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Originally published In Press as doi:10.1074/jbc.M400795200 on March 3, 2004

J. Biol. Chem., Vol. 279, Issue 19, 20480-20489, May 7, 2004
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Crystal Structure of Human Thimet Oligopeptidase Provides Insight into Substrate Recognition, Regulation, and Localization*

Kallol Ray{ddagger}, Christina S. Hines§, Jerry Coll-Rodriguez, and David W. Rodgers

From the Department of Molecular and Cellular Biochemistry and Center for Structural Biology, University of Kentucky, Lexington, Kentucky 40536

Received for publication, January 23, 2004 , and in revised form, February 27, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Thimet oligopeptidase (TOP) is a zinc metallopeptidase that metabolizes a number of bioactive peptides and degrades peptides released by the proteasome, limiting antigenic presentation by MHC class I molecules. We present the crystal structure of human TOP at 2.0-Å resolution. The active site is located at the base of a deep channel that runs the length of the elongated molecule, an overall fold first seen in the closely related metallopeptidase neurolysin. Comparison of the two related structures indicates hinge-like flexibility and identifies elements near one end of the channel that adopt different conformations. Relatively few of the sequence differences between TOP and neurolysin map to the proposed substrate-binding site, and four of these variable residues may account for differences in substrate specificity. In addition, a loop segment (residues 599-611) in TOP differs in conformation and degree of order from the corresponding neurolysin loop, suggesting it may also play a role in activity differences. Cysteines thought to mediate covalent oligomerization of rat TOP, which can inactivate the enzyme, are found to be surface-accessible in the human enzyme, and additional cysteines (residues 321,350, and 644) may also mediate multimerization in the human homolog. Disorder in the N terminus of TOP indicates it may be involved in subcellular localization, but a potential nuclear import element is found to be part of a helix and, therefore, unlikely to be involved in transport. A large acidic patch on the surface could potentially mediate a protein-protein interaction, possibly through formation of a covalent linkage.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Thimet oligopeptidase (TOP,1 3.4.24.15 [EC] ) is a 77-kDa zinc metalloendopeptidase that bears the His-Glu-Xaa-Xaa-His (HEXXH) active site sequence motif characteristic of a large superfamily of metallopeptidases (1-4). It is widely distributed in mammalian tissues with the highest expression levels in the brain, pituitary gland, and testis (5-8). TOP is present in different subcellular locations depending on cell type, with reports of secreted and cytosolic forms (5-16), membrane association (5-7, 17, 18), and nuclear localization (7, 12, 14). Consistent with its broad tissue and subcellular compartment distribution, TOP appears to play a variety of physiological roles. It has been implicated in the metabolism of a number of small peptides active in the central nervous system and the periphery including neurotensin, bradykinin, somatostatin, opioids, and angiotensin I (4, 6, 9, 16, 19-26). In addition, recent reports demonstrate that TOP is primarily responsible for degrading peptides released from proteasomes, thereby limiting the extent of antigen presentation by MHC class I molecules (27-29). TOP has also been linked to amyloid precursor protein processing (30), and it promotes increased degradation of the A{beta} peptide, a key component of amyloid plaques in Alzheimer's disease (31). Expression of TOP activity is regulated at the level of transcription (32-34), but activity may also be regulated by posttranslational modification. TOP oligomerizes by intermolecular disulfide bond formation in the absence of reductant, resulting in a decrease or loss of activity, and this modulation by multimerization may be a physiological regulatory mechanism (35-37). The activity of rat TOP can also be modulated by phosphorylation, and phosphorylated forms of the enzymes have been isolated from cultured cells (38, 39).

TOP belongs to the M3 family of metallopeptidases (2), which has eight other known members that all share some sequence similarity with TOP. The member of this family most closely related to TOP, the peptidase neurolysin, has more than 60% sequence identity and a similar tissue distribution (1, 40, 41). Both TOP and neurolysin hydrolyze only short peptides, generally less than 20 residues in length, and they recognize cleavage sequences that vary widely, with no consistent preferences at any position relative to the site of hydrolysis (1, 19, 20, 40, 42). TOP and neurolysin cleave most bioactive or synthetic peptides at the same peptide bond, as might be expected from their high level of sequence similarity. Interestingly though, the two peptidases hydrolyze some peptides at different sites (1, 19, 20, 40), and systematic studies of sequence preferences show considerable differences at positions close to the cleavage site (43-48). For example, although both enzymes play an important role in metabolizing the 13-residue neuropeptide neurotensin, TOP cleaves between Arg-8 and Arg-9, whereas neurolysin cleaves between Pro-10 and Tyr-11. Comparing TOP and neurolysin will, therefore, provide insight into the mechanisms for the complex substrate specificity in these enzymes, which is frequently found in other enzymes that metabolize bioactive peptides, the neuropeptidases.

The neurolysin crystal structure (49) showed that the enzyme active site is at the bottom of a deep, narrow channel, explaining why the M3 family members are restricted to unstructured peptide substrates. Subsequent modeling indicated that relatively few of the residue changes between TOP and neurolysin mapped to the walls and floor of the channel, allowing identification of a small number of positions that likely mediate differences in specificity (50). Here, we present a 2.0-Å crystal structure of human thimet oligopeptidase. This structure validates the specificity model and provides additional insights into the basis for differences between TOP and neurolysin. The structure also allows us to evaluate biochemical data concerning regulatory modification and subcellular localization of TOP, providing a framework for further study of this important enzyme.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Preparation of Overexpression Constructs—The cDNA for human thimet oligopeptidase (TOP) was obtained from an expressed sequence tag clone (clone ID 2507774, Research Genetics) and subcloned into the pET32 (Novagen) expression vector. A construct for expressing TOP missing the first 15 residues, disordered in the related peptidase neurolysin, was produced by PCR amplification of the full-length clone using site-specific primers. The PCR product was subcloned into pET32a vector (TOP-N15). The construct was further modified (vector TOP-N15-C2S) by PCR-based mutagenesis (QuikChange, Stratagene) to produce a version of TOP with cysteines 246 and 253 replaced by serines to prevent covalent aggregation of the protein.

Expression and Purification of Native Human Thimet Oligopeptidase Constructs—All TOP variants were expressed in Escherichia coli BL21(DE3)RP cells (Stratagene) to overcome poor protein production caused by differences in codon usage. Cells were grown in Terrific Broth media at 37 °C to an A600 of 0.6, and transcription was induced by adding 1 mM isopropyl-1-thio-{beta}-D-galactopyranoside to the culture after lowering the growth temperature to 16 °C to maintain protein solubility. After 6 h, cells were centrifuged and lysed, and TOP was purified by metal affinity chromatography using nickel nitrilotriacetic acid beads (Qiagen). The protein was eluted from the beads by incubation with enterokinase (Invitrogen) to cleave it from the polyhistidine-containing N-terminal fusion sequence. Purified TOP constructs were dialyzed against 50 mM Tris, pH 8.0, containing 1 mM {beta}-mercaptoethanol and 1% glycerol and then concentrated for crystallization trials. The activities of the purified constructs were determined by monitoring cleavage of a fluorogenic peptide substrate with the sequence of the 13-residue peptide neurotensin.

Crystallization of Thimet Oligopeptidase—Modified human thimet oligopeptidase was crystallized by hanging drop vapor diffusion (51) at 4 °C. The best crystals were grown in 13% polyethylene glycol 6000, 100 mM sodium cacodylate, pH 6.5, and 25 mM magnesium acetate beginning with protein at a concentration of 10 mg/ml. Crystals typically grew to 0.25 x 0.1 x 0.03 mm. They were prepared for data collection by transfer to a solution containing 25% glycerol, 20% polyethylene glycol 6000, 100 mM sodium cacodylate, pH 6.5, and 25 mM magnesium acetate for ~10 s, mounting in a nylon loop, and plunging into liquid nitrogen (52).

Data Collection and Structure Determination—X-ray data were collected at the Advanced Photon Source beamline 22-ID (Southeast Regional Collaborative Access Team), Argonne National Laboratory. Data were reduced with HKL2000 (53). The space group of the crystals is P212121 with unit cell dimensions of a = 77.17 Å, b = 99.1 Å, c = 105.5 Å. Initial phases were determined by molecular replacement with the CNS software package (54) using neurolysin (Protein Data Bank code 1I1I [PDB] ) as a search object. Model building and analysis were done using the program O (55), and structure refinements was carried out in CNS.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Overview of the Structure and Comparison with Neurolysin—We have determined the crystal structure of recombinant human TOP at 2.0-Å resolution (Table I). To obtain crystals, we used a modified version of thimet oligopeptidase, which begins at residue 16 and has residues 246 and 253 mutated from cysteine to serine. The N terminus was removed because the corresponding residues of the closely related neurolysin were disordered in the crystal structure (49), and the cysteines were mutated to prevent covalent oligomerization of the enzyme (35). Full-length native thimet oligopeptidase (both human and rat orthologs) did not crystallize despite extensive screening. As expected, the modified recombinant thimet oligopeptidase had activity similar to native full-length enzyme (data not shown).


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  		TABLE I
Summary of crystallographic data and refinement

 
TOP is composed of 24 {alpha} helices and 9 short {beta} strands (Fig. 1A). A deep narrow channel, open at one end but closed off by helices 3-6 at the other, runs the length of the enzyme, dividing it into two distinct domains (I and II). The active site is located at the bottom of the channel about midway along its length, and the zinc ion cofactor is present at high occupancy (refined at full occupancy with a final thermal factor of 16.98 Å2) in the crystal structure. This overall fold was first seen in neurolysin (49) and is likely conserved within the entire M3 metallopeptidase family (2), which has a total of nine members including TOP and neurolysin. Surprisingly, however, this fold has also been found recently in angiotensin-converting enzyme (56, 57) and Pyrococcus furiosus carboxypeptidase (58), metallopeptidases that were not thought to be closely related to the M3 family based on sequence comparisons. Residues 16-24 of the TOP construct are disordered in the crystals, with no identifiable electron density, suggesting that even more of the N terminus than in neurolysin is disordered or that removing the first 16 residues caused adjacent residues to lose conformational stability.



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  		FIG. 1.
Overview of the thimet oligopeptidase structure and active site density. A, stereo ribbon diagram of thimet oligopeptidase. Individual {alpha} helices and {beta} strands are numbered, and the region structurally similar to other zinc metallopeptidases is in gold. The active site zinc is shown as a blue sphere. All molecular figures were created using RIBBONS (71) unless otherwise indicated. B, active site of thimet oligopeptidase. Density (2 Fo - Fc) is shown in wire-frame representation at a contour level of 0.8 r.m.s.d. of the map. Key residues of the HEXXH sequence motif are marked along with the third zinc ligand from the protein, Glu-502. The active site zinc ion and catalytic water molecule are shown as blue and red spheres, respectively. The image was produced with Swiss-PDBViewer (72).

 
The active site of TOP contains an HEXXH sequence motif characteristic of a large class of zinc metallopeptidases (2). As in other metallopeptidases of this type, the two histidines (residues 473 and 477) in the TOP sequence coordinate the zinc ion cofactor (Fig. 1B). The side chain of a downstream glutamate (Glu-502) from helix 16 serves as the third zinc ligand contributed by the protein, a characteristic of enzymes grouped into metallopeptidase clan MA (2). The glutamate residue of the HEXXH motif is believed to play an important role in the reaction mechanism, polarizing and orienting a zinc-coordinating water molecule that is the attacking nucleophile (59). In TOP, the water molecule coordinating the zinc is well defined by the density, and the glutamate (Glu-474) makes the expected hydrogen bonds with this water (Fig. 1B). The active site is contained within a region of the enzyme that has strong structural similarity to the active site regions of other clan MA metallopeptidases (see Fig. 1A), including the prototypical group member thermolysin (60).

Neurolysin is the closest homolog of thimet oligopeptidase in the M3 metallopeptidase family (Fig. 2), and as noted, TOP and neurolysin (49) adopt the same overall fold. Superposition of the two enzymes on C{alpha} atoms (Fig. 3) yields an overall root mean square deviation (r.m.s.d.) of only 1.19 Å, indicating a high degree of structural conservation. The backbones superimpose particularly well in the closed end and the floor of the channel. At the open end and near the top of the channel, however, the elements in TOP are displaced outward relative to the corresponding elements in neurolysin (Fig. 3), widening the channel. To some extent, this difference results from a hinge-like motion of the two domains about an axis located in the floor of the channel and running parallel to the long axis of the molecule. We have observed this hinge-like motion when comparing different crystal packings of neurolysin,2 and it is likely that here it also reflects the effect of packing environment on the relatively flexible channel walls in the two molecules. Elements near the open end of the channel (from the C terminus of {alpha}6 through {beta} strands 1 and 2 to the N terminus of {alpha}8 in domain I and the extended loop between {beta}10 and {alpha}15 in domain II), however, show differences beyond the hinge-like motion, and the outward movement of these elements in TOP reflects a true conformational difference from neurolysin. These changes would be unlikely to affect substrate specificity or catalytic activity given their location near the top of the channel.



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  		FIG. 2.
Sequence alignment of human TOP and neurolysin. Sequence differences are highlighted in green. Structural elements of TOP are shown schematically above the TOP sequence. Accessible cysteines in TOP are indicated with red triangles, and a key residue difference that affects an active site loop is indicated by a red star.

 



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  		FIG. 3.
Backbone superposition of TOP and neurolysin. Stereo view of superimposed thimet oligopeptidase (blue/red) and neurolysin (gold) C{alpha} traces. Elements near the open end of the channel that show the greatest conformational differences are colored red in the TOP backbone representation.

 

Model for Substrate Binding—As in neurolysin (49), the shielding elements erected over the active site of TOP clearly account for its inability to hydrolyze large, folded substrates. The small oligopeptides hydrolyzed by TOP are known to be largely unstructured in solution (61, 62), and the long narrow channel leading to its active site suggests that these substrates bind in an extended conformation consistent with the known binding of peptide analogs to other metallopeptidases (63, 64).

We modeled the 13 residue peptide neurotensin into the TOP channel in a manner similar to our previous work on neurolysin (49, 50). Because TOP cleaves neurotensin between Arg-8 and Arg-9, we positioned the carbonyl carbon of Arg-8 to coordinate with the active site zinc, and the N terminus of the peptide was oriented toward the closed end of the channel based on the binding orientation seen in other metallopeptidases with structurally similar active sites (63, 64). The peptide was adjusted to lie along strand {beta}7, making antiparallel sheet-like interactions with the main chain, and individual residues were placed to make plausible interactions with the protein surface. After manual fitting, the modeled complex was subjected to energy minimization using a CHARMM force field (65).

We used the modeled peptide simply as a ruler to analyze the features of the binding site and to gauge the possible modes of peptide binding. The fact that the peptide in extended conformation fits entirely within the binding site (Fig. 4) suggests that the relatively basic peptide substrates could extend along the floor of the channel, maximizing contact with its negatively charged surface. The N terminus extends near but not to the closed end of the channel, and additional residues could be accommodated N-terminal to the cleavage site. As in neurolysin, however, it is unlikely that more than a few additional residues could be added to the N terminus without substantially altering the peptide conformation. This limitation on the number of N-terminal residues is consistent with limitations on the activity of another M3 family member, mitochondrial intermediate peptidase, which can only cleave at its preferred site in N-terminal mitochondrial-targeting sequences if the sequence is first trimmed by another enzyme (66, 67).



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  		FIG. 4.
Model of substrate peptide binding. A stereo view of the molecular surface representation of thimet oligopeptidase sectioned to show the floor of the active site channel is shown with the 13-residue substrate neurotensin modeled as described under "Model for Substrate Binding." The overall orientation is similar to Fig. 1A, and the N terminus of neurotensin is at the top of the figure. The image was produced with GRASP (73).

 
Differential Substrate Recognition by TOP and Neurolysin—TOP and neurolysin cleave many bioactive peptides at the same site or sites, a consequence of their nearly identical structures and the high degree of sequence identity in the residues that line their substrate binding channels (50). Indeed the current structure confirms our earlier modeling study, which suggested that only 11% of the sequence differences between TOP and neurolysin map to the walls and floor of the channel, which accounts for more than 23% of the total molecular surface. The two enzymes are known to cleave some peptides at different positions, however, and understanding the determinants of this specificity should provide insight into the mechanism of broad sequence recognition.

As noted above, TOP cleaves the 13-residue peptide neurotensin at Arg-8-Arg-9, whereas neurolysin cleaves the peptide at between Pro-10 and Tyr-11. A possible reason for this sequence specificity is evident when comparing the electrostatic potentials at the channel floors of the two enzymes (Fig. 5A). In TOP, the surface of the floor near the open end of the channel is strongly electronegative, whereas the corresponding surface in neurolysin has several basic patches. The large acidic patch in TOP may cause neurotensin to bind with its two adjacent arginines shifted toward the open end of the channel to optimize electrostatic interactions. The basic patches in neurolysin may have the opposite influence, causing neurotensin to shift its binding site toward the closed end of the channel to minimize electrostatic repulsion between the enzyme and the basic residues in the peptide.




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  		FIG. 5.
Determinants of cleavage site differences between TOP and neurolysin. A, surface electrostatic potential in the substrate binding channels of unliganded TOP and neurolysin (49). Standard colors are used to indicate electrostatic potential (blue for positive and red for negative), and the neurotensin sequences shown are offset to represent the different binding positions in the two enzymes. Molecular surfaces and electrostatic potentials were calculated using GRASP (73). pGlu, cyclic Glu. B, key enzyme residues likely to mediate differences in substrate specificity. The TOP backbone structure and key residue side chains are shown with the side chains from neurolysin superimposed. Residues shown for TOP/neurolysin are Glu-469/Arg-470, Met-490/Arg-491, His-495/Asn-496, and Arg-498/Thr-499. The residue positions of the substrate peptide neurotensin are indicated by numbers next to the C{alpha} positions, and the active site zinc and conserved residues from the active site sequence motif are shown for reference. Images were produced with Swiss-PDBViewer (72). NT, neurotensin. C, summary of neurotensin binding in thimet oligopeptidase and neurolysin relative to the four key residue differences between the two enzymes. Sequences of neurotensin in the modeled registration for TOP and neurolysin binding are shown on the top and bottom, respectively, with color coding for residue type. The key residue differences between TOP and neurolysin are shown in the center with the position numbers in each enzyme. Enzyme residues are aligned relative to the substrate peptide sequences based on models of substrate binding described under "Model for Substrate Binding," with the primary cleavage position indicated by the vertical line. D, schematic representation of residue preferences in TOP and neurolysin relative to the four key residue differences between the enzymes. The residue preferences were derived by systematic variation at each residue position of the fluorogenic substrate ortho-aminobenzoyl-Gly-Phe-Ser-Pro-Phe-Arg-Gln- N-[2,4-dinitrophenyl]-ethylenediamine in a study by Juliano and co-workers (48). Residue preferences, reflected by low Km values, in positions near the cleavage site (heavy vertical line) are indicated for TOP (above) and neurolysin (below). Key differences in TOP and neurolysin are shown in the center, aligned according to models of substrate binding.

 
The differences in electrostatic potential near the open end of the channel floor are due in large part to a single residue change in the region, Glu-469 in TOP converting to an arginine in neurolysin (Fig. 5B). Arg-9 in the model of bound neurotensin could hydrogen-bond to Glu-469 of TOP, suggesting that this specific contact, in addition to general electrostatic complementarity, may contribute to differences in substrate recognition. In fact, the TOP structure supports earlier work (50) that identified four key positions, including 469/470, which likely determine the specificity differences between TOP and neurolysin. As shown in Fig. 5B, the three other key residue changes are Met-490/Arg-491, His-495/Asn-496, and Arg-498/Thr-499 in TOP and neurolysin. These three positions all arise from the extended turn between helices 15 and 16, the helices that contribute the zinc coordinating residues in the active site.

The extended turn spans most of that region of the channel floor that runs from the active site to the closed end of the channel, and a substrate peptide like neurotensin, with a relatively large number of residues N-terminal to the cleavage site, could potentially interact with the enzyme at all of the key positions. In particular, Met-490 of TOP is positioned to favorably interact with Leu-2 (at position P7 using the standard nomenclature for peptidase substrates) of neurotensin in the current model, and His-495 could make a salt bridge to Glu-4 (P5) of the peptide. Also, the peptide registration places the strongly electropositive adjacent arginines at positions 8 (P1) and 9 (P1') of the peptide, away from Arg-498, reducing any unfavorable interaction. In neurotensin, on the other hand, where the peptide shifts toward the closed end of the channel, changes in these key residues appear complementary to the altered peptide position. Instead of Met-490, Arg-491 at the equivalent neurolysin position can now make a salt bridge to Glu-4 (P7) of neurotensin. And the equivalent residue to His-495 in TOP is now an asparagine, a change that avoids an electrostatic clash with Lys-6 (P5) of the peptide and makes possible hydrogen-bond interactions with that residue or possibly Asn-5 (P6). Finally, Arg498 of TOP is a threonine in neurolysin (Thr-499), and this change avoids an electrostatic clash with the electropositive region of the peptide, which is now near this position, and possibly allows a direct interaction with one of the arginine side chains. The model for difference in neurotensin specificity is summarized in Fig. 5C.

This relatively simple picture of differential substrate specificity appears to hold for a systematic series of sequence variants studied by Juliano and co-workers (48). Substitution of every possible amino acid except glycine at each position in the fluorogenic substrate ortho-aminobenzoyl-Gly-Phe-Ser-Pro-Phe-Arg-Gln- N-[2,4-dinitrophenyl]-ethylenediamine identified preferred residues at each position. For this short peptide, the 490/491 sites on the two enzymes are not close enough to interact with substrate in our model. The other three key positions, however, are largely consistent with the observed substrate Km values, the most relevant parameter for this analysis (Fig. 5D).

On the C-terminal side of the scissile bond, neurolysin shows a strong preference for aspartate at P2', which could interact with the Arg-470 of the enzyme. TOP on the other hand, does not show a strong preference at P2'. Instead, Glu-469, which replaces Arg-470 or neurolysin, could interact with the preferred positive residues at P1' or possibly P3'. On the other side of the scissile bond, TOP preference for negatively charged residues at P3 is consistent with the positively charged residues present at positions 495 and 498 of the enzyme, both of which could interact with the P3 residue in our model. Arg-498 is also in position to interact with the preferred asparagine at P2 of the substrate or possibly the preferred phenylalanine at P1. The polar residues at the corresponding two positions in neurolysin are compatible with the preference for positively charged residues at P4 and the ability to accommodate a positively charged residue at P1. Also, the preferred phenylalanine at P3 might interact with either Asn-496 or Thr-499. The preference of neurolysin for hydrophobic residues at positions P1 and P2 is more difficult to rationalize with this model, and an understanding will likely require visualization by structure determination of substrate-enzyme complexes.

Possible Role of an Active Site Loop in Differential Substrate Recognition—We proposed in earlier work that at least some of the many loop and open coil segments lining the active site channel of neurolysin might rearrange when binding different substrate peptides, a possible mechanism for the ability of the enzyme to recognize a range of peptide sequences (49). One loop in particular (residues 600-612) contains five potentially flexible glycine residues and is partially disordered in the neurolysin crystal structure, suggesting high mobility. In TOP, the corresponding loop (residues 599-611) has only four glycine residues, and the change from glycine to alanine at position 607 might be expected to affect the conformation and mobility of this segment. The loop is in close proximity to the active site (Fig. 6A) and would likely interact with bound substrate. Mutating Tyr-606 at the tip of the neurolysin segment differentially affects the substrate recognition of the enzyme, supporting a role for this loop in substrate binding.2 It is possible, therefore, that in addition to the key residue differences in the floor of the channel, changes in this loop segment may contribute to differences in cleavage site specificity between TOP and neurolysin.



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  		FIG. 6.
Changes in the loop just opposite the active site in TOP and neurolysin. A, conformations of the loops. The backbone trace of the TOP loop (residues 599-611) together with the adjacent Tyr-612 is in gray with the conformations of large side chains indicated in atom colors. The corresponding side chains from superimposed neurolysin are in yellow, and the position the alanine substitution in the TOP loop is indicated. The TOP active site residues and zinc ion are shown for reference. B, active site loop order in TOP and neurolysin. Backbone traces of the loops opposite the active site in the two enzymes are colored according to the thermal factors of main chain atoms in the loop residues. The key indicates the color coding scheme, with yellow representing the highest thermal factor (66 Å2) and dark blue representing the lowest thermal factor (9 Å2). The arrow indicates the alanine residue in TOP (Ala-607) that is changed to a glycine in neurolysin.

 
Changes in loop conformation and mobility are indeed found in the TOP crystal structure (Fig. 6). Backbone and side chain rearrangements result in different placement of residues that might interact with substrate, including His-600 and Tyr-605 as well as Tyr-612, which is immediately C-terminal to the loop. His-600 adopts a different side chain rotomer conformation, pointing toward the active site in TOP and away from it in neurolysin. Tyr-605 shifts position by 2.1 Å in C{alpha} position and also adopts a different side chain rotomer conformation in the two enzymes but in both cases still points in toward the active site. Interestingly, Tyr-612 rotates away from the active site. Mutations at this position affect catalysis in both TOP and neurolysin (68), suggesting that it may play a role by contributing a hydrogen bond to the oxyanion transition state intermediate (49, 68). The tyrosine in neurolysin is well positioned to serve in this function, but the TOP loop would need to change main chain conformation upon substrate binding to place the Tyr-612 within hydrogen bonding distance of the substrate carbonyl oxygen.

The differences in loop conformation between the two enzymes are accompanied by an overall increase in order of the TOP loop relative to the same segment in neurolysin (Fig. 6B). The average backbone thermal factors for the 12 residues in the TOP loop is 19 Å2 compared with a much larger value of 45 Å2 in neurolysin. Clearly the loss of one glycine has a considerable effect on both mobility as well as conformation. Given the differences between the loops in the two crystal structures, it seems likely that this single residue change plays a role in creating specificity differences. Recently, the glycine at position 607 in TOP was mutated to the alanine found in neurolysin (68), and the altered TOP has kinetic parameters more like neurolysin for hydrolysis of a peptide that is not normally a good TOP substrate. Additional mutagenesis and characterization will be necessary to fully test the model for differences in cleavage site selection.

Modification and Subcellular Localization—TOP activity can be modulated by posttranslational covalent modification (3). The enzyme loses activity in the absence of reducing agents, and this inactivation occurs in part by the formation of disulfide-linked multimers, which appear to lose affinity for substrate (35, 37). Shrimpton et al. (35) suggest that polymerization may provide a mechanism for modulating TOP activity based on the redox characteristics of its environment. Recently, an apparently proteinaceous component of cerebrospinal fluid has been shown to affect the multimerization state of TOP, indicating another possible level of regulation for the secreted pool of the enzyme (36).

Of the 14 cysteines in human TOP, 7 are solvent-accessible in the crystal structure, and 6 of those 7 are on the outside of the enzyme where they could participate in intermolecular disulfide links (Fig. 7). In addition, Cys-18 and Cys-682 are located in the disordered N and C termini, respectively (not included in the crystal structure), and are, therefore, also likely to be solvent-accessible. In the rat ortholog, cysteines 246, 248, 253, and 682 have been implicated in multimer formation (35, 37), and all are among the surface-accessible residues in the crystal structure of the human enzyme. Cysteines at positions 46 and 687 in rat TOP are also thought to be involved in multimer formation. Human TOP has an arginine at position 46. This residue is in an exterior portion of {alpha}1, and it is likely that the S{gamma} of a cysteine at this position would be accessible and in a good position to mediate dimer contacts. Position 687 (glutamine in human TOP) is in the disordered C terminus of the crystal structure and would likely also be available for intermolecular disulfide bond formation. Interestingly, the other accessible cysteine residues outside the channel, positions 321, 350, and 644, are not present in the rat homolog and might be additional sites of covalent linkage in human TOP. As noted previously based on a TOP model (50), cysteines 246, 248, and 253, which appear particularly important in disulfide bond formation, cluster tightly on the molecular surface and could themselves mediate only dimer formation. In fact, these three cysteines are located in a strongly electronegative region of the TOP surface (Fig. 7, lower right panel), suggesting that dimerization via only these residues may be rare. Multimer formation, particularly the formation of aggregates larger than dimers, must therefore involve cysteines other than this tightly clustered set.



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  		FIG. 7.
Covalent modification of TOP. Surface-accessible cysteine residues. Cysteines that form part of the molecular surface of the TOP crystal structure are indicated by pink patches for three different orientations of the molecule in the upper and lower left panels. Residue numbers for each cysteine are given. In the lower right panel, the surface electrostatic potential near cysteines (246, 248, 253) known to be involved in the disulfide mediated polymerization of TOP (35) is shown in the same molecular orientation as the lower left panel. The images in this figure were created with GRASP (73).

 
Given the locations of the accessible cysteines, it is not immediately clear why multimerization inhibits the enzyme, since covalent linkage would be unlikely to prevent substrate entry into the active site channel. The decrease in activity has only been shown with the rat homolog, and it is possible that multimer formation involving Cys-46, which is located at the top of one channel wall, restricts substrate access to the active site. Indeed, mutation of this position improves activity under oxidizing condition (37). Mutation of Cys-682 and Cys-687, on the other hand, does not improve activity, consistent with their location on the backside of the enzyme far away from the channel opening. Mutating Cys-46 is not sufficient to prevent all loss of activity, and it seems clear that other cysteines, most prominently cysteines 246, 248, and 253, also mediate inactivation by multimerization (35, 37). It is difficult to imagine how multimerization involving these residues and any of the other surface cysteines would occlude the active site channel, and determining if loss of activity is a steric or a conformational effect awaits further experimentation.

Both TOP and neurolysin are inactivated by thiol-modifying reagents, and a conserved cysteine at the base of the channel near the active site, Cys-427 in TOP, would almost certainly affect substrate binding if modified by a bulky reagent (50). This cysteine is not surface-accessible in the TOP crystal structure, however, and the only solvent-exposed cysteine inside the channel, Cys-175, is located high up on one channel wall where modification is unlikely to affect activity. It appears then that either local dynamics allows transient access to Cys-427 or modification of another cysteine causes a conformational change that affects activity. The early suggestion (69) that thiol-modifying reagents or high concentrations of reductant might disrupt intramolecular disulfide bonds is incorrect since there are no disulfide bonds or appropriately positioned cysteine pairs in the crystal structure.

The rat homolog of TOP is a substrate for protein kinase A, and phosphorylation at Ser-644 affects activity on at least one substrate peptide (38). In addition, rat TOP expressed in cultured cells is phosphorylated, suggesting that this modification may be an additional form of TOP regulation. The serine at position 644 is a cysteine in the human form of the enzyme, so this regulatory mechanism is not completely conserved. In any case, whereas Cys-644 is solvent-accessible in the crystal structure, it is located in {alpha}22 on the side of the enzyme opposite the channel opening, more than 20 Å from the active site (see Fig. 7). The remote location of this residue might explain the modest and inconsistent effect of modification in the rat enzyme (38). Interestingly, the residue at position 643 in human TOP is a serine, but it is not known if this residue can be phosphorylated.

A key remaining question about TOP is the mechanism by which it is localized to different compartments despite the absence of known signal sequences. In this regard, our finding that the N terminus is disordered as far back as residue 25 in the crystal structure suggests that it may play a role in localization by one or more yet unrecognized sequence motifs. TOP has been reported to be primarily nuclear in rat brain neuronal cells, in apparent contradiction with its proposed functions, and residues 234-240 (PETRRKV) were identified as a possible nuclear localization sequence (12-14). This sequence forms the N-terminal segment of helix 8 in one channel wall of the enzyme, however, and a major rearrangement would be required for it to adopt the extended conformation recognized by nuclear import carrier molecules (70). Several reports have also suggested that TOP associates with membranes of the secretory apparatus and the plasma membrane, perhaps even the specialized substructures known as lipid rafts (5-7, 12, 13, 17, 18). There are no basic regions of the molecular surface that might mediate direct electrostatic association. The one prominent surface electrostatic feature, the large acidic patch (Fig. 7), might mediate membrane association by interaction with another protein, a possibility that could be tested by site-directed mutagenesis.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1S4B) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by United States Public Health Service Grant NS38041 (to D. W. R.), National Science Foundation Grant MCB-9904886 (to D. W. R.), and American Chemical Society Petroleum Research Fund 37135-AC4 (to D. W. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} A predoctoral fellow of the American Heart Association Ohio Valley Affiliate. Back

§ A fellow of the National Institute on Drug Abuse (Grant DA14596). Back

To whom correspondence should be addressed. Tel.: 859-257-5205; Fax: 859-323-1037; E-mail: david.rodgers{at}uky.edu.

1 The abbreviations used are: TOP, thimet oligopeptidase; r.m.s.d., root mean square deviation. Back

2 K. Ray, C. S. Hines, J. Coll-Rodriguez, D. W. Rodgers, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank the staffs of Advanced Photon Source beamlines 22ID (SER-CAT), 19ID (SBC), and 14-BMC (BioCARS) for help with data collection. Use of the Advanced Photon Source is supported by the United States Department of Energy.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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