Structures of Two Melanoma-Associated Antigens Suggest Allosteric Regulation of Effector Binding

Abstract

The MAGE (melanoma associated antigen) protein family are tumour-associated proteins normally present only in reproductive tissues such as germ cells of the testis. The human genome encodes over 60 MAGE genes of which one class (containing MAGE-A3 and MAGE-A4) are exclusively expressed in tumours, making them an attractive target for the development of targeted and immunotherapeutic cancer treatments. Some MAGE proteins are thought to play an active role in driving cancer, modulating the activity of E3 ubiquitin ligases on targets related to apoptosis. Here we determined the crystal structures of MAGE-A3 and MAGE-A4. Both proteins crystallized with a terminal peptide bound in a deep cleft between two tandem-arranged winged helix domains. MAGE-A3 (but not MAGE-A4), is predominantly dimeric in solution. Comparison of MAGE-A3 and MAGE-A3 with a structure of an effector-bound MAGE-G1 suggests that a major conformational rearrangement is required for binding, and that this conformational plasticity may be targeted by allosteric binders.

A Comprehensive Guide to the MAGE Family of Ubiquitin Ligases

“…Structural studies have revealed that the MHD consists of two tandem winged-helix (WH) motifs, referred to as WH-A and -B (Figures 3C, D) [26, 27]. Each WH features a characteristic helix-turn-helix motif packed against a three-stranded antiparallel β-sheet “wing”, however, WH-B also contains additional α-helices [26, 27].…”

“…Each WH features a characteristic helix-turn-helix motif packed against a three-stranded antiparallel β-sheet “wing”, however, WH-B also contains additional α-helices [26, 27]. Overall, the MAGE-A3 and -A4 MHD structures share the same relative orientation and both exhibit a peptide extension binding into the conserved cleft between the two WH motifs; however, the C-terminus of MAGE-A4 is more closely associated with the rest of the molecule and forms a longer section of α-helix [27]. …”

“…Intriguingly, while the WH-A and WH-B motifs of NSE1-bound MAGE-G1 and free MAGE-A4 are similar, with rmsd values of 1.05Å and 1.07Å, respectively, their relative orientations are very distinct in the two structures (Figure 4C) [26, 27]. The WH motifs in MAGE-G1 are distantly separated, such that WH-B rotates approximately 170° and translates about 30Å relative to WH-A [26, 27].…”

“…The WH motifs in MAGE-G1 are distantly separated, such that WH-B rotates approximately 170° and translates about 30Å relative to WH-A [26, 27]. Moreover, the peptide extension bound between the two WH motifs in the MAGE-A3 and -A4 structures is not present in the MAGE-G1-NSE1 structure [26, 27].…”

“…The WH motifs in MAGE-G1 are distantly separated, such that WH-B rotates approximately 170° and translates about 30Å relative to WH-A [26, 27]. Moreover, the peptide extension bound between the two WH motifs in the MAGE-A3 and -A4 structures is not present in the MAGE-G1-NSE1 structure [26, 27]. Therefore, the two different conformational states demonstrate that the MHD undergoes extensive rearrangement for MRL complex formation and the structural changes required to accommodate association with RING ligases may confer binding specificity.…”