Generally glycines and other small residues such as alanine
Generally, glycines (and other small residues such as alanine) present in a helical GxxxG-motif that are facing the same side of the helix form a uniform surface that enables direct contact with a second helix. Crystal structures of multipass membrane proteins have demonstrated that GxxxG-motifs are used in various constellations to pack TM helices. In the T. thermophilus transhydrogenase domain II  or the sodium-proton antiporter NhaA  some GxxxG-motifs (and extended repeats thereof) are facing towards each other (in some cases with a shifted register), whereas other GxxxG-motifs are only present in one of the interacting helices, but still forming a dimer interface with the neighbouring TM helix. In the sodium-potassium pump  a variant of the GxxxG-motif (AxxxA) is found at the TM interface between the α- and γ-subunits. This AxxxA-motif is present in both interacting helices and both motifs are facing towards each other (again a shift in register is observed). In the β-/α-subunit binding interface, the extended motif GxxxGxxxG in the β-subunit faces the lipid environment and thus away from the interface . This might allow for binding of interaction partners or simply demonstrates that not all GxxxG-motifs are involved in direct helix-helix interactions.
The GxxxG-motif in MARCH-1/8 Sequence alignment of MARCH-1/8 homologues reveals a strongly conserved GxxxG-motif in the central part of the TM2 helix (Fig. 3). A third glycine residue is placed at the n+3 position, which might allow for the accommodation of bulkier sidechains or intertwined helices. The canonical GxxxG-motif is placed centrally within the TM2 helix and it would not come as a surprise if this motif is involved in helix-helix interactions either by mediating oligomerisation or substrate recognition. However, structural information is needed to confirm this hypothesis. Interestingly, TM1 does not contain any GxxxG or related motif. In addition to the motif found in TM2, a second GxxxG-motif is present in the L1-loop in MARCH-1/8. The motif is less conserved within MARCH-1/8 homologues and is present in the middle of the L1-loop. The L1-loop is generally rich in amino LDC000067 residues with high turn propensities , suggesting lack of secondary structure in this loop. However, as the length of the L1-loop in e.g. human MARCH-1 with 14 amino acid residues is longer than needed for just a simple turn, one could speculate that the motif-surrounding residues create flexibility and that the motif engages interaction partners on the luminal side, e.g. via parallel extension of a β‐sheet.
The GxxxG-motif in the other MARCH proteins The GxxxG-motif is also present in TM helices of the other MARCH proteins (Fig. 3). In MARCH-2/3, the GxxxG-variants are found both in TM1 and TM2. Interestingly, the two motifs (AxxxG in TM1 and AxxxA in TM2) are positioned at similar distance from the luminal membrane. This would allow for a classical dimerisation interface involving the GxxxG-like motifs in MARCH-2/3. MARCH-4/9/11 have an AxxxG-motif in TM1 close to the cytoplasmic membrane, while there is no such motif in TM2. MARCH-5 even has two GxxxG-motif variants (GxxxG and AxxxG) in TM1, but none in TM2. Interestingly, mutation of this GxxxG motif to LxxxL prevented oligomerisation of MARCH-5 molecules, while the same mutation of another GxxxG motif in the L1-loop between TM1 and TM2 did not interfere with oligomerisation . March-6 does not contain any GxxxG- or related motifs in TM1, but a CxxxS motif is found in TM2. Cysteines and serines are in this context also defined as small residues that can promote helix-helix interactions .
Conclusions A large number of membrane proteins use GxxxG-motifs (and variants thereof) in their TM helices for intra- and intermolecular recognition of other TM helices. Here we describe that this GxxxG-motif is also found in different variations in all membrane-embedded MARCH E3 ligases. Presence, number and positioning of these motifs in the two core TM-helices (TM1 and TM2) differ between the phylogenetic MARCH subgroups. However, when the motif is present it is always strongly conserved, suggesting a role in oligomerisation and/or substrate recognition for these MARCH-proteins. However, structural data on the MARCH proteins in context with the TM helices is completely lacking, but would be beneficial for the design of drugs that regulate MARCH-induced ubiquitination.