Structural basis for tetraspanin functions as revealed by the cryo-EM structure of uroplakin complexes at 6-A resolution

Abstract

Tetraspanin uroplakins (UPs) Ia and Ib, together with their single-spanning transmembrane protein partners UP II and IIIa, form a unique crystalline 2D array of 16-nm particles covering almost the entire urothelial surface. A 6 A-resolution cryo-EM structure of the UP particle revealed that the UP tetraspanins have a rod-shaped structure consisting of four closely packed transmembrane helices that extend into the extracellular loops, capped by a disulfide-stabilized head domain. The UP tetraspanins form the primary complexes with their partners through tight interactions of the transmembrane domains as well as the extracellular domains, so that the head domains of their tall partners can bridge each other at the top of the heterotetramer. The secondary interactions between the primary complexes and the tertiary interaction between the 16-nm particles contribute to the formation of the UP tetraspanin network. The rod-shaped tetraspanin structure allows it to serve as stable pilings in the lipid sea, ideal for docking partner proteins to form structural/signaling networks.

Figure 1.

The 3D structure of the mouse UP tetraspanin complexes at 6-Å resolution. (A) The top view of the hexagonal 16-nm particle shows that it consists of six subunits (one of them is outlined in blue) that encircle a central area of ∼6 nm in diameter filled with lipids. Each subunit in turn consists of an inner subdomain (arrowhead) and an outer subdomain (arrow) that are connected at the top of the particle (Fig. 3 C). Bar, 2 nm. (B) The UP particle has a cylindrical shape and a height of ∼12.5 nm. The vertical dimension of the particle can be approximately divided into four zones: from the top, the joint (JT; ∼3 nm in height), the trunk (TK; ∼5 nm), the TM (∼4 nm), and cytoplasmic domain (CT).

Figure 2.

The TM of the primary UP tetraspanin pair as a five-TM helix bundle. (A) The transmembrane helices of the UP Ia/II tetraspanin pair viewed from the cytoplasmic side. The electron density is represented by a green mesh contoured at the 2.0-σ level. The four helices of the tetraspanin UP Ia are labeled 1–4, whereas the single helix of UP II is labeled 1' (see text for details). (B) The section of the electron density in the middle of the transmembrane region of the UP Ia/II pair. Models of the five transmembrane helices are shown as red ribbons. The yellow, red, and green meshes represent the electron density contoured at the 1.0-, 1.5-, and 2.0-σ levels, respectively. The inset shows the relative positions of the TM regions in the 16-nm particle.

Figure 3.

The subdomains of the 16-nm particle and the relation between the UP tetraspanins and their partners. The top and bottom panels represent the side and top views, respectively, of the electron densities contoured at the 2-σ level. (A and B) The two primary UP pairs, i.e., the inner (A; UP Ia/II) and the outer (B; UP Ib/IIIa) subdomains, viewed separately. The tetraspanin UPs (inner and outer subdomains are shown in brown and yellow, respectively) are segmented from that of their partners (inner and outer subdomains are shown in blue and green, respectively). (C) A subunit of the 16-nm UP particle is made of an inner and an outer subdomain linked through the top “joint.” A 1.0-σ level contour of the electron density is shown in the top view (bottom) to illustrate the connection between the inner and outer domains. A possible FimH binding site on UP Ia is indicated by an arrow (top; see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200602086/DC1). (D) Two neighboring inner domains are connected through a weak link at the exoplasmic boundary of the lipid bilayer. This link joins the tetraspanin UP Ia with the UP II of the neighboring primary UP Ia/II pair. The arrowhead indicates the NH₂-terminal end of TM1. The two parallel lines (yellow) in the top panels approximately mark the boundaries of the TM, and the four vertical zones are indicated on the right as the joint (JT), trunk (TK), transmembrane (TM), and cytoplasmic (CT) domains. The inset in the top right corner of each top panel illustrates the position of the shown subdomains within the 16-nm particle.

Figure 4.

Molecular model of the UP tetraspanins. (A) A molecular model of UP Ia tetraspanin is fitted into the electron density map. (B) The density of the UP Ia tetraspanin is removed to show the model's relation to the density of the single-spanning transmembrane protein. The model is colored so that the color spectra start with blue at the NH₂ terminus and end with red at the COOH terminus.

Figure 5.

Interactions in the UP complexes and the formation of the tetraspanin networks. (A) Primary interaction. A UP tetraspanin (UP Ia or Ib; yellow) interacts with its partner (UP II or IIIa; green) via both their transmembrane and head domains to form primary pair complexes Ia/II or Ib/IIIa (Fig. 3, A and B; and Fig. 4), which occupy the inner or outer subdomains of the 16-nm particle, respectively. (B) Two types of secondary interactions. (left) Two primary tetraspanin pairs interact mainly via the extracellular head domains of the single-spanning transmembrane partners to form a heterotetramer complex (Fig. 3 C), one of the six subunits of the 16-nm particle. (right) The tetraspanin of one primary pair interacts with the single-spanning transmembrane protein of another primary pair, as occurring in the inner ring of the 16-nm particle (Fig. 3 D). Note that only the complexes formed by the second type of interaction (right) can extend to incorporate more than two primary pairs. (C) Formation of the urothelial tetraspanin network. The formation of the 16-nm particle involves the secondary interactions between two primary heterodimeric complexes (red bars) and between the neighboring UP Ia/II heterodimers of the inner subdomains (red arrowheads). Tertiary interactions between the particles (dotted red lines) further extend the UP tetraspanin network. For clarity, only one particle is colored.