The Architecture of CopA from Archeaoglobus fulgidus ... · PDF fileStudied by Cryo-Electron Microscopy and Computational Docking ... cryo-electron microscopy of a new ... and metallochaperones

Structure

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TheArchitectureofCopA fromArcheaoglobus fulgidusStudied by Cryo-Electron Microscopyand Computational DockingGregory S. Allen,1 Chen-Chou Wu,1 Tim Cardozo,3 and David L. Stokes1,2,4,*1Skirball Institute2Department of Cell Biology3Department of PharmacologyNew York University School of Medicine, 540 First Avenue, New York, NY 10016, USA4New York Structural Biology Center, 89 Convent Avenue, New York, NY 10027, USA

*Correspondence: [email protected]

DOI 10.1016/j.str.2011.05.014

SUMMARY

CopA uses ATP to pumpCu+ across cell membranes.X-ray crystallography has defined atomic structuresof several related P-type ATPases. We have deter-mined a structure of CopA at 10 A resolution bycryo-electron microscopy of a new crystal form andused computational molecular docking to study theinteractions between the N-terminal metal-bindingdomain (NMBD) and other elements of the molecule.We found that the shorter-chain lipids used toproduce these crystals are associated with move-ments of the cytoplasmic domains, with a noveldimer interface and with disordering of the NMBD,thus offering evidence for the transience of its inter-action with the other cytoplasmic domains. Dockingidentified a binding site that matched the location ofthe NMBD in our previous structure by cryo-electronmicroscopy, allowing a more detailed view of itsbinding configuration and further support for itsrole in autoinhibition.

INTRODUCTION

Copper is essential to cells as a cofactor for a wide variety of

enzymes such as superoxide dismutase and cytochrome

oxidase. If not carefully controlled, however, Cu can be toxic,

due to its redox potential and its ability to produce free radicals

(Rae et al., 1999). As a result, a system of pumps, transporters,

and metallochaperones has evolved to control the delivery and

distribution of Cu. In general, intracellular Cu scavengers, such

as metallothioneins, ensure that there is a vanishingly low

concentration of free Cu in the cytoplasm (Rae et al., 1999),

and metallochaperones, such as Atox1, CCS, and Sco1/2, are

used to carry the Cu through the cytoplasm and deliver it to

specific targets (Lutsenko et al., 2007). In mammals, Ctr1 is

a secondary transporter on the cell surface that facilitates entry

into the cell (Lee et al., 2001), and two transmembrane

ATPases, ATP7A and ATP7B, pump Cu across the endoplasmic

reticulum and plasma membrane, respectively (Lutsenko et al.,

Structure 19, 1219–12

2007). Mutations in ATP7A give rise to Menkes disease, resulting

from insufficient delivery of Cu in brain and other tissues,

whereas mutations to ATP7B are responsible for Wilson’s

disease, where Cu overload is responsible for liver and brain

dysfunction (Barry et al., 2010; Veldhuis et al., 2009a).

ATP7A andATP7B are homologous to CopA frombacteria and

belong to the family of P-type ATPases (Lutsenko and Kaplan,

1995). This family comprises ATP-dependent, transmembrane

ion pumps that share a common reaction mechanism, mem-

brane topology, domain organization, as well as conserved

sequence motifs implicated in ATP hydrolysis and in formation

of their hallmark phosphoenzyme intermediate (Kuhlbrandt,

2004; Møller et al., 1996). Phylogenetic analysis of P-type

ATPases delineates five subgroups, denoted PI–PV (Axelsen

and Palmgren, 1998). CopA, ATP7A, and ATP7B are in the PIB

subfamily together with transporters of a diverse array of

transition and heavy metal ions such as Cu+, Cu2+, Zn2+, Cd2+,

Co2+, and Pb2+.

PII ATPases are themost thoroughly studied and have become

paradigms for the family. They includeCa2+-ATPase andNa+/K+-

ATPase, which have ten transmembrane helices (M1–10) and two

main cytoplasmic loops inserted between M2/M3 and M4/M5.

The larger cytoplasmic loop (between M4/M5) folds into two

separate domains, thus composing the nucleotide-binding (N)

domain and phosphorylation (P) domain, whereas the smaller

loop (between M2/M3) composes the so-called actuator (A)

domain.Eachdomain ischaracterizedbysequencemotifs related

to their individual roles in the reaction cycle, namely DKTGTLT at

the phosphorylation site of the P domain, isolated residues

surrounding the ATP binding site in the N domain, and the

conserved TGE in the A domain (Olesen et al., 2007). The well-

characterized reactioncyclealternatesbetweenE1andE2states,

in which transmembrane ion binding sites are oriented toward the

intracellular or extracellular milieu, respectively. Switching

between these two states is controlled by phosphorylation of

the catalytic aspartate in the conserved DKTGTLT sequence,

which instigatescoordinatedmovementsofN,P, andAand trans-

membrane domains. By coordinating changes in the affinity and

accessibility of the ion binding sites, ions are transported against

a concentration gradient: that is, E1 binds cytoplasmic ions with

high affinity, whereas E2 binds extracellular (or luminal) ions with

much lower affinity (Jørgensen and Andersen, 1988).

32, September 7, 2011 ª2011 Elsevier Ltd All rights reserved 1219

mailto:[email protected]

http://dx.doi.org/10.1016/j.str.2011.05.014

Structure

Cryo-EM and Computational Docking of CopA

PIB ATPases such asCopA, although functionally and structur-

ally related to PII ATPases, are distinguished by having only eight

transmembrane helices and by bearing one or more N-terminal

metal-binding domains (NMBDs). These NMBDs are homolo-

gous to soluble metallochaperones that carry copper through

the cytoplasm (e.g., Atox1). Both NMBDs and metallochaper-

ones bind Cu+ with high affinity via CxxC sequence motifs.

Although it has been postulated that NMBDs mediate transfer

of Cu+ from the metallochaperones to transport sites, there is

increasing evidence that these domains are instead involved in

autoregulation and, in the case of ATP7A, in targeting the mole-

cule to the basolateral membrane (see reviews by Arguello et al.,

2007; Lutsenko et al., 2007). The structural basis for this autore-

gulation remains to be determined.

X-ray structures have been solved for several intact PII

ATPases, including Ca2+-ATPase from sarcoplasmic reticulum

(SERCA1) (Toyoshima et al., 2000), Na+/K+-ATPase (Morth

et al., 2007), andH+-ATPase (Pedersen et al., 2007). These struc-

tures have demonstrated the nature of the E1 and E2 states as

well as the role of conformational changes in coordinating the

multiple steps of the enzymatic cycle (Olesen et al., 2007).

X-ray structures of PIB-type ATPases have so far only involved

isolated cytoplasmic domains of CopA from Archeaoglobus

fulgidus: specifically, a construct containing the N and P

domains (Sazinsky et al., 2006b; Tsuda and Toyoshima, 2009)

and another construct of the A domain alone (Sazinsky et al.,

2006a). NMR has revealed the binding of ATP to the isolated N

domain (Banci et al., 2010) and has characterized the structural

homology between various NMBDs and the soluble metallocha-

perones (e.g., Banci et al., 2006). Our previous reconstruction of

CopA using cryo-electron microscopy (cryo-EM) is the only

structure of an intact PIB-type ATPase. Although the resolution

was limited (�17 A), this reconstruction served as a template

for building a CopA homology model that defined the location

of the NMBD relative to the other cytoplasmic domains and sug-

gested that the NMBD could regulate CopA activity by holding

the enzyme in an inactive state in the absence of copper and

ATP (Wu et al., 2008). A different conclusion was reached by

a more recent study employing chemical crosslinking, which

suggests that the NMBD binds to the opposite side of the mole-

cule and represents a static structural element of the cyto-

plasmic domain (Lubben et al., 2009).

To reevaluate the location and role of the NMBD, we have

determined a higher-resolution structure using cryo-EM and

helical reconstruction of tubular crystals of CopA from

A. fulgidus. This 10 A resolution structure reveals well-defined

cytoplasmic domains that are readily fit with the A, N, and P

domains from X-ray crystallography. Although the transmem-

brane domain is less distinct, the new map leads us to propose

a novel location for the extra two N-terminal helices that charac-

terize the PIB subfamily. Surprisingly, the NMBD is disordered

in these crystals, presumably due to dramatic changes in the

molecular packing of CopA, thus supporting the idea that the

NMBD is not a static structural element of the cytoplasmic

domain. Based on an all-atom homology model for the cyto-

plasmic domains of CopA, we used computational methods

to dock the NMBD, thus identifying the most favorable site

of interaction. The results are consistent with our previous

structure from cryo-EM and with the proposed autoinhibitory

1220 Structure 19, 1219–1232, September 7, 2011 ª2011 Elsevier Lt

role for NMBD, which would act by retaining CopA in the E2

conformation.

RESULTS

New Crystal Form for CopAOne of our primary goals was to obtain a higher-resolution struc-

ture of CopA in order to understand better the architecture of the

various domains that compose the molecule. As in earlier work,

we used Escherichia coli to express DC-CopA from A. fulgidus

that carried the functionally important N-terminal MBD, which

is common to all PIB ATPases, but not the C-terminal MBD,

which is a unique feature of this particular CopA homolog.

Expression levels were quite high, producing a final yield of

6–9 mg of detergent-purified protein from a 6 l expression with

purity >98% (see Figure S1 available online).

By altering the lipid species and the temperature used for

crystallization, we obtained wider tubular crystals with stronger

diffraction compared to our previous work. Previously, tubular

crystals of DC-CopA (Wu et al., 2008) were produced at 50�Cwith a reconstituted membrane of DOPC (1,2-dioleoyl-sn-glyc-

ero-3-phosphocholine) and in a solution containing the Cu+

chelator BCDS (bathocuproinedisulfonic acid) at pH 6.1. These

tubes were 35 nm in diameter and the corresponding structure

resembled that of SERCA in the E2 enzymatic state, consistent

with the absence of Cu+ in the crystallization media. For the

current work, the lipid was changed to a mixture of DMPC/

DOPE (1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dio-

leoyl-sn-glycero-3-phosphoethanolamine) (4:1 weight ratio),

which produced substantially wider (60 nm diameter) tubular

crystals at 30�C under otherwise identical conditions (Figure 1).

Layer line data from these wider crystals were substantially

stronger and were consistent with the presence of several

related helical symmetries, characterized by systematic differ-

ences in diameter and by the Bessel orders of principal layer

lines: (n1,0, n0,1) equal to either (12,�8), (14,�8), or (14,�10).

Among tubes with (12,�8) symmetry, the variability of unit cell

dimensions for the underlying 2D lattice was small (SD <0.5 A

and <0.4� for the included angle) (Table 1), making the Fourier-

Bessel approach to helical reconstruction viable (Diaz et al.,

2010). Based on two-fold related phase residuals, the final

map from an average of 12 tubes was judged to have 10 A reso-

lution, compared to 17 A from a similar number of tubes in DOPC

(Figure 2C). The visibility of several individual a helices in themap

(discussed below) is consistent with this estimate of resolution.

Packing of DC-CopA in Tubular CrystalsThe reconstruction shows that the orientation of DC-CopA in

DMPC/DOPE tubes is inverted relative to DOPC tubes (Figure 2;

Figure S2). Similar to helical crystals of Ca2+-ATPase (Xu et al.,

2002) and nicotinic acetylcholine receptor (Toyoshima and Un-

win, 1988), DC-CopA molecules in DOPC tubes were oriented

with their cytoplasmic domains facing the outside of the highly

curved bilayer. In these cases, the positive curvature is presum-

ably induced by the larger linear dimension required to accom-

modate the much larger cytoplasmic domains relative to the

transmembrane and luminal domains (Young et al., 1997).

Surprisingly, the large cytoplasmic domains of DC-CopA face

the inside of tubes produced from DMPC/DOPE. This inverted

d All rights reserved

Figure 1. Images of Tubular Crystals of DC-CopA

(A–C) Tubes grown in a 4:1 mixture of DMPC/DOPE at 30�C.(D–F) Tubes grown in DOPC at 45�C. Except for the differences in lipid composition and temperature, the crystallization conditions were otherwise identical.

Low-magnification images of negatively stained crystals are shown in (A) and (D), where the scale bars represent 200 nm. Higher-magnification images of

frozen-hydrated tubes used for helical reconstruction are shown in (B) and (E), where the scale bars represent 50 nm. Computed Fourier transforms in (C) and (F)

reveal layer lines characteristic of helical symmetry, which are indexed according to their Miller index and Bessel order in the right margin (h,k;n). The edge of

these transforms is at �20 A resolution.

See also Figure S1.

Structure


orientation was also observed for tubular crystals of Na+/K+-

ATPase grown in native membranes from the duck supraorbital

salt gland, although in this case the b subunit added substantial

mass to the outside of these tubes (Rice et al., 2001).

Table 1. Comparison of Unit Cell Dimensions for Tubular Crystals

of DC-CopA

Cytoplasmic Domaina Membraneb

DMPC/DOPE DOPC DMPC/DOPE DOPC

Radius (A) 136 120 200 75

a (A) 63.9 ± 0.24 97.5 ± 1.38 82.9 ± 0.29 78.7 ± 0.92

b (A) 69.8 ± 0.40 61.1 ± 0.76 78.5 ± 0.42 48.7 ± 0.47

g (�) 78.6 ± 0.38 101.0 ± 0.66 99.0 ± 0.40 74.4 ± 0.65

Area (A2) 4370 ± 30.3 5851 ± 69.9 6427 ± 44.6 3656 ± 43.7aUnit cell dimensions measured at a radius corresponding to the middle

of the A domain, where major intermolecular contacts occur.bUnit cell dimensions in the middle of the membrane, that is, halfway

between the density peaks in the mean radial density profile that corre-

spond to the lipid phosphate head groups (see Figure 3).


The packing of DC-CopA molecules is also substantially

different in DMPC/DOPE tubes. In both crystal forms, CopA

forms dimers that are stabilized by reciprocal interactions

between N and A domains. In DOPC tubes, the dimers pack

into ‘‘dimer ribbons’’ reminiscent of Ca2+-ATPase crystals (Tay-

lor et al., 1986), in which contacts between A and N domains

form a continuous ribbon of molecules related by two-fold

symmetry, with another ribbon running in the opposite direction

along the b axis of the unit cell (Figure 2B). In contrast, DMPC/

DOPE tubes lack dimer ribbons; instead, the lattice is character-

ized by a more isotropic set of contacts between neighboring

dimers. As will be discussed in more detail below, the geometry

of the dimer itself is also quite different, with a 50� angle between

the molecules. As a result of these differences, the density of

cytoplasmic domains in DMPC/DOPE tubes is 33% higher

than in DOPC tubes, based on the area of the unit cell at radii cor-

responding to the middle of these domains (Table 1).

In contrast to the cytoplasmic domains, the transmembrane

domains in DMPC/DOPE tubes are less densely packed, with

a density at the center of the membrane that is <60% relative

to DOPC tubes (Table 1). Another way to look at this difference


A

B

resolution (Å)

25.00 12.50 8.30 6.25

2-fo

ld p

hase

resi

dual

(deg

)

0

10

20

30

40

50

DMPC/DOPEDOPC

DMPC/DOPE

DOPC

C

ab

a

b

N

NA A

Figure 2. Packing of DC-CopA in the Two Types of Tubular Crystals

(A) Cytoplasmic domains of DC-CopA face the inside of DMPC/DOPE tubes

and are tightly packed with an isotropic set of intermolecular contacts.

(B) Cytoplasmic domains face the outside of DOPC tubes and are packed in

‘‘dimer ribbons’’ that run along the b axis of the array. Both types of crystals are

composed of DC-CopA dimers, and two-fold related molecules have been

colored red and blue in this figure with the unit cell outlined in black.

Structure



is to compare membrane and cytoplasmic domains within each

crystal: the unit cell area within the membrane of DOPC tubes is

considerably smaller (64%) than in the region of the cytoplasmic

domains, as one would expect given the smaller size of themole-

cule within the membrane. In contrast, the inverted nature of

the DMPC/DOPE tubes provides a much larger area to the

membrane domains relative to cytoplasmic domains (150%).

This excess membrane area in DMPC/DOPE tubes may

explain the diffuse density seen on the outer surface of the

reconstruction (Figure 3). When images of individual tubes are

projected down their cylindrical axes, several discrete peaks

are observed in the resulting 1D profile (Figure 3C). At the inner-

most radii, these peaks correspond to cytoplasmic domains,

followed by two peaks from the phosphate head groups of the

lipid bilayer. Unlike previous tubes of CopA and of Ca2+-ATPase,

there is an additional density peak on the outer surface of the

membrane in DMPC/DOPE tubes (arrow in Figure 3C). Helical

reconstruction of these tubes produces a considerable amount

of low, diffuse density in this region. More organized features

with somewhat higher density are visible outside the tubes, but

these features lie directly along symmetry axes, where noise

builds up during the reconstruction process (e.g., compare

sections shown in Figures 3A and 3B). From these observations,

we speculate that there is a population of disordered CopAmole-

cules with their cytoplasmic domains facing the outside of the

DMPC/DOPE tubes. Indeed, a symmetric distribution of mole-

cules across the membrane is expected from reconstitution,

and the inverted geometry of the CopA crystals within DMPC/

DOPE tubes produces excess membrane area that could readily

accommodate extra molecules facing the outside of the tubes.

These extra molecules would be unable to participate in regular

crystal contacts given the long distance between their cyto-

plasmic domains. Furthermore, the presence of disordered

molecules would explain why, in the helical reconstruction, the

protein domains within the membrane have much lower contrast

relative to the cytoplasmic domains on the inside of the tube.

Functional Analysis of CopABecause of the dramatic effect of bilayer composition on the

morphology and molecular packing of the tubular crystals, we

used ATPase assays to assess whether lipid had an effect on

DC-CopA function. In particular, we characterized the depen-

dence of ATPase activity on Cu+ or Ag+ concentrations and on

temperature, reasoning that bilayer composition might affect

either the mobility or the stability of the enzyme. The results (Fig-

ure 4) indicate that DC-CopA has an apparent Kd of 0.44 mM for

Cu+ and 0.26 mM for Ag+ in DMPC/DOPE and corresponding

Vmax of 1.97 and 1.76 mmol/mg/min. For comparison, affinities

in DOPC were 0.23 and 0.22 mM for Cu+ and Ag+ with Vmax of

0.94 and 1.48 mmol/mg/min, respectively. These values are

similar to those previously measured by Rice et al. (2006) from

detergent-solubilized DC-CopA: Kd of 0.11 and 0.2 mM for Cu+

and Ag+, respectively, with Vmax of 1.8 and 2.4 mmol/mg/min.

In all cases, ATPase activity was partially inhibited at high

concentrations of either Cu+ or Ag+, probably reflecting the

(C) Two-fold phase statistics for the two helical reconstructions, documenting

a resolution of 17 and 10 A for DOPC and DMPC/DOPE crystals, respectively.

See also Figure S2.


z=0 z=12

0 54 108 162 216 270 324

radius (Å)

proj

ecte

d de

nsity

radius (Å)

0 100 200 300

mea

n ra

dial

den

sity

BA

DC

Figure 3. Radial Distribution of Mass in DMPC/DOPC Tubes

(A and B) Contour plots of individual sections from the density maps at z = 0 and z = 12 A, where green dashed contours indicate low density and black contours

start at a threshold corresponding to the molecular envelope. The boundaries of the bilayer and protein domains inside the tube are clearly delineated. The inside

of the tube is devoid of density, but diffuse density is evident on the outside of the membrane. These outer densities are prominent at four-fold and two-fold

symmetry axes present in the section at z = 0, but are less prominent at z = 12, which does not have these symmetry axes.

(C) One-dimensional projection of an individual image along the helical axis. Peaks in this projection correspond to the cytoplasmic domains (at radii of�120 and

150 A) and to the phosphate head groups of the bilayer (at radii of 170 and 210 A). An additional peak at 230 A (arrow) is inconsistent with CopA’s lack of an

extracellular domain.

(D) Mean radial density distribution from the helical reconstruction of DC-CopA. The differing height of the peaks reflects the contrast transfer function correction

that has been applied to the data. Even considering this correction, the peak at the outer radius (arrow) has been significantly suppressed during the recon-

struction process. The transparent gray rectangles in (C) and (D) delineate the membrane.

See also Figure S2.

Structure


low-affinity ion binding sites involved in ion release to the extra-

cellular side of the membrane. For both lipids, maximal activities

were obtained at 65�C, which compares well with published

studies of detergent-solubilized CopA by Mandal et al. (2002)

and to our own unpublished observations of detergent-solubi-

lized CopA.

Atomic Model for CopAThe new density map from DMPC/DOPE tubes was used as

a template for building a model of CopA that revealed the juxta-

position of cytoplasmic domains and the organization of the

transmembrane helices. The first step was to build an all-atom

model for the core of CopA using an X-ray crystallographic struc-

ture of SERCA1a as a template. For the cytoplasmic domains,


X-ray crystal structures for the isolated A domain of CopA

(Protein Data Bank [PDB] ID code 2HC8) (Sazinsky et al.,

2006a) and for the isolated pair of N and P domains of CopA

(PDB ID codes 3A1C and 2B8E) (Sazinsky et al., 2006b; Tsuda

and Toyoshima, 2009) were aligned with SERCA1a in the E2

conformation (PDB ID code 1IWO) (Toyoshima and Nomura,

2002). For the transmembrane helices and their connections to

the cytoplasmic domains, the sequence of CopA was aligned

with SERCA1a and used tomodel by homology. Two extra trans-

membrane helices are predicted in the N-terminal region of

CopA and, after building a helices with the corresponding

sequences (see Experimental Procedures), they were inserted

between M1 and M2 of SERCA1a, as suggested by previous

reports (Hatori et al., 2007; Lutsenko and Kaplan, 1995; Wu


Cu concentration ( M)

0.1 1 10

activ

ity (

mol

es/m

g/m

in)

0.0

0.4

0.8

1.2

1.6

2.0

Ag concentration ( M)

0. 11 100.0

0.4

0.8

1.2

1.6

A B

temperature (°C)0 20 40 60 80 100

activ

ity (

mol

es/m

g/m

in)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

temperature (°C)0 20 40 60 80 100

0.0

0.5

1.0

1.5

2.0

C D

DMPC/DOPEDOPC

Figure 4. ATPase Activity of Reconstituted DC-CopA

(A and B) Concentration dependence of activity for Cu+- and Ag+-dependent activity. A background level of �0.3 mmol/mg/min has been subtracted from these

data, which have been fitted with the Michaelis–Menten equation for a single binding site. Because there is inhibition at higher ion concentrations, the last two or

three points of each curve have been omitted for this fit. The resulting values for apparent Kd and Vmax are cited in the text.

(C and D) Temperature dependence of activity for Cu+ (C) and Ag+ (D) shows a peak at�65�C, which is consistent with previous work with detergent-solubilized

CopA. In all plots, the open circles represent DC-CopA reconstituted in DMPC/DOPE and the filled circles represent DC-CopA reconstituted in DOPC. Error bars

correspond to the standard error of the mean.

Structure


et al., 2008). The resulting hybrid homology model lacked the

N-terminal and C-terminal metal-binding domains, which have

no counterpart in the SERCA1 template. This hybrid model

was used for the docking studies described below.

The density map from DMPC/DOPE tubes was used to

reposition the individual domains of this DC-CopA homology

model to produce a fitted model. Specifically, after placing the

homology model roughly into the envelope from the helical

reconstruction, the loops connecting the domains were broken

and the position of each cytoplasmic domain was refined using

cross-correlation (using the Fit-in-Map feature of Chimera). The

density map accounted well for these domains, with evidence

for individual a helices in several locations (Figure 5A; Movie

S1). Surprisingly, the A, N, and P domains of CopA accounted

for all of the densities in this region of the map, indicating that

the NMBD was disordered. In our previous map from DOPC

tubes, the NMBD was seen near the dimer interface, and its

disordering in the current DMPC/DOPE tubes may be due

to changes in this interface. Indeed, Figure 6 shows that there

is an �50� angle between two-fold related molecules in the


DMPC/DOPE tubes (defined by axes running from the middle

of the transmembrane helices to catalytic aspartate), whereas

these molecules were almost parallel in DOPC tubes. There is

also a shift in the cytoplasmic domains relative to one another

(Figure 7), which may explain the absence of the NMBD in the

map from DMPC/DOPE tubes.

Protein densities were less distinct within the membrane than

in the extramembranous regions of the map, although the

general outline of the transmembrane domain could be dis-

cerned (Figure 5B). This loss of contrast is common in helical

reconstructions and may be exacerbated in this case by the

extra, disordered molecules hypothesized to face the outside

of the tubes, which would contribute to the background density

within themembrane. Despite the poorer definition of membrane

domains, we were able to use our all-atommodel of DC-CopA to

interpret the transmembrane densities. Specifically, the six

conserved transmembrane helices (M1–M2, M5–M8) fell within

strong density. Similar to maps of SERCA from helical crystals

(Xu et al., 2002; Young et al., 2001; Zhang et al., 1998), a density

was observed at the cytoplasmic surface above M1 (arrowhead


Figure 5. Fitting an Atomic Model to the Map from DMPC/DOPE

Tubes

(A) Domains are shown in various shades of red and are labeled accordingly.

Isolated densities for individual a helices are visible in A and P domains, and

other a helices at the periphery of N and P domains fit snugly within the iso-

surface density envelope. The envelope for the transmembrane domain (TM)

shows a distinct bulge at the top right, consistent with the kinked cytoplasmic

end of M1.

(B) Section through the transmembrane region of the densitymap overlaid with

transmembrane helices, which are numbered. This is a view from the extra-

cellular side of the membrane.

See also Movie S1.

Structure


in Figure 5A) that matches the bent extension of this helix seen in

atomic structures of SERCA, Na+/K+-ATPase, and H+-ATPase.

Additional membrane density was observed surrounding M4,

which we fitted with the extra M2 and M3 helices that are char-

acteristic of PIB ATPases. The location of these helices is slightly

displaced from our previous model from DOPC tubes, with M2

taking the position of M3 in the previous map and M3 falling

Figure 6. Comparison of the DC-CopA Dimer Seen in the Two Helical

(A) Dimer from the DMPC/DOPE tubes viewed parallel to themembrane surface. T

the membrane normal.

(B) The dimer from DMPC/DOPE tubes viewed normal to the membrane surface

(C) Dimer from our previous map from DOPC tubes (PDB ID code 2VOY; Wu et a

transmembrane domain is closely aligned to the membrane normal. The NMBD,

purple for the blue monomer.

(D) Dimer from DOPC tubes viewed normal to the membrane surface. Note that (B

correspond to the membrane.


between M4 and M8. Given the poor contrast within the trans-

membrane domains, the precise location of these two extra

helices remains tentative.

Computational Docking of the NMBDBecause our previous density map from the thinner DOPC tubes

revealed the binding site of the NMBD next to the A domain, we

were disappointed that the current map from DMPC/DOPE

tubes did not provide further information at higher resolution.

Nevertheless, we hypothesized that the NMBD binding site

previously identified next to the A domain, although perturbed,

still represented the most structurally compatible location for

docking of the NMBD. To test this hypothesis, we performed

in silico docking of the NMBD to the other cytoplasmic domains

of CopA models obtained either by homology to the E2 state

of SERCA or by fitting to our map from DMPC/DOPE tubes.

This procedure uses Monte Carlo sampling to identify favor-

able docking sites on the molecular surface and ranks them

energetically.

For our initial docking, we used the hybrid homology model of

DNDC-CopA based on SERCA1 in the E2 conformation as

a template, due to its close resemblance to the structure from

DOPC tubes in which the NMBD was seen. For a search target,

we made a homology model of NMBD based on the structure of

a CopZ metallochaperone from Bacillus subtilis. This particular

template was chosen for its high resolution (1.5 A) and for its

high sequence identity (41%) relative to the CopA NMBD from

A. fulgidus. Although the CopZ template structure had Cu+

bound to its CxxC motif, NMR structures of Cu+-free NMBD

homologs indicated root-mean-square deviations of only �1 A,

suggesting that structural changes induced by Cu+ binding are

minimal. For docking, the homology models for Cu-free NMBD

Crystals

his view illustrates the >30� inclination of the transmembrane domain relative to

.

l., 2008) viewed parallel to the membrane surface showing that in this case the

which is visible in these crystals, is shaded in orange for the red monomer and

) and (D) are at a smaller scale than (A) and (C); the transparent gray rectangles


Figure 7. Docking of the NMBD to an All-

Atom Model for DC-CopA

(A and B) Two views of the result obtained by

computational docking related by an 180� rotationabout the vertical axis. The NMBD is colored

purple, the A domain is in yellow, the N domain is

in blue, the P domain is in green, and the trans-

membrane helices are in cyan. Key residues

shown are C27 from the GMTCAMC30 Cu-binding

loop of the NMBD, H462 from the ERRSEHP463

ATP-binding loop in the N domain, and the cata-

lytic D424 from the DKTGTLTT431 phosphorylation

loop in the P domain.

(C and D) Equivalent views of an overlay between

the homology model used for docking (same as in

A and B but colored gray and lacking the NMBD)

and the atomic model fitted to the density map

from DMPC/DOPE tubes (colored shades of red).

The P domains are aligned, revealing relative

movements of the N and A domains as well as

a different angle of the transmembrane domain.

See also Figure S3.

Structure


and CopA were treated as independent, unconstrained mole-

cules free to rotate and translate in all directions in space, and

the ICM docking routine evaluated numerous alternative binding

interfaces, ultimately ranking them according to their predicted

binding energy. Table 2 shows that the lowest-energy solution

was well separated from the other solutions, making it the clearly

preferred location for the NMBD on the molecular surface of

CopA. Significantly, this binding pose is consistent with the loca-

tion of the NMBD in DOPC tubes, despite the lack of physical

constraints or any other information related to the EM structure.

This docking result places the CxxC Cu-binding loop of the

NMBD next to a conserved loop in the N domain that controls

ATP binding and, when bound, the Cu+ ion would lie within

13 A of theMg2+ ion at the catalytic site of the Pdomain (Figure 7).

Unlike the model previously derived directly from the EM struc-

ture, the docking solution packed the two a helices of the NMBD

against the A domain. Specific interactions at this interface also

involve the loops between b strands composing the body of the

A domain (residues 252–256, 270–274, 278–281, 295–298). The

surface area buried in this interaction is 1943 A2, which is divided

between the three domains as follows: 1040 A2 for the A domain,

507 A2 for the P domain, and 374 A2 for the N domain.

For comparison, we also used the fitted model corresponding

to the conformation observed in DMPC/DOPE tubes as a tem-

plate for docking, although in this case the apparent disordering

of the NMBD in the corresponding crystals led us to expect

1226 Structure 19, 1219–1232, September 7, 2011 ª2011 Elsevier Ltd All rights reserved

a lower binding energy. Indeed, the ICM

docking routine determined that the

total energy for this pose was almost

17 kcal/mol higher for the DMPC/DOPE

tube model compared to the SERCA1

homology model (�28.2 versus �44.9).

These energy units are not absolute,

due to inaccuracy in the force field and

the coarseness of the grid on which

potential energies were calculated, but

are indicative of the relative binding

strength. Accordingly, calculations of buried surface area

showed a >40% decrease to 1176 A2. The reason for this

reduced binding energy is evident in Figures 7C and 7D, where

the homology model for CopA is overlaid with the fitted model

for CopA fromDMPC/DOPE tubes. This reveals a significant shift

in the A and N domains relative to the P domain, and the loss of

binding energy is consistent with an alteredmolecular surface for

NMBD docking due to the disrupted interface between A, N, and

P domains. Indeed, intermediate values for binding energy were

obtained in docking studies with other permutations of the

DNDC-CopA model in which the N domain was either moved,

modified, or removed altogether (data not shown). These results

illustrate that all three cytoplasmic domains contribute to the

NMBD binding site and that the binding was strongest when

the conformation resembled the E2 state adopted by SERCA1.

Finally, to complete our model of DC-CopA, we constructed

a linker between the M1 and the NMBD (Figure 7). In SERCA,

twoN-terminal a helices are attached to the front of the A domain

and are connected to the cytoplasmic end of M1 via a flexible

linker. Although the NMBD is associated with the side of the A

domain in our model, the CopA linker sequence is long enough

to wrap under the A domain and to connect to the kinked

extension of M1. Thus, our model of DC-CopA is consistent

with the idea that the physical couplings observed between

SERCA transmembrane helices and the cytoplasmic domains

are conserved in the PIB subfamily of pumps.

Table 2. Results of Computational Docking

RankaEnergy Terms

Totalb vdW Hc vdW Cd H Bonde Electrostaticf Hydrophobicg

1 �44.9 �4.71 �37.83 �4.71 �1.10 �12.32

2 �40.6 �5.76 �32.46 �3.68 �1.31 �11.48

3 �39.4 �5.76 �31.49 �7.75 �2.43 �8.80

4 �36.2 �3.55 �39.56 �2.65 0.42 �13.89

5 �35.9 �4.56 �21.10 �17.63 �4.53 �8.71

6 �35.6 �3.30 �27.97 �7.93 �0.51 �9.68

7 �35.3 0.07 �25.46 �9.20 �1.05 �9.59

8 �35.3 �0.30 �26.78 �3.53 �0.55 �11.70

9 �35.2 �3.05 �28.23 �8.29 �3.38 �11.30

10 �35.2 �7.25 �33.14 �10.48 �2.26 �10.74a Each docking result was ranked according to its total energy. In our application, the lowest-energy solution (rank 1) was well separated from the suc-

ceeding solutions, lending confidence to the result.b Total energy of the conformation. These values are reported as kcal/mol by ICM but should only be taken as relative binding energies due to various

approximations during the calculation.c van der Waals grid potential using a hydrogen probe.d van der Waals grid potential using a carbon probe.eHydrogen-bonding grid potential.f Electrostatic grid potential.g Hydrophobic grid potential.

Structure


DISCUSSION

Using cryo-EM and image reconstruction, we have produced

a new structure of DC-CopA from A. fulgidus at 10 A resolution

from a new form of helical crystal grown in membranous tubes

composed of shorter-chain lipid (DMPC). Although DC-CopA

forms a dimer as in previous DOPC tubes, there are marked

differences in the intermolecular contacts stabilizing this dimer

and in the packing of this dimer in the unit cell; furthermore,

the transmembrane domain is strongly tilted relative to the

thinner DMPC membrane. At 10 A resolution, X-ray structures

of the three isolated cytoplasmic domains could be fitted

precisely into themap. However, density for theNMBD is absent,

indicating that this domain is disordered. We then used compu-

tational molecular docking to explore the site of NMBD binding

relative to the other CopA cytoplasmic domains and its confor-

mational determinants. Using a homology model based on the

E2 conformation of SERCA1, the lowest-energy pose was

consistent with the site previously identified in tubular crystals

composed of DOPC. Although there were differences with our

previous, manually docked model (i.e., an �180� rotation of the

NMBD relative to the A domain), this new model is nevertheless

consistent with an autoinhibitory interaction between the NMBD

and the N and P domains in the E2 conformation. Comparison of

this CopA homology model with a model fitted to the map from

DMPC/DOPE tubes reveals a distinct shift in N and A domains

that is predicted to reduce the binding energy of the NMBD by

�17 kcal/mol. This result suggests that the affinity of the

NMBD for its preferred site has been lowered to the level of

a weak, Brownian association, explaining why it has become

disordered in the crystal.

Although our previous model of DC-CopA from DOPC tubes is

consistent with the E2 enzymatic state of SERCA1, the new

structure from DMPC/DOPE tubes reveals a significant confor-


mational change. As shown in Figure 7, the A domain has shifted

away from the P domain in the new map, leaving room for the N

domain to swing closer to the catalytic site. Such movement is

generally consistent with conformational changes of SERCA1

during its reaction cycle, which depends on an interplay between

the A and N domains to regulate access to the catalytic aspar-

tate. The overarching idea is that the A domain interacts with

elements of the P domain in E2 states, thus preventing the

approach of the N domain and its associated ATP. In E1 states,

the A domain swings out of the way to enable transfer of

phosphate. Figure S3 compares the position of the A domain

in E2-P and E2 states of SERCA1. In E2-P, the conserved (T/S)

GE278 sequence in the A domain is closest to the catalytic aspar-

tate in the P domain (DKTGTLT430), making direct interactions

with the associated Mg2+ ion. In E2, the (T/S)GE loop has moved

back and interacts with the conserved DG(V/I)ND622 loop at the

periphery of the P domain. In our model fitted to DMPC/DOPE

tubes, this (T/S)GE loop has moved back even more, although

it still appears to interact with residues in the DG(V/I)ND loop.

The latter configuration of cytoplasmic domains has not previ-

ously been seen in structures of P-type ATPases and could

represent a transition from the E2 to the E1 state as the A domain

begins to disengage from the P domain. The increased angle of

the transmembrane domain relative to the P domain (Figures 7C

and 7D) is consistent with this transition, as it eventually results in

a 30� upward rotation of the P domain in the E1 state relative

to the E2 state (Toyoshima and Nomura, 2002). Normally, the

E2-to-E1 transition would be initiated by ions binding to their

transport sites in the membrane. In the current situation, we

speculate that a shearing of the transmembrane domain caused

by its oblique angle relative to the membrane might be respon-

sible for inducing this conformation.

Factors that may contribute to the unusually high tilt of the

membrane domain are (1) the negative curvature of the crystal


Structure


contacts between cytoplasmic domains, (2) the intermolecular

contacts that stabilize the dimer, and (3) the thinner bilayer

produced by the shorter aliphatic chains of DMPC, which

composes the bulk of the bilayer. Indeed, the hydrophobic

core thickness of DMPC has been experimentally measured as

23 A compared to 27 A for DOPC (Lewis and Engelman, 1983),

and this difference would be consistent with a tilt of�30� in orderto accommodate a hydrophobic domain with a fixed thickness

(cos�1(23/27)). Given the corresponding difference in the tilt of

the CopA molecule in DMPC/DOPE versus DOPC tubes, it

seems plausible that the thinner DMPC bilayer could be the

driving force for altering the geometry of the dimer and thus

producing the negative curvature of the crystal. Despite the

dramatic effect on crystallization, however, the DMPC/DOPE

bilayer had only modest effects on ATPase activities. It should

be noted that the lipid-to-protein ratio was six times higher for

this assay, where no crystalline arrays were observed to form.

Nevertheless, the approximately 2-fold increase in Kd and Vmax

is comparable to the 2-fold changes in these parameters in

detergent micelles (Rice et al., 2006) and can therefore be

considered to represent the normal range of behavior for CopA

in different hydrophobic environments. Furthermore, this range

is in line with measurements of other P-type ATPases in lipid

and detergent environments (e.g., Cornea and Thomas, 1994;

Warren et al., 1974).

From a structural point of view, a shearing of the transmem-

brane domain would be required tomaintain the upper and lower

boundaries of the transmembrane helices as the domain was

tilted in the bilayer. In other words, one would expect a relative

movement of transmembrane helices along their helical axes.

Such movements have in fact been described for the M1/M2

helical pair of SERCA, both during the transition from E2 to E1

(Toyoshima and Nomura, 2002) and during Ca2+ occlusion by

E1�P (Olesen et al., 2004). In the case of CopA, M2 and M3

are inserted between this M1/M2 pair and placed at the

periphery of the transmembrane domain; accordingly, the entire

M1–M4 bundle would likely slide relative toM5–M8, thus accom-

modating the shear force. By analogywith the structural changes

during the E2-to-E1 transition in SERCA, this movement of

M1–M4would pull the A domain away from the P domain, poten-

tially inducing the conformation seen in DMPC/DOPE tubes. A

corresponding force on the NMBD, which is directly linked to

M1, could also help disengage it from its binding site on the A

domain. According to this hypothesis, M1 would no longer be

tethered to the cytoplasmic domain in the E1 state. This implies

that the more rigid connection between M4 (M2 in SERCA) and

the A domain may be the important element that drives changes

to the transmembrane domain and leads to ion occlusion during

the transition to E1�P.

The site for the NMBD identified by in silico docking is consis-

tent with the corresponding density in our previous map from

DOPC tubes (Wu et al., 2008). The specifics of the interface

between the NMBD and the A domain are different in our pre-

vious model, presumably reflecting our earlier neglect of binding

energy and molecular surface compatibility during manual

assembly and fitting. Nevertheless, in both cases, the NMBD

binds to the side of the A domain with additional interactions

between the GMTCAMC30 Cu-binding loop and the conserved

ERRSEHP463 loop on the N domain. This ERRSEHP463 loop


has been shown in NMR and X-ray crystal structures of isolated

domains to be intimately involved in ATP binding (Banci et al.,

2010; Dmitriev et al., 2006). In particular, His462 makes critical

interactions with the a- and b-phosphates and with the adenine

ring of ATP (Sazinsky et al., 2006b; Tsuda and Toyoshima, 2009);

its mutation in ATP7A almost completely prevents ATP binding

(Morgan et al., 2004) and the H462Q mutation is the most

common cause of Wilson’s disease (Thomas et al., 1995).

Interaction between the NMBD and the N domain has been

demonstrated experimentally by Gonzalez-Guerrero et al.

(2009) using pull-down assays. Specifically, they showed that

Cu-free NMBD bound to an N/P-domain construct whereas

Cu-loaded NMBD did not. Furthermore, these investigators

showed that interaction between the Cu-free NMBD and the

N/P-domain construct was disrupted by ADP-Mg2+. Although

our docking result indicates that the majority of binding energy

for NMBD comes from its interface with the A domain, the inter-

face between the NMBD and the N and P domains still accounts

for 891 A2 of buried surface (out of a total of 1934 A2), which

seems sufficient to explain the pull-down results. It is interesting

to note that the altered conformation represented in our DMPC/

DOPE crystals results in a loss of 767 A2 of buried surface (i.e.,

almost the entire interaction with the N and P domains). This

observation, together with the disordering of the NMBD in these

crystals, suggests that the interaction with the A domain alone is

not enough to retain NMDB at its binding site and that its asso-

ciation with the cytoplasmic domains is transient.

This transience is also implied by interaction between the

NMBD andmetallochaperones. Physiologically relevant metallo-

chaperones have been shown to transfer Cu+ to MBDs of both

ATP7A (Walker et al., 2002, 2004) and CopA (Gonzalez-Guerrero

and Arguello, 2008), and NMR structures of the complex

between the HAH1 metallochaperone and related MBDs of

ATP7A indicate that the a-helical face of NMBD interacts with

the corresponding face of the metallochaperone during Cu+

transfer (Achila et al., 2006; Banci et al., 2005a, 2005b). Accord-

ing to our model, these a helices are packed against the A

domain, implying that the NMBD must dissociate prior to inter-

acting with metallochaperones. This dissociation step may be

the kinetic barrier to activation that has been revealed by kinetic

analysis of the transport cycle (Hatori et al., 2009). An alternative

binding site for the NMBD on the opposite side of the A domain,

where it would not have interactions with either the N or P

domain, has been proposed on the basis of chemical crosslink-

ing (Lubben et al., 2009). This alternative is not compatible with

our previous density map or with the results of molecular dock-

ing, and it is difficult to explain how the NMBD would play an

autoinhibitory role from that location.

There is a growing consensus that NMBDs play a regulatory

role for Cu pumps and are not involved in transferring Cu+ to

the transport sites within the transmembrane domain (Arguello

et al., 2007). This conclusion applies both to the human pumps

(ATP7A and ATP7B), which carry six MBDs on their N terminus,

and to CopA from bacteria, which generally have a single

N-terminal MBD. In particular, several studies have shown Cu

transfer from metallochaperones to MBDs (Achila et al., 2006;

Banci et al., 2005a, 2005b; Gonzalez-Guerrero and Arguello,

2008; Larin et al., 1999; Ralle et al., 2004; Singleton et al.,

2009; Strausak et al., 2003) and from metallochaperones to


Structure


transport sites (Gonzalez-Guerrero et al., 2009). However,

disruption of MBDs by either truncation or mutation does not

affect transport activity of bacterial pumps (Bal et al., 2001;

Fan and Rosen, 2002; Mana-Capelli et al., 2003; Mandal and

Arguello, 2003; Mitra and Sharma, 2001; Rice et al., 2006), and

disruption of MBD on human pumps mainly affects enzyme acti-

vation and trafficking (Cater et al., 2007; Huster and Lutsenko,

2003; Strausak et al., 1999; Tsivkovskii et al., 2001; Veldhuis

et al., 2009b; Walker et al., 2002). An enzymatic study of CopA

from Thermotoga maritima concluded that the NMBD was

responsible for kinetic inhibition of the reaction cycle and that

this inhibition was alleviated by its binding of Cu+ (Hatori et al.,

2008). Our structure offers a structural explanation for this auto-

inhibition. Specifically, the NMBD has substantial interactions

with both the A and N domains in our hybrid model of the E2

conformation. Proteolytic digestion patterns of CopA indicate

that these domains undergo large movements during the transi-

tion from the E2 state to the E1�P state, similar to SERCA (Hatori

et al., 2009), and the binding energy of the NMBD would resist

these movements. Cu+ could have a role in disrupting this inter-

action in three ways: (1) by binding to transport sites and

driving a conformational change; (2) by binding directly to the

GMTCAMC30 loop at the interface between the NMBD and the

N domain; and (3) by promoting interaction between the NMBD

and metallochaperones, which would require dissociation of

the NMBD from the A domain in order to form the corresponding

complex.

EXPERIMENTAL PROCEDURES

Protein Expression and Purification

Plasmids containing the C-terminal truncation of the copA gene (Wu et al.,

2008) behind a pBAD promoter were expressed in E. coli (strain LMG1940)

using LB broth with 0.1 mg/ml ampicillin at 37�C. After allowing 1 l cultures

to reach an OD of 1, expression of CopA was induced with 0.02% arabinose

for 2 hr at 30�C. Cells were then harvested by centrifugation at 10,000 3 g

for 10 min and the pellet was suspended in 50 mM Tris-Cl (pH 7.5), 20% glyc-

erol, 50 mM NaCl, 2 mM b-mercaptoethanol, 0.05 mg/ml DNase I, and

complete protease inhibitor cocktail (Hoffmann-La Roche). Cells were broken

by French press at 20,000 psi and, after removing large debris by centrifuga-

tion at 10,000 3 g for 20 min, membranes were collected by centrifugation at

150,000 3 g for 1 hr and stored at �80�C. Membrane pellets were solubilized

in 10 mg/ml n-dodecyl-b-D-maltoside (DDM; Anatrace), 25 mM Tris-Cl

(pH 7.5), 100 mM NaCl, 20 mM imidazole, 10% glycerol, 2 mM b-mercaptoe-

thanol, and protease inhibitor cocktail at a protein concentration of 10 mg/ml.

After gentle mixing for 30 min, insoluble material was removed by centrifuga-

tion at 150,000 3 g for 30 min and the supernatant was loaded onto a 5 ml

HiTrap Chelating HP column (GE Healthcare), which was charged with 0.1 M

NiCl2 and equilibrated with 25 mM Tris-Cl (pH 7.5), 100 mM NaCl, 20 mM

imidazole, 10% glycerol, 2 mM b-mercaptoethanol, and 0.1% DDM. After

washing, purified DC-CopA was eluted with 25 mM Tris-Cl (pH 7.5), 100 mM

NaCl, 400 mM imidazole, 10% glycerol, 2 mM b-mercaptoethanol, and 0.1%

DDM. Overnight incubation with thrombin (1 U/mg of protein) was used to re-

move the polyhistidine tag. A benzamidine-Sepharose column (Amersham

Biosciences) equilibrated with 25 mM Tris-Cl (pH 7.5), 100 mM NaCl, 10%

glycerol, 2 mM b-mercaptoethanol, and 0.1% DDM was used to remove the

thrombin. Finally, purified DC-CopA protein was dialyzed against 25 mM

Tris-Cl (pH 7.5), 100 mM Na2SO4, 10% glycerol, 2 mM DTT, and 0.1% DDM.

All the steps in this purification were done at 4�C.

ATPase Activity Assays

For measuring ATPase activity, proteoliposomes were made with a 2.5:1 lipid:

protein weight ratio using either DOPC or a 4:1weight ratio of DMPC/DOPE. To


make these proteoliposomes, DC-CopA (solubilized in DDM) and the relevant

lipid were dialyzed against 50 mM Tris-SO4 (pH 7.5), 50 mM Na2SO4, 50 mM

K2SO4, 10 mM MgSO4, and 2 mM b-mercaptoethanol for 5 days at 4�C. Asdescribed in our previous work on CopA (Rice et al., 2006), themalachite green

assay (Kimura et al., 1996) was used to quantitate phosphate production in

50 mM Tris-SO4, 200 mM Na2SO4, 3 mM MgSO4, 5 mM glutathione, 2.4 mM

ATP, 10% glycerol, and 0.025% NaN3. To test dependence on either Cu+ or

Ag+, assays were performed at 70�C; to test temperature dependence, assays

were performed with either 2 mM Cu+ or 1 mM Ag+. In order to prevent buildup

of transport ions inside the vesicles, a small, subsolubilizing amount of deter-

gent (0.01% DDM) was added to make the vesicles leaky. All nonreducing

buffers were pretreated with 100 mg/ml Chelex (Bio-Rad) for at least 1 hr.

Aliquots of DC-CopA proteoliposomes at 0.02 and 0.04 mg/ml were prepared

and stored on ice, where the enzyme is completely inactive. Using a program-

mable PCR thermocycler (Eppendorf North America), duplicate 45 ml aliquots

of the reaction mixture were heated to 70�C for 3, 5, 7, and 9 min and then

cooled to 4�C to stop the reaction. Phosphate was then quantified in 96-well

microtiter plates using a development time of 3 min; absorbance was read

at 630 nm. In order to maintain a pH of 6.3 at a range of different temperatures,

the following buffers were adjusted to the corresponding pHs at room temper-

ature: MES-SO4 (pH 6.2) for 15�C, MES-SO4 (pH 6.3) for 25�C, MES-SO4

(pH 6.4) for 35�C, MES-SO4 (pH 6.5) for 45�C, MES-SO4 (pH 6.6) for 55�C,MOPS-SO4 (pH) 6.9 for 65�C, Tris-SO4 (pH 7.7) for 75�C, Tris-SO4 (pH 8) for

85�C, and Tris-SO4 (pH 8.3) for 95�C. Like in previous work, we observed

a background of 0.3–0.35 mmol/mg/min in the absence of added Cu+ or Ag+;

this background activity was abolished by the Cu chelator BCDS, suggesting

that it arises from trace Cu+ in the solutions. After subtracting the background

activity, the data were fitted with the Michaelis–Menten binding model to

determine the apparent dissociation constant (Kd).

Crystallization and Cryo-Electron Microscopy

DC-CopA tubular crystals were grown with a 4:1 mixture of DMPC/DOPE at

a protein concentration of 1 mg/ml and at a lipid:protein weight ratio of 0.4.

Dialysis was carried out for 5 days in 50 ml dialysis buttons (Hampton Research)

at 30�C against 500ml of 50mMMES (pH 6.1), 25 mMNa2SO4, 25mMK2SO4,

200 mMBCDS, 10 mMMgSO4, and 2 mM b-mercaptoethanol. Stock solutions

of lipid for both crystallization and ATPase assays were made in dodecyl octa-

ethylene glycol ether (C12E8) at 1 mg lipid per 2 mg detergent.

For cryo-EM, a suspension of crystals was deposited on a holey carbon grid

and rapidly frozen in liquid ethane. Samples were imaged at �175�C with a

CT3500 cryoholder (Gatan) at 50,0003 nominal magnification with a CM200

FEG electron microscope (FEI) operating at 200 kV. Electron micrographs

were recorded on Kodak SO-163 film and, after screening by optical diffrac-

tion, suitable images were digitized at 14 mm intervals with a Zeiss SCAI micro-

densitometer (Intergraph).

Helical Reconstruction

Tubular crystals with a diameter of�600 A, displaying symmetric layer lines by

optical diffraction and with defocus values between 1.5 and 2.5 mm, were

selected for helical processing using Fourier-Bessel methods (Diaz et al.,

2010). Layer lines were cataloged with a consistent set of Miller indices, which

reflect the underlying 2D lattice within the membrane. To define the helical

symmetry, Bessel orders were assigned to all of these layer lines (Figure 1).

Initial assignments were based on phase relationships across the meridian,

the radial location of the diffraction maximum, and the self-consistency of

assignments across the transform (e.g., the construction of a plausible n,l plot).

To verify the Bessel order assignments, several alternative indexing schemes

were tested, using EMIP helical reconstruction software (http://cryoem.nysbc.

org/EmIP.html) to compare phase residuals for the determination of out-of-

plane tilt (U). For highly tilted tubes (U�5�), this phase residual was definitive

in identifying the correct indexing. Near-/far-phase residuals and two-fold

phase residuals were also consistently lower with the correct indexing. In

the end, EMIP was used to apply the unbending methods of Beroukhim and

Unwin (1997) to 12 tubes. Correction for the contrast transfer function was

based on an amplitude contrast of 7% (Toyoshima et al., 1993) and the

resolution of the final maps was truncated at 9 A resolution, where the two-

fold related phase residual approached 43� (random data produce a 45�

residual).


http://cryoem.nysbc.org/EmIP.html

http://cryoem.nysbc.org/EmIP.html

Structure


Modeling and Docking

All modeling and docking procedures were carried out using the ICM protein

docking software (v3.6-1f; Abagyan and Totrov, 1994; Cardozo et al., 1995)

as previously described (Allen and Stokes, 2011). Briefly, the sequences of

CopA and SERCA1a were aligned by Needleman and Wunsch global align-

ment using zero end-gap penalties (Abagyan and Batalov, 1997). A 3D model

of CopA (omitting N- and C-terminal metal-binding domains) was then con-

structed by threading its sequence onto the X-ray crystallographic structure

for SERCA1a in the E2 conformation (PDB ID code 1IWO) using the alignment

as a guide. For this homology model, the geometries of conserved residues

were fixed, whereas the nonconserved residues were set to their standard

geometries and a local minimization was performed to relieve any clashes.

Structural databases were consulted for probable configurations of the non-

conserved loops or, in the event no reasonable solution was found, the loop

was modeled freely to its lowest-energy conformation (Cardozo et al., 1995).

Existing X-ray crystal structures of CopA cytoplasmic domains (the isolated

P/N-domain pair, PDB ID code 3A1C, and the isolated A domain, PDB ID

code 2HC8) were then aligned with the initial homology model, and this model

was adjusted to exactly match the X-ray structures in overlapping regions.

Thus, the resulting hybrid homology model of CopA inherits X-ray crystallo-

graphic information from both the SERCA1a E2 conformation and the isolated

structures of theCopA cytoplasmic domains. A homologymodel for the NMBD

of CopA based on the X-ray crystallographic structure of the metallochaper-

one CopZ (PDB ID code 2QIF) was built in ICM using the same approach as

above. Finally, we used Coot (Emsley et al., 2010) to make minor adjustments

or refinements to the model and Phyre (Bennett-Lovsey et al., 2008) to build

the extra M2 and M3 helices of CopA, and then several rounds of energy mini-

mization were performed in ICM. Finally, Chimera (Pettersen et al., 2004) was

used to visualize results and to make figures.

After building these atomic models, the NMBD was docked to the hybrid

model of CopA (N, P, and A domains) using ICM protein-protein docking

(Fernandez-Recio et al., 2005). ICM first predicted surface patches on the

cytoplasmic domains that were likely to produce energetically favorable

protein interactions using the optimal docking area method. Next, ICM

computed electrostatic, van der Waals, hydrogen-bond, solvation, and hydro-

phobic potentials on a coarse grid over these surface regions. Interaction

energies were determined for a Monte Carlo sampling of NMBD poses at

each of the surface patches. The 400 most favorable results were then sub-

jected to side-chain energy minimization and ranked according to their overall

binding energy. Buried surface area was determined in CNS (Brunger, 2007)

with a 1.4 A probe radius.

ACCESSION NUMBERS

Coordinates for the two models have been deposited in the Protein Data Bank

under ID codes 3J08 and 3J09, and the map has been deposited in the

Electron Microscopy Databank under accession number 5271.

SUPPLEMENTAL INFORMATION

Supplemental Information includes three figures and one movie and can be

found with this article online at doi:10.1016/j.str.2011.05.014.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the facilities at the New York Structural

Biology Center and support from NIH grants R01 GM56960 and U54

GM094598 to D.L.S. as well as NIH grant DP2 OD004631 to T.C.

Received: April 4, 2011

Revised: May 9, 2011

Accepted: May 12, 2011

Published online: August 4, 2011

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