Can ERAP1 and ERAP2 Form Functional Heterodimers? A Structural Dynamics Investigation

Abstract

Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2) play important roles in the generation of antigenic peptides presented by Major Histocompatibility Class I (MHCI) molecules and indirectly regulate adaptive immune responses. Although the discrete function of these enzymes has been extensively characterized, recent reports have suggested that they can also form heterodimers with functional consequences. However, lack of structural characterization of a putative ERAP1/ERAP2 dimer has limited our understanding of its biological role and significance. To address this, we employed computational molecular dynamics calculations to explore the topology of interactions between these two, based on experimentally determined homo-dimerization interfaces observed in crystal structures of ERAP2 or homologous enzymes. Our analysis of 8 possible dimerization models, suggested that the most likely ERAP1/ERAP2 heterodimerization topology involves the exon 10 loop, a non-conserved loop previously implicated in interactions between ERAP1 and the disulfide-bond shuffling chaperone ERp44. This dimerization topology allows access to the active site of both enzymes and is consistent with a previously reported construct in which ERAP1 and ERAP2 were linked by Fos/Jun zipper tags. The proposed model constitutes a tentative structural template to help understand the physiological role and significance of ERAP1/ERAP2 molecular interactions.

Keywords: aminopeptidase; MHC class I; adaptive immunity; antigen presentation; antigen processing; binding free energy; enzyme mechanism; molecular dynamics.

Figure 1

(A, B) Crystallographic structures of ERAP2 homodimers in the two different topologies formed within the unit cell, and which were used as templates for the 4 heterodimeric ERAP1/ERAP2 models. For each dimer, both possible topologies of ERAP1/ERAP2 (A1, B1) and ERAP2/ERAP1 (A2, B2) heterodimers were investigated. Domains are color-coded as indicated for each enzyme with Latin numbers.

Figure 2

Crystallographic structures of the homodimeric IRAP (C) and APN (D), which were employed as templates for the construction of the corresponding ERAP1/ERAP2 (C1, C2) and ERAP2/ERAP1 (D1, D2) heterodimeric topologies. Domains are color-coded as indicated for each enzyme with Latin numbers.

Figure 3

Bar plots of the estimated binding affinity (ΔG ^est, upper panels) and the buried surface area (BSA, lower panels) for each crystallographic homodimer (left panels) and the corresponding heterodimeric models of ERAP1/ERAP2 (right panels). Mean values were calculated from 3 independent 100-ns MD simulations, and error bars indicate standard deviation.

Figure 4

Ribbon representations (upper panels) and surface representations (lower panels) of 3 frames from one MD simulation of the most favorable heterodimeric ERAP1/ERAP2 topology B2, illustrating the initial, energy minimized and two snapshots at 15 and 90 ns of the simulation. Domains are designated with Latin numbers and surfaces are colored with cyan C atoms for ERAP1 and orange C atoms for ERAP2. The position of the interacting exon 10 loop and the buried surface area (BSA) of each complex are indicated.

Figure 5

(A) The proposed heterodimeric ERAP2/ERAP1 model B2 from a representative snapshot taken from a 100-ns MD simulation. The snapshot is the centroid of the highest populated cluster of conformations that represents 64% of the trajectory within 2 Å RSMD of all Cα atoms. (B) Close-up view showing two key salt–bridge interactions between helices 8 of ERAP1 (orange C atoms) and ERAP2 (cyan C atoms). The disulfide bridges that stabilize exon 10 loops are shown as sticks. (C) Another close-up view of the dimeric interface illustrating hydrophobic/aromatic interactions between ERAP1 (orange C atoms) and ERAP2 (cyan C atoms).

Figure 6

Models of ERAP1–c-Jun/ERAP2–c-Fos based on two different topologies. (A) Initial, energy minimized dimer based on model B2 and (B) representative structure from a MD simulation, calculated as the centroid of the highest populated cluster of conformations (40% fraction of the trajectory) with respect to the RMSD of the C^α atoms excluding the Jun/Fos linker residues. (C) Initial, energy minimized structure of the IRAP-based model C2, and (D) representative structure of the highest populated cluster of conformations (43% fraction of the trajectory) calculated from a MD simulation. Arrows indicate the substrate entrance site.