Implications for tetraspanin-enriched microdomain assembly based on structures of CD9 with EWI-F.

Tetraspanins are eukaryotic membrane proteins that contribute to a variety of signaling processes by organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster partner receptors into tetraspanin-enriched microdomains is unknown. Here, we present crystal structures of the large extracellular loop of CD9 bound to nanobodies 4C8 and 4E8 and, the cryo-EM structure of 4C8-bound CD9 in complex with its partner EWI-F. CD9-EWI-F displays a tetrameric arrangement with two central EWI-F molecules, dimerized through their ectodomains, and two CD9 molecules, one bound to each EWI-F transmembrane helix through CD9-helices h3 and h4. In the crystal structures, nanobodies 4C8 and 4E8 bind CD9 at loops C and D, which is in agreement with the 4C8 conformation in the CD9-EWI-F complex. The complex varies from nearly twofold symmetric (with the two CD9 copies nearly anti-parallel) to ca. 50° bent arrangements. This flexible arrangement of CD9-EWI-F with potential CD9 homo-dimerization at either end provides a "concatenation model" for forming short linear or circular assemblies, which may explain the occurrence of tetraspanin-enriched microdomains.

Site-specific functionality and tryptophan mimicry of lipidation in tetraspanin CD9.

Lipidation of transmembrane proteins regulates many cellular activities, including signal transduction, cell-cell communication, and membrane trafficking. However, how lipidation at different sites in a membrane protein affects structure and function remains elusive. Here, using native mass spectrometry we determined that wild-type human tetraspanins CD9 and CD81 exhibit nonstochastic distributions of bound acyl chains. We revealed CD9 lipidation at its three most frequent lipidated sites suffices for EWI-F binding, while cysteine-to-alanine CD9 mutations markedly reduced binding of EWI-F. EWI-F binding by CD9 was rescued by mutating all or, albeit to a lesser extent, only the three most frequently lipidated sites into tryptophans. These mutations did not affect the nanoscale distribution of CD9 in cell membranes, as shown by super-resolution microscopy using a CD9-specific nanobody. Thus, these data demonstrate site-specific, possibly conformation-dependent, functionality of lipidation in tetraspanin CD9 and identify tryptophan mimicry as a possible biochemical approach to study site-specific transmembrane-protein lipidation.

High frequency of inactivating tetraspanin C D37 mutations in diffuse large B-cell lymphoma at immune-privileged sites.

Tetraspanin CD37 is predominantly expressed on the cell surface of mature B lymphocytes and is currently being studied as novel therapeutic target for B-cell lymphoma. Recently, we demonstrated that loss of CD37 induces spontaneous B-cell lymphoma in Cd37-knockout mice and correlates with inferior survival in patients with diffuse large B-cell lymphoma (DLBCL). Here, CD37 mutation analysis was performed in a cohort of 137 primary DLBCL samples, including 44 primary immune-privileged site-associated DLBCL (IP-DLBCL) samples originating in the testis or central nervous system. CD37 mutations were exclusively identified in IP-DLBCL cases (10/44, 23%) but absent in non-IP-DLBCL cases. The aberrations included 10 missense mutations, 1 deletion, and 3 splice-site CD37 mutations. Modeling and functional analysis of CD37 missense mutations revealed loss of function by impaired CD37 protein expression at the plasma membrane of human lymphoma B cells. This study provides novel insight into the molecular pathogenesis of IP-DLBCL and indicates that anti-CD37 therapies will be more beneficial for DLBCL patients without CD37 mutations.