Endoplasmic reticulum aminopeptidase 1 (ERAP1) plays a key role in controlling the immunopeptidomes available for presentation by MHC (major histocompatibility complex) molecules, thus influences immunodominance and cell-mediated immunity. It carries out this critical function by a unique molecular ruler mechanism that trims antigenic precursors in a peptide-length and sequence dependent manner. Acting as a molecular ruler, ERAP1 is capable of concurrently binding antigen peptide N- and C-termini by its N-terminal catalytic and C-terminal regulatory domains, respectively. As such ERAP1 can not only monitor substrate’s lengths, but also exhibit a degree of sequence specificity at substrates’ N- and C-termini. On the other hand, it also allows certain sequence and length flexibility in the middle part of peptide substrates that is critical for shaping MHC restricted immunopeptidomes. Here we report structural and biochemical studies to understand the molecular details on how ERAP1 can accommodate side chains of different anchoring residues at the substrate’s C-terminus. We also examine how ERAP1 can accommodate antigen peptide precursors with length flexibility. Based on two newly determined complex structures, we find that ERAP1 binds the C-termini of peptides similarly even with different substrate sequences and/or lengths, by utilizing the same hydrophobic specificity pocket to accommodate peptides with either a Phe or Leu as the C-terminal anchor residue. In addition, SPR (surface plasmon resonance) binding analyses in solution further confirm the biological significance of these peptide-ERAP1 interactions. Similar to the binding mode of MHC-I molecules, ERAP1 accommodates for antigenic peptide length difference by allowing the peptide middle part to kink or bulge at the middle of its substrate binding cleft. This explains how SNP coded variants located at the middle of ERAP1 substrate binding cleft would influence the antigen pool and an individual’s susceptibility to diseases.

内质网氨肽酶 1 (ERAP1) 在控制可用于 MHC（主要组织相容性复合物）分子呈递的免疫肽组中起关键作用，从而影响免疫优势和细胞介导的免疫。它通过一种独特的分子标尺机制执行这一关键功能，该机制以肽长度和序列依赖性方式修剪抗原前体。作为分子标尺，ERAP1 能够分别通过其 N 端催化域和 C 端调节域同时结合抗原肽 N 端和 C 端。因此，ERAP1 不仅可以监测底物的长度，还可以在底物的 N 端和 C 端表现出一定程度的序列特异性。另一方面，它还允许在肽底物的中间部分具有一定的序列和长度灵活性，这对于塑造 MHC 限制性免疫肽组至关重要。在这里，我们报告结构和生化研究，以了解 ERAP1 如何适应底物 C 端不同锚定残基侧链的分子细节。我们还研究了 ERAP1 如何适应具有长度灵活性的抗原肽前体。基于两个新确定的复杂结构，我们发现即使具有不同的底物序列和/或长度，ERAP1 也可以相似地结合肽的 C 末端，通过利用相同的疏水特异性口袋来容纳具有 Phe 或 Leu 作为 C-的肽末端锚定残基。此外，溶液中的 SPR（表面等离子体共振）结合分析进一步证实了这些肽-ERAP1 相互作用的生物学意义。与 MHC-I 分子的结合模式相似，ERAP1 通过允许肽中间部分在其底物结合裂隙的中间扭结或隆起来适应抗原肽长度差异。这解释了位于 ERAP1 底物结合裂隙中间的 SNP 编码变体如何影响抗原库和个体对疾病的易感性。