Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2) cooperate to trim antigenic peptide precursors for loading onto MHC class I molecules and help regulate the adaptive immune response. Common coding single nucleotide polymorphisms in ERAP1 and ERAP2 have been linked with predisposition to human diseases ranging from viral and bacterial infections to autoimmunity and cancer. It has been hypothesized that altered Ag processing by these enzymes is a causal link to disease etiology, but the molecular mechanisms are obscure. We report in this article that the common ERAP2 single nucleotide polymorphism rs2549782 that codes for amino acid variation N392K leads to alterations in both the activity and the specificity of the enzyme. Specifically, the 392N allele excises hydrophobic N-terminal residues from epitope precursors up to 165-fold faster compared with the 392K allele, although both alleles are very similar in excising positively charged N-terminal amino acids. These effects are primarily due to changes in the catalytic turnover rate (k_cat) and not in the affinity for the substrate. X-ray crystallographic analysis of the ERAP2 392K allele suggests that the polymorphism interferes with the stabilization of the N terminus of the peptide both directly and indirectly through interactions with key residues participating in catalysis. This specificity switch allows the 392N allele of ERAP2 to supplement ERAP1 activity for the removal of hydrophobic N-terminal residues. Our results provide mechanistic insight to the association of this ERAP2 polymorphism with disease and support the idea that polymorphic variation in Ag processing enzymes constitutes a component of immune response variability in humans.

Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias.

Significance: Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels involved in fast neurotransmission. Here, we present the crystal structure of the homopentameric assembly of the extracellular domain (ECD) of α2 nAChR subunit in complex with an agonist. The structure provides a unique opportunity to probe the interactions involved in the formation of the ligand binding site of a WT nAChR and their role in stabilizing an agonist. Furthermore, functional studies revealed the role of additional residues in the activation and desensitization of the α2β2 nAChRs. High sequence identity of α2 ECD with other neuronal subunits signifies the importance of the structure as a template for modeling several neuronal nAChR ECDs and for designing nAChR subtype-specific drugs against related diseases.

The α9α10 nicotinic acetylcholine receptor (nAChR) was first identified in the auditory system, where it mediates synaptic transmission between efferent olivocochlear cholinergic fibers and cochlea hair cells. This receptor gained further attention due to its potential role in chronic pain and breast and lung cancers. We previously showed that α-conotoxin (α-CTx) RgIA, one of the few α9α10 selective ligands identified to date, is 300-fold less potent on human versus rat α9α10 nAChR. This species difference was conferred by only one residue in the (−), rather than (+), binding region of the α9 subunit. In light of this unexpected discovery, we sought to determine other interacting residues with α-CTx RgIA. A previous molecular modeling study, based on the structure of the homologous molluscan acetylcholine-binding protein, predicted that RgIA interacts with three residues on the α9(+) face and two residues on the α10(−) face of the α9α10 nAChR. However, mutations of these residues had little or no effect on toxin block of the α9α10 nAChR. In contrast, mutations of homologous residues in the opposing nAChR subunits (α10 Ε197, P200 and α9 T61, D121) resulted in 19- to 1700-fold loss of toxin activity. Based on the crystal structure of the extracellular domain (ECD) of human α9 nAChR, we modeled the rat α9α10 ECD and its complexes with α-CTx RgIA and acetylcholine. Our data support the interaction of α-CTx RgIA at the α10/α9 rather than the α9/α10 nAChR subunit interface, and may facilitate the development of selective ligands with therapeutic potential.

The α9 subunit of nicotinic acetylcholine receptors (nAChRs) exists mainly in heteropentameric assemblies with α10. Accumulating data indicate the presence of three different binding sites in α9α10 nAChRs: the α9(+)/α9(−), the α9(+)/α10(−), and the α10(+)/α9(−). The major role of the principal (+) side of the extracellular domain (ECD) of α9 subunit in binding of the antagonists methyllylcaconitine and α-bungarotoxin was shown previously by the crystal structures of the monomeric α9-ECD with these molecules. Here we present the 2.26-Å resolution crystal structure of α9-ECD in complex with α-conotoxin (α-Ctx) RgIA, a potential drug for chronic pain, the first structure reported for a complex between an nAChR domain and an α-Ctx. Superposition of this structure with those of other α-Ctxs bound to the homologous pentameric acetylcholine binding proteins revealed significant similarities in the orientation of bound conotoxins, despite the monomeric state of the α9-ECD. In addition, ligand-binding studies calculated a binding affinity of RgIA to the α9-ECD at the low micromolar range. Given the high identity between α9 and α10 ECDs, particularly at their (+) sides, the presented structure was used as template for molecular dynamics simulations of the ECDs of the human α9α10 nAChR in pentameric assemblies. Our results support a favorable binding of RgIA at α9(+)/α9(−) or α10(+)/α9(−) rather than the α9(+)/α10(−) interface, in accordance with previous mutational and functional data.