Chat with us, powered by LiveChat BIOL 3410 Cell Biology Fall 2021 Section 3. Assignment 2 (34 points) Question 1 To stimu - Study Help
  

BIOL 3410 Cell Biology Fall 2021 Section 3.

Assignment 2 (34 points)

Question 1

To stimulate an immune response, a T lymphocyte must come into physical contact with an antigen-presenting cell. At the site of contact, the T-lymphocyte rearranges multiple proteins to form an immune synapse. Panels A and B (left) summarize FRAP experiments exploring the lateral mobility of the membrane-bound protein CD81. To do this, the authors tagged CD81 with the auto-fluorescent protein mCherry and used fluorescence microscopy to image the cells. Panel A (top row) shows T lymphocytes alone. The middle and bottom rows show T lymphocytes in contact with a antigen-presenting cell (not fluorescent, but annotated with an asterisk). The white circles show the regions bleached in the FRAP experiment (the green regions are unbleached control).

(a) What does FRAP stand for? (2 point)

(b) Describe CD81 fluorescence in pre-bleach unstimulated T lymphocytes (2 points).

(c) What happens to CD81 when the T lymphocyte is in contact with an antigen presenting cell? (2 points)

Panel B shows FRAP data on the above cells.

(d) After 200 seconds of recovery, what percentage of CD81 fluorescence is mobile in an unstimulated cell? (2 points)

(e) After 200 seconds of recovery, what percentage of CD81 fluorescence is mobile in the center of the cell-to-cell contact zone? (2 points)

(f) What do you think is happening to CD81 at the cell-to-cell contact zone? (4 points)

(g) What is the purpose of measuring fluorescence in the red circles (panels A)? (2 points)

Question 2.

CD81 is a membrane-associated protein required for B cell function in the immune system.

A. Using your favorite interweb search engine, find and download the human CD81 protein sequence and paste it below. (4 points)

B. Using the online tool EMBOSS (see URL below), copy/paste the above sequence into the submission window then hit “Submit”. (2 points)

https://www.ebi.ac.uk/Tools/seqstats/emboss_pepwindow/

C. Using your scientific spidey sense, label what you think are candidate transmembrane domains. Hint: take a look at the accompanying paper by Zimmerman et al. (2016) and use this to refine your answer. (4 points)

D. Are the N and C-termini of CD81 on the intracellular or extracellular face of the plasma membrane? (2 points)

E. Based on the CD81 crystal structure and the CD81 FRAP data from question 1, what is likely to be happening to CD81 at an immune synapse. Use one or more diagrams to illustrate your answer. (4 points).

F. CD81 can bind to small molecules. What’s its ligand? (2 points)

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.

mailto:[email protected]

mailto:[email protected]

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

http://crossmark.crossref.org/dialog/?doi=10.1016/j.cell.2016.09.056&domain=pdf

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

mailto:[email protected]

mailto:[email protected]

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

http://crossmark.crossref.org/dialog/?doi=10.1016/j.cell.2016.09.056&domain=pdf

(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

http://evfold.org

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
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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

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