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    • ERAP has been crystallized in

    2022-11-17

    ERAP1 has been crystallized in two different conformations, wherein the C-terminal domain IV is either closely interacting with domains I/II and enclosing the active site (termed ‘closed’), or is rotated away in a hinge motion which exposes the active site (‘open’). This large motion correlates with a reorganization of the active site, with Tyr438 oriented in a catalytically active orientation only in the closed state. In the open structure, Tyr438 is rotated such that the hydroxyl group is displaced 5.5 angstroms relative to the closed structure, and is no longer oriented to interact with a putative bound substrate (Kochan et al., 2011; Nguyen et al., 2011). This correlation between ERAP1 conformation and active site organization suggests that the enzyme might Ruxolitinib phosphate between these states in order to bind substrate, catalyze hydrolysis, and release product. This is notable within the entire M1 aminopeptidase family, of which several members have been crystallized in the closed conformation, including the homologous ERAP2 and IRAP (Mpakali et al., 2015a; Birtley et al., 2012; Mpakali et al., 2017).
    Conformational change and conformational dynamics Conversion between structural conformations is a widely studied feature of many enzymes. Such structural alterations can range from small loop reorganizations to large domain motions (Gutteridge and Thornton, 2004). One longstanding model of enzyme function assumes a structural change upon substrate binding referred to as a ‘induced fit’ (Koshland, 1958). If these conversions are required for catalysis and occur slowly relative to the enzyme catalytic rate, small perturbations in conformational dynamics can have notable effects on enzyme’s function (Wu and Xing, 2012). The identification of two ERAP1 conformations by crystallography raises the possibility that conformational interconversion may occur for ERAP1 and possibly for other members of the M1 aminopeptidase family. Using molecular dynamics simulations, an energy landscape connecting the open and closed structures has been identified with several local minima. Simulations of polymorphic variants of ERAP1 indicate that Q730E and K528R can each alter the population of conformations (Stamogiannos et al., 2015). The relative stability of the closed conformation requires electrostatic interactions between domains I/II and IV (Stamogiannos et al., 2017). The molecular dynamics model was supported by concordance with small angle X-ray scattering data of mutants designed to disrupt these interdomain interactions (Stamogiannos et al., 2017).
    Although the importance for ERAP1-mediated peptide generation has been well established in the literature, the physiological substrate for ERAP1 trimming is controversial. As early as 1990, Falk et al. proposed several models to explain MHCI-restricted generation of antigenic peptides from extended precursors (Falk et al., 1990), generally encompassed by two main ideas: that the peptide is generated by proteolysis in solution before binding to MHCI, or it is generated while bound onto MHCI, using the latter as a template. Thus, in one possible pathway ERAP1 trims precursor peptides in solution and relies on its unique enzymatic properties to optimize them for MHCI; mature peptides then can bind onto MHCI at which point they are protected from any further ERAP1 trimming (Fig. 1, left pathway). Alternatively, peptide precursors can bind onto the MHCI with N-terminal extensions such that ERAP1 can trim them while still bound onto the MHCI (Fig. 1, right pathway). Competition between ERAP1 trimming and optimal binding of the peptide onto MHCI is the main driving force behind MHCI cargo optimization in either pathway. Below, we describe each pathway in more detail and outline the main supporting experimental evidence.
    Interplay of trimming models with crystal structures and conformational dynamics To date, the known experimental structures of ERAP1 and MHCI are more consistent with pathway #1 than pathway #2. Crystal structures of ERAP1, even in the open conformation, cannot dock onto MHCI such that a peptide N-terminus could reach into the ERAP1 active site in a canonical configuration for catalysis (Nguyen et al., 2011). The same appears to apply for ERAP2 and IRAP (Mpakali et al., 2015a,b). If ERAP1 has the ability to open further, however, this may allow docking onto MHC to form a ternary structure (Papakyriakou and Stratikos, 2017). Indeed, molecular dynamics simulations have suggested a continuous but rugged energy terrain between different conformations of ERAP1, suggesting that other conformations are possible, albeit of high energy (Stamogiannos et al., 2017). Even if such a “wide-open” structure could form however, then for efficient processing, Tyr438 would have to properly orient for catalysis when ERAP1 is in the open state. Interestingly, in a recent “semi-closed” crystal structure of the homologous IRAP, the catalytic Tyr was found to properly orient towards a transition-state analogue bound in the catalytic site (Mpakali et al., 2015b; Hermans et al., 2015).