The membrane-associated RING-CH (MARCH) proteins are a family of E3 ubiquitin ligases that regulate the surface expression of proteins by attaching ubiquitin to their cytoplasmic tails, thus tagging them for internalization by endocytosis. The viral MARCH proteins, also known as modulators of immune recognition (MIRs), 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 65-amino-acid fragment containing the two TM domains and the extracellular loop of MARCH9, a member of the mammalian MARCH family, which can be stably reconstituted into lipid micelles for structure determination using solution nuclear magnetic resonance (NMR) spectroscopy. Backbone assignments for this fragment were completed using a standard suite of transverse-relaxation-optimized (TROSY) triple resonance experiments. We have also utilized a stereo-specific methyl-labeling scheme to facilitate the assignment of methyl protons, as well as the detection and measurement of long-range inter-helical backbone-to-methyl and methyl-to-methyl nuclear Overhauser effects (NOEs) that can be used as physical restraints for structure calculations. 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.