The X-ray Crystal Structure of Human Aminopeptidase N Reveals a Novel Dimer and the Basis for Peptide Processing^*

Protein Expression and Purification

A soluble form of hAPN (residue 66–967) was expressed and purified from a stably transfected HEK 293S GnT1⁻ cell line (40) essentially as previously described (41). Cells were grown in DMEM/F-12 supplemented with 3% FBS (Invitrogen), 1× penicillin-streptomycin (Invitrogen), 1 mg/liter of doxycycline (Sigma), and 1 mg/liter of aprotinin (Bioshop Canada). The harvested medium was concentrated 10-fold and the fusion protein was purified by IgG-Sepharose affinity chromatography. The protein A tag was removed by on-column tobacco etch virus protease digestion and the liberated hAPN was further purified by Q-Sepharose ion exchange chromatography. The resultant hAPN was treated with endo-β-N-acetylglucosaminidase A (42) in 10 mm HEPES, 50 mm NaCl, pH 7.5, at 37 °C, followed by jack bean α-mannosidase (Prozyme) in 50 mm MES, 0.4 mm ZnSO₄, pH 5.5. The deglycosylated hAPN was then purified by Q-Sepharose ion exchange chromatography and Superdex 200 gel filtration chromatography in 10 mm HEPES, 50 mm NaCl, pH 7.4, and concentrated to 20 mg/ml. The selenomethionine-labeled protein was expressed by supplementing methionine-free media with 30 mg/liter of seleomethionine as previously described (41).

Protein Crystallization

The deglycosylated native and seleomethionine-labeled hAPN was crystallized by the hanging drop method. Protein stock solutions at 20 mg/ml in 10 mm HEPES, pH 7.5, and 50 mm NaCl were mixed 1:1 with well solution containing 2 m (NH₄)₂SO₄, 10% glycerol, and 100 mm sodium acetate, pH 5.0. Crystals were cryoprotected with well solution containing 25% glycerol. For crystallization of the AngIV complex, hAPN was preincubated for 3 days with 2.5 mm EDTA, and AngIII (Anaspec) was then added at 300 μm. Crystals were grown for approximately 1 week before cryoprotection and data collection. Complexes of bestatin (300 μm) (Bioshop Canada) and amastatin (300 μm) (Bioshop Canada) with the zinc-bound native enzyme were obtained by co-crystallization.

Data Collection, Structure Determination, and Refinement

Data were collected at the Canadian Light Source, Saskatoon (Beamline CMCF-08ID-1). A single-wavelength anomalous dispersion experiment was performed at the peak (0.9795 Å) of the selenium absorption edge. Diffraction images were processed and scaled using HKL2000 (43); 5% of each dataset was flagged for the calculation of R_free. A summary of statistics is provided in Table 1. The SHELX (44) program suite was used to determine the selenium atom positions and to determine phases. Automated model building using ARP/wARP resulted in a model that was 95% complete. Alternate rounds of manual rebuilding using COOT (45) and automated refinement using REFMAC (46) and Phenix (47) were performed. Geometric parameters for bestatin and amastatin were obtained from the Ligand Expo database. Ramachandran analysis of all four structures (native, AngIV, bestatin, and amastatin complex) showed that 92% of the residues are in the most favored region, with 8% in the additionally allowed region. All of the residues in the substrate/inhibitor structured loop also fall in the most favored and additionally allowed regions of Ramachandran space. Figures were generated using the program PyMOL. Interface calculations were done using the PISA server.

Surface Plasmon Resonance Analysis, Analytical Ultracentrifugation, and Enzyme Kinetics

The ectodomain was used without deglycosylation for the C18 HPLC-based kinetics assay, the sedimentation equilibrium analysis, the surface plasmon resonance peptide binding assay, and the colorimetric enzyme assay. hAPN enzymatic activity was assayed using l-leucine-ρ-nitroanalide (Sigma) in 10 mm MES, pH 6.5. Initial velocities were obtained at 298 K over a range of substrate concentrations at an enzyme concentration of 10 nm. The generation of ρ-nitroanalide was monitored at 405 nm. Kinetic analysis of the removal of the first amino acid from AngIII, AngIV, and the peptides, VVYIHPF and RYIHPF, was performed by measuring the loss of the substrate using a C18 reverse phase HPLC assay. Various concentrations of peptides were mixed with hAPN (0.5 nm) in 10 mm MES, pH 6.5, at 298 K, and the digest was stopped with 5% phosphoric acid at various time points to obtain initial velocities. Each stopped reaction was loaded onto a C18 column (Vydac 218TP) and eluted isocratically with 85 mm phosphoric acid, adjusted to pH 3 with triethanolamine, containing 15 (VVYIHPF) or 17% (the others) acetonitrile. Peptides were quantitated at 195 nm based on a standard curve generated with known peptide concentrations. AngIII, AngIV, AngI/II(4–8), VVYIHPF, and RYIHPF are all baseline separated. Sedimentation equilibrium analysis of hAPN at concentrations of 0.1, 0.25, and 0.5 mg/ml were performed at speeds of 7000 and 9000 rpm at 277 K. Surface plasmon resonance was performed on CM-5 chips coupled with hAPN that had been preincubated with 2.5 mm EDTA (1 day) and analysis was performed in buffer containing 2.5 mm EDTA. Binding plateau values as a function of AngIII and AngIV concentration were used to compute the dissociation constants assuming a 1:1 binding model.

Overall Structure of the hAPN Dimer

hAPN is a 967-residue type-2 membrane glycoprotein as shown in Fig. 1a. The ectodomain was expressed and shown to possess a K_m of 0.3 ± 0.05 mm for the hydrolysis of leucine-ρ-nitroanilide, a value similar to that obtained for the rabbit and porcine enzymes purified from tissue (48). The intact membrane protein exists as dimers and monomers on the cell surface in rabbit (13) and using analytical ultracentrifugation we have determined that the ectodomain expressed alone dimerizes with a K_D of 0.8 μm. The ectodomain is also dimeric in the crystal (data collection and refinement statistics in Table 1). As shown in Fig. 1b, each monomer possesses the four-domain structure (domains I-IV) characteristic of the four-domain M1 metallopeptidases whose structures have been determined to date (30–36, 49). Domain II possesses the thermolysin-fold and contains both the zinc binding site and the catalytic site, as well as the characteristic consensus motifs, ³⁸⁸HEXXHX₁₈E⁴¹¹ and ³⁵²GXMEN³⁵⁶. The dimer interface is mediated by hydrophobic interactions and a hydrogen bond and salt-bridge network, and it buries ∼840 Ų of surface area on each monomer (Fig. 2). In each monomer, the catalytic site is exposed to a large internal cavity (∼2800 ų), which is inaccessible to the bulk solvent. The native, peptide bound, and inhibitor bound structures are very similar (root mean square deviation over all protein atoms of 0.13–0.17 Å) except for a flexible loop in domain IV that is structured by substrate and inhibitor binding as discussed below. Fig. 1b shows a model for the orientation of hAPN on the cell surface. The dimer possesses dimensions of 131 Å × 62 Å in projection, values very close to those measured by negative stain electron microscopy (135 Å × 55 Å) for intact porcine APN in reconstituted lipid vesicles (50).

Peptide Binding and the Catalytic Site

To shed light on the structural basis for substrate binding and catalysis we determined the x-ray crystal structure of hAPN in its native form and in complex with peptide substrate. The latter was obtained by the co-crystallization of zinc-depleted hAPN with 300 μm AngIII (RVYIHPF), although the clearly defined electron density of the first three residues shows that only AngIV (VYIHPF) is bound in the catalytic site (Fig. 3a). Zinc-depleted hAPN binds both AngIII (5 ± 0.4 μm) and AngIV (15 ± 4 μm) with similar affinity (Table 2 and supplemental Fig. S1). Fig. 3b shows an overlay of the zinc-bound native enzyme with that of the zinc-free AngIV complex in the vicinity of the zinc binding site. In the native enzyme the zinc ion is coordinated by His³⁸⁸, His³⁹², and Glu⁴¹¹ (of the ³⁸⁸HEXXHX₁₈E⁴¹¹ motif) and both oxygen atoms of an acetate molecule from the crystallization buffer (supplemental Table S1). In the AngIV complex the N-terminal Val residue is deeply buried, an observation consistent with the fact that these M1 enzymes are exopeptidases. The carbonyl oxygen atom of the valine occupies the same position as that of the OD1 acetate oxygen atom in the native structure (there is no bound acetate in the peptide complex) and at the same time it accepts a hydrogen bond from Tyr⁴⁷⁷, a residue proposed to stabilize the oxyanion generated in the transition state (51). In addition, the α-amino group of the Val residue is hydrogen bonded to Glu³⁵⁵ (of the ³⁵²GXMEN³⁵⁶ motif), Glu⁴¹¹ and Gln²¹³ (a cis peptide conserved among members of the family), residues all implicated in substrate binding and/or transition state stabilization (51). Taken together, it is clear that the bound peptide is poised for catalysis and represents a substrate complex. Further support for this suggestion stems from the fact that the scissile bond (between the Val (P1) and Tyr (P1′)) straddles the side chain of Glu³⁸⁹, a residue proposed to shuttle a proton from the hydrolytic water molecule to the amide nitrogen of the scissile bond (51). Indeed, the side chain of Glu³⁸⁹ forms a hydrogen bond to the more weakly coordinated OD2 acetate oxygen atom (supplemental Table S1), an atom whose position approximates that of the hydrolytic water molecule prior to nucleophilic attack. Because the zinc ion is thought to both activate the hydrolytic water molecule and coordinate the oxyanion in the transition state (supplemental Fig. S2), we propose that our native acetate-bound zinc structure, in conjunction with the zinc-free peptide complex, serves as a good model for that of the Michaelis complex.

Peptide Binding, Loop Ordering, and Specificity

Both the electron density and temperature factors show that the first three amino acids (residue positions P1-P1′-P2′) of the bound AngIV peptide are the most well defined, whereas the last three (residue positions P3′-P5′) are increasingly disordered (Fig. 3a). The binding of AngIV buries ∼470 Ų of hAPN, and the binding site is mainly composed of residues from domain II (336Ų) and domain IV (134Ų) (Fig. 3c). In addition, comparison of the peptide complex with that of the native enzyme shows that peptide binding leads to the ordering of an eight-residue flexible loop (⁸⁹¹YGGGSFSF⁸⁹⁸) that is not observed in the electron density maps of the native structure. The loop further buries the bound substrate and although the interactions between the loop and the bound peptide are not that extensive, the electron density describing it is strong and its temperature factors (25–35 Ų) are comparable with that of non-loop residues in the vicinity (supplemental Fig. S3a).

The Val (P1) and Tyr (P1′) residues make extensive interactions with hAPN and both are completely buried (Fig. 3, b and c). In addition to the key interactions between the Val amino group and its carbonyl oxygen atom (as discussed above), its side chain sits in an apolar pocket formed by Gln²¹¹, Gln²¹³, Ala³⁵¹, Met³⁵⁴, and Phe⁴⁷² and the side chain of loop residue Phe⁸⁹⁶, which serves to cap the pocket (supplemental Fig. S4a). Notably, the observed loop conformation would not be able to accommodate the bulkier N-terminal Arg residue found at substrate position P1 in AngIII (supplemental Fig. S4b). With regard to the Tyr at position P1′, its amide nitrogen donates a hydrogen bond to the carbonyl oxygen of Ala³⁵³ and its carbonyl oxygen atom accepts a hydrogen bond from Gly³⁵², residues found in the ³⁵²GXMEN³⁵⁶ motif. The Tyr side chain also makes stacking interactions with the side chains of hAPN residues Val³⁸⁵ and His³⁸⁸, of the S1 pocket, and its hydroxyl group makes a water-mediated hydrogen bond to the carboxyl group of Glu⁴¹⁸. In contrast to that observed for the first two amino acids, the main chain atoms of the remaining peptide residues are not hydrogen bonded to hAPN and their interaction with hAPN lacks structural/chemical complementarity as reflected in a buried surface calculation that shows that on complex formation 715 Ų of surface area is buried on the peptide, whereas only 470 Ų is buried on the surface of hAPN.

In addition to the interactions that the structured loop makes with the bound peptide it also makes interactions with domain II. Loop residue Phe⁸⁹⁶ makes a stacking interaction with Phe⁴⁷², and loop residues Gly⁸⁹⁴ and Ser⁸⁹⁵ make hydrogen bonds with Ser⁴⁶⁹ and Asn³⁵⁰, respectively. As a result, a total surface area of 402 Ų is buried between the loop and domain II.

Inhibitor Binding and an Alternate Loop Conformation

To provide further insight into how hAPN might bind different peptide substrates we determined the structure of native zinc-bound hAPN in complex with amastatin (LeuβN[αOH]-Val-Val-Asp) and bestatin (PheβN[αOH]-Leu) (52), peptidomimetic inhibitors each possessing a nonhydrolyzable α-hydroxyl-β-amino acid at its N terminus (Fig. 4a). Comparison of the amastatin complex (Fig. 4, b and c) with that of the native enzyme and the AngIV complex shows that the LeuβN[αOH] moiety makes essentially the same interactions as that of the N-terminal Val residue (P1) of the AngIV substrate complex (supplemental Table S1). The α-hydroxyl group of this moiety also coordinates the zinc ion and as such provides an additional model for activation of the hydrolytic water molecule. The second residue (Val) of amastatin makes backbone interactions with hAPN similar to those of the second residue (Tyr, P1′) of AngIV in the AngIV complex, and the side chains of the second (Val) and third (Val) residues of amastatin occupy the same sites as those in the AngIV complex. The electron density for the fourth amino acid (Asp) of amastatin is weak. The interactions between hAPN and the first two residues of amastatin are very similar to those observed in the bestatin complexes of other M1 enzymes (30–34, 53). Amastatin binding also structures the flexible loop (average temperature factors are 25–30 Ų) around the side chain of the LeuβN[αOH] moiety in a fashion very similar to that seen for Val (P1) in the AngIV complex (supplemental Fig. S3b and S4c). In addition, the hydrogen bonding pattern between the loop and other hAPN residues is essentially the same as that observed in the AngIV complex.

In contrast to that observed for the amastatin complex, the interactions between bestatin and hAPN do not correspond to that of a substrate complex (Fig. 4, b and d). The PheβN[αOH] moiety of bestatin is pushed deeply into the S1 pocket that accommodates the Val (P1) side chain in the AngIV complex and its amino group, carbonyl oxygen atom, and α-hydroxyl group make only water-mediated hydrogen bonds to hAPN. The carboxyl group of the C-terminal Leu residue coordinates the zinc ion in a fashion similar to that of acetate in the native complex (supplemental Table S1), and a tetrahedrally coordinated water molecule, also observed in the native structure, occupies the pocket filled by the Val (P1) α-amino group in the AngIV complex. The side chain of the C-terminal Leu residue occupies the S1′ pocket that accommodates the Tyr (P1′) side chain of AngIV and the Val (P1′) side chain of amastatin, in their respective complexes. Despite the differences in binding geometry, both bestatin and amastatin would block substrate binding, an observation consistent with the fact that they are both competitive inhibitors.

The flexible loop in the bestatin complex is well ordered (average temperature factors are 30–40 Ų) (supplemental Fig. S3c) but now found to assume a conformation very different from that observed in the AngIV and amastatin complexes (Fig. 5). Loop residue Phe⁸⁹⁶, which formerly capped the S1 pocket has been repositioned to accommodate the Phe side chain of the PheβN[αOH] moiety (supplemental Fig. S4d) and the interactions involving loop residues Gly⁸⁹⁴ and Ser⁸⁹⁵, observed in the AngIV and amastatin complexes, are replaced by both direct and water-mediated interactions between loop residue Ser⁸⁹⁵ and non-loop residues Asp¹⁸⁹, Leu¹⁹⁰, Asp²¹⁶, and Gln²¹³ in domain II. The Phe side chain sits in a pocket formed by both loop and non-loop residues. Although the bestatin binding geometry does not mimic a true substrate complex, the loop conformation observed does provide a model for how substrates with bulky side chains at the P1 position might be accommodated (supplemental Fig. S4e).

Kinetic Analysis of Angiotensin III and IV and Related Peptides

To gain insight into the basis for the ability of hAPN to processes both AngIII and AngIV we measured the kinetic parameters for the removal of the first amino acid from AngIII, AngIV, and two related peptides where the first amino acids of AngIII and AngIV were swapped for that of the other (Table 2 and supplemental Fig. S5). The data show that although k_cat for the removal of Arg from AngIII is about 3-fold higher than that of Val from AngIV, a compensatory increase in K_m leads to similar catalytic efficiencies. Comparison of the control peptide pairs which differ only in the N-terminal amino acid shows that k_cat for the removal of Arg is 3–5-fold higher than that of Val and that the nature of the N-terminal amino acid does not significantly affect K_m.

Access to the Catalytic Site and a Model for the Open Conformation

As discussed above, the catalytic site of hAPN is exposed to a large internal cavity that is not connected to bulk solvent by an appreciable channel or opening (Fig. 6a). Moreover, structural alignment shows that both monomers of the hAPN dimer correspond to that of ERAP1 in its closed conformation (33, 34). To explore the possibility that hAPN might be able to access the ERAP1 open conformation, domains (I + II) and (III + IV) of hAPN were treated as rigid bodies and superimposed on domains I and IV of ERAP1 in the open conformation. Each monomer in the open conformation was then superimposed on the hAPN dimer through domain IV. The resultant models are free of steric clashes and with both monomers in the open conformation, the dimer takes on an S-shaped configuration, each lobe of which corresponds to a monomer whose internal cavity and catalytic site are exposed to bulk solvent (Fig. 6b). Although a large protein interface between domain (I + II) and IV is broken on conversion from the closed to the open form, the interface possesses a large percentage of polar residues. Conversion to the open form pulls the catalytic residues in domain II away from both the loop and non-loop residues in domain IV (see Fig. 3c), in this way pulling apart the residues that serve to sandwich the peptide in the binding site. In the open form of ERAP1 the equivalent of Tyr⁴⁷⁷ in hAPN is rotated away from its catalytically active conformation, a perturbation thought to render the open form catalytically inactive (33–35) and it is likely that the same would occur in hAPN.