Critical Role of Interdomain Interactions in the Conformational Change and Catalytic Mechanism of Endoplasmic Reticulum Aminopeptidase 1

Abstract

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that is important for the generation of antigenic epitopes and major histocompatibility class I-restricted adaptive immune responses. ERAP1 processes a vast variety of different peptides but still shows length and sequence selectivity, although the mechanism behind these properties is poorly understood. X-ray crystallographic analysis has revealed that ERAP1 can assume at least two distinct conformations in which C-terminal domain IV is either proximal or distal to active site domain II. To improve our understanding of the role of this conformational change in the catalytic mechanism of ERAP1, we used site-directed mutagenesis to perturb key salt bridges between domains II and IV. Enzymatic analysis revealed that these mutations, although located away from the catalytic site, greatly reduce the catalytic efficiency and change the allosteric kinetic behavior. The variants were more efficiently activated by small peptides and bound a competitive inhibitor with weaker affinity and faster dissociation kinetics. Molecular dynamics analysis suggested that the mutations affect the conformational distribution of ERAP1, reducing the population of closed states. Small-angle X-ray scattering indicated that both the wild type and the ERAP1 variants are predominantly in an open conformational state in solution. Overall, our findings suggest that electrostatic interactions between domains II and IV in ERAP1 are crucial for driving a conformational change that regulates the structural integrity of the catalytic site. The extent of domain opening in ERAP1 probably underlies its specialization for antigenic peptide precursors and should be taken into account in inhibitor development efforts.

Caspase-9 CARD : core domain interactions require a properly formed active site

“…Alternative modes of caspase-2 activation have since been proposed depending on the type of cellular death signals (review (50)); in these cases it is tempting to speculate that CARD:core interactions may play a role in regulating caspase-2 function, should they be present. Interdomain interactions have been demonstrated to be critical in controlling the different conformational states and in regulating the catalytic activity of several proteins including the deubiquitinating enzyme USP4 (51), phenylalanine hydroxylase (52) and ERAP-1 (endoplasmic reticulum aminopeptidase-1) (53). …”

A systematic re-examination of processing of MHCI-bound antigenic peptide precursors by endoplasmic reticulum aminopeptidase 1

“…Sodium ions were added to neutralize the total charge of the systems and ff14SB parameters were applied using the LEaP module of AMBER (67). The equilibration method of 10 ns and simulation parameters were as described in (68). After 1 ns equilibration of the systems at 300 K under isothermal-isobaric ensemble (NPT) conditions, production runs of 500 ns were performed in the canonical ensemble (NVT).…”

The Role of Conformational Dynamics in Antigen Trimming by Intracellular Aminopeptidases

“…Recent experimental analysis of the solution structure of ERAP1 using small-angle X-ray scattering (SAXS) revealed that, in solution, the average structure of ERAP1 corresponds well with the crystallographically observed open conformation ( 32 ). This analysis was performed with ligand-free enzyme and is, therefore, consistent with the proposal that “open” ERAP1 is the substrate-capture conformation and that substrate binding promotes the conformational shift to “closed” ERAP1 that facilitates catalysis ( 17 , 29 ).…”