The membrane-associated RING-CH (MARCH) family of proteins are membrane-embedded E3 ubiquitin ligases that regulate the cell surface expression of substrates by ubiquitination, 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 or substrate have been identified, and the determinants of substrate specificity have yet to be defined.

This study employs a combination of biochemical and biophysical techniques aimed at characterizing the structural features of the TM-embedded portion of MARCH in order to uncover potential interaction sites that are responsible for substrate specificity. 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 are currently establishing a Förster Resonance Energy Transfer (FRET)-based flow cytometry assay that will provide a physical readout of MARCH-substrate interaction in human cell lines, and will be used to validate structural insights gained from our NMR studies.

Structural characterization of the MARCH-substrate interface will reveal important elements required for intra-membrane interactions, providing a substrate ‘signature’ with which other physiological substrates can be revealed. This will allow us to uncover the full range of biological processes that are regulated by the MARCH family of E3 ligases.