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Article

Crystal Structure of a Full-Length Human

Tetraspanin Reveals a Cholesterol-Binding Pocket

Graphical Abstract

Highlights

d The structure of full-length CD81 reveals a cone-like

architecture

d A large intramembrane cavity exists within the

transmembrane region

d Cholesterol binds CD81 within the intramembrane cavity

d MD simulations suggest that CD81 can adopt open and

closed conformations

Zimmerman et al., 2016, Cell 167, 1041–1051 November 3, 2016 ª 2016 Elsevier Inc. http://dx.doi.org/10.1016/j.cell.2016.09.056

Authors

Brandon Zimmerman, Brendan Kelly,

Brian J. McMillan, Tom C.M. Seegar,

Ron O. Dror, Andrew C. Kruse,

Stephen C. Blacklow

Correspondence [email protected] (A.C.K.), [email protected] (S.C.B.)

In Brief

The tetraspanin CD81 contains a large

intramembrane cavity occupied by

cholesterol, indicating the potential for

functional modulation by small

molecules.

Article

Crystal Structure of a Full-Length Human Tetraspanin Reveals a Cholesterol-Binding Pocket Brandon Zimmerman,1,2 Brendan Kelly,3 Brian J. McMillan,1,2 Tom C.M. Seegar,1,2 Ron O. Dror,3 Andrew C. Kruse,1,* and Stephen C. Blacklow1,2,4,5,* 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA 2Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA 02215, USA 3Departments of Computer Science and of Molecular and Cellular Physiology and Institute for Computational and Mathematical Engineering,

Stanford University, Stanford, CA 94305, USA 4Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02115, USA 5Lead Contact *Correspondence: [email protected] (A.C.K.), [email protected] (S.C.B.)

http://dx.doi.org/10.1016/j.cell.2016.09.056

SUMMARY

Tetraspanins comprise a diverse family of four-pass transmembrane proteins that play critical roles in the immune, reproductive, genitourinary, and audi- tory systems. Despite their pervasive roles in human physiology, little is known about the structure of tet- raspanins or the molecular mechanisms underlying their various functions. Here, we report the crystal structure of human CD81, a full-length tetraspanin. The transmembrane segments of CD81 pack as two largely separated pairs of helices, capped by the large extracellular loop (EC2) at the outer mem- brane leaflet. The two pairs of helices converge at the inner leaflet to create an intramembrane pocket with additional electron density corresponding to a bound cholesterol molecule within the cavity. Mo- lecular dynamics simulations identify an additional conformation in which EC2 separates substantially from the transmembrane domain. Cholesterol bind- ing appears to modulate CD81 activity in cells, suggesting a potential mechanism for regulation of tetraspanin function.

INTRODUCTION

Tetraspanins comprise a large family of four-pass transmem-

brane proteins arising evolutionarily in protists (Huang et al.,

2005). In early unicellular eukaryotes, tetraspanins are believed

to play a role in the dynamic regulation of membrane

morphology, an activity thought to be subsequently co-opted

for cell-cell interactions. As a result, there is an evolutionary

link between the emergence of tetraspanins and the develop-

ment of multicellularity (Huang et al., 2005). The development

of specialized cell-cell interactions and new cell types likely

selected for the duplication and differentiation of tetraspanins

that evolved to effect specific functions. Thus, primitive fungi

have a single tetraspanin, while the size of the tetraspanin family

has grown to ten representatives in the sea urchin Strongylocen-

trotus purpuratus, 17 in the tunicate Ciona intestinalis, and 33 in

Homo sapiens (Garcia-España et al., 2008).

Despite their common ancestry, the vertebrate tetraspanins

have acquired a variety of discrete and important biological func-

tions, which highlight their critical, though underappreciated,

role in mammalian physiology (Hemler, 2005). Studies of tetra-

spanin knockouts in mice and other organisms have identified

essential functions for tetraspanins in the immune, reproductive,

genitourinary, and auditory systems. For instance, mice lacking

CD151/tspan24 have abnormalities in hemostasis and lympho-

cytes that respond abnormally to mitogenic stimulation (Le

Naour et al., 2000; Wright et al., 2004). Patients with frameshift

mutations in CD151 present with a similar bleeding disorder,

along with hereditary nephritis, deafness, and epidermolysis bul-

losa (Karamatic Crew et al., 2004). Additionally, CD63/tspan30

knockout mice exhibit altered water homeostasis with increased

urinary flow and water intake (Schröder et al., 2009), and CD9/

tspan29 null mice are sterile due to failure of sperm-egg fusion

(Le Naour et al., 2000).

The biological effects of many tetraspanins appear to be

attributable to their activities as modulators of central signal

transduction pathways. There is accumulating evidence that

the six tetraspanins in the C8 subclass regulate Notch signaling

by promoting ADAM10 trafficking and enzymatic maturation in

both flies and mammals (Dornier et al., 2012; Haining et al.,

2012; Jouannet et al., 2016; Noy et al., 2016). A number of other

tetraspanins appear to exert their effects by influencing integrin

signaling, either indirectly, by modulating responses of integrins

to their ligands (van Spriel et al., 2012; Wee et al., 2015), or by

directly and stably associating with particular integrin hetero-

dimers (Yauch et al., 1998). Moreover, knockout of CD37 in

mice predisposes them to development of B cell lymphoma, a

phenotype attributed to unrestrained interleukin 6 (IL-6) signaling

in the absence of CD37 (de Winde et al., 2016).

CD81/TAPA-1 (target of antiproliferative antibody 1)/tspan28

was first identified as the target of an antibody discovered in

a screen for the inhibition of proliferation in a lymphoma cell

line (Oren et al., 1990). CD81, together with ME491/CD63 and

CD37, became the founding members of the tetraspanin

(TM4SF) protein family in mammals (Hemler, 2005; Huang

et al., 2005). CD81 is also among the most well characterized

tetraspanins because of its essential role in B cell biology

Cell 167, 1041–1051, November 3, 2016 ª 2016 Elsevier Inc. 1041

(Cherukuri et al., 2004b; Mattila et al., 2013), forming a multi-

protein complex with CD19, CD21/CR2, and CD225 to regulate

B cell receptor function. The importance of CD81 in B cell func-

tion is further highlighted by the recent report of a common var-

iable immunodeficiency patient who has a germline mutation

that produces an alternatively spliced, truncated form of CD81

(van Zelm et al., 2010), which appears to sequester CD19 intra-

cellularly (Vences-Catalán et al., 2015). CD81 is also a host factor

that interacts with the hepatitis C virus (HCV) E2 envelope protein

and is required for efficient HCV entry (Bradbury et al., 1992;

Pileri et al., 1998).

Tetraspanins are the largest family of transmembrane proteins

in mammals for which detailed structural information about the

intramembrane domain remains unavailable. This lack of infor-

mation, together with a limited understanding about the struc-

tural relationship between the intramembrane domain and the

extracellular region of the protein, has greatly hindered efforts

to understand the molecular mechanisms of action of these pro-

teins. The tetraspanins are predicted to contain a number of

shared structural features, including intracellular N and C termini,

small (EC1) and large (EC2) extracellular loops, four transmem-

brane regions (TMs), and a short intracellular loop between

TM2 and TM3 (Yáñez-Mó et al., 2009). All tetraspanins also

possess intracellular cysteine residues, which are typically pal-

mitoylated (Yáñez-Mó et al., 2009). These cysteines and their

palmitoylation are required for efficient interaction with certain

associated proteins and the formation of tetraspanin microdo-

mains known as the tetraspanin web (Hopf et al., 2012), which

is thought to be generated by tetraspanin-tetraspanin interac-

tions as well as by heterologous interactions with other associ-

ated proteins in ‘‘signaling hubs’’ (Levy and Shoham, 2005).

Although structures have been reported for the extracellular

EC2 regions of Schistosoma mansoni TSP-2 and human CD81,

they provide limited insight into other family members because

of the poor conservation of the EC2 region, and they offer no in-

formation about the intramembrane portion of the protein, which

is the most highly conserved part of the molecule (Stipp et al.,

2003).

We report here the structure of human CD81 (PDB: 5TCX) as

a representative example of a full-length tetraspanin. The protein

contains a bound cholesterol molecule in a large intramembrane

pocket between two largely independent pairs of transmem-

brane helices capped by the EC2 domain, and specific

cholesterol binding to this site is observed in vitro. The EC2

domain appears to more readily adopt an ‘‘open’’ conformation

in molecular dynamics simulations when cholesterol is not pre-

sent in its binding site, consistent with the modulation of tetra-

spanin function by mutations that compromise cholesterol bind-

ing. Together, the structural, computational, and biochemical

studies suggest a model for tetraspanin function, as well as a

route to modulating tetraspanin activity as a therapeutic strategy

in a variety of different diseases.

RESULTS

Crystal Structure of CD81 To define the overall architecture of an intact tetraspanin, eluci-

date interdomain relationships between the EC2 and the intra-

1042 Cell 167, 1041–1051, November 3, 2016

membrane region, and gain insight into tetraspanin-ligand inter-

actions and the tetraspanin web, we purified full-length CD81

from insect cells (Figures S1A and S1B), crystallized the protein

using the lipidic cubic phase method, and determined its struc-

ture. As is common for integral membrane protein crystals, the

diffraction pattern was anisotropic, with strong scattering along

two reciprocal space axes and weaker scattering along the third.

Prior to refinement, we performed ellipsoidal truncation with res-

olution limits of 5.5 Å along the a* axis, 2.95 Å along the b* axis,

and 2.95 Å along the c* axis (Table S1). To obtain phase informa-

tion, we utilized a fragment-based iterative molecular replace-

ment approach (Kruse et al., 2013). Briefly, we first located

EC2 of CD81 by molecular replacement using a previously re-

ported structure of the CD81 EC2 region (PDB: 1IV5, chain A)

as a search model (Kitadokoro et al., 2002) (Figure S1C). After

placing this domain, we used four more rounds of iterative mo-

lecular replacement using a polyalanine alpha helix search

model to locate the four alpha helices comprising the transmem-

brane domain. This model was then used as a starting point for

model building and refinement (Figure S2).

The overall structure of CD81 resembles a waffle cone in which

the EC2 domain covers an intramembrane cavity bounded by the

four transmembrane helices (Figure 1A). No electron density is

visible for the small extracellular loop (EC1), suggesting this re-

gion is disordered. The overall fold of the four transmembrane

helices does not resemble that of any other integral membrane

protein of known structure. The transmembrane region consists

of two largely separated pairs of antiparallel helices: one pair

comprises TM1/TM2 and the other TM3/TM4. The two pairs of

helices only converge close to the cytoplasmic side of the

membrane through contacts between TM2 and TM3. The central

cavity bounded by the four transmembrane helices and the bot-

tom face of EC2 encloses a total volume of 3,300 Å3 (Figure 1B).

Evolutionary Conservation of Tetraspanin Structure To confirm the relative positions of the transmembrane helices

and assess our assignments of the residues in this region, we

performed an evolutionary coupling analysis, which compares

homologous sequences to determine amino acid residues that

are correlated through evolution (Marks et al., 2012). The corre-

lation map for CD81 indicates that strong evolutionary couplings

occur between helices one and two, between helices three

and four, and within the EC2 domain, as predicted by the X-ray

structure. We extended this analysis to additional tetraspanin

proteins dispersed throughout phylogeny to examine whether

this unusual fold is evolutionarily conserved, or a specific feature

of CD81 (Figure 2). CD81 (Figure 2A), hypothetical protein

FGSG_08695 from the fungus Fusarium graminearum PH-1

(Figure 2B), tetraspanin 3A from the fruit fly (Drosophila mela-

nogaster) (Figure 2C), and AX4 tetraspanin family protein from

the eukaryotic slime mold Dictyostelium discoideum (Figure 2D)

all exhibit the same pattern of evolutionary coupling, providing

support for the inference that the fold seen in the CD81 structure

is conserved among all tetraspanins.

A more detailed analysis of sequence conservation patterns for

CD81 homologs across species (Figures 3A and S3A) and for tet-

raspanin paralogs within humans (Figures 3B and S3B) highlights

the tight evolutionary constraints within the transmembrane

A

B

TM1

TM4

TM3 TM2

60º

Viewed from extracellular side

90º

Viewed from intracellular side

60º

Viewed from intracellular side

Viewed from extracellular side

90º

º 90

EC2

Intracellular

Negative Positive

A

TM1

TM4

TM3 TM2

EC2

Intracellula

Extracellular

Figure 1. Overall Structure of Human CD81

(A) Cartoon representation viewed parallel to the

membrane plane. Helix one (TM1) is blue, helix two

(TM2) is cyan, helix three (TM3) is green, and helix

four (TM4) is magenta. The large extracellular re-

gion (EC2) between TM3 and TM4 is red.

(B) Surface representation colored by electrostatic

surface potential on a sliding scale from blue

(basic) to red (acidic).

See also Figures S1, S2, and S6.

region and the greater variability of the second extracellular loop

by comparison. Strikingly, highly conserved residues within EC2

are located on extracellular helices one and two at their points of

contact with the junction between EC1 and the first two trans-

membrane segments, accounting for the ‘‘closed’’ conformation

of the EC2 cap. Contacts between the EC2 and the transmem-

brane region include hydrophobic interactions between L35 of

TM1 and V146 of EC2 and of F56 of TM2 with F126 of EC2

(Figure S3C).

Analysis of Intramembrane Binding Pocket The most striking feature of the CD81 structure is the large, hy-

drophobic pocket bounded by the four transmembrane helices

and the EC2 cap. We observe unexpected additional Fo-Fc elec-

tron density within this pocket. On the basis of the shape of the

electron density, the presence of cholesterol in the crystallization

mix, and the chemical features of the pocket, we tentatively iden-

tified the additional density as a bound cholesterol molecule

(Figures 4 and S4, related to Figure 4). Within 4 Å of the bound

cholesterol are a number of hydrophobic and aromatic residues,

including F21 of TM1; I64, V68, V71, M72, and V75 of TM2; F94,

L98, and L101 of TM3; and V212 and M216 of TM4 (Figure 4A).

N18 and E219, two polar residues belonging to TM1 and TM4,

respectively, form hydrogen bonds to the cholesterol hydroxyl

group. Analysis of the CD81 sequences from all 37 available ho-

mologs reveals that these 13 amino acids are nearly 100%

conserved (Figure S4). Comparison of the CD81 sequence with

its 32 human paralogs shows N18 is conserved in 27 of the 33

human tetraspanins. However, E219 is only present in CD81

and tspan10, with the majority of mammalian tetraspanins hav-

Ce

ing a polar glutamate or glutamine residue

(82%) on the preceding turn of the helix

and a glycine residue (64%) at this posi-

tion instead.

CD81 Binds Specifically to Cholesterol To determine directly whether CD81

specifically binds cholesterol, we as-

sessed the cholesterol-binding ability of

wild-type CD81 immunopurified from

HEK293T cells using a radioactive binding

assay and compared it with the Beta-

lactam binding receptor BlaR, a negative

control four-pass transmembrane protein

prepared similarly (Figure 4B). Wild-type

CD81 immunoprecipitates recover �15-fold more cholesterol than immunoprecipitates from untransfected control cells or

from cells expressing BlaR. To determine whether cholesterol

binding by CD81 relies on specific interactions seen in the struc-

ture of CD81, we mutated residue E219 to either alanine or gluta-

mine, as it is a critical polar contact with the cholesterol hydroxyl

in our structure (the hydrogen bond between E219 and choles-

terol is formed 90% of the time in the simulations described in

the following section). The E219A and E219Q point mutants of

CD81 recover �50% less cholesterol than wild-type (Figure 4C), whereas a G26F/G30F double mutant on the external face of helix

one does not detectably affect cholesterol recovery (Figure S4).

Finally, to address whether CD81 and the transmembrane pocket

are capable of binding other lipids, we explored the binding of

radioactive estradiol and palmitate using the same radioligand-

binding assay. The recovery of both estradiol and palmitate is

dramatically reduced in comparison to cholesterol (Figure 4D).

Moreover, the amount of bound palmitate is unaffected by the

E219Q mutation (though it does appear that the amount of bound

estradiol is reduced somewhat upon introduction of the E219Q

mutation, suggesting that it might have very weak affinity for

the cholesterol-binding pocket, consistent with the fact that it is

a cholesterol derivative).

Molecular Dynamics Simulations Identify an Open Conformation of EC2 We performed molecular dynamics simulations of CD81 in a hy-

drated lipid bilayer, both with and without cholesterol bound in

the intramembrane pocket. In three of nine simulations of the

apoprotein (i.e., with cholesterol removed), EC2 transitioned to

ll 167, 1041–1051, November 3, 2016 1043

20 40

220 200 180 160 140 120

100 80 60

Residue 100 20050 150

TM1 TM2 TM3 TM4

TM1

TM2

TM3

TM4

EC2

EC2

TM1/TM2

TM3/TM4

TM3/TM4

EC2

50 100 150 200

20

40

60

80

100

120

140

160

180

200

220

TM1 TM2 TM3 TM4 EC2

TM1

TM2

TM3

TM4

EC2

TM1/TM2

EC2

TM3/TM4

TM3/TM4

Residue

A B

C D

TM1

TM2

TM3

TM4

EC2

TM1 TM2 TM3 TM4EC2

TM1/TM2

EC2

20 40 60

80 100 120 140 160 180 200

220

50 100 150 200

TM3/TM4

TM3/TM4

TM1 TM2 TM3 TM4EC2

TM1

TM2

TM3

TM4

EC2

EC2

50

100

150

200

250

300 50 100 150 200 250 300

TM3/TM4

TM3/TM4

Residue

TM1/TM2

Residue

Figure 2. Evolutionary Coupling Map of Tetraspanins

(A–D) The top 90 amino acid evolutionary coupling pairs of (A) human CD81, (B) hypothetical protein FGSG_08695 from Fusarium graminearum PH-1, (C) tet-

raspanin 3A from Drosophila melanogaster, and (D) AX4 tetraspanin family protein from Dictyostelium discoideum. Hotspots include couplings between residues

of TM1 and TM2, TM3 and TM4, the junction between TM2 and TM3, and intradomain coupling within EC2. Analysis was performed using the EVFold server

(http://evfold.org). See also Figure S2.

an open conformation in which it disengaged from TM1 and TM2

(Figure 5A). EC2 remained in this open conformation for the

remainder of these three simulations. After opening, EC2 is flex-

ible and dynamic relative to the TM domain (Figure 5C); however,

the same fully open conformation is observed in all three simula-

tions that display opening.

The opening motion involves a substantial straightening of

TM3 and TM4. During EC2 opening, a salt bridge between

D196 on EC2 and K201 on TM4 that stabilizes the closed state

breaks, leading to extension and straightening of TM4. A newly

formed salt bridge between K116 and D117 stabilizes the

extended form of TM3 observed in the open state (Figure 5B).

Interestingly, cholesterol-bound simulations consistently

maintained a closed conformation, similar to thecrystal structure,

1044 Cell 167, 1041–1051, November 3, 2016

in which EC2 remained in contact with TM1 and TM2 (Figure 5A).

In two of the nine simulations that we initiated with cholesterol

bound, however, cholesterol dissociated from the binding pocket

into the membrane, exiting through the gap between TM1 and

TM4. In one of these simulations, EC2 transitioned to the fully

open conformation after cholesterol dissociated. EC2 remained

in that conformation for the rest of the simulation. The fact that

we only observed opening when cholesterol was absent from

the binding pocket suggests that the presence of cholesterol

may stabilize the closed conformation and that the absence of

cholesterol may favor opening (p = 0.03; see STAR Methods).

TM1 and TM2 undergo substantial motion in simulations both

in the presence and in the absence of cholesterol (Figure S5). The

intracellular end of TM1 remains in close contact with TM4 in

Figure 3. Sequence Conservation of CD81

(A and B) Cartoon representation of CD81 versus

the top 50 CD81-related sequences determined by

Consurf (A) or CD81 versus the 32 human tetra-

spanin paralogs (B) colored on a sliding scale

from teal (poorly conserved) to maroon (highly

conserved) (Landau et al., 2005). Residues with

insufficient information for analysis are yellow. The

high degree of conservation of the transmembrane

region contrasts with the high divergence at the

surface of the extracellular domain. The large

pocket within the membrane bounded by the ec-

todomain and the TM helices is �3,300 Å3 in vol- ume. See also Figure S3.

simulations of the apoprotein, stabilized by a hydrogen bond be-

tween the side chains of N18 and E219. When cholesterol is

bound, it competes for interaction with E219, often leading the

intracellular end of TM1 to separate from TM4.

Cholesterol Binding Regulates CD81 Function Numerous reports have highlighted the importance of CD81 in

CD19export to the cell surface, anda CD81 truncation in ahuman

patient results in a combined variable immunodeficiency pheno-

type and intracellular retention of CD19 in cell-based assays (van

Zelm et al., 2010; Vences-Catalán et al., 2015). Here, we used a

flow cytometry assay to measure the amount of CD19 at the sur-

face of transfected cells and the ability of CD81 to increase the

amount of CD19 detected at the cell surface. In the absence of

added CD81, 293T cells transfected with FLAG-tagged CD19

show a minimal increase in surface staining compared to un-

transfected cells. Upon co-transfection of wild-type CD81, we

see a 10-fold increase in surface staining compared to CD19

with control vector (Figure 6A). Moreover, when cholesterol bind-

ing is compromised by either the E219A or E219Q mutation, the

amount of CD19 surface staining increases further by an addi-

tional 50% when comparable amounts of CD81 protein are ex-

pressed and present at the cell surface, whereas no effect is

seen when the G26F/G30F mutant is tested (Figure 6B).

DISCUSSION

We have solved the crystal structure of a full-length human tetra-

spanin and defined an unknown intramembrane binding pocket

Ce

for tetraspanins. The discovery of the

pocket with cholesterol bound was then

used to design experiments for interroga-

tion of cholesterol binding and its poten-

tial role in tetraspanin function.

Our structure of CD81 revealed a

monomeric form of a tetraspanin, which

contrasts with the dimeric structure of

the isolated EC2 fragment. The putative

dimerization interface seen in the struc-

ture of the isolated EC2 fragment is

located on its bottom face in the full-

length protein within 3.5 Å of the TM1/

TM2 bundle, indicating that the dimer is

likely a non-native interaction driven by lattice packing effects

in the absence of the transmembrane regions of the protein.

Early modeling work proposed that the four TM helices would

form a tightly associated four-helix bundle (Seigneuret, 2006),

yet our structure reveals the transmembrane region to fold as

two largely separated pairs of antiparallel helices, a conclusion

supported by evolutionary coupling analysis (Figure 2). The

overall similarity seen among diverse family members argues

strongly that all tetraspanin proteins possess this transmem-

brane fold. The intramembrane pocket seen in the structure be-

tween TM1/4 and TM2/3 is likely to be accessible only through

lateral diffusion within the membrane plane, because entrance

from the extracellular space is precluded by the presence of

EC2 above the pocket.

Much attention has been previously given to the concept of a

tetraspanin web, where tetraspanins form homooligomers or het-

erooligomers with other tetraspanins to form higher-order com-

plexes and protein-rich microdomains in the cell membrane

(Charrin et al., 2003a; Horváth et al., 1998; Levy and Shoham,

2005; Rubinstein et al., 2013). Recently, the existence of the tet-

raspanin web has come under more scrutiny, as early experi-

ments done with detergents did not effectively disrupt secondary

interactions (Dornier et al., 2012; Zuidscherwoude et al., 2015). In

our CD81 crystals, individual monomers in the lattice pack such

that adjacent subunits in the membrane plane lie in antiparallel

orientations (Figure S6), an arrangement that is necessarily

non-physiological. Though the absence of lateral homotypic

packing interactions among adjacent subunits does not in itself

exclude the possibility of higher-order CD81 complexes or the

ll 167, 1041–1051, November 3, 2016 1045

90 º 90 º

A

B *** **

***

26kDa

Wb: Anti-FLAG (Rabbit)

C on

tro l

W ild

ty pe

C D

81 B

la R

E 21

9Q

WCL Sol IP-FLAG (Mouse)

C

Extracellular

Intracellular

C E P C E P C E P

Control CD81 CD81

E219Q Co

nt ro

l CD

81

CD 81

E 21

9Q

CD 81

E 29

1A Bl

aR 0

4000

8000

12000

3 H

c ho

le st

er ol

b in

di ng

(C P

M )

0

4000

8000

12000

3 H

li ga

nd b

in di

ng (C

P M

) D

C on

tro l

W ild

ty pe

C D

81 B

la R

E 21

9Q

C on

tro l

W ild

ty pe

C D

81 B

la R

E 21

9Q

F94

F21

I64

V68

V71

E219

M216

L101

L983.0 Å

2.6 Å

N18

V212

M72

V75

Extracellular

Intracellular

Figure 4. CD81 Binds Cholesterol within Its Intramembrane Cavity

(A) CD81-cholesterol interactions. CD81 residues within 4 Å of the bound cholesterol molecule are rendered as sticks and labeled in the zoomed in view (right). An

Fo-Fc omit map of electron density contoured at 2.0 s is shown for the bound cholesterol. N18 and E219 coordinate the cholesterol hydroxyl group. Views of the

pocket in surface representation are shown in open-book form projecting onto the TM1/2 bundle (left) and the TM3/4 bundle (right). CD81 residues at the ligand

interface are colored orange.

(B) CD81 wild-type, CD81 mutant proteins (E219A, E219Q), and BlaR were immunopurified from HEK293T cells. Proteins captured on FLAG beads were used for

radioactive cholesterol binding experiments. WCL, whole-cell lysate; Sol, solubilized protein; IP, immunoprecipitation.

(C) Cholesterol binding by immunopurified proteins. 1,2-3H-cholesterol was incubated with immunopurified FLAG-CD81 or FLAG-BlaR from 293T cells and

bound cholesterol was measured. The figure represents three independent experiments performed in duplicate. Statistical analysis was performed using ANOVA,

and a Bonferroni post hoc test was performed comparing all columns. **p < 0.01; ***p < 0.001.

(D) Specificity of lipid binding to CD81. Immunopurified FLAG-CD81 from 293T cells prepared as in (C) was incubated with 1,2-3H-cholesterol (C), 2,4,6,7-3H(N)-

estradiol (E), and 9,10-3H(N)-palmitic acid (P). Bound 3H-lipid was measured in a scintillation counter.

The figure represents three independent experiments performed in duplicate. See also Figure S4.

potential for assembly of a tetraspanin web, there is also evi-

dence among the uroplakin tetraspanins that tetraspanin mono-

mers do not interact with one another. Uroplakins Ia and Ib, in

complex with their accessory proteins uroplakin II and IIIa, form

hexameric lattices. Despite the close proximity of the six tetra-

spanin molecules in the lattice, the lattice is entirely bridged by in-

1046 Cell 167, 1041–1051, November 3, 2016

teractions between the non-tetraspanin partners, uroplakin II and

IIIa (Min et al., 2006). A recent study using super-resolution micro-

scopy also shows that tetraspanins, including CD81, lie in closer

proximity to their non-tetraspanin binding partners than to other

tetraspanins in the membrane, again suggesting that tetraspa-

nins need not be constitutively oligomeric in the cellular milieu

F58

F126

F58 F126

closed conformation (cholesterol-bound simulation)

open conformation (apoprotein simulation)

BA

TM1

TM2

TM3

TM4

C

closed conformation

open conformation

K201

D196 K116

D117

K116

D117

K201

D196

TM3 TM3TM4 TM4

EC2 EC2

EC2

EC2

TM1

TM2

TM3

TM4

F58

F1266666666666666

EEC2

TTTTM1T

TTM2

TM3M3

TM4

F58 F126

A

TM1

TM2

TM3

TM4

EC2 K201K20K2KK222K2222222K222

D196D1DDD K11

D117

TTTM4M4M4M4M4M4M44M444444444444M4M444M4M4MMMM4M44MM4M4

EC2

K111111111111611111111

D117

K201

D196

TMTTTM4TM4TTTTTTTTTTTTTTTTTTTT 4T 4TTTT 4TT 4TT 44TT 444TT 4T 4

EC2

Figure 5. Molecular Dynamics Simulations Reveal an Open Conformation in which EC2 Separates from the Transmembrane Domain

(A) Closed (blue) and open (red) states of CD81, as observed in cholesterol-bound and apoprotein simulations, respectively. The open conformation is

characterized by substantial domain separation and straightening of TM helices 3 and 4.

(B) The salt bridge from EC2 to TM4 (D196 – K201) stabilizes the closed conformation and breaks during opening, while a different salt bridge (K116 – D117)

formed upon opening helps stabilize the open conformation.

(C) Interdomain distance (measured between alpha carbons of F58 and F126) as a function of time for an apoprotein simulation in which the domains separate and

a cholesterol-bound simulation in which they do not. Thin traces show values every 1 ns, and thick traces are smoothed.

See also Figure S5.

(Zuidscherwoude et al., 2015). An important caveat from our

structure is the fact that four intracellular cysteine residues

were mutated to prevent disulfide crosslinking during purifica-

tion. These sites are expected to be palmitoylated, and palmitoy-

lation of tetraspanins has been implicated for at least some

tetraspanin-tetraspanin interactions (Yang et al., 2002) and tetra-

spanin functions (Cherukuri et al., 2004a). However, it now seems

at least equally likely that other factors are more responsible for

the creation of tetraspanin-enriched microdomains than direct in-

teractions between tetraspanins themselves.

The relationship between cholesterol and tetraspanins has

been discussed in numerous studies, often in the context of the

tetraspanin web connecting lipid rafts to other membrane

signaling domains. A study examining the role of CD82 in the actin

cytoskeleton of T lymphocytes revealed that removal of choles-

terol altered the cellular distribution of CD82 and furthermore dis-

rupted all CD82-dependent signaling events (Delaguillaumie

et al., 2004). Early work demonstrated a physical link between

cholesterol and CD81 as well as CD9 and CD82 using photoacti-

vatable cholesterol crosslinking, demonstrating the close prox-

imity of cholesterol and tetraspanins within the membrane (Char-

rin et al., 2003b). Perhaps the most compelling evidence

highlighting the central role of cholesterol to tetraspanin biology

comes from a study showing that cholesterol in the host cell

membrane was required for both maintenance of a CD81 mono-

clonal antibody epitope and for CD81-dependent infection by

Plasmodium falciparum sporozoites, but not by sporozoites

that were CD81 independent (Silvie et al., 2006). Our results

reveal that CD81 binds cholesterol and that E219 is an important

residue in the binding pocket, offering a potential mechanism for

how tetraspanins might detect cholesterol or other membrane

lipids (whereas CD81 and tspan10 both possess a polar residue

in this position, most other tetraspanins have a polar residue one

helical turn earlier, with other cholesterol-interacting residues

highly conserved throughout evolution).

Our molecular dynamics simulations suggest that CD81 may

exist in both closed and open conformations and that the equi-

librium between closed and open conformations may be shifted

toward the closed state when cholesterol is bound. Mutations

that interfere with cholesterol binding by CD81 result in

enhanced delivery of its binding partner (CD19) to the cell sur-

face. On the basis of these observations, we speculate that the

open conformation may bind partner proteins more tightly and

that the closed state, favored when cholesterol is bound, disfa-

vors partner binding and, hence, protein export (Figure 6C).

Modulation of tetraspanin conformation in response to differ-

ences in cholesterol concentration could explain how tetraspa-

nins regulate the subcellular localization of their partner proteins

Cell 167, 1041–1051, November 3, 2016 1047

A

C

B

Figure 6. Cholesterol Binding Regulates CD81-Mediated Export of CD19

(A and B) HEK293T cells were transfected with cDNA encoding the indicated proteins, and cell-surface amounts of CD19 (A) and CD81 (B) were assessed by flow

cytometry. Histograms shown represent four independent experiments done in triplicate. Statistical analysis was performed using ANOVA, and a Bonferroni post

hoc test was performed comparing all columns. ***p < 0.01.

(C) Proposed model for modulation of cargo binding in response to cholesterol. CD81 favors a closed conformation when cholesterol is bound (left) and more

readily accesses an open conformation, which allows more efficient export of its cargo CD19 (modeled in cartoon form), when not bound to cholesterol (right).

by loading and unloading cargo in response to variation in lipid

composition among different membrane compartments.

Accumulating evidence that tetraspanins such as CD81 have

growth-promoting activity in certain human cancers has led to

their emergence as potential therapeutic targets (Hemler,

2014). Indeed, monoclonal antibodies directed at the tetraspa-

nin26/CD37 have moved into phase 1 or phase 2 clinical trials

for the treatment of chronic lymphocytic leukemia and non-

Hodgkin lymphoma (Deckert et al., 2013; Zhao et al., 2007).

The unexpected discovery of the intramembrane ligand-binding

pocket represents a potential targetable site for modulating tetra-

1048 Cell 167, 1041–1051, November 3, 2016

spanin function using small molecules and provides a route for-

ward for elucidation of the molecular mechanism of action of

this enigmatic class of important but poorly understood proteins.

STAR+METHODS

Detailed methods are provided in the online version of this paper

and include the following:

d KEY RESOURCES TABLE

d CONTACT FOR RESOURCE AND REAGENT SHARING

d EXPERIMENTAL MODEL AND SUBJECT DETAILS

d METHOD DETAILS

B Expression and Purification

B Crystallography and Data Collection

B Phasing and Structure Refinement

B Evolutionary Coupling Analysis

B Sequence Conservation Analysis

B Radioactive Lipid Binding Assay

B Molecular Dynamics Simulation: System Setup

B MD Simulation Protocol

B MD Simulation Analysis

B Flow Cytometry Assay

d QUANTIFICATION AND STATISTICAL ANALYSIS

d DATA AND SOFTWARE AVAILABILITY

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

six figures, and one table and can be found with this article online at http://

dx.doi.org/10.1016/j.cell.2016.09.056.

AUTHOR CONTRIBUTIONS

The overall project was designed and developed by B.Z., T.C.M.S., A.C.K.,

and S.C.B. Molecular cloning, protein purification, and crystallization experi-

ments were conducted by B.Z. Data collection was performed by B.Z. and

A.C.K. Data processing, phase calculation, and structure refinement were car-

ried out by B.Z., B.J.M., and A.C.K. B.K. and R.O.D. performed and analyzed

molecular dynamics simulations. The manuscript was written by B.Z. and

S.C.B. with input from B.K., R.O.D., and A.C.K.

ACKNOWLEDGMENTS

We would like to thank beamline staff at APS GM/CA beamline 23ID-B for their

superb technical assistance and support and N. Latorraca for helpful sugges-

tions regarding simulation analysis. Financial support for this work was provided

by NIH grants NCI 5 RO1 CA092433 (S.C.B.) and 1DP5 OD021345 (A.C.K.) and

by a Terman Faculty Fellowship (R.O.D.). B.Z. was supported by a CIHR post-

doctoral fellowship, and T.C.M.S. was supported by training grant 2T32

HL007627.

Received: May 2, 2016

Revised: July 12, 2016

Accepted: September 29, 2016

Published: October 27, 2016

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Cell 167, 1041–1051, November 3, 2016 1051

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies

Alexa Fluor 488 anti-human CD19 Antibody BioLegend 302219; RRID: AB_389313

APC anti-human CD81 (TAPA-1) Antibody BioLegend 349510; RRID: AB_2564021

ANTI-FLAG M2 Affinity Gel Sigma Aldrich A2220; RRID: AB_10063035

Chemicals, Peptides, and Recombinant Proteins

[1,2-3H]-cholesterol Perkin Elmer NET139250UC

2,4,6,7-3H(N)-estradiol Perkin Elmer NET013250UC

9,10-3H(N)-palmitic acid Perkin Elmer NET043001MC

Benzonase recombinant nuclease Sigma Aldrich E1014

Iodoacetamide Sigma Aldrich I1149

In-Fusion HD Cloning Plus Clontech 638911

ESF 921 Insect Cell Culture Medium, Protein-Free Expression systems 96-001

BestBac 2.0, v-cath/chiA Deleted Linearized

Baculovirus DNA

Expression systems 91-002

Fugene HD Promega E2311

Lipofectamine 2000 Invitrogen 11668019

n-Dodecyl-b-D-Maltoside (Anagrade) Anatrace D310

5-Choesten-3b-ol Hemisuccinate Steraloids C6823-000

Monoolein Hampton Research HR2-435

Cholesterol Sigma C8667

PEG300 Hampton Research HR2-517

Deposited Data

CD81 Structure This paper PDB: 5TCX

Experimental Models: Cell Lines

293T (ATCC CRL-3216) ATCC CRL-3216

Spodoptera frugiperda ovarian tissue (SF9) Expression systems 94-001S

Recombinant DNA

pVL1392 Expression systems 91-030

pCDNA3.1 (+) hygro Invitrogen V87020

gBlocks Integrated DNA Technologies N/A

Software and Algorithms

Amber14 Case et al., 2014 http://ambermd.org/

GraphPad Prism 7.0 N/A http://www.graphpad.com/scientific-software/prism/

EVFold conservation server Hopf et al., 2012 http://evfold.org/evfold-web/evfold.do

BD Accuri C6 software BD Accuri C6 https://www.bdbiosciences.com/instruments/accuri/

features/software.jsp

UCLA-DOE LAB — Diffraction Anisotropy Server Strong et al., 2006 https://services.mbi.ucla.edu/anisoscale/

SBGrid Consortium Morin et al., 2013 https://sbgrid.org/software/

XDS Kabsch, 2010 https://sbgrid.org/software/

Phenix (1.10_2155) Afonine et al., 2012 https://sbgrid.org/software/

Coot Emsley and Cowtan, 2004 https://sbgrid.org/software/

Pymol Schrödinger Team, 2010 https://sbgrid.org/software/

e1 Cell 167, 1041–1051.e1–e5, November 3, 2016

CONTACT FOR RESOURCE AND REAGENT SHARING

For additional information about reagents and resources, contact the Lead Contact, Stephen Blacklow, at Stephen_blacklow@hms.

harvard.edu.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

CD81 protein for crystallographic studies was expressed in Spodoptera frugiperda ovarian tissue (SF9) cells grown in grown in ESF

921 Insect Cell Culture Medium, Protein-Free media at 27�C. Protein was harvested 42 hr after baculovirus infection and isolated from the Sf9 membrane fraction.

Radioactivity experiments were performed with protein isolated from HEK293T cells. HEK293T cells were grown in DMEM media

supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. HEK293T cells were also used in the flow cytometry

assays.

METHOD DETAILS

Expression and Purification The sequence encoding full-length human CD81 was assembled using synthetic gene blocks (gBlocks, Integrated DNA Technolo-

gies) and inserted into the baculovirus transfer vector pVL1392 with an amino-terminal FLAG epitope tag followed by a 3C protease

cleavage site. Four intracellular cysteine residues at positions 6, 9, 227 and 228 were mutated to serine to prevent disulfide cross-

linking during purification. This protein was expressed in Sf9 insect cells using the BestBac system (Expression Systems) according

to the manufacturer’s instructions with Fugene HD as the transfection reagent. Infection was performed when cells reached a density

of 4 3 106 cells/ml, and flasks were then shaken at 27�C for 42 hr prior to harvest. Cells were harvested by centrifugation and frozen at �80�C until purification. After frozen cell paste was thawed, cells were lysed by osmotic shock in 20 mM HEPES (pH 7.4), 2 mM mag- nesium chloride, 2 mg/ml iodoacetamide (Sigma Aldrich) and 1:100,000 (v:v) benzonase nuclease (Sigma Aldrich). Lysed cells were

centrifuged at 18,000 RPM in a Sorvall RC 5C Plus centrifuge with an SS-34 rotor for 15 min. CD81 was then extracted from the pellet

using a glass dounce tissue grinder to homogenize lysed cells in a solubilization buffer consisting of 250 mM NaCl, 20 mM HEPES

(pH 7.5), 10% (v/v) glycerol, 1% (w/v) n-Dodecyl-b-D-Maltoside (DDM - Anagrade; Anatrace), 0.1% (w/v) cholesterol hemisuccinate

(CHS; Steraloids) and 2 mg/ml iodoacetamide. Samples were stirred for 2 hr at 4�C, then centrifuged at 20,000 RPM for 20 min. Su- pernatant containing solubilized protein was filtered through a glass microfiber prefilter and loaded by gravity flow onto 3 mL of M2

anti-FLAG antibody affinity resin (Sigma Aldrich). The resin was then washed with 50 mL of buffer containing 100 mM NaCl, 20 mM

HEPES (pH 7.4), 1% glycerol, 0.1% DDM, and 0.01% CHS. Bound protein was eluted in the same buffer supplemented with 0.2 mg/

mL FLAG peptide. 3C protease was added (1:100 w:w) and incubated with CD81 at 4�C overnight. CD81 was further purified by size exclusion chromatography (SEC) on a Sephadex S200 column (GE Healthcare) in buffer containing 0.1% DDM, 0.01% CHS, 100 mM

NaCl, and 20 mM HEPES (pH 7.4). CD81 was biochemically pure but consistently eluted as two broad peaks during SEC. The SEC-

purified protein was concentrated to 30 - 40 mg/mL and flash frozen with liquid nitrogen in aliquots of 8 mL. Samples were stored at

�80�C until use for crystallography. Purity and monodispersity of crystallographic samples was evaluated by SDS-PAGE and analyt- ical SEC, respectively (Figure S1).

Crystallography and Data Collection Purified CD81 was reconstituted into lipidic cubic phase by mixing with a pre-made 10:1 (w:w) mix of monoolein (Hampton Research)

with cholesterol (Sigma Aldrich) at a ratio of 1.5:1.0 lipid:protein by mass, using the coupled syringe reconstitution method (Caffrey

and Cherezov, 2009). All samples were mixed at least 100 times. The resulting phase was dispensed in 40 - 50 nL drops onto a glass

plate, and overlaid with 650 nL of precipitant solution using a Gryphon LCP robot (Art Robbins Instruments). Crystals grew in precip-

itant solution containing 35% - 45% PEG 300, 100 - 400 mM dibasic sodium citrate, 0.1 M Tris (pH 8). Initial crystallization hits grew

slowly, with crystals appearing after more than three weeks. Crystals were harvested using mesh loops and stored in liquid nitrogen

until data collection.

Data collection was performed at Advanced Photon Source GM/CA beamline 23ID-B. An initial grid raster with 80 3 30 mm beam

dimensions was performed, followed by a sub-raster using a 20 mm beam to locate crystals in the loop. Additional rastering was

performed using a 10 mm beam diameter to optimally position crystals for data collection. Data collection used a 10 mm beam

and diffraction images were collected in 1 degree oscillations at a wavelength of 1.033 Å. The final dataset for CD81 was compiled

by merging data from five crystals using XDS (Kabsch, 2010). Data are summarized in Table S1, using the format of Harrison and

colleagues (Corbett et al., 2010).

Visual inspection of diffraction frames revealed strong anisotropy, with weaker diffraction in the reciprocal a* axis and stronger

diffraction in the other two directions. We thus performed ellipsoidal truncation on the merged dataset using the UCLA

anisotropy diffraction server (Strong et al., 2006). Resolution limits along reciprocal space axes were chosen based on an

Cell 167, 1041–1051.e1–e5, November 3, 2016 e2

F/sigF > 2 criterion, giving resolution limits of 5.50 Å, 2.95 Å and 2.95 Å along the reciprocal space a*, b* and c* axes, respec-

tively. Anisotropic B scaling was also applied using the server to compensate for differences in intensity along each reciprocal

space axis.

Phasing and Structure Refinement To obtain phases, molecular replacement was performed in Phenix with Phaser using chain A from a CD81 ectodomain X-ray

structure, PDB ID 1IV5 (Kitadokoro et al., 2002) (Figure S1C). A subsequent molecular replacement search was conducted using

a polyalanine helix as a search model, with the ectodomain included as a fixed partial model. This procedure, which located one

transmembrane helix, was performed three more times, in order to locate all four transmembrane helices. For refinement, reciprocal

space optimization of XYZ coordinates and individual atomic B-factor parameters was performed with standard Phenix restraints in

phenix.refine (Afonine et al., 2012); optimization for X-ray/stereochemistry weight and X-ray/ADP weight was also performed. Exten-

sive additional model building was carried out manually in Coot (Emsley and Cowtan, 2004) and refined following each round of model

building. Near the end of refinement, custom geometric restraints were placed on the two disulfide bonds present in the EC2. As a

control for register assignment, the structure was built and register assigned using two independent approaches. First, the model

was manually built and register assigned by inspection of electron density in combination with consideration of evolutionary conser-

vation of residues interacting within the transmembrane domains (Marks et al., 2012). In parallel, sequence register was indepen-

dently assigned automatically with phenix.autobuild, which resulted in the same register assignment. Representative composite

omit map density is shown in Figure S2. Cholesterol was manually placed into an Fo-Fc difference map. Following refinement, struc-

ture quality was assessed using MolProbity (Chen et al., 2010), and figures were prepared in PyMOL (Schrödinger Team, 2010). All

crystallographic data processing, refinement, and analysis software was compiled and supported by the SBGrid Consortium (Morin

et al., 2013).

Evolutionary Coupling Analysis For evolutionary coupling analysis, the EVcouplings option on http://evfold.org was used (Marks et al., 2012). The sequence of the

indicated tetraspanins was used as a template and default search settings were used, except that alpha helical TM domain was set to

‘‘Yes.’’ The top 90 evolutionary coupling pairs were chosen for display.

Sequence Conservation Analysis For conservation analysis, a multiple sequence alignment aligning human CD81 to its most similar 150 homologs was analyzed using

the Consurf server (Ashkenazy et al., 2010; Landau et al., 2005). The multiple sequence alignment was generated using a protein

sequence BLAST on the NCBI database. For conservation analysis among the 33 human tetraspanins, the sequences were aligned

using Clustal Omega and then analyzed using the Consurf server (Sievers et al., 2011).

Radioactive Lipid Binding Assay [1,2-3H]cholesterol (45 Ci/mmol), 2,4,6,7-3H(N)-estradiol (94 Ci/mmol), and 9,10-3H(N)-palmitic acid (30 Ci/mmol) were purchased

from PerkinElmer Life Sciences. FLAG-tagged CD81 was subcloned into the mammalian expression vector pcDNA3.1 using NheI

and BamHI restriction sites and mutations were made using gBlocks from IDT. FLAG-BlaR was constructed using PCR, and subcl-

oned into the mammalian expression vector pcDNA3.1 using the same restriction sites. HEK293T cells were transfected with empty

vector, FLAG-tagged CD81 (wild-type, E219A, E219Q), or FLAG-tagged BlaR. Cells were lysed 36 hr later with 20 mM HEPES (pH 7.4),

2 mM MgCl2, 1 ml benzonaze and 2 mg/ml iodoacetamide. Lysed cells were harvested by centrifugation at 15,000 RPM for 10 min at

4�C. Pellets were resuspended in 1% DDM, 0.1% CHS, 20 mM HEPES (pH 7.4) and 250 mM NaCl and solubilized for 2 hr. The samples were clarified by centrifugation at 15,000 RPM for 10 min at 4�C. The soluble fraction was then incubated with 15 ml of anti-M2 FLAG beads per sample for 1 hr. Beads were recovered and washed three times with 0.1% DDM, 0.01% CHS, 20 mM HEPES (pH 7.4) and

100 mM NaCl. FLAG-CD81 conjugated beads were then incubated with 1 mCi of 3H-lipid for 1 hr. Beads were washed three times used

a spin filter column and remaining 3H-lipid was counted using a Beckman Coulter LS6500 scintillation counter.

Molecular Dynamics Simulation: System Setup Simulations of CD81 were based on the cholesterol-bound crystal structure described in this manuscript. The receptor was simulated

in two distinct conditions: (1) the cholesterol-bound crystal structure; (2) the same structure with cholesterol removed.

Prime (Schrödinger) was used to model in missing side chains and the missing residues E86, S87 and 41–54 of EC1. The crystal-

lized protein construct with cysteine mutations at S6, S9, S227 and S228 was employed during simulation. S-(2-amino-2-oxoethyl)-

cysteines 80 and 89 were returned to natural cysteines. Hydrogen atoms were added, and protein chain termini were capped with the

neutral groups acetyl and methylamide. Titratable residues were left in their dominant protonation state at pH 7.0. An alternative side-

chain orientation of K11 was used to prevent early formation of potentially artifactual interactions with E219 that was observed in

initial simulations. For apoprotein simulation conditions, cholesterol was removed.

The prepared protein structures were aligned on the transmembrane helices to the z-axis, and internal waters added with Dowser

(Hermans et al., 2003; Zhang and Hermans, 1996) (in addition to internal waters resolved in the crystal structure). The structures were

then inserted into a pre-equilibrated palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayer, and solvated with 0.15 M NaCl in explicitly

e3 Cell 167, 1041–1051.e1–e5, November 3, 2016

represented water, then neutralized by removing chloride ions. Final system dimensions were approximately 80 3 70 3 96 Å3,

including about 124 lipids, 29 sodium ions, 25 chloride ions, and 10600 water molecules.

MD Simulation Protocol We used the CHARMM36 parameter set for protein molecules, lipid molecules, cholesterol, and salt ions, and the CHARMM TIP3P

model for water; protein parameters incorporated CMAP terms (Best et al., 2012a, 2012b; Huang and MacKerell, 2013; Klauda et al.,

2010; MacKerell et al., 1998).

Simulations were performed on GPUs using the CUDA version of PMEMD (Particle Mesh Ewald Molecular Dynamics) in Amber14

(Case, 2014; Le Grand et al., 2013; Salomon-Ferrer et al., 2013).

Prepared systems were minimized, then equilibrated as follows: The system was heated using the Langevin thermostat from 0 to

100 K in the NVT ensemble over 12.5 ps with harmonic restraints of 10.0 kcal$mol�1$�2 on the non-hydrogen atoms of lipid, protein, and ligand, and initial velocities sampled from the Boltzmann distribution. The system was then heated to 310 K over 125 ps in the

NPT ensemble with semi-isotropic pressure coupling and a pressure of one bar. Further equilibration was performed at 310 K with

harmonic restraints on the protein and ligand starting at 5.0 kcal$mol�1$�2 and reduced by 1.0 kcal$mol�1$�2 in a stepwise fashion every 2 ns, for a total of 10 ns of additional restrained equilibration.

We performed nine simulations of cholesterol-bound CD81, and another nine simulations of unliganded CD81, for a total of 18 sim-

ulations. These simulations were conducted in the NPT ensemble at 310 K and 1 bar, using a Langevin thermostat and Monte Carlo

barostat. In each of these simulations, we performed 5 ns of unrestrained equilibration followed by a production run of 1.0–2.5 ms.

Simulations initiated with cholesterol bound totaled 13.7 ms, and simulations initiated with cholesterol removed totaled 14.8 ms.

Simulations used periodic boundary conditions, and a time step of 2.5 fs. Bond lengths to hydrogen atoms were constrained using

SHAKE. Non-bonded interactions were cut off at 9.0 Å, and long-range electrostatic interactions were computed using the particle

mesh Ewald (PME) method with an Ewald coefficient b of approximately 0.31 Å and B-spline interpolation of order 4. The FFT grid size

was chosen such that the width of a grid cell was approximately 1 Å.

MD Simulation Analysis Trajectory snapshots were saved every 100 ps during production simulations. Trajectory analysis was performed using VMD (Hum-

phrey et al., 1996) and CPPTRAJ (Roe and Cheatham, 2013), and visualization was performed using VMD and PyMol (Schrödinger

Team, 2010). Trajectories were aligned to the crystal structure, along TM3 and TM4.

To determine how frequently the hydrogen bond between the hydroxyl group of cholesterol and the carboxylate side-chain of E219

was formed in simulation, we calculated the percentage of time the distance between the cholesterol hydroxyl hydrogen and either

carboxylate oxygen of E219 was 3.3 Å or less during simulations.

To calculate a p-value associated with the alternative hypothesis that the absence of cholesterol in the binding pocket favors open-

ing, we proceed as follows. We define the p-value as the probability that we would have observed data that favors the alternative

hypothesis at least as strongly as the data we observed under the null hypothesis that opening is equally likely when simulating

CD81 with or without cholesterol in the binding pocket. Given that we observed no opening events when cholesterol was bound

and four opening events when it was not, we calculate the probability, under the null hypothesis, of observing no opening events

when cholesterol was bound and four or more opening events when it was not. Because we do not have an a priori estimate of

the probability of opening in a given simulation, we maximize the calculated p-value over all possible opening probabilities—in other

words, we report the largest of a family of possible p-values.

More specifically, we assume that, under the null hypothesis, the number of opening events observed in t ms of simulation of

closed-state CD81 is given by a Poisson distribution with mean qt, where q is a proportionality or rate constant. The value of q is

unknown but assumed to be the same in simulations with or without cholesterol bound. We calculate, as a function of q, the prob-

ability of observing no opening events when cholesterol is bound (in 11.04 ms of simulation of closed, cholesterol-bound CD81) and

four or more opening events when cholesterol is not bound (in 12.55 ms of simulation of closed CD81 with no cholesterol bound,

including the relevant portion of simulations in which cholesterol dissociated). Our calculated p value, 0.027, is the maximum value

of this probability over all possible values of q (p = 0.03; see STAR Methods).

Flow Cytometry Assay HEK293T cells were transfected with empty vector, Flag-tagged CD19 or ProtC-tagged CD81 (wild-type, E219A, E219Q). Cells were

washed with PBS 36 hr post-transfection and resuspended with 20mM HEPES (pH7.4), 150mM NaCl, and 0.1% BSA. Cells were

incubated for 15min with Alexa 488-anti-CD19 (Molecular probes) and APC-anti-CD81 (BioLegend) per manufacturers recommen-

dation. Cells were washed and plated into a 96 well U-bottom dish and sorted on a BD Accuri C6 flow cytometer. Experiments were

done in triplicate or quadruplicate at least 4 independent times.

QUANTIFICATION AND STATISTICAL ANALYSIS

Bar graphs display mean ± SD. p values were calculated by one-way ANOVA followed by post hoc Bonferroni tests where applicable

using GraphPad Prism. All radioactivity experiments were performed three independent times with duplicate measurements

Cell 167, 1041–1051.e1–e5, November 3, 2016 e4

(Figure 4C). Flow cytometry experiments were performed four independent times with triplicate measurements (Figures 6A and 6B).

Statistical analyses relevant to the structural model are included in Table S1, related to Figure 1.

DATA AND SOFTWARE AVAILABILITY

The structure reported in the paper is deposited in the PDB under code: 5TCX. GraphPad Prism 7.0 is available from an institutional

license at Harvard Medical School. The EVfold server is a freely available online tool created by the Marks lab at Harvard Medical

School and the Sander lab at Memorial Sloan Kettering Cancer Center. All software used in structure determination (XDS, Phenix,

Pymol and Coot) are accessible via the Structural Biology Grid (SBGrid) at Harvard Medical School.

e5 Cell 167, 1041–1051.e1–e5, November 3, 2016

Supplemental Figures

0 5 10 15 20 25 0

10

20

30

Retention (mL)

A 28

0 ( m

A u)

BA

75 kDa 50 kDa 37 kDa 25 kDa 20 kDa 15 kDa

CC

Figure S1. Assessment of CD81 Biochemical Quality and Phase Calculation, Related to Figure 1

(A) SDS-PAGE analysis of purity. Left lane shows molecular weight standard, right lane is FLAG-purified CD81.

(B) Analytical size exclusion profile of purified CD81 in DDM detergent buffer on a Superdex 200 column.

(C) Overlay of CD81 ECL structures. Ribbon diagrams of EC2 of CD81 from this structure superimposed on five other EC2 structures. Red: EC2 from this

structure, blue: 1G8Q, chain A, green: 3X0E, chain A, cyan: 3X0E, chain B, orange: 1IV5, chain A and magenta: 1IV5, chain B.

Figure S2. Assessment of Helix Register Assignment, Related to Figures 1 and 2

(A–D) Composite omit 2Fo-Fc electron density map contoured at 1.0 s for TM1 (A), TM2 (B), TM3 (C) and TM4 (D).

A

B

1 2 3 4 5 6 7 8 9 Variable Average Conserved

L35

V146 F56

F126

TM2 TM1

EC2

L35

V146 F56

F126

TM2 TM1

E

C

º180

º180

1 2 3 4 7 8 9

º0

Figure S3. Sequence Conservation among CD81 Homologs and Paralogs and ECL2 and TM1/TM2 Homolog Interface, Related to Figure 3 (A and B) Surface representation of CD81 homologs (A) and mammalian paralogs (B). Analysis was performed using Consurf, colored in a sliding scale from teal

(poorly conserved) to crimson (highly conserved). Yellow indicates residues with insufficent data for assignment.

(C) Interface between TM1/TM2 and EC2. Residues at the interface between the TM1/2 bundle and EC2 are shown as sticks colored by conservation.

Figure S4. Electron Density, Cholesterol Binding, and Conservation of Pocket Residues, Related to Figures 4 and 6

(A) Composite omit map (2Fo-Fc) of protein density contoured at 2 s (blue) and difference map (Fo-Fc) showing difference electron density (green) modeled as

cholesterol (yellow sticks). The protein model is in magenta.

(B) Immunopurified proteins. Wild-type and mutant (E219Q, G26F/G30F) CD81 proteins were immunopurified from HEK293T cells. Proteins captured on FLAG

beads were used for radioactive cholesterol binding experiments. WCL: whole cell lysate; Sol: solubilized protein; IP: immunoprecipitated protein.

(C) Cholesterol binding. 1,2-3H-cholesterol was incubated with immunopurified FLAG-CD81 proteins and bound cholesterol was measured.

(D) Sequence conservation in the cholesterol binding pocket. Sequence conservation was determined using ConSurf as described in the STAR Methods.

Residues within 4 Å of the cholesterol binding site are denoted by a red asterisk. Percentage conservation is denoted in the table at the right.

N18 E219

Cholesterol-bound simulation

Crystal structure

Apoprotein simulation

Crystal structure

TM1

TM4

TM1

TM4

N18

E219

TM1

TTM4

N1888888888888888

E2E 19 N188 EEEEEEEEEEEEEEEEEEEE2E2EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE 19 TM11

TTM4

Figure S5. Movement of Transmembrane Domains during Molecular Dynamics Simulation, Related to Figure 5

In both cholesterol-bound and apoprotein simulations, the transmembrane helices undergo substantial motion. Snapshots from cholesterol-bound and apo-

protein simulations are shown in blue and red, respectively, while the crystal structure is shown in gray for comparison. In the apoprotein simulations, a hydrogen

bond forms between N18 and E219 that keeps TM1 and TM4 in close proximity. In cholesterol-bound simulations, as in the crystal structure, cholesterol forms a

hydrogen bond with E219, disrupting interaction between N18 and E219 and often leading to separation of TM1 from TM4.

Figure S6. Lattice Contacts, Related to Figure 1

(A–C) Lattice packing of CD81 viewed parallel to the membrane plane (A). (B) and (C) show a view normal to the membrane and a second parallel view,

respectively.