F. Hoffmann-La Roche Ltd., Pharma Preclinical Research, CH-4070 Basel, Switzerland
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Abstract |
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Keywords: endothelin-converting enzyme/modeling/metalloproteinase/neutral endopeptidase/phosphoramidon
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Introduction |
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Two major forms of ECE have been identified so far in human hECE-1 and hECE-2. Moreover four different splice variants of ECE-1 could be found in humans (hECE1ad) (Schweizer et al., 1997; Valdenaire et al., 1999a
) and they are all most active at neutral pH, whereas ECE-2 shows the highest activity at pH 5.5. There is an average sequence identity of 60% between the two human ECE forms. The isoforms of hECE-1 differ in the cytoplasmic and transmembrane domains only whereas the four ectodomains are identical. Since the ectodomain of human NEP (hNEP) shares an overall sequence identity of 40 and 36%, respectively, with those of hECE-1 and hECE-2 and all three ectodomains are similar in length, it is very likely that these enzymes share a common fold.
Endothelin exists in three isoforms (ET-1, ET-2 and ET-3), but interestingly both hECE-1 and hECE-2 seem to process ET-1 preferentially. Only recently was a solubilized form of Kell blood group protein described to convert correctly big ET-3 into ET-3 at slightly acidic pH (Lee et al., 1999). Under physiological conditions, hNEP is involved in the metabolism of a number of hormone peptides such as bradykinin, atrial natriuretic factor and endothelin whereas ECE-1 preferentially cuts the bond between Trp21 and Val22 in big ET-1 to produce active endothelin and the residual `C-terminal fragment'. ECE-1 is thereby performing the last and rate-limiting step in the conversion cascade of preproendothelin to endothelin, one of the most active vasoconstrictors known to date. Regulation of endothelin biosynthesis might have beneficial effects in a number of diseases in the cardiovascular system.
Phosphoramidon, a metabolite produced by Streptomyces tomashiensis, is a generic metalloproteinase inhibitor with broad enzyme specificity that has IC50 values for hECE-1, hNEP and human ACE of 3.5, 0.034 and 78 µM, respectively (Kukkola et al., 1995). Unfortunately, many other metalloproteinase inhibitors (e.g. thiorphan) do not inhibit ECE. This creates a need for new synthetic, potent and selective inhibitors of ECE that can be converted into a drug, thus structural information of this enzyme will be essential in order to understand fully the mechanism of action and to guide inhibitor design.
Since the structure of ECE-1 has not yet been solved, a number of attempts were made to model the active site region of ECE (Sansom et al., 1995, 1996
, 1998
; Hoang et al., 1996
). However, until recently model building was hampered by the lack of structural data for members of this metalloproteinase family and therefore significantly shorter structures like that of thermolysin and related enzymes had to be utilized as templates for model building. Since hNEP and hECE are similar in length and have a fairly high sequence identity (40%), a significantly more accurate model might be expected with hNEP being the template for model building of hECE. Better predictions for the binding pockets on both sides of the cleavage site might be expected and therefore better predictions for inhibitors should be possible.
Recently, we succeeded in solving the structure of the ectodomain (amino acids 52749) of human NEP inhibited by phosphoramidon (Oefner et al., 2000). This puts us for the first time in the very comfortable position of being able to build models of complete ectodomains of members of the metalloproteinase family M13 utilizing a well-resolved structure of a member of this family as template. Here we describe the building of a model of human ECE-1 and the subsequent docking of phosphoramidon into its active site. We also discuss structural differences between hNEP and hECE-1 that might explain the 100-fold stronger binding of this inhibitor to the former enzyme.
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Materials and methods |
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Alignment
A global multialignment was obtained with help of our in-house program Xsae (C.Broger, unpublished work) using a modified version of CLUSTAL V (Higgins et al., 1992). Sequences were obtained from SWISS-PROT: human ECE-1, P42892; rat ECE-1, P42893; guinea pig ECE-1, P97739, bovine ECE-1, P42891. The sequence of human NEP was read directly from the hNEP structure. Note that there is a shift of +16 amino acids between the human ECE file in the OWL database and that in SWISS-PROT. This is due to different hECE-1 isoforms stored in the respective databases but has no effect on model building since the sequences of the ectodomains are identical for all ECE-1 isoforms. Sequence identities between the ectodomain of hNEP and those of human, rat, guinea pig and bovine ECE are in the range 3941%.
Model building
All modeling calculations were made on a Silicon Graphics Octane with a single R12000 processor using our in-house modeling package Moloc (Gerber and Mueller, 1995; Gerber, 1998
). An initial C-alpha model of hECE-1 was built by fitting the aligned hECE-1 sequence (Figure 1
) on the hNEP template C-alpha structure. This model was subsequently improved by searching a Moloc internal database of loops obtained from highly resolved protein structures. Loop selections were made on the basis of minimal steric interactions with the rest of the model. Subsequently newly introduced loops were optimized with the Moloc C-alpha force field. In a next step a full atom model of the complete ectodomain was generated. Phi and psi angles were obtained for aligned amino acids from the NEP template. Chi angles were also adopted from the hNEP structure where possible or in case of non-identical amino acids generated by using the most probable value applying the PonderRichards method (Ponder and Richards, 1987
).
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Refinement of the model
Refinement of the hECE-1 model was performed using Moloc. Neither water nor inhibitor molecules were introduced at this point. In a first step only amino acid side chains were allowed to move while all backbone atoms were kept in fixed positions. This step greatly removed repulsive interactions between side chains, further improved chi angles of non-conserved amino acids and revealed regions with unfavorable interactions. In a following optimization step only C-alpha atoms were kept in a fixed position while all other atoms were allowed to move. In a third round of optimization no atoms were kept stationary but constraints were applied to C-alpha atoms. The quality of the model was then checked (i) with Moloc internal programs, (ii) with a program by Luthy et al. (Luthy et al., 1992) and (iii) with PROCHECK (Laskowski et al., 1993
).
Docking of inhibitor
Coordinates of phosphoramidon were adopted from the respective NEP complex (Oefner et al., 2000) and manually docked into the active site of the hECE-1 model. Where necessary non-conserved amino acid side chains in the hECE-1 model were rotated such that no vdW conflicts with the inhibitor occurred. All amino acid side chains reaching within a 5 Å distance of the inhibitor were subsequently included in a round of optimization. One water molecule was introduced in order to mimic a water-mediated interaction observed in hNEP between inhibitor and the conserved amino acid side chain of R102 (R145 in hECE-1).
Membrane bound ECE-1 dimer
A copy of the hECE-1 model was made and manually translated and rotated such that the two bridging C428 of each monomer came to minimal distance without other parts of the enzymes forming repulsive interactions. Subsequently a model of the transmembrane helices and the five missing amino acids of the ectodomain were built and connected to the ectodomain model (Figure 2).
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Results and discussion |
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The ectodomains of hECE-1 and human NEP share a sequence identity of 40% that is statistically highly indicative of a common origin and almost certainly a common fold. Until recently only fragments of members of the M13 metalloproteinase family could be modeled owing to lack of an appropriate structural template. Thermolysin (from Bacillus stearothermophilus) shares statistically significant similarity with the C-terminal half of this enzyme family and was typically used as structural template for modeling studies of hECE-1 (Sansom et al., 1995, 1996
, 1998
; Hoang et al., 1996
) or hNEP (Tiraboschi et al., 1999
). Three characteristic motifs HExxH, ExxxD and NAArAr (x = any amino acid, Ar = aromatic amino acid), which are all part of the active site, are conserved between the bacterial template and the mammalian sequences. Other short sequences around these three motifs are identical or show high similarity between the three sequences and can be aligned straightforwardly.
However, the complete N-terminal half of the ectodomain with the important residue R102 in hNEP (Beaumont et al., 1991; Kim et al., 1992
) equivalent to R145 in hECE-1 could not be modeled with this template and left any model of hECE-1 or hNEP incomplete. Those models were largely devoid of reliable structural information concerning the S2' pocket or even more remote subsites. Moreover, no equivalent to R203 in thermolysin could be identified unequivocally in the sequences of either hNEP or hECE-1. The recently determined structure of the hNEP ectodomain (Oefner et al., 2000
) allows for the first time modeling of the complete hECE-1 ectodomain and therefore rationalizing effects found for mutations as well as a better understanding of differences in SAR for phosphoramidon and future inhibitors. Additionally, comparing the model of hECE-1 with the experimentally determined structure of hNEP allows a better understanding of structural differences and therefore could aid in the design of potent and selective inhibitors.
A multiple sequence alignment of hNEP and ECE-1 sequences from human, rat, guinea pig and bovine was done automatically with subsequent manual corrections in order to position insertions and deletions in loop regions and to preserve secondary structure of the template. Using the sequences of ECE-2, PEX or Kell blood group protein did not lead to an improved result. The final alignment comprised two gaps in hNEP and seven gaps in the hECE-1 sequence, none of them longer than six amino acids (Figure 1). The model was subsequently built and refined as described in Materials and methods. A Ramachandran plot created in PROCHECK with a hypothetical resolution of 3.0 Å revealed that 87% of the amino acid residues are in the most favorable region whereas all others were found in additional allowed areas. An r.m.s.d. value of 0.26 Å was obtained for matching 622 out of 673 amino acids of the hECE-1 model on the hNEP template. Two cis amide bonds can be found in the hNEP structure (Lys318Pro319, Pro561Pro562), but probably only the latter one is conserved in hECE-1 (Ala585Pro586) since there is a deletion of six amino acids in hECE-1 where the first cis amide is found in hNEP.
Unlike NEP, Kell blood group protein or the bacterial-derived thermolysin hECE-1 is a disulfide-linked dimer, as was shown by SDSPAGE under reducing and non-reducing conditions and by cross-linking experiments (Takahashi et al., 1995). Mutagenesis experiments identified C428 (C416 in Hoang et al., 1996) to be the linking residue. In our model this cysteine is located at the surface of the protein in a sequence stretch with little secondary structure and it is in a very good position to interact readily with its counterpart from a second monomer and form a disulfide bridge (Figure 2
). The model of the hECE-1 dimer also allows a search for a second potential entrance/exit of the enzyme through which substrates/products can access/leave the interior of the enzyme.
Description of active site and phosphoramidon complex
The conserved active site motifs HExxH and ExxxD (Figure 3a) from template and model superimpose very nicely. The two histidines and the leading glutamate in the second motif contact the catalytically important zinc ion with their side chains. The NAYY motif in hECE-1 aligns nicely with the NAFY sequence in hNEP (Figure 3a
) and is located in an equivalent position along the crest of the non-prime half of the active site. The highly conserved H732 (H711 and H231 in hNEP and thermolysin, respectively) is H-bonding in template and model to the side chain of an aspartate two amino acids upstream. While there is a Val in hNEP between these two residues, hECE-1 has a proline in this position. The bulkier and more rigid proline side chain leads to a shift of the upstream aspartate side chain and thereby induces a second H-bond with the side chains of R718 (Y697 in hNEP). The shorter and less flexible tyrosine side chain in hNEP does not allow a direct H-bond but forms two water-mediated contacts with R102 and Y701 instead.
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Striking differences between hNEP template and hECE-1 model are found in the region of the S2' pocket where the indole moiety of phosphoramidon is located. In hNEP the side chains of R102, F106, D107, R110 and N566 (Figure 3b) form this pocket. It hosts the tryptophan side chain of the inhibitor favoring an H-bond with the backbone carbonyl of V565. The indole moiety stacks on the side chain of F106 and is surrounded by the other mentioned residues that are connected either via direct or water-mediated H-bonds and thereby completely embrace the heteroaromatic bicyclus. In hECE-1 this pocket has a significantly different shape since D107 is a serine in hECE-1 and R110 is a tryptophan, leading to a much more open pocket. The individual side chains are no longer connected via H-bonds and therefore do not embrace the indole moiety of the inhibitor so tightly. The reduced binding of phosphoramidon to hECE-1 might be partly the result of decreased interactions of the inhibitor with the protein in this area. Additional enzymeinhibitor interactions in hNEP result from a water-mediated contact between the carboxylate group of phosphoramidon and the side chain of the highly conserved R102. This contact is very likely to be conserved in hECE-1.
In structures of thermolysin inhibitor complexes a tight interaction between R203 and the S2' carbonyl group of inhibitors can be observed. There is no equivalent arginine found between the ExxxD motif and the catalytically important H711 in hNEP. However, the hNEP structure revealed that the side chain of the highly conserved R717 forms a similar H-bond interaction with the S2' carbonyl group as R203 in the bacterial enzyme. Interestingly, in the mammalian M13 metalloproteinases this arginine is not located 28 amino acids upstream of the catalytically important histidine (H711 and H732 in hNEP and hECE-1 respectively) as found in thermolysin but seven amino acids downstream.
At some stage it was speculated that R102 and R747 in hNEP might be involved in substrate binding (Beaumont et al., 1991). Wherease R102 is conserved in hECE-1 and clearly in a position to interact with the substrate, R747 is a glutamate (E768) in hECE-1 and therefore not very likely to be involved in comparable interactions. In fact, this amino acid side chain is located far from the active site at the surface of the protein. While the arginine side chain in hNEP is forming a salt bridge with the C-terminal carboxylate, the negatively charged side chain in hECE-1 is in position to interact with K676, which is a glycine (G655) in hNEP. Arginine 680 that is conserved in hNEP (R659) can make up for this lost salt bridge since T458 in hNEP is a serine (S478) in hECE-1 and the missing methyl group allows R680 to assume an extended conformation and get in H-bond contact with the terminal carboxylate.
Possible reasons for substrate specificity of ECE-1
The overall shape of the hNEP ectodomain that served as target structure for homology modeling of hECE is an ellipsoid with a major axis of ~95 Å and a minor axis of ~55 Å (Oefner et al., 2000). The enzyme consists of two domains of about equal sequential length. Whereas the domain following the transmembrane helix comprises all residues important for zinc binding and catalysis, the second one forms a dome-shaped roof covering the active site, thereby preventing access for large molecules. The two domains embrace a spherical cavity of about 20 Å diameter, which bears the active site. In both hNEP and the hECE-1 model there exists only one small opening that allows access to the interior of the enzyme and to its active site. It can be speculated whether big endothelin forces a second opening through the enzyme wall or adapts a conformation allowing the C-terminal part of the substrate to fit into the interior of hECE-1. The fact that big endothelin-1 and -2 have identical residues from P7 to P4' but processing speed is significantly different (Waxman et al, 1994
) supports the conclusion that substrate selectivity must be achieved by recognizing sequence differences fairly far from the cleavage site. This allows speculations that big endothelin has to thread through this loophole in order to gain access to the interior of the enzyme. Productive binding that is necessary for substrate cleavage must be achieved when the cleavable bond is close to the catalytic zinc and specific interactions are formed between hECE-1 and its respective substrate. In order to understand better the reasons for the high preference of hECE-1 for its substrate big endothelin-1, crystallization of either hNEP or hECE-1 in complex with a large substrate or inhibitor will be necessary. Additional structural information is needed to shed more light on the dynamic behavior and conformational changes of hECE-1 in the presence of a substrate or extended inhibitor.
Conclusions
Molecular modeling of the complete ectodomain of human ECE-1 was performed using the recently solved structure of hNEP as a template. The metalloproteinase inhibitor phosphoramidon could be docked into the active site and for the first time the importance of selected amino acid residues (e.g. R145, R738) could be explained and the interactions with phosphoramidon were rationalized. Our hECE-1 model is more complete and probably more accurate than any previous one owing to the superior template structure utilized. This model also puts us in a position to design new potent and selective inhibitors that might eventually lead to a drug.
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Notes |
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Acknowledgments |
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References |
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Received October 10, 2000; revised January 19, 2001; accepted February 15, 2001.