Importance of accessibility to the extracellular juxtamembrane stalk region of membrane protein for substrate recognition by viral ubiquitin ligase K5

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is a carcinogenic virus that latently infects B cells and causes malignant tumors in immunocompromised patients. KSHV utilizes two viral E3 ubiquitin ligases, K3 and K5, in KSHV-infected cells to mediate the polyubiquitination-dependent down-regulation of several host membrane proteins involved in the immune system. Although K3 and K5 are members of the same family and have similar structural topologies, K3 and K5 have different substrate specificities. Hence, K5 may have a different substrate recognition mode than K3; however, the molecular basis of substrate recognition remains unclear. Here, we investigated the reason why human CD8α, which is known not to be a substrate for both K3 and K5, is not recognized by them, to obtain an understanding for molecular basis of substrate specificity. CD8α forms a disulfide-linked homodimer under experimental conditions to evaluate the viral ligase-mediated down-regulation. It is known that two interchain disulfide linkages in the stalk region between each CD8α monomer (Cys164–Cys164 and Cys181–Cys181) mediate homodimerization. When the interchain disulfide linkage of Cys181–Cys181 was eliminated, CD8α was down-regulated by K5 with a functional RING variant (RINGv) domain via polyubiquitination at the cytoplasmic tail. Aspartic acid, located at the stalk/transmembrane interface of CD8α, was essential for K5-mediated down-regulation of the CD8α mutant without a Cys181–Cys181 linkage. These results suggest that disulfide linkage near the stalk/transmembrane interface critically inhibits substrate targeting by K5. Accessibility to the extracellular juxtamembrane stalk region of membrane proteins may be important for substrate recognition by the viral ubiquitin ligase K5.

A basic charge cluster near the C‐terminus of the cytoplasmic tail contributes to the molecular stability of human herpesvirus 8 E3 ubiquitin ligases

“…Protein stability was measured using cycloheximide chase analysis, as described previously 15 . Briefly, HeLa cells were transfected with the pTracer PA‐ligase vector using PEI Max.…”

“…Protein stability was measured using cycloheximide chase analysis, as described previously. 15 Briefly, HeLa cells were transfected with the pTracer PA-ligase vector using PEI Max. Forty-eight hours after transfection, the cells were exposed to 10 µg/mL cycloheximide (Nacalai Tesque) for 0, 60, 120, and 180 min.…”

“…11 They recognize substrate membrane proteins via both TM helices and inter-TM loops. 11,14,15 In the cytoplasm, they have a RINGv domain at the N-terminal end, which is essential for ubiquitin ligase activity because the domain is required to recruit activated ubiquitin on the E2 ubiquitin-conjugating enzyme to lysines on the target protein. 11,16 In contrast, they have a long tail predicted to be intrinsically disordered in the cytoplasmic area of the C-terminal end.…”

“…They have two transmembrane (TM) helices and a short extracellular inter‐TM loop connecting the C‐terminal end of TM1 and the N‐terminal end of TM2 11 . They recognize substrate membrane proteins via both TM helices and inter‐TM loops 11,14,15 . In the cytoplasm, they have a RINGv domain at the N‐terminal end, which is essential for ubiquitin ligase activity because the domain is required to recruit activated ubiquitin on the E2 ubiquitin‐conjugating enzyme to lysines on the target protein 11,16 .…”