Regulation of T Cell Receptor Activation by Dynamic Membrane Binding of the CD3ɛ Cytoplasmic Tyrosine-Based Motif

SummaryMany immune system receptors signal through cytoplasmic tyrosine-based motifs (ITAMs), but how receptor ligation results in ITAM phosphorylation remains unknown. Live-cell imaging studies showed a close interaction of the CD3ɛ cytoplasmic domain of the T cell receptor (TCR) with the plasma membrane through fluorescence resonance energy transfer between a C-terminal fluorescent protein and a membrane fluorophore. Electrostatic interactions between basic CD3ɛ residues and acidic phospholipids enriched in the inner leaflet of the plasma membrane were required for binding. The nuclear magnetic resonance structure of the lipid-bound state of this cytoplasmic domain revealed deep insertion of the two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor ligation thus needs to result in unbinding of the CD3ɛ ITAM from the membrane to render these tyrosines accessible to Src kinases. Sequestration of key tyrosines into the lipid bilayer represents a previously unrecognized mechanism for control of receptor activation

A structure-based designed small molecule depletes hRpn13Pru and a select group of KEN box proteins

Proteasome subunit hRpn13 is partially proteolyzed in certain cancer cell types to generate hRpn13Pru by degradation of its UCHL5/Uch37-binding DEUBAD domain and retention of an intact proteasome- and ubiquitin-binding Pru domain. By using structure-guided virtual screening, we identify an hRpn13 binder (XL44) and solve its structure ligated to hRpn13 Pru by integrated X-ray crystallography and NMR to reveal its targeting mechanism. Surprisingly, hRpn13Pru is depleted in myeloma cells following treatment with XL44. TMT-MS experiments reveal a select group of off-targets, including PCNA clamp-associated factor PCLAF and ribonucleoside-diphosphate reductase subunit M2 (RRM2), that are similarly depleted by XL44 treatment. XL44 induces hRpn13-dependent apoptosis and also restricts cell viability by a PCLAF-dependent mechanism. A KEN box, but not ubiquitination, is required for XL44-induced depletion of PCLAF. Here, we show that XL44 induces ubiquitin-dependent loss of hRpn13Pru and ubiquitin-independent loss of select KEN box containing proteins

Spatial domain organization in the HIV-1 reverse transcriptase p66 homodimer precursor probed by double electron-electron resonance EPR

Significance HIV-1 reverse transcriptase, the enzyme that catalyzes the conversion of viral RNA into DNA, is initially released from the Gag-Pol polyprotein as a p66 homodimer precursor. Further cleavage by HIV-1 protease removes the RNase H domain of only a single subunit to yield the mature p66/p51 heterodimer. We have determined the spatial subunit organization within the p66 homodimer from a large number of distances between spin labels obtained by EPR. We show that the structural subunit asymmetry of the p66/p51 heterodimer is preserved in the p66 homodimer and that only one of the RNase H domains is accessible to HIV-1 protease.</jats:p