orals 5th Asia-Pacific NMR Symposium 2013

Intra-Membrane Substrate Recognition by Membrane-Tethered E3 Ligases (#18)

Cyrus Tan 1 , Melissa J Call 1 , Matthew E Call 1
  1. Walter and Eliza Hall Institute, Parkville, VIC, Australia

The membrane-associated RING-CH (MARCH) proteins are a family of E3 ubiquitin ligases that regulate the levels of cell-surface proteins by attaching ubiquitin to their cytoplasmic tails, thus tagging them for internalization by endocytosis. The viral MARCHs proteins, also known as modulators of immune recognition (MIR), down-regulate the surface expression of major histocompatibility complex (MHC)-class I molecules, allowing viral pathogens to evade the immune system. The mammalian MARCH proteins regulate cell surface proteins involved in antigen presentation, such as MHC-class I and class II molecules, and in lymphocyte activation, such as the T cell co-receptor CD4. The viral MIRs and most mammalian MARCH proteins contain two α-helical transmembrane (TM) domains that not only tether them to the cell membrane, but also control substrate recognition through direct TM-TM interactions. However, no ‘recognition motifs’ within the TM domains of MARCH and substrate have been identified, and the determinants of substrate specificity remain unclear.

In order to characterize the MARCH-substrate TM-TM domain interface, we have generated a MARCH fragment containing the two TM domains and extracellular loop that can be stably reconstituted into lipid micelles for structure determination using solution nuclear magnetic resonance (NMR) spectroscopy. Backbone assignments for a 65-amino-acid, lipid-embedded MARCH-9 fragment were completed using a standard suite of transverse-relaxation-optimised (TROSY) triple resonance experiments. A stereospecific methyl-labeling scheme is being implemented to facilitate the assignment of methyl protons and the measurement of inter-helical methyl-to-methyl nuclear Overhauser effects (NOEs) for structure calculation. Several strategies for generating stable MARCH-substrate-TM complexes are also currently being pursued for further NMR studies of the MARCH-substrate TM-TM interface. Structural characterization of this interface will reveal important elements required for intra-membrane interactions, providing a substrate ‘signature’ and thus the ability to uncover the full range of biological processes that are regulated by MARCH E3 ligases.

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