map. EM the of features lobe the facing side its cytoplasmic with placed was × AQP0 (40 The 35 Å). tetramer cles whereas Å), of fit CaM well into in views the the parti side two features seen lobe 40 × 65 × (65 map EM the of body main the into well Fig. 2 Fig. ( of CaM AQP0 with model in complex respectively. views, cytoplasmic and extracellular the as Å ~10–15 wide ( cleft a by separated and reader the toward oriented map EM the lobes of two the showing reconstruction, the of ‘bottom’ the displays ( side the from averages projection ( the in observed features bilobed the with well ( tall Å 35 and wide Å 40 each domain, main the from extending lobes two the and reveals 75 Å tall the along we ( that averages views projection in square-shaped observed the with consistent is view top This 65 × from 65 the ‘top’Å viewed when ( of the reconstruction extending off the face of the main body. The square-shaped domain is of consisting domain a structure with two square-shaped protrusions nature structural & molecular biology molecular & structural nature Fig. 1 Fig. -helices were connected to the transmembrane domain of AQP0 AQP0 of domain transmembrane the to connected were -helices ITC studies used 20-residue peptides corresponding to the bovine bovine to the corresponding peptides 20-residue used studies ITC We previously demonstrated that CaM binds two cytoplasmic cytoplasmic two binds CaM that demonstrated previously We of a pseudoatomic We construction the EM map the to guide used The 3D reconstruction of the AQP0–CaM complex shows a bilobed 46 2+ ). The crystal structure of the AQP0 tetramer (PDB (PDB AQP0 tetramer of the structure crystal The ). , CBD 4 c 7 –CaM ( –CaM ). The main square-shaped domain is 40 Å tall when viewed viewed when tall Å 40 is domain square-shaped main The ). . . Only the compact of structure the complex ptGAD–CaM fit 4 5 x sequence shown in in shown sequence and the voltage-gated calcium channel (Ca axis gives a side view of the reconstruction that stands at stands that reconstruction the of view side a gives axis

uses Fig. 1 Fig. Fig. 2 Fig. 2+ Fig. Fig. 1

a –CaM bound to two adjacent antiparallel helices. helices. antiparallel adjacent two to bound –CaM

hydrophobic d a d ). ). An additional 90° rotation along the the along rotation 90° additional An ). ). The resulting pseudoatomic model of the the of model pseudoatomic resulting The ). ). To determine which of these residues are are residues these of which determine To ). ). ). In this work, we refer to the top and bottom h t t p Fig. Fig. 2 : Figure 2 Figure / Fig. 1 Fig. Fig. 1 Fig. / w

‘bind-and-capture’ Fig. 1 Fig. w b w ). The stepwise binding showed binding ). The stepwise f d . ). After energy minimization, minimization, energy After ). r ). These features correspond correspond features These ). Fig. 1e Fig. c a c α 3 s . The wild-type AQP0 wild-type The . ). Rotation of the map 90° 90° map the of Rotation ). 5 2+ α b -helices of -helices AQP0 (residues . . The only conformational CBD . -helices -helices as templates. The –CaM molecules located located molecules –CaM o r Fig. 1 Fig. g , ) contains an an contains ) f /

and ), there are only two two only are there ), advance online publication online advance e V1.2 Supplementary Supplementary ).

Fig. 2a Fig. mechanism )–CaM )–CaM com 2 B Fig. Fig. 1 Fig. Fig. 1 6 α – P K -helix -helix x c ) a1 axis axis and and 3 4 CBD 0 4 of of d fit fit to to e ). ). ). ). - - ,

increased by ~410% over wild type ( type wild over by ~410% increased ( event binding second the during interaction favorable mol kcal −0.87 energy binding enhanced an produced alanine to Ile236 an in overall resulted in reduction all ~83% an in reduction in resulted mutation L234A The CaM. with complex 2:1 a maintain could mutants these but type, wild of that to compared affinity binding reduced showed also L237A and L234A mutations AQP0 by the wild-type displayed stoichiometry 2:1 the to contrast in peptides, mutant two these and respectively. Only a 1:1 stoichiometric complex was formed with CaM penalties, energetic ( CaM to binding on effects thermodynamic unique in resulted tions modynamic parameters obtained by ITC.) Each of the AQP0 kcal −16.4 mol ( energy free overall an yielded model binding state ( 10 × ~1.4 loop residue Leu83 (ref. (ref. Leu83 residue loop AQP0 hydrophobic the between formed interactions Waals der van by part, in orientation, of in each monomer AQP0 this and beneath stabilized are positioned exposed. solvent primarily is or N- the ( CaM of toward pockets binding C-lobe displayed faces hydrophobic the with modeled AQP0 the complex, AQP0–CaM the of structure our In formation. complex during CaM of pockets binding C-lobe AQP0 the of face hydrophobic ( residues hydrophilic the of side opposing on the located is to alanine mutated when affinity binding enhanced the of face AQP0 same the along cluster all Leu237) and (Leu227, Leu234 Val230, alanine to mutation upon penalties energetic in resulted ( representation AQP0 the mapped C terminus could inhibit water-channel permeability. In our our In permeability. water-channel inhibit could terminus C AQP0 the to binding CaM which by mechanisms several are There Calmodulin complex. AQP0–CaM the forming for mechanism bind-and-capture two-step this during roles unique and separate in participate sites hydrophobic these that suggesting result a event, binding each for contributions energetic ( arrangement AQP0 second a capture readily to CaM allow AQP0 neighboring a of proximity the formed, between AQP0 configuration neighboring antiparallel preformed a of absence tetramer the AQP0 in the bind to CaM allow would interaction binding mecha nism driven by hydrophobic 1:1 The interactions. high-affinity initial bind-and-capture stepwise a through CaM by stabilized is stabilized by CaM. Our binding studies suggest that this configuration rotate and come together to form the antiparallel configuration that is to monomers neighboring of termini C the allow to broken be must Fig. 2 Fig. K To assess the structural context of these mutational effects, we we effects, mutational these of context structural the assess To In the CaM–free AQP0 crystal structure, the C-terminal C-terminal the structure, AQP0 crystal In CaM–free the a2 ∆ of ~7.8 × 10 of ~7.8 ∆ CBD G c ). Mutations L227A and V230A resulted in the largest overall overall in largest the resulted and ). Mutations V230A L227A 1+2

7

α M of 1.3 kcal mol kcal 1.3 of -helix. In contrast, the residue Ile236 that resulted in an an in resulted that Ile236 residue the contrast, In -helix.

K stabilizes K −1 a1 Fig. 2 Fig. −1 a1 −1 ) followed by a second lower-affinity binding event event binding lower-affinity second a by followed ) , whereas , whereas and a 38% reduction in in reduction 38% a and . . ( 4 i. 2 Fig. . This enhanced affinity is primarily due to a more more a to due primarily is affinity enhanced This . M Supplementary Fig. 3 Supplementary CBD CBD f −1 ∆ α ). The mutations studied here revealed distinct distinct revealed here studied mutations The ). ∆ ∆ ). Fitting of the energetic parameters to a two- parameters energetic of the Fitting ). Fig. 2 Fig. -helix, which is characterized by a mixture of of mixture a by characterized is which -helix, residues to a two-dimensional helical-wheel helical-wheel two-dimensional a to residues

d ∆ AQP0 oan ( domains G 3 CBD ). Notably, the hydrophobic residues that that residues hydrophobic the Notably, ). G 1+2 0 K 1+2 ). ). For to CaM bind AQP0, interaction this a2 −1 eiu Lu3 ad h cytoplasmic- the and Leu237 residue of 8.4 kcal mol kcal 8.4 of d was essentially unaffected ( unaffected was essentially of 0.5 kcal mol kcal of 0.5 . The L237A mutation showed a 63% 63% a showed mutation L237A The .

). This analysis suggests that it is this this is it that suggests analysis This ). and CBD CBD Fig. 2 Fig.

closes i. 2 Fig. that interacts with the N- and and N- the with interacts that Fig. 2 Fig. ( Fig. 2 Fig. e ) while the hydrophilic face face hydrophilic the while ) contains a table of full ther K

f the ). Once a 1:1 complex is is complex 1:1 a Once ). c a2 ). c −1 ( −1 ). Hydrophobic-residue ). Hydrophobic-residue

Fig. 2 Fig. CBD channel . . Notably, of mutation and 7.75 kcal mol kcal 7.75 and CBD in the antiparallel antiparallel the in s e l c i t r a CBD c domain would would domain ∆ ), with an over an with ), G

domains are are domains gate K 1+2 Fig. Fig. 2 a2 ∆ CBD ) equal to to equal ) ) that ) was that α ∆ -helices -helices G muta c 1+2 ) ) and of of −1  - - - - ,

© 2013 Nature America, Inc. All rights reserved. in the CaM–free system mapped to the extracellular loops and the the and loops extracellular the to mapped system CaM–free the in AQP0 the monomers of in the tetramer.each of The highest r.m.s.dynamics fluctuation overall values the limits binding CaM that shows ( fluctuations per-residue the by AQP0 of dynamics formational into the mechanism of water-channel inhibition, we the assessed con AQP0 results in water-channel to inhibition binding CaM that showing studies functional previous our with 10 × 0.035 as low as to reduced, simulation, AQP0 CaM-bound studies functional and dynamics molecular previous cm trajectory) each from pores four × 10 2.1 was AQP0 tetramer CaM-free ns. ~500 for ran simulations dynamics 4 Fig. lipid bilayer with or without the two CaM molecules ( (POPC) palmitoyl-oleoyl-phosphatidylcholine a in tetramer AQP0 points of the two simulations contained identical conformations of the and CaM-bound AQP0 tetramer. The starting CaM-free the on study dynamics molecular comparative a conducted we bound, is CaM when occurs that dynamics conformational channel the in change the into insight gain ( bound is CaM when even to water bulk and accessible open remain could subunits of these vestibule mic plug a analysis with HOLE as profile act pore and density water 3D However, potentially could CaM and of ( monomers pores AQP0 four the of cytoplasmic two the below directly positioned are molecules CaM two the com plex, AQP0–CaM the of model structural complex. AQP0–CaM 2:1 the of assembly for mechanism bind-and-capture ( representations. stick as shown CaM with interface, hydrophobic proposed the forming residues AQP0 with helices) orange and (yellow in as (colored CaM to bound monomers AQP0 two ( recognition. CaM in involved face a hydrophobic identifying ( models. binding fitted following uncertainties wild-type over ( mutant as calculated are Ratios alanine. to in (orange site hydrophobic each AQP0 for ITC by obtained ( model. binding a two-state with fit isotherm binding Right, Ca AQP0 the within residues hydrophobic conserved indicate dots) by marked (and (AQP0 domain binding calmodulin- AQP0 the of alignment sequence primary- and (yellow) structure secondary the of ( formation. complex AQP0–CaM 2 Figure s e l c i t r a  residues whereas binding, CaM of site the terminus, C cytoplasmic f

) Schematic illustrating the two-step two-step the illustrating ) Schematic h cluae snl-hne wtr emaiiy ( permeability water single-channel calculated The 2+ 2 –CaM to the wild-type AQP0 wild-type the to –CaM Fig. Fig. CBD ). After equilibration of the two systems, the two molecular molecular two the systems, two the of equilibration After ). f ). This arrangement suggested that that suggested arrangement This ).

. . ( Hydrophobic interactions involved in involved interactions Hydrophobic 1 b ). Zoom view shows the AQP0 the shows view Zoom ). ) Left, ITC raw heats of binding of binding of heats raw ITC ) Left, e ) Structure of AQP0–CaM showing showing AQP0–CaM of ) Structure K Fig. 3c Fig. a c values. Error bars indicate indicate bars Error values. ) Ratio of binding affinities affinities binding of ) Ratio i. 3a Fig. 4 8 CBD – show that the cytoplas χ d 2 e ) Helical-wheel analysis analysis ) Helical-wheel CBD minimization of the the of minimization ). Clustering analysis of r.m.s. fluctuations fluctuations r.m.s. of analysis Clustering ). ). Residues in orange orange in Residues ). , b peptides when when peptides ). Therefore, to to Therefore, ). α a CBD a ) Schematic ) Schematic -helical -helical ) was mutated mutated ) was −15 p peptide. peptide. f values within the tetramer were were tetramer the within values cm

Figs. 1f 1f Figs. CBD 3

s −15 3 s

−1

K 14 −1 ( 3 a (ref. (ref. , * ± 5 2 . These results are in line line in are results These . - - ) . 6 1.8 × 10 1.8

. . To insight gain further 4 a c 9 Human Mouse Sheep ), consistent with with consistent ), Ratio of binding affinities Cow TM-6 0.25 0.50 0.75 1.00 1.25 4.00 4.25 Rat α Supplementary Supplementary −15 28 0 -carbon r.m.s. r.m.s. -carbon Wild type 223 s.e.m. of the of the s.e.m. , 50– p f advance online publication online advance ) for the the for ) 5 K e f 2 a1 CaM-binding domain . In the the In . L227A */ K a1 AQP0 tetramer V230A CaM bindsasingleAQP0 - K a2 Initial bindingevent: */ Phe75, His66 and Tyr149 form CSII in AQP0 ( displayed large movements AQP0 during the time course of the in simulations. CSII form Tyr149 and His66 Phe75, pro been gate has channel dynamic a as site act to posed this because notable was simulation dynamics region. CSII stabilizing the by particularly AQP0, of dynamics the constrains CaM ( CSII forming residues to and (TM6) helix transmembrane last the of base the to mapped residues ( CaM by confined when dynamics molecular C-terminal AQP0 the of tuations CaM-free the during ( simulation dynamic most the channel the of vestibule mic cytoplas the at located CSII, termed site, constriction second a ing membrane lipid a of AQP0 in crystallography by obtained electron factors temperature tions ( fluctua thermal low exhibited domain transmembrane the within L234A K a2 Stabilization of the AQP0 CSII in the CaM-bound molecular molecular CaM-bound the in CSII AQP0 the of Stabilization contain pore channel a has AQP0 aquaporins, other most Unlike I236A Fig. 3c CaM Step L237A Two-step bind-and-capturemechanism 242 Fig. 3 Fig. , d CBD 1 ). ). These results are in agreement with the experimental –1 b

40 Heat input (µcal s ) –0.6 –0.4 –0.2 3 d ° 0

. ). In the CaM-bound simulation, the r.m.s. fluc r.m.s. the simulation, CaM-bound the In ). 0 nature structural & molecular biology molecular & structural nature 1 0 0 N lobe Fig. 3 Fig. Time (min) Molar ratio 30 d 2 3 CaM capturesasecondAQP0 3 Hydrophobic . Residues forming CSII were among among were CSII forming Residues . 4 3 Fig. 3 Fig. e Secondary bindingevent: Step ). In addition, the most stabilized stabilized most the addition, In ). 60 V α L 23 L 234 237 0 -helices were markedly reduced reduced markedly were -helices Leu227 L 2 22 5 e 9 ). These results indicate that that indicate results These ). 7 Leu234 33 R 0 AQP0–CaM complex 233 , 5 K

0 Integrated heats 238 Leu237

. The conserved residues residues conserved The . –1 Val230 (kcal mol ) –4 –2 S binding interface S 0 240 231 Hydrophobic Wild type 0 0 Fig. 4a Fig. C lobe CBD S 1 229 Time (min) Molar ratio 30 S 7.8 K 2 235 a2 I 1.4 K , 236

× b K = a1 4 3 10 228 E G ). Tyr149 Tyr149 ).

× 232 = 60 23 10 4

9 M 7 –1

M 5

–1 9 Hydrophilic 0 - - - - -

© 2013 Nature America, Inc. All rights reserved. tions ( and tions Tyr149 simula two the of course between time the over Phe75 distance residue opposed the the monitored we complex, CaM molecules. water Å ~1.5 to ( CSII at diameter pore the narrowed Tyr149 orientation was states Å, ~2.5 the and open and closed in the closed eter between downward a ( between ( oscillated orientation conformation ‘open’ chain side The in Asterisks Å). −0.8 blue, dark Å; −0.4 blue, light 0 Å; (white, color to according structure AQP0 the to mapped system CaM-free the to compared ( Å). 1.2 red, Å; 0.6 white, Å; 0.3 ( ribbon). gray and (white protomer ( stick). (gray 3 Figure nature structural & molecular biology molecular & structural nature and and Fig. 4 Fig. Fig. 4 Fig. a d a Normalized population AQP0– the for CSII at dynamics the in differences the Toassess 0.2 0.4 0.6 0.8 1.0 e Cytoplasm indicate the position of the AQP0 cytoplasmic constriction site II (CSII). II site constriction cytoplasmic AQP0 the of position the indicate 0 Extracellular Fig. 4 Fig. b c

4 2 and ). This tight closure would effectively block the passage of of passage the block effectively would closure tight This ). Calmodulin restricts the dynamics of AQP0. ( AQP0. of dynamics the restricts Calmodulin CaM bound CaM free b d Supplementary Video 1 Video Supplementary ) Pore profile analysis with HOLE with analysis profile ) Pore ). In the CaM-free system, the distance between these these between distance the system, CaM-free the In ). Extracellular Cy CSII gatedisplacement(Å) t oplasm i. 4 Fig. Pore profile 8 6 b

Percentage of orientation ‘closed’ upward an and )

population d 10 15 20 c ) Zoom view of the cytoplasmic domain of AQP0. ( AQP0. of domain cytoplasmic the of view ) Zoom 0 5 <5 (blue, color to according structure AQP0 the to mapped values (r.m.s.f.) fluctuation r.m.s. per-residue ) CaM-free 10 Å CaM >8 free CSII

Å diam in pore difference ). The

<5 bound

Å CaM 12 >8

Å 4 8 b Cy Extracellular showing water permeation pathway (blue surface) in the CaM-bound conformation of the AQP0 AQP0 the of conformation CaM-bound the in surface) (blue pathway permeation water showing t

oplasm c b e advance online publication online advance

Water permeability (P ) Pore diameter (Å) a Tyr149 f bilayer a lipid in ribbon) (gray AQP0–CaM equilibrated the for isosurface) (blue density ) Water 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 10 20 30 40 50 60 0 –15 Wa Uninjecte Open CSII 1.8 mMCa 0 mMCa t er per pathw Phe75 –10 d meation ay Ca 2+ AQP0 2+ His6 Oocyte expression M * CSII –5 - - 6

z Y149G axis(Å) Tyr149 centered at 6.6 Å and a second smaller population centered at 9 Å 9 at centered population smaller second a and Å 6.6 at centered distribution main a with Å, 12 and Å ~3.5 between varied residues in the presence of CaM. of presence the in indicated that the probability of CSII adopting a closed state increases system was essentially absent in the CaM-bound system. This analysis CaM-free the in displacement 9-Å large at the centered of structures ( displacement inter-residue shorter with structures of population new a observed we in but addition, Å, 6.4 at centered population major a exhibited also system ( closure pore toward more shifted dues ( 2.5 Fig. 4 Fig. c 5 0 Closed CSII Å Extracellular UI Cy Y149L t Phe75 oplasm e Y149 d ) Change in protein dynamics ( dynamics protein in ) Change ). In the CaM-bound system, the distance between the resi the between distance the system, CaM-bound the In ).

G149 C aM-binding Closed His6 domain Open Y149S L149 10 6 S149 immunoblot in in immunoblot (uncropped membrane plasma the to trafficked correctly and expressed was construct each that verifying immunoblot anti-AQP0 t Ca by inhibited Ca mM 1.8 or (blue) 0 mM of conditions buffer under obtained Y149S) and Y149L (Y149G, mutants CSII and AQP0 wild-type ( ( Å. >8 or Å <5 displacement CSII with structures of population the showing plot Inset, Phe75. and Tyr149 residues CSII their of displacement the to according clustered simulations, dynamics molecular (red) AQP0 CaM-bound and (blue) AQP0 CaM-free the during obtained structures ( (red). CSII closed the and (blue) CSII AQP0 open ( sticks. as displayed are His66 and Phe75 in ( conformation closed upward, an in and ( conformation open a downward, in Tyr149 shown is simulation. dynamics molecular the during AQP0 of analysis ( (CSII). II gate site constriction cytoplasmic 4 Figure test ( test P c b a ) Plot indicating the pore diameter of the the of diameter pore the indicating ) Plot , , right). The location of CSII is indicated indicated is CSII of location The , right). f 2+ a b ; ; . Side chains of the CSII residues Tyr149, residues CSII the of chains . Side ) Structures illustrating pore profile profile pore illustrating ) Structures µ (yellow). The AQP0 AQP0 The (yellow). m m s Fig. 4 Fig. n d = 10 biological replicates)). Inset, Inset, replicates)). biological = 10 ) Plot indicating the population of population the indicating ) Plot

−1 CaM binding closes the AQP0 AQP0 the closes binding CaM e ) Water channel permeability rates rates permeability channel ) Water ) obtained from oocytes expressing expressing oocytes from ) obtained d ∆ ). Furthermore, the population population the Furthermore, ). r.m.s.f.) of the CaM-bound system system CaM-bound the of r.m.s.f.) 2+ Supplementary Fig. 5 Fig. Supplementary ( P Fig. 4 Fig. = 0.012 by two-sided two-sided by = 0.012 e d P d bound Ca fr Ca f ee is significantly significantly is ). The CaM-bound CaM-bound The ). M M s e l c i t r a a ; ; b ). , left) , left)

∆

r

* * .m. r. m.

0 1 0.6 0.3 –0.8 –0.4

.2

s.

Å

s. d

f. Å

f. Å Å Å Å

 - © 2013 Nature America, Inc. All rights reserved. fected by calcium ( calcium by fected unaf also were that permeabilities low only displayed channels the (Y149L), leucine to mutated was Tyr149 eliminates When and sensitivity. calcium altogether gate the removes essentially CSII the of position this at size chain side the of reduction drastic a that gested a complete loss of ( to response calcium high ( calcium) low (at rates permeability water increased displayed channels type, to wild as compared mutant, to In Y149G the conditions. respond high-calcium to ability the lost they however, flux; water facilitate to ability their maintained channels the serine, or leucine glycine, a to dynamic gating residue at CSII. When this residue was mutated either dependent CaM be to shown been previously has effect results experimental previous with consistent replicates)), µ ( halved approximately was permeability Ca mM (1.8 calcium high of presence ( ( rates permeability bulk water exhibited AQP0 wild-type expressing oocytes EGTA), mM 2 concentrations. calcium varying of swelling cell by mutants monitoring point CSII AQP0 various and AQP0 wild-type of rates permeability water-channel bulk assayed we hypothesis, To this permeability. test CSII residues should abrogate Ca the of mutation present, indeed is relationship this If gate. CSII the forming residues of conserved the dynamics and conformational the AQP0 of terminus C the to binding CaM between relationship mate inti an suggested result This channel. the of states closed and open the between equilibrium the in shift a causes terminus C AQP0 the results dynamics that indicated Our molecular Ca Mutation s e l c i t r a  to determine the pseudoatomic structure of AQP0 the water channel structure pseudoatomic the to determine methods CaM hybrid used we study, this In which function. by channel modulates mechanisms molecular and structural underlying the about known is little very regulation, channel in role central this Ca intracellular in changes to response in channels membrane numerous of activity the regulates Calmodulin DISCUSSION at CSII. closure water-channel to terminus C AQP0 at the a dynamic channel gate couples that Ca allosterically The modulation. residue calcium data also demonstrate that Tyr149 as serves its and CSII permeability water AQP0 the of magnitude the the both alters that Tyr149 suggesting results dynamics–based by Tyr149. mediated mechanism gating the mimic ( ( calcium high under rates n low-calcium conditions ( under channels wild-type of those to comparable were that rates ity permeabil water in resulted mutant Y149S The mechanism. gating group that it has in common with tyrosine might be important for the hydroxyl the that we hypothesized because (Y149S) Tyr149 to serine Ca abolishes consequently and gating channel restricts chain side a hydrophobic large with tion n n ± ±

= 10) under low Ca = under 10) = 10)) and displayed marginal reduction (~30%) in permeability permeability in (~30%) reduction marginal displayed and 10)) = = 10) under high Ca high under 10) = m s m s.e.m.; s.e.m.; s.e.m.; s.e.m.; When calcium was removed from the buffer (0 mM Ca mM (0 buffer the from removed was calcium When Our molecular dynamics studies suggested that Tyr149 acts as the the as Tyr149 acts that suggested studies dynamics molecular Our These experimental data are consistent with our molecular molecular our with consistent are data experimental These −1 ( ± n

n P s.e.m.); s.e.m.); of = 10)) ( 10)) = = 10), well above those of control cells ( cells control of those above well 10), = f = 50.6 × 10 = 50.6

the

AQP0 P Fig. 4 Fig. P = 0.012 by two-sided two-sided by 0.012 = 2+ f = 19.2 × 10 × 19.2 = and 17.2 × and 10 17.2

2+ −5 gate P e ) ( )

f ), thus suggesting that serine can partially partially can serine that suggesting thus ), ± = 35.7 × 10 2+ P 0.5 × 10 0.5 Fig. 4 Fig. P f

–CaM modulation. Lastly, we mutated mutated we Lastly, modulation. –CaM abolishes o 4. × 10 × 44.6 of ) f 2. × 10 × 24.4 = 2+ e –CaM –CaM regulation of water-channel −5 ), thus suggesting that substitu that suggesting thus ), −5

−5 ± −5

2+ 1.2 × 10 × 1.2 µ

Xenopus laevis Xenopus

± Ca

± m m s P ), the rate of water-channel water-channel of rate the ), 0.8 × 0.8 10 6.1 × 10 f 2 2+ = × 20.2 10 + −1 −5 −5 t Fig. Fig. 4 concentration. Despite Despite concentration. –CaM test ( test (

± ± ± −5 . × 10 × 5.3 . × 10 × 2.8 s.e.m.; s.e.m.; −5 2+ −5 e

µ n

). ). This result sug

–CaM –CaM binding to regulation

µ 2+ µ = 10 biological biological 10 = m s m Fig. 4 Fig. m m s m m s oocytes under under oocytes –CaM –CaM binding −5 1 n −1

4 ± −1 −1 . = 10)) and = 10)) −5 5.2 × 5.2 10 −5 ( advance online publication online advance 14 ( ( e ±

± ). In the the In ). ± , 2 2+ µ µ s.e.m.; s.e.m.; s.e.m.; s.e.m.; s.e.m.; 6 s m s m . This This . with with −1 −1 −5 - - - - -

(AQP0/CaM) stoichiometry, using chemical cross-linking, structural structural stoichiometry, (AQP0/CaM) using cross-linking, chemical Ca a in permeability water modulating closed dynamically a thereby favor conformation, that interactions allosteric through gate CSII the to communicated is terminus C AQP0 the at binding CaM that show blocked when the CSII gating residue Tyr149 is mutated. effectively Together, is our results permeability water AQP0 of regulation CaM that showed that studies functional and mutagenesis by supported ( channel each of helix transmembrane last the and linker cytoplasmic the through Å at to over site CSII ~15–20 the AQP0 the is propagated C terminus binding CaM of effect stabilizing the that shows analysis dynamics molecular Our AQP0 CSII. of the dynamics on the acts that tension, or force, mechanical a provides AQP0 of subunits neighboring two across CaM of bridging The closure. channel in resulting thus site, constriction pore CSII the at dynamics the modulates terminus C allosterically AQP0 the to binding CaM that showed which lations, simu dynamics molecular using regulation, CaM into insight nistic mecha obtain to able Wewere molecules. CaM two with complex CaM. with complex in channel membrane any full-length for model structural complete first the providing CaM, with complex in shown as stick representations. stick as shown are Tyr149Phe75 and residues CSII clarity. for removed is AQP0 of helix transmembrane last the Likewise, clarity. and purposes display for figure the in deleted were molecules water Some red. in shown are molecules water and ribbon, gray as shown is AQP0 simulations. dynamics molecular the from obtained were models structural The permeability. water-channel restricting thereby state, CSII closed the of stabilization allosteric by Ca of Binding His66. and Tyr149, Phe75 residues by formed CSII the at changes conformational dynamic by formed are states These (right). state closed and (left) state open an between equilibrium in exists channel 5 Figure with association AQP0, stepwise CaM appears to bind both ptGAD the Unlike dimer conformation. ptGAD compact the a of in intersection the around wraps CaM single a complex, In ptGAD–CaM the interactions. coiled-coil dimeric other antiparallel adopt Ca α two bridging CaM of structures high-resolution two only currently by ( complex used AQP0–CaM 2:1 the mechanism assemble to CaM bind-and-capture stepwise a proposed have orientation antiparallel an in together come to subunits neighboring from domains CaM-binding two for is interaction of type this facilitate to way only the spectroscopy NMR using studies previous ( studies binding ITC and analysis EM Cytoplasm Extracellular -helical peptides, belonging to the ptGAD–CaM complex to the ptGAD–CaM belonging peptides, -helical We demonstrated that CaM binds full-length AQP0 with a 2:1 2:1 a with AQP0 full-length binds CaM that demonstrated We in tetramer AQP0 the shows structure AQP0–CaM resulting The 2+ V1.2 –CaM at the C terminus of AQP0 results in a shift in this equilibrium equilibrium this in a shift in results AQP0 of C terminus the at –CaM –CaM complex –CaM 2+

Mechanism of Ca of Mechanism -dependent manner ( manner -dependent Open state Fig. Fig.

3 α ). This allosteric mechanism was experimentally was experimentally mechanism ). allosteric This -helical orientations that intersect similarly to to similarly intersect that orientations -helical nature structural & molecular biology molecular & structural nature 46 , 4 2+ 7 . In either case, the CaM-binding domains domains CaM-binding the case, . In either –CaM regulation of AQP0. The AQP0 water water AQP0 The AQP0. of regulation –CaM 3 Fig. Fig. 5 Ca . On the basis of these results, we we results, these of basis the On . 2+ –CaM 5 ). Figs. 1 1 Figs. α 3 -helices -helices simultaneously 5 . In the AQP0 structure, structure, AQP0 the In . and Closed state Fig. 2 Fig.

2 ), confirming confirming ), f ). There are are There ). 4 α 5 and the -helical -helical 5 3 - - .

© 2013 Nature America, Inc. All rights reserved. erativity between AQP subunits is not a functional requirement requirement functional pore water active a fully own its forms monomer not each because is subunits AQP between erativity closure. channel drive allosterically to used force mechanical the provides stoichiometry the in which case is another that this able conceiv it is complex, on this information structural no currently is inactivation channel in to result stoichiometry 2:1 a with CaM bind receptor NMDA the of subunits NR1 the example, For to functions. their regulate cooperativity CaM-driven use similar force required to open the channel gate the domain C-terminal of the SK channel and provide the mechanical α three across extended CaM with structure a unique in resulting thus complex, 2:2 a form to subunit neighboring a from domain channel Ca binds CaM When ometry. stoichi a in 1:1 of SK channels domain a C-terminal with associated Ca a in of SK the channel activation the to drive proposed were that changes Ca of absence to or bound presence the channel in SK CaM the from domain C-terminal the of studies channel (SK) potassium as conductance such small the CaM, by modulated are properties gating whose channels ( state closed its in channel the stabilize and gate channel the restrain Ca which through nel of chan of the reins as the thought be can AQP0 The gate. C termini CSII the including channel, the of region cytoplasmic the stabilizes mechanism. regulatory the role in AQP0 functional to have an is for thought important stoichiometry However, this CaM. of use economical stoichiometry more 2:1 a a provide may cases, both In permeation. water restrict that gate channel the in changes allosteric induces binding CaM AQP0, ceso codes. Accession the in v available are references associated any and Methods M targets. membrane-channel unveil undoubtedly many its will regulate to CaM by used here, mechanisms unexpected and new applied those as such biology structural techniques, complementary using studies Future channels. in full-length interactions these characterize ally and mechanistically of membrane channels. This functions possibility emphasizes the the need to structur modulate to CaM by used theme common a be to prove subunits. may subunits that neighboring between between cooperativity scaffold interactions Facilitating regulatory necessary the cooperative provide facilitates to appears Ca as tetramerization such proteins regulatory of the binding for prerequisite a is tetramerization AQP0 that suggest studies activation functional in resulting thus enzyme, the of domain autoinhibitory an relieves binding CaM ptGAD, For different. quite are mechanisms regulatory the interaction, ptGAD-CaM the to lar our studies structural suggest that the AQP0-CaM interaction is simi consistent with that of the complex. compact Although ptGAD–CaM most was CaM of AQP0 with interaction the that suggested data EM Ca the bridges helical dimer, with each CaM adopting an extended conformation that Ca the In nature structural & molecular biology molecular & structural nature (EMD- database Fig. Fig. e -helices ethods r Unlike other multimeric channels, such as ion channels, coop channels, ion as such channels, multimeric other Unlike domains C-terminal AQP0 neighboring two to binding CaM s i o 5 n ). ). This may be similar to the mechanisms used to regulate other

2+ o f 3 -dependent manner -dependent

6 V1.2 t . This Ca This . h e

CM ope, w CM oeue bn a Ca a bind molecules CaM two complex, –CaM p V1.2 a 5 p 6 e The EM map has been deposited with the EM EM the with deposited been has map EM The dimer distant from the helical intersection. Our Our intersection. helical the from distant dimer 7 r . 9 2+ ). The pseudoatomic model of the AQP0–CaM AQP0–CaM of the model pseudoatomic The ). -induced interaction was proposed to rotate rotate to proposed was interaction -induced 2+ –CaM provides the mechanical force to to force mechanical the provides –CaM 2+ 36 , it associates with an additional SK- additional an with associates it , , 5 6 . Ca . 36 2+ , 5 2+ -free CaM is constitutively constitutively is CaM -free 6 . . Many other channels may revealed conformational conformational revealed 2+ –CaM. Thus, AQP0 AQP0 Thus, –CaM.

5 5 advance online publication online advance . Crystallographic Crystallographic . 5 7 . Although there there . Although 5 o 5 8 4 . Our Our . n . For . l V1.2 i n e ------

and analysis of molecular dynamics simulations. D.M.C. and K.L.N.-C. performed on the AQP0–CaM complexes. D.M.C., J.A.F., M.H. and D.J.T. performed setup manuscript. performed proteinS.L.R. purification, EM and ITC binding studies for this work. All authors contributed to data analysis and preparation of the D.M.C.,S.L.R., J.E.H. and T.G. conceived of and the designed experiments funded by the HHMI (T.G.). part by NIH grant R01 GM079233 (T.G.). Research in the laboratory of T.G. is from the German Academy of This Leopoldina. Sciences work was supported in FoundationScience grant (D.J.T.).CHE-0750175 M.H. is supported by a fellowship National Institute of Medical General grantSciences GM86685 and US National of D.J.T. is supported by NIH National Institute of Neurological Disorders– Kirschtein National Award Research Service from NIH. Research in the laboratory Program Award, no. LM007443. Research by was S.L.R. supported by the Ruth L. the NIH National ofLibrary Medicine Biomedical Informatics Research Training National Eye Institute grant EY5661 (J.E.H.). Research by D.M.C. was supported by the laboratory of J.E.H. is supported by US National Institutes of Health (NIH) Janelia Farm Research Campus) for help with various ofaspects EM. Research in (HHMI), Janelia Farm Research Campus) for help with ITC and D. Shi (HHMI, The authors would like to thank M. Sarhan (Howard Hughes Medical Institute the in available onlin are files Data Source and Information Supplementary Any Note: (PDB Bank Data Protein the with deposited been has density EM the to fit complex 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. r at online available is information permissions and Reprints The authors declare no competing interests.financial and analysis ofpermeability data. oocyte permeability measurements, oocyte construction ofexpression oocyte constructs 17. AUTH A C e ckn O p

r

MPETING FINANCIAL INTERESTS FINANCIAL MPETING au Y.S. Babu, water R.T.aquaporin Mathias, of & Regulation A. Shiels, S., Kumari, Varadaraj,K., permeability water the regulate calcium and pH J.E. Hall, & K.L. Németh-Cahalan, and pH calcium, of roles channels; junction gap of gating Chemical C. Peracchia, A. Sotkis, Calmodulin A. Persechini, & L.L. Peracchia, X.G., Wang, A., Sotkis, C., Peracchia, Ca other and calmodulin of roles Multiple M.X. Zhu, Z. Zhang, Scott, K., Sun, Y., Beckingham, K. & and Zuker, C.S. receptor Calmodulin regulation of ryanodine the of Modulation A.R. Marks, & S.E. single Lehnart, R., of Zalk, modulation Calmodulin G. Meissner, & E. Rousseau, J.S., calcium Smith, of regulation the in calmodulin for subunit. role channel a ion of Evidence an G. as Meissner, Calmodulin C. Kung, & Y. Saimi, H.L. Tan, Calmodulin H. Reuter, & R.W. Tsien, K., Deisseroth, G.S., Pitt, R.D., sodium Zühlke, calcium-dependent of activation Calmodulin K.Y. Ling, & Y. Saimi, hn, . Tnk, . Iua M Climidcd ofrainl transition conformational Calcium-induced M. Ikura, & T. Tanaka, M., Zhang, (1995). lens. the in permeability 0. aquaporin of calmodulin. gating. channel junction gap channels. junction gap gates directly channels. TRP of regulation functional USA Sci. Acad. domain. binding common a from calmodulin inhibitory of displacement light the of response termination mediates function receptor and channels light-activated calcium. intracellular Res. Circ. Ca reticulum sarcoplasmic (1986). reticulum. sarcoplasmic muscle skeletal from release Physiol. excitability. (1999). 159–162 channels. calcium L-type of facilitation and inactivation both supports (1990). of patches membrane excised in channels revealed by the solution structure of apo calmodulin. apo of structure solution the by revealed (1985). i n e t

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© 2013 Nature America, Inc. All rights reserved. Thetransmembrane domain ofbovine the AQP0 structure (PDB crystal subunits were used as templates to model the AQP0 C-terminal menthelices was fromoptimized each to avoid steric clashes with AQP0. The two AQP0placemonomers.neighboringtransmembraneThisof last helixptGAD the of cal axes of the two ptGAD subunits were oriented toward the cytoplasmic ends thatwas inconsistent with thedimensions of our EMreconstruction. The heli Ca conformationthe however,extended in anadopts CaM (Ca nel AQP0 tetramer. The structure of CaM bound to the voltage-gated calcium chan mapfashion,symmetricofEMbinding thea in CaM with the clefts facing the (ptGAD–CaM, PDB Ca of structure lationally optimized by computational minimization routines in Chimera lobe domains of the EM map. This initial placement was rotationally and trans EM map with the cytoplasmic face of the AQP0 tetramer facing the two vacant 9–222) residues into the AQP0–CaM EM density map as follows and in AQP0–CaMcomplex builtwasbystructuresinitiallyavailabledocking crystal Molecular modeling of the AQP0–CaM complex. resolution, as suggested by Fourier shell correlation (FSC) analysis. final 3D density map was reconstructed with C2 symmetry and filtered to 25-Å Final refinement of the 3D density map was performed with FREALIGN initial three-dimensional (3D) reconstruction with random conical tilt methods. SPIDER with CTFTILT trast transfer function (CTF) parameters were determined for each micrograph only select micrographsthose freeofor drift noticeable astigmatism. Thecon final pixel size of 3.98 Å per pixel. Thon rings in the power spectra were used to a Nikon Coolscan 9000 with a 6.9- (Kodak SO-163) as tilted-pair images ( film on level werespecimen the at ×52,000 images ofmagnification nominal a atrecordedand (FEI), TEM kV 120 a on visualized were particles stained flow.Negativelyair laminar by dried and paper filter on blotted w/v), (0.75% meshcarbon-coated400 (Ted a to grid EM Pella), staineduranylwith formate 25 mM HEPES, pH 7.4, 5 mM CaCl ml mg 0.02 todiluted AQP0–CaMcomplex was fied Electron microscopy and three-dimensional reconstruction. tions were determined by BCA assay (Pierce) or UV absorbance at 280 nm. of AQP0–CaM was specifically stabilized by the EDC reaction.weight Protein aggregates concentra in SDS-PAGE demonstrates that theabsenceAQP0–CaM2:2of a cross-linked complex 2:1orother higher-molecular- stoichiometric complex was used to monitor each step of the purification ( 25 mM HEPES, pH 7.4, 5 mM CaCl chromatography (S200, GE Lifescience) pre-equilibrated with buffer containing size-exclusionby CaM excessfrom purified further complexcross-linkedwas productbyapplication gradientofNaCl.a mM of 750 elutedThe AQP0–CaM and 0.3% DM. Unreacted AQP0 was separated from the adhere,AQP0–CaM GE Lifescience) equilibrated with cross-linked20 mM HEPES, pH 5.5, 1 mM CaCl on ice. The reaction mixture was loaded onto an anion-exchange column (Capto and was quenched by the addition of 10 mM hydroxylamine (Sigma) and placed cross-linkingreaction wasallowed toproceed for atmin90 room temperature HEPES,pH 7.4, 5 mM CaCl mM 25 containing buffer cross-linking in CaM) of excess molar 2× a to ing CaM (2 mg ml unreacted EDC was quenched with 20 mM activation reaction was allowed to proceed at room temperature for 15 min, and (EDC, Pierce) and 20 mM wasperformed 1-ethyl-3-(3-dimethylaminopropyl)withmM10 carbodiimide buffer containing 25 mM MES, pH 6.0, and 5 mM CaCl purified as previously described and the vertebrate calmodulin (CaM) was heterologously expressed in length aquaporin-0 (AQP0) was purified from lenses obtained from AQP0–CaMyoungpurificationthe andofCross-linkingcomplex. sheep, ONLINE doi: AQP0 C-terminal The 227–241). (residuessubunit 10.1038/nsmb.2630 V1.2 4 2 for generation of multivariate reference-free projection averages and

)–CaM complex )–CaM METHODS −1 5 2+ 9 3 ) was added to freshly purified AQP0 (1.5 mg ml . Subsequently, 11,720 particles were selected and processed in 0 –CaM bound to the plant glutamate decarboxylase peptides peptides decarboxylase glutamateplant the to bound –CaM was manually placed into the 65 × 65 × 40–Å region of the of region 40–Å × 65 × 65 the into placed manually was 1NW N D 46 -hydroxysulfosuccinimide (sulfo-NHS, Pierce). The ) 2 4 ,5 mM , 4 5 7 was manually placed within the two vacant lobes was also assessed for fitting into the EM map; EM the intofitting for assessed also was 3 5 . CaM was prepared at 8 mg ml µ 2 2 and 0.3% DM. SDS-PAGE and silver staining m step size and binned three times to yield a and 0.3% DM. A volume of 2 β α Meand 0.3% decylmaltoside (DM). The of 0° and 50°). Images were digitized with β -mercaptoethanol ( A pseudoatomic model of the Supplementary Fig. 1 α −1 Supplementary Figure 2 2 -helical residues Leu234 Leu234 residues -helical . The activation reaction with buffer containingbuffer with V1.2 The freshly puri The nativeThe full- −1 β –CaM complex –CaM −1 Me). Activated ) (correspond µ in activation l was applied E. coli α -helical 4 4 ). The 3 4 2B6 . The . The and P 2 ------, .

POPC-embedded AQP0: one for AQP0 in complex with CaM and one for AQP0 Moleculardynamicssimulations. parameters were obtained by fitting the data to a two-state binding isotherm. Ca heatswithobtainedfrommixingalonefrom (OriginLab) by subtraction of the Origin background in heats processed of werepeptide mixingdata to ITCbuffer °C. 25 at held and only buffer dialysis or 500 to cutoff membrane (Spectra/Por). AQP0 CaCl mM 5 and 7.0 pH HEPES, AQP0 The (Biomatik). V230A,L234A, I236A or L237A were synthesized and purified to >98% purity L227A, mutants point the or 223–242) (residues sequence bovine wild-type AQP0 Lifescience). (GE VP-ITCMicroCal calorimetry. titration Isothermal experimental map ( the ofthat cross-correlationcompared to 0.95a gaveof model final the to ing and remove steric interactions. A map calculated at 25 Å ( jugate gradient minimization routines in Chimera to regularize the geometries 223–241) were subjected to steepest decent minimization and subsequent con molecules and the AQP0 C-terminal linker and CaM binding domains (residues (residues 223–226) were manually built and minimized with COOT subunit each oftransmembrane helixlast the to helices C-terminalAQP0 the connecting linkers cytoplasmic respectively.The Ile482, andTrp485 residues ptGADhydrophobicwerethewith andLeu237 structurallyaligned anchoring Ecocyte and injected with 10 ng of RNA encoding wild-type, Y149G, Y149L Y149G, wild-type, encoding RNA of ng 10 with injected and Ecocyte Waterassays. permeability tory. Pore profile analysis was performed with HOLE shown, were calculated as the average converted to the single-channel osmotic permeability constant. Statistics, where each trajectory was converted to a collective diffusion constant, which was then coordinateresultantforcollectiveThe trajectories. the of equilibratedportion the over repeated was process the and pore, the in remained they while lated calcu werewaters these of velocities the selected, pore the within watersand previously of each trajectory. Water permeabilities were calculated with a method described (ref. 1.8.7 VMD with performed were analyses trajectory and graphics Molecular control, and a Noseì–Hoover–Langevin piston was used for pressure controlatoms were held fixed. A Langevin dynamics scheme was used for temperature bonded forces and 1 fs for bonded forces. All bond lengths involving hydrogen of motion with a time step of 4 fs for electrostatic forces, 2 fs forA reversibleshort-range multiple-time-step nonalgorithm function. switching a with Å, 12 at off cut were interactions real-space range method Ewald mesh ids, respectively, and the TIP3P model was used for water CHARMM22andCHARMM32 force fields for 560 ns for the AQP0–CaM complex and 495 ns for the apo-AQP0 system. Unrestrained simulations were run at constant temperature and pressure (1 atm)The protein backbone atoms were then released in a stepwise manner over 4 ns. proteinfixed.backboneatomsthe held volumewith and temperature K) (300 conjugate-gradient energy minimization followed by 4 of ns189,933 of atoms.simulation These starting at configurationsconstant were minimized by 1,000 AQP0steps tetramer,of 410 POPC molecules, 40,175 waters ( and 32 counterions, for a total systems,the CSII sites of AQP0 were unaltered from startingconformations the simulationstwoAQP0ofthe in wereoriginal identical. bothIn PDB deletion of the CaM coordinates from the AQP0–CaM complex. In this way, the tion of the AQP0 tetramer (residues 5–239) in the absence of CaM waters,was created forby a total of 244,011 atoms. For theAQP0 CaM–free tetramer, system, twoa startingCaM molecules,conforma 8 Ca 239)and PDB tothestructure presented in AQP0–CaMThecomplexconstructedfree). similarlywassystem (CaM alone SupplementaryFig.4 The simulations were performed with the NAMD 2.7b1 (ref. (ref. 2.7b1 NAMD the with performed were simulations The 6 9 µ ) over the last 519 ns (AQP0–CaM system) or 474 ns (CaM-free system) M andtitratedM containingintoMicroCalcell the Ca 4 9 and implemented in VMD using tcl. In brief, the pore was defined 1NW σ D 6 = 4.96) in Chimera. for CaM. The complex inour simulations consisted of one 5 was used to calculate electrostatic interactions. Short- interactions. electrostatic calculate to used was ). Thesimulation). forCaM-freethe system included the CBD nature structural & molecular biology molecular & structural nature peptides and CaM were dialyzed against 20 mM mM 20 against dialyzed were CaM and peptides Oocytes from Oocytes Figure 2 over 72 h with a 1,000-Da molecular-weight1,000-Da a with h over72 Two simulation systems weregenerated for T eprmns ee efre o a on performed were experiments ITC 1 ± , with,PDB CBD s.e.m. of the four pores from each trajec 2+ 6 peptide concentrations were adjusted ions, 410 POPC molecules and 56,701 6 Xenopuslaevis was used to integrate the equations CBD 62 , 6 3 peptides corresponding to the the to corresponding peptides werefor used protein andlip 2+ 2B6 –CaM, and thermodynamicand –CaM, 4 P 8 for AQP0 (residues to5 . 6 4 . The smooth particle σ were obtained fromobtained were = 3.5) correspond 2+ –CaM at20 –CaM 6 2B6 0 . The CaM 6 P 1 model ). The The ). 67 µ , 6 M 8 ------.

© 2013 Nature America, Inc. All rights reserved. Santa Cruz Biotech AQP0 (H-44) rabbit polyclonal IgG; sc-99059) followed by a Levels of AQP0 abundance were probed with an AQP0 primary antibody (1:500 (Life Technologies) and transferred to PVDF (Life Technologies) for 1 h to at approximately100 V. half an oocyte) was separated on a NuPAGE 4–12% Bis-Trisgel in gel Membrane Protein Extraction Kit (Calbiochem/EMD Millipore). The uncroppedblot after isolation of plasma-membrane proteins with the ProteoExtract Native initial swelling rate. Total membrane expression levels were assayed by western d V the water.ofof slope initial The oocyte at time 0, time, V the formula permeability,calculatedwetime, intrinsicmembrane,anproperty theof from area.powerofFromestimatesof3/2 function volumethethese ofa as asume from a stage micrometer. These cross-sectional data were used to calculate vol number was converted to area with the scale factors of the microscope measured automatically by ImageJ and transferred to an Excel spreadsheet in which pixel a few seconds). The number of pixels in the oocyte cross-section was calculated acquisition of images of the oocyte at appropriate time intervals (on the order of function of time. The cross-sectional area was measured as a function of time for surements of the cross-sectional area of the osmotically challenged oocytes as a a dilution of ND96. Swelling rates due to water flow were estimated from mea to response in oocyte the of area cross-sectional in increase the ofsurements automatedmea from estimated was which increase,volume of rate the from previously Technologies) described as orY149S AQP0 generated with the mMessage mMachine T3 kit (Ambion/Life nature structural & molecular biology molecular & structural nature ( V/V Figure o o )/ is the volume at time 0, S dt in the formula above. Thus P 4 f is shown in = [ ∆ d osm ( V/V is the osmotic gradient and V o )/ Supplementary Figure 5 dt ][ V o /S o is the estimated geometric surface area of the o (t) ]/[ curve was used to determine the value of value the determine to used was curve P ∆ 14 f osm

is directly proportional to the observed , 2 6 . Water permeability was calculated Waterwas . permeability V w ]. V is the volume as a function of . Each sample (corresponding w is the partial molar volume ­ ­ - 68. 67. 66. 65. 64. 63. 62. 61. 60. 59. protein abundance from immunoblots can be problematic. LAS-4000 gel documentation system. We note that the accurate assessments of Fuji a on exposed Westand Signal (Thermo)Super Femto kit a with detected antibody secondary (1:10,000 Promega anti-Rabbit IgG HRP; cat. no. W4011), 69.

elr SE, hn, . Pso, .. Bok, .. osat rsue molecular pressure Constant B.R. Brooks, & R.W. Pastor, Y., Zhang, S.E., Feller, dynamics molecular pressure Constant M.L. Klein, & D.J. Tobiax, G.J., Martyna, Verlet Generalized K. Schulten, & A. Windermuth, H., Heller, H., Grubmuller, U. Essmann, M.L. Klein, & R.W. Impey, J.D., Madura, J., Chandrasekhar, W.L., Jorgensen, R.W. Pastor, & R.M. Venable, Jr., A.D. MacKerell, B.R., Brooks, J.B., Klauda, A.D. MacKerell, graphics. molecular for J.C. tools Phillips, model-building Coot: K. Cowtan, & P. Emsley, Mindell, J.A. & Grigorieff, N. Accurate determination of local defocus and specimen Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. molecular visual VMD: K. Schulten, & A. Dalke, W.,Humphrey, oprsn f ipe oeta fntos o smltn lqi water. liquid simulating for functions potential simple of Comparison (2005). bilayer. DPPC a and alkanes of simulations An proteins. of studies dynamics yais iuain Te agvn itn method. piston Langevin (1995). 4613–4621 The simulation: dynamics algorithms. Simul. Mol. interactions. long-range with simulations dynamics molecular efficient for algorithm (1995). 8577–8593 Phys. Chem. Crystallogr. Biol. D Crystallogr. Acta microscopy. electron in tilt Graph. ab initio ab

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