Crystal Structure of a Full-Length Human Tetraspanin Reveals a Cholesterol-Binding Pocket

Abstract

Tetraspanins comprise a diverse family of four-pass transmembrane proteins that play critical roles in the immune, reproductive, genitourinary, and auditory systems. Despite their pervasive roles in human physiology, little is known about the structure of tetraspanins 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 membrane 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. Molecular dynamics simulations identify an additional conformation in which EC2 separates substantially from the transmembrane domain. Cholesterol binding appears to modulate CD81 activity in cells, suggesting a potential mechanism for regulation of tetraspanin function.

Keywords: CD19; CD81; X-ray crystallography; membrane protein; protein structure; protein trafficking.

Figure 1

Overall structure of human CD81. (A) Cartoon representation viewed parallel to the membrane plane. Helix one (TM1) is blue, helix two (TM2) cyan, helix three (TM3) green and helix four (TM4), magenta. The large extracellular region (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.

Figure 2

Evolutionary coupling map of tetraspanins. The top 90 amino acid evolutionary coupling pairs of (A) human CD81, (B) hypothetical protein FGSG_08695 from Fusarium graminearum PH-1, (C) tetraspanin 3A from Drosophila melanogaster, and (D) AX4 tetraspanin family protein from Dictyostelium discoideum. Hot spots include couplings between residues of TM1 and TM2, TM3 and TM4, the junction between TM2 and TM3, and intradomain coupling within EC2. Analysis performed using the EVFold server (http://www.evfold.org). See also Figure S2.

Figure 3

Sequence conservation of CD81. Cartoon representation of CD81 versus the top 50 CD81-related sequences determined by Consurf (A) or CD81 versus the 32 human tetraspanin 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 ectodomain and the TM helices is almost 3300 ų in volume. See also Figure S3.

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 σ 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.

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 new electrostatic interaction K116 – D117 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 cholesterolbound simulation in which they do not. Thin traces show values every 1 ns, and thick traces are smoothed. See also Figure S5.

Figure 6

Cholesterol binding regulates CD81-mediated export of CD19. 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).