The crystal structure of ERAP1 bound to the aminopeptidase inhibitor bestatin reveals an open, four-domain arrangement with a large solvent-accessible chamber contiguous to a zinc-based aminopeptidase active site. The large cavity appears to be an adaptation specific to ERAP1 and related to its ability to efficiently process peptides that are longer than the substrates of typical aminopeptidases. The ability of short peptides to allosterically activate ERAP1 towards the fluorogenic aminopeptidase substrate, L-AMC, suggests conversion between (at least) two different forms of the enzyme dependent on peptide binding, with one more and one less active. Kinetic analysis suggests that conversion between the forms is regulated by occupancy of a regulatory subsite that is distinct from but near the enzyme active site. Comparison with a recent crystal structure of ERAP1 in a closed form suggests that the structure of ERAP1 bound to bestatin represents an open state not optimized for catalysis, but which could adopt a more closed arrangement by simple rigid-body domain motions. The concave surface of the C-terminal helical domain ensures that after closure there would still be a cavity sufficient to accommodate a peptide substrate of up to 13–16 residues.
A model for how subsite activation within the ERAP1 binding cleft can lead to length-dependent cleavage of peptide substrates is shown in . In this model, binding to a regulatory site promotes a conformational change to a closed conformation with increased enzymatic activity. Short peptide substrates cannot reach from the catalytic site to the regulatory site (), and so are not able to stabilize the closed conformation. As a result, they will be processed at a slower rate. Peptides longer than ~8–9 residues can concurrently occupy the catalytic and regulatory sites (), leading to the stabilization of the closed, active conformation and efficient trimming. Short fluorogenic substrates cannot reach from the catalytic site to the regulatory site (), and are processed inefficiently, unless catalysis is accelerated by occupancy of the regulatory site (). The regulatory site can be occupied by the fluorogenic substrate itself if present at high concentration, leading to positive cooperativity and sigmoidal activation kinetics, or by binding of an activator peptide, provided that the peptide does not interfere sterically with substrate binding. For a full-length substrate that occupies both catalytic and regulatory sites, no cooperativity and hyperbolic (Michaelis-Menten) kinetics are observed. Similarly a non-hydrolyzable peptide activator of L-AMC kinetics acts as an inhibitor for hydrolysis of a full-length peptide, since in this case occupancy of the regulatory site prevents substrate binding. This model explains the efficiency of ERAP1 in trimming long substrates since such substrates can activate their own trimming by binding to both the regulatory and catalytic sites. Furthermore, this model can explain the slow trimming rate for small substrates that are too short to self-activate the enzyme. Occupancy of both catalytic and regulatory sites within the overall extended peptide binding site is thus an important determinant for efficient trimming. Similar subsite activation models have been proposed to explain the length preference of bovine RNAse A43
, and the sigmoidal velocity plots observed for the monomeric B. subtilis
PI-PLC when assayed with short-chain lipid analog substrates44
. The location of the regulatory site(s) within the ERAP1 structure is not yet clear in the absence of structural data for a bound peptide complex, but one possible site might be the inner surface of top of helices H20 and H22, which in the closed conformation would contact an extended version of the tripeptide model shown in , influencing interactions between helix H22 and helix H5 near the active site (see below). Overall, this model is consistent with the biological role of ERAP1 in efficiently removing 1–7 residues from the N-terminus of antigenic peptide precursors, with broad specificity but sparing many mature antigenic peptides.
Figure 7 Model for ERAP1 length dependent cleavage activity. (a) A short peptide (5-mer shown) cannot reach from the catalytic site to the regulatory site. ERAP1 remains in the lower-activity open conformation and the peptide is processed inefficiently. (b) A (more ...)
How could conversion between open and closed conformations regulate ERAP1's enzymatic activity? The arrangement of key functional groups in the active site of ERAP1 is identical to that observed for other M1 family members, except for Tyr438, which is oriented away from the catalytic zinc in ERAP1 but towards it in most other M1 aminopeptidase structures (). The one other exception is TIFF3, which like ERAP1 is in an open conformation45
, and which also has the Tyr438 analog also oriented away from the active site32
. In the closed form of ERAP1, a small ~30–40° rotation of helix H5 orients Tyr438 towards the active site, in a position to participate in peptide bond hydrolysis chemistry. Helix H5 is oriented towards the C-terminal domain, sandwiched between helix H3, which carries the second Glu of the HExxHx18
E motif, and helix H4, which packs against helix H10 of the C-terminal domain. In the open conformation, no direct contacts between helix H5 and the C-terminal domain are observed, although a disordered region for which we do not observe electron density extends from the end of helix 5 towards the C-terminal domain. Two other disordered regions are found directly across the cavity, between the tops of helices H21 and H22, and between the tops of helices H23 and H24 (). In the closed conformation, these regions make contact with the disordered region upstream of helix H5 folding and packing against the loops between helices H21 and H22 and between H23 and H24. This interaction appears to result in reorientation of helix H5, motion of Tyr438, and capping of the S1 site, all changes which would favor increased catalytic activity. Structural studies of other enzymes related to ERAP1 suggest that domain motions triggered by peptide binding could induce active site changes to enhance peptidase activity as suggested here. In two multidomain zinc metallopeptidases distantly related to ERAP1, human angiotensin-converting enzyme-related carboxypeptidase (ACE2)46
and E. coli
dipeptidyl carboxypeptidase (Dcp)47
, domain closure motions have been associated with active site reorganizations favoring catalysis. In two single-domain zinc metallopeptidases more closely related to ERAP1, thermolysin48
, subdomain motions similarly reorient active site residues for hydrolysis.
The open conformation of ERAP1 may be important for catalysis because it can facilitate long substrate approach and capture. Low-affinity, low-specificity binding sites on the interior surface of the cavity, similar to those present on nuclear importin-α, another armadillo/HEAT repeat protein50
, might enable efficient peptide capture in the open form. Cavity closure could help to align peptides towards the active site for amino-terminal processing, and differential exposure of interior binding sites between open and closed forms could facilitate peptide binding and release cycles, which would appear to be necessary for sequential removal of peptide N-termini as a long antigenic precursor is processed. This type of mechanism has been suggested for insulin degrading enzyme, a dimeric chambered zinc endopeptidase with “exosites” for binding entrapped peptide at positions distant from the hydrolytic site51,52
. Recently, ERAP1's paralog, IRAP, has been shown to process relatively long peptides53
. If this model is correct, then IRAP also would be expected to adopt open and closed conformations during catalysis.
Based on the ERAP1 crystal structures, length and sequence specificity, allosteric regulation by peptides, and comparison with other M1-family proteases, we can describe a likely mechanism for ERAP1 processing of antigenic precursors. (1) Antigenic precursor peptides can access and bind to the groove while the protein is in an open conformation, using interactions at N-terminal S1 and S1′ sites but also interactions at other sites in the cavity. Active site residues are not in the optimal arrangement. (2) Peptide binding with occupancy of the regulatory site causes domain closure around the bound peptide and conversion to the optimal active site constellation. Interactions responsible for triggering this change are not yet clear, but are likely to involve portions of the substrate ~7–8 residues away from the N-terminus, and (directly or indirectly) ERAP1 residues located near domain boundaries. (3) Catalysis. Based on studies of similar metalloproteases, catalytic steps would include: attack of the scissile amide bond by a zinc-bound water to form a tetrahedral intermediate stabilized by interactions with active site residues, followed by collapse of the tetrahedral intermediate with proton transfer to generate a free amino acid and new peptide N-terminus. (4) Release of products after hydrolysis and substrate rebinding for next cycle. Most likely liberation of the cleaved N-terminal amino acid requires at least partial conversion to the open form after hydrolysis. Interaction of the cleaved peptide product with low-specificity binding sites could facilitate rebinding in a shifted frame in preparation for the next catalytic cycle. (5) When peptide substrates are trimmed beyond ~8–9 residues, they are too short to engage both regulatory and active sites, and cannot be processed efficiently.
Ankylosing spondylitis (AS) is a chronic inflammatory joint disease with a strong genetic component that is strongly linked to the MHC allelic variant HLA-B27. Genome-wide studies in many human populations have shown strong associations outside the MHC region (reviewed in 12
), that encompasses the ERAP1 gene54
. Identified disease-associated ERAP1 polymophisms include residues located near putative substrate binding and regulatory sites. shows sites of eight single nucleotide polymorphisms that are present in allelic variants of ERAP1 associated with altered risk of developing AS54,55
. Potential effects of some of these variants can be identified by examination of the structure of ERAP1. M349V is located in the active site, and R725Q and Q730E are exposed on the inner surface of the C-terminal cavity that could affect substrate sequence or length specificity. Other polymorphisms, R127P, K528R, D575N and V647I, at domain junctions could indirectly affect specificity or enzymatic activity by altering the conformational change between open and closed forms. Altered peptide trimming by ERAP1 variants may lead to altered levels of arthritogenic self peptides or of their mimics, or may lead to altered generation of T cell clones in the thymus. Structure-guided studies of the specificity and activity of these ERAP1 variants are clearly warranted.
Ankylosing spondylitis-associated mutations mapped on surface of ERAP1 Surface of ERAP1 in gray, with polymorphisms associated with ankylosing spondylitis shown in red. Underlined residues are hidden in this view.
The crystal structure of ERAP1 bound to bestatin provides insight into ERAP1's unusual length specificity and its peptide sequence preferences. These intrinsic properties of ERAP1 with MHC-binding protection contribute to the length and sequence distribution of MHC class I bound antigens. Overall, it appears that both the overall conformation of ERAP1 as well as its ability to be activated by long substrates are features that the enzyme has evolved in order to trim large antigenic precursor peptides, while at the same time sparing peptides of length suitable for MHC binding.