Bioinformatic Comparison of Structures and Homology-Models of

Bioinformatic Comparison of Structures and Homology-Models of Matrix Metalloproteinases Claudia Andreini,† Lucia Banci,†,‡ Ivano Bertini,*,†,‡ Claudio Luchinat,†,‡ and Antonio Rosato†,‡ Magnetic Resonance Center (CERM), University of Florence, Via L. Sacconi 6, 50019 Sesto Fiorentino, Italy, and ProtEra s.r.l., Viale delle Idee 26, 50019, Sesto Fiorentino, Italy Received July 1, 2003

The entire family of human matrix metalloproteinases (MMPs) was investigated using phylogenetic trees and homology modeling. The phylogenetic analysis indicates that individual domains of each MMP have evolved in a correlated manner. Despite their high sequence similarity, the phylogenetic tree of the catalytic domains already allows functional (e.g., linked to regulation and substrate recognition) homologies between different MMPs to be identified. The same pattern of functional homologies is confirmed by the phylogenetic analysis of the mature proteins. Structural models were built for the catalytic domains of the entire MMP family, for twelve hemopexin domains and for twelve mature proteins. The surface properties around the active site cleft of the modeled and experimental structures are quite conserved, whereas the hemopexin domains are more differentiated, possibly indicating a role in determining substrate specificity. The analysis of mature MMPs showed that the area of the interface between the catalytic and hemopexin domains is essentially conserved, with both hydrophobic and hydrophilic amino acids at the interface. The absence of specific conserved interdomain contacts suggests that the interface is tolerant to amino acid replacements, and that there may be a certain degree of plasticity with respect to the reciprocal orientation of the two domains. Keywords: matrix metalloproteinase • MMP • hemopexin • collagen • cancer

Introduction

of MMP regulation, cellular function and interactions at the time the trials were designed.17-19

Extracellular matrix (ECM) components are crucial in modulating the cellular environments during development and morphogenesis. Degradation/remodeling of ECM components has therefore an important impact on a number of fundamental physiological processes, both under normal and pathological conditions.1,2 Indeed, disturbances in ECM processing may cause a number of diseases,3-13 and play a pivotal role in cancer development and tumor metastasis.8,14 Matrix metalloproteinases (MMPs) form one of the most important families of proteases that participate in ECM degradation under a variety of conditions.2 The activities of MMPs are precisely regulated at various levels, e.g., transcription, activation of precursor zymogens, and inhibition by endogenous inhibitors.9,15,16 Loss or imperfections in activity control may induce pathological conditions, e.g., by affecting cell proliferation, adhesion, and migration, as well as angiogenesis and apoptosis (see ref 2 and references therein). The important role of MMPs in tumor progression has prompted the development of several MMP inhibitors as cancer therapeutics.8,17,18 To date, clinical trials of these inhibitors have proven quite disappointing, possibly also because of the relatively poor comprehension of the details

Along with ever-increasing interest in MMPs came a number of insightful studies on various aspects of MMP biochemistry and physiology.9,20-25 MMPs are multidomain proteins, which are secreted as inactive zymogens (ref 26 and references therein). An 80-residue pro-domain extending from the catalytic domain to the protein N-terminus is responsible for enzyme latency. In the majority of MMPs, the C-terminus of the catalytic domain is connected to a hemopexin-like domain via a linker of variable length. Different MMPs may also contain additional domains, e.g., a C-terminal trans-membrane region, or fibronectin inserts within the catalytic domain. Outstanding efforts in a number of laboratories worldwide have yielded high-resolution three-dimensional structures of the catalytic domain of several members of the family, also in complex with a variety of inhibitors.1,27-30 The structures of some isolate hemopexin domains are also available,31-34 as well as the structure of two multidomain constructs (for MMP1 from Sus scrofa31 and human MMP235) In addition, some structures of MMPs in complex with their tissue inhibitors (TIMP’s) are available,36-38 which have provided important indications on the regulation mechanisms of MMPs.

* To whom correspondence should be addressed. Tel.: +39 055 4574272. Fax: +39 055 4574271. E-mail: [email protected]. † Magnetic Resonance Center (CERM), University of Florence. ‡ ProtEra s.r.l.

Until now, most investigations have focused on the catalytic domain, in an attempt to unravel the determinants of substrate specificity at and around the active site cleft, also with the aim to obtain information useful to design inhibitors that can be

10.1021/pr0340476 CCC: $27.50

 2004 American Chemical Society

Journal of Proteome Research 2004, 3, 21-31

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research articles selective for specific MMPs.24,39-42 From the structural point of view, the results on the catalytic domain currently available in the literature provide a very extended picture of its properties also across different MMPs. However, it is more and more appreciated that a deeper understanding of the structural features of the other domains and of the higher multidomain construct is needed.25 Indeed, from the point of view of drug design, targeting sites contributing to the interaction with the substrates but far from the active site could be a good strategy to enhance the selectivity of candidate drugs.18 Here, we have applied bioinformatic tools such as phylogenetic analyses and homology modeling to expand the relatively limited structural knowledge on full-length mature MMPs with the aim to obtain more general insights on the properties of most of the members of MMP family. The relationship and the relative differentiation within the family of the catalytic vs the hemopexin domains are discussed. Note that, at variance with previous literature,26 the analysis is extended to the entire family of the human proteins, exploiting the recent availability of the whole genome sequence.

Materials and Methods Completeness of the Human MMP Family. The human genome was searched for proteins having homology to the catalytic domain of MMPs, using the BLAST program.43 The search results were not affected by the choice of the input MMP sequence. The most different sequence with respect to those already annotated as MMPs (mmplike-1) was used for a further search with Blast, to ensure that the search was as complete as possible. However, no additional sequences were retrieved by this last search. The sequences obtained from BLAST were divided into domains using the SMART database,44,55 and then manually corrected. Sequence alignments were then performed: (i) on the whole sequences, (ii) on the catalytic domain only and (iii) on the hemopexin-like domain only. In all cases, the sequences were aligned with the program ClustalW.46 The sequence alignments were used to build phylogenetic trees with the program PHYLIP.47 An alignment of mature MMP proteins, which has been used for amino acid numbering in the work, is given as Supporting Information. 3D Structural Models. Structural models of the various domains as well as of full-sequence proteins were generated using the program Modeller-6v2.48 The input alignment for Modeller was obtained with ClustalW and every model was created using as template the structure with the closest sequence. If other structures with sequence identity within 5% to the first template were available, then up to two other templates were used. The templates were selected among the structures of the catalytic domain and of the hemopexin-like domain deposited in the Protein Data Bank49 at May 2003 (Table 1). When more than one structure was available for the same construct, only that with the highest resolution was used. This corresponds to 12 experimental structures of different proteins for the catalytic domain, to four for the hemopexinlike domain and to two for the mature MMPs (Table 1). The 29 catalytic domains of the full human MMP family were built with three different methods: (i) without restraints, (ii) adding restraints for only the zinc ions, and (iii) adding restraints for all the metal ions (2 Zn2+ and from 1 to 3 Ca2+) present in the structure. No restraints were used to generate the structural models of the hemopexin-like domains. The latter choice is based on the observation that the calcium ions present in the latter protein are not necessary for the correct fold.38 For MMPs, 22

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Andreini et al. Table 1. Templates Chosen to Build Structural Models of the MMP Domains PDB code

1hfc 1fbl 1eak 1hy7 1mmq 1i76 1gkd 1hv5 1jk3 830c 1cxv 1buv 1fbl 1gen 1itv 1pex 1fbl 1gxd

MMP

source

catalytic domains MMP-1 Homo sapiens MMP-1 Sus scrofa MMP-2 Homo sapiens MMP-3 Homo sapiens MMP-7 Homo sapiens MMP-8 Homo sapiens MMP-9 Homo sapiens MMP-11 Mus musculus MMP-12 Homo sapiens MMP-13 Homo sapiens MMP-13 Mus musculus MMP-14 Homo sapiens hemopexin domains MMP-1 Sus scrofa MMP-2 Homo sapiens MMP-9 Homo sapiens MMP-13 Homo sapiens mature proteins MMP-1 Sus scrofa MMP-2 Homo sapiens

reference

63 31 to be published 64 65 66 67 68 69 70 71 37 31 32 34 33 31 38

with known structure, the accuracy of the models built using different templates was evaluated by determining the RMSD values of these models with respect to the experimental structure. In the case of mature MMPs, the templates input to Modeller consisted of the structure of a mature MMP (Table 1) together with the templates used to model the individual catalytic and hemopexin domains. This was done to ensure the correct reciprocal orientation of the domains while maintaining the best possible templates for modeling the domains themselves. All the structural models calculated were validated by checking their quality with the programs PROCHECK50 and PROSA.51 The program MOLMOL52 was used to analyze the structural models in terms of per-residue solvent accessibility and surface properties (shape, electrostatics). The coordinate files of the models are available at http://www.cerm.unifi.it/ MMPstructures/MmpModels.htm. Inter-domain interaction energies were calculated with the program AMBER v.6.0.53 The AMBER energies, separated into electrostatic, van der Waals and hydrogen bond contributions and also into the different inter-domain interactions, are given as Supporting Information (Table S2).

Results and Discussion Searches with the Program BLAST. BLAST searches output 29 proteins already annotated as MMPs and 29 proteins with other annotations. The first ensemble, on which the rest of this article focuses, contains 23 different MMPs in agreement with the most recent literature9,25 plus five additional shorter isoforms for MMPs 16, 19, 21, 23, 28, and one protein annotated “MMP-like1” (as described in the remainder, this latter protein does not contain the binding site for the catalytic zinc, and thus should not be considered a MMP). For all MMPs occurring as two isoforms, there is 100% identity in the catalytic domain. All MMPs contain, besides a N-terminal signal peptide to direct the protein to the appropriate cellular location (not present in MMP23), a pro-domain that maintains the protein as an inactive zymogen until it is cleaved off and the catalytic ZnMc (SMART nomenclature) domain. Linked at the C-terminus of the catalytic domain most MMPs also feature four hemopexinlike repeats (HX). Note that four HX repeats are needed for the

Structural Comparison of MMPs

Figure 1. Phylogenetic tree of entire MMP sequences (including the N-terminal signal peptide and the pro-domain). Continuous line: GPI-anchored MMPs; broken line: trans-membrane MMPs; line-dotted: gelatinases; dotted: non furin-regulated MMPs (aka simple hemopexin domain).

hemopexin domain to fold properly. Isoforms II of MMP16 and MMP28 are the only ones with a single HX domain, suggesting that they may not be completely folded in vivo. The only proteins lacking the hemopexin domain altogether are MMPlike1, MMP7 and MMP26 (so-called “minimal domain”), while MMP23 has an immunoglobulin-like domain instead of the hemopexin repeats. In the pro-domain of some MMPs (here after called furin-regulated or furin-activated) it is possible to identify a recognition sequence for furin-like serin proteases. This recognition sequence permits cleavage of the pro-domain and thus activation of the MMP by furin-like convertases in the trans-Golgi network.2 MMPs 14, 15, 16, 24 have an additional trans-membrane domain followed by a cytoplasmic domain that is C-terminal to the hemopexin domain, whereas MMPs 17 and 25 are anchored to the membrane through a C-terminal glycosylphosphatidylinositol (GPI) moiety. MMP23 is instead anchored to the membrane at the N-terminus. Analysis of MMP Phylogenetic Trees. Historically, MMPs have been grouped into different classes (and named) based on their substrate specificity. However, it later became apparent that often there is a broad overlap in binding capabilities between MMPs originally thought to bind different substrates, and that each MMP may be capable of binding different substrates almost equally well.2,25 Therefore, in the latest studies the classification of MMPs has been based mostly on domain organization, and the presence of specific inserts and/or recognition sites.8,9,25 Phylogenetic trees can be of help to analyze the evolutionary inter-relationships among the various domains, also taking into account the contribution of individual domains. This is done by constructing phylogenetic trees on the basis of multiple alignments of entire MMP sequences or of domain sequences. The phylogenetic tree (Figure 1) for entire sequences permits the identification of a few different groups, which correlate well, as expected, with the domain-based classification, but with some interesting exceptions. The group of transmembrane MMPs is well separated from the rest, and comprises MMP14, MMP15, MMP16, and MMP24. Gelatinbinding MMPs (MMP2 and MMP9), which contain three fibronectin-like repeats inserted within the catalytic domain,

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Figure 2. Phylogenetic tree of catalytic domain sequences. Continuous line: GPI-anchored MMPs; broken line: transmembrane MMPs; line-dotted: gelatinases; dotted: non furinregulated MMPs (aka simple hemopexin domain).

are also well separated. A third group of proteins that can be identified from the tree of Figure 1 comprises all non furinregulated MMPs, except MMP19 and the minimal-domain MMP26. Another distinct group observable in Figure 1 comprises the two GPI-anchored MMPs (MMP17 and 25, both of which contain also a furin recognition sequence), and the furinactivated MMP11. Finally, all other MMPs not previously mentioned form a separate ensemble comprising MMP19 (non furin-regulated), MMP21 (containing a vitronectin-like insert and a furin recognition sequence), MMP26 (minimal domain), MMP28 (furin-activated), and MMP23 (whose domain organization is quite unique among MMPs, but contains a furin recognition sequence). Note that the differentiation of the sequences within this last group is the highest among all groups identified above. Overall, it appears that there is a significant separation among trans-membrane MMPs, MMPs containing a furin recognition sequence and MMPs without a furin recognition sequence. Trans-membrane MMPs form a separate group on their own. MMPs without a furin recognition sequence sub-group into so-called gelatin-binding (gelatinases), which contain fibronectin inserts, and all others, which do not contain such inserts. Sub-grouping of MMPs containing a furin recognition sequence is less straightforward, and segregates GPI-anchored MMPs together with MMP11 from all others MMPs of this type. MMP19 and MMP26 are the only exceptions to the above schematization, as they are non furing-regulated but cluster together with MMPs containing a furin recognition sequence. The phylogenetic tree of only the catalytic domains (Figure 2) essentially maintains the groupings described above. It is to be noted that this latter tree highlights even more the substantial similarity of the catalytic domains of non furinregulated MMPs. Note, however, that at variance with the behavior of Figure 1, the catalytic domain of MMP12 now forms a group by itself. Interestingly, this group now includes also the gelatinases, MMP2 and MMP9, notwithstanding the fact that they contain fibronectin inserts (however not considered when building the tree); indeed, gelatinases do not contain any recognition sequence for furin-like proteases. On the other hand, the important differentiation of furin-activated MMPs Journal of Proteome Research • Vol. 3, No. 1, 2004 23

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Metal Binding Sites. Entire MMP sequences show an average identity of 35%, whereas alignment of only the catalytic domains results in an average sequence identity of 47%. The analogous figure for the alignment of only PEX domains is 35%. The values of identity range from 21% to 67% for the whole sequences, from 33% to 86% for the catalytic domains and from 20% to 86% for the hemopexin-like domains. Note that when evaluating the range of identity, isoforms are not taken into account given their complete identity to the corresponding fulllength proteins.

Figure 3. Phylogenetic tree of hemopexin-like domain sequences. Continuous line: GPI-anchored MMPs; broken line: trans-membrane MMPs; line-dotted: gelatinases; dotted: non furin-regulated MMPs (aka simple hemopexin domain).

is also more evident, with the exception of the two GPIanchored MMP17 and MMP25. Finally, it is to be noted that MMP19 and MMP26, which do not contain the furin recognition sequence, are somewhat more distant in the tree of the catalytic domains than in that of the entire sequences. The substantial agreement between the findings based on these two trees is particularly noteworthy if we consider that the furin recognition sequence is contained in the pro-domain, and thus not taken into account when building the tree of the catalytic domains; the same consideration holds for transmembrane segments or the other anchors. This is thus an indication that the different regulation mechanism (furin-related or not furinrelated), or the different protein interaction with the membrane (induced by the presence of the trans-membrane anchor and the cytoplasmic tail) actually correlate with different features of the catalytic domain and thus possibly of catalytic activity. Finally, Figure 3 shows the phylogenetic tree built upon the sequences of the hemopexin domains. The analysis of this tree reinforces the conclusions above, but serves also to highlight some differentiation of MMP2 and MMP9 between each other, and, perhaps more interestingly, from the other MMPs lacking a furin recognition sequence. Once again, it is important to keep in mind that MMP2 and MMP9 are differentiated from the others because they additionally possess fibronectin inserts, but this differentiation is intrinsically removed in the comparison of the hemopexin domains. It is also interesting to observe that in Figure 3, a relationship between MMP19 and MMP28 is apparent, which can be tracked also in the other trees. In summary, the above considerations indicate a strong correlation between the evolution of individual MMP domains, in keeping with a previous analysis of MMP sequences.26 There is significant support to the use of a domain-based classification of MMPs, but noteworthy exceptions can be highlighted (MMP19, MMP26, and the differentiation of MMP11 and MMP28) together with general patterns. Finally, these comparative phylogenetic analyses suggest a correlation between regulation mechanisms, catalytic properties, and recognition features (insofar as hemopexin domains play a role in determining partner and/or substrate recognition). 24


Inspection of the residues constituting the binding site of the catalytic zinc(II) ion in the alignment of the catalytic domains shows that the three histidines binding the metal are conserved in all the MMPs analyzed, with the exception of MMP-like1 that lacks all of them. This implies that MMP-like1 cannot bind the catalytic zinc and consequently suggests that in vivo it does not function as a proteolytic enzyme: thus, MMP-like1 will no longer be considered in the present work. The same alignment additionally shows that the binding site of the first calcium(II) ion (which is the one with highest affinity) is always conserved. Instead, the site of the structural zinc(II) ion presents, as already noted,26 some variations for MMP19, MMP21, and MMP27. In MMP19, the first His and the Asp binding to the zinc ion are replaced respectively by Gln and Ser; in the other two above proteins the Asp is replaced respectively by Gly and Ser. These replacements are likely to abrogate binding of the metal ion. Figure 4 shows a portion of the alignment of the catalytic domains around the binding sites of the calcium ions conventionally indicated as II and III. Only some MMPs can bind these two ions. Indeed, the site of the second calcium (bold and italics) is not always conserved. It appears that when Asp238, which binds the second calcium ion with its side chain, is replaced by another amino acid (even Glu), amino acids 233 and 236 (both most frequently Gly and binding with their carbonyl oxygen) in the remainder of the binding site are either lost (most commonly) or nonconservatively substituted (in the case of MMP26). The other charged amino acid in the site is Asp195, which binds the calcium(II) ion with its backbone oxygen and is always strictly conserved, regardless of whether the other ligands are substituted/deleted, which may be due to this residue playing an additional functional role besides metal binding. Only for MMP21, amino acids 233 and 236 are deleted but Asp238 (and Asp195) is preserved. However, it is still most likely that this protein is unable to bind the second calcium ion. The site of the third calcium ion (bold in Figure 4) comprises the side chains of residues 151 (Asp) and 243 (Asp or Glu), and the backbone oxygen of the strictly conserved Glu245, whose side chain additionally binds the first calcium ion. In roughly 60% of the cases in which either residue 151 or residue 243 is replaced by a nonacidic side chain, also the other residue is similarly replaced, suggesting that the substitution of either side chain compromises calcium binding capabilities, therefore removing evolutionary pressure to maintain the companion carboxylate moiety. With respect to the various groupings identified through the analysis of phylogenetic trees, it appears that the only consistent pattern is that trans-membrane type MMPs lack the binding site of the third calcium ion, while preserving that of the second calcium. In summary, our analyses indicate that MMPs 8, 11, 14, 15, 16, 20, 24, and 25 can bind the second but not the third calcium ion; MMP26 can bind the third but not


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Figure 4. Sequence alignment for the binding site of the second (bold and italics) and third (bold) calcium ions.

the second calcium ion; and MMPs 19, 21, 23, and 28 bind neither the second nor the third ion. Structure Modeling: Catalytic Domains. From the preceding analysis, it appears that there is enough sequential identity and metal binding consensus sequence conservation among all human MMPs to allow a meaningful structural modeling approach to all of those constructs (catalytic, hemopexin, and mature proteins) for which experimental structural information is lacking. We are of course aware that structural modeling may not be reliable in the details that are important e.g., for in silico docking experiments of candidate drugs. However, we are interested here in the overall features of the surfaces of the separate domain and possibly on their spatial relationships in the mature proteins. These features may be important for the recognition of large molecules such as the most common substrates of MMPs. Structural models were thus built for the catalytic domains of all MMPs (i.e., including those for which the experimental structure of the human construct is available), using the templates summarized in Table 2. PROSA and PROCHECK were used to evaluate the quality of the models (Table 3). For comparison, an analogous analysis was performed also on the experimental templates (Table S1 in the Supporting Information). The PROCHECK results are quite satisfactory, with most models featuring over 80% of residues in the most favored regions of the Ramachandran plot. Z-scores computed by PROSA are also good, with only two models (MMP7 and MMP23) having a score worse than -6 (experimental structures have Z-scores between -7 and -8, see Table S1). In addition, the RMSD of the coordinates of backbone atoms between each model and the corresponding experimental structure, when available, was calculated (Table 4). In all cases, RMSD values were below 2 Å, with two-thirds of the models actually being within 1 Å from the experimental structure. This latter quality assessment method also allowed us to easily evaluate different approaches to model the metal sites, which indicated that use of explicit constraints, in particular for the zinc ion, was beneficial (Table 4). For the following analyses, the ensemble of structures taken into account comprised the experimental

structures of MMPs of Table 4 and the structural models for all other MMPs not characterized experimentally. The solvent accessibility of individual amino acids was calculated for every structure: the output allows us to identify the amino acids belonging to the core as those with an accessibility lower than 10% of amino acid total surface. For the catalytic domain, the number of core residues averages to 65-90% of the amino acids which are conserved throughout MMP sequences; overall, the core residues have an average conservation of 66%. The conservation of surface residues is much lower, about 30%. Among the amino acids with a solvent accessibility larger than 10% the following patterns of substitution are noteworthy (Figure 5): Arg144 is always conserved, but in trans-membrane MMPs, where it is replaced by a buried Ser or Cys, and in MMP9, where this position contains a Trp (with 10% accessibility). At position 151, all trans-membrane MMPs feature a Lys, whereas other MMPs never do, actually showing a distinct preference for Asp. At position 160, all MMPs without a furin recognition sequence grouped together by phylogenetic trees feature Asp/Glu, whereas the others never do, and 11 out of 13 sequences have Lys/Arg. An opposite charge reversal (positive to negative) is observed for the same MMPs at position 180. Residue 220, which is within the binding site of the first calcium ion, is most commonly Pro or a hydrophobic, with the exceptions of MMP12 (Lys), of the two gelatinases MMP2 and MMP9 (Lys), of MMP21 (Ser) and of trans-membrane MMPs (three Glu and one Thr). Residue 223, a ligand to the first calcium ion with its backbone, is somewhat variable, but it is consistently a Phe in trans-membrane MMPs. Residue 229 is again quite variable, but it is consistently a Phe in trans-membrane MMPs and in all membrane-anchored MMPs. Residues 233 and 236 are part of the binding site for the second calcium ion, and are simultaneously mutated or lacking in most of membrane-anchored and furin-activated MMPs. Residue 246 is somewhat variable, but it is consistently a Pro in trans-membrane MMPs. Residue 451 is Asp/Asn in non furin-activated and trans-membrane MMPs, with the exception of MMP9 (Val), but is substituted by other residues in furin-activated MMPs. Residue 457 is aromatic in non furinJournal of Proteome Research • Vol. 3, No. 1, 2004 25

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Table 2. Templates Used to Model Catalytic Domains, Hemopexin Domains and the Mature Human MMPsa MMP

PDB code

catalytic domains MMP-1 1fbl MMP-2 830c - 1cxv MMP-3 830c - 1fbl - 1hfc MMP-7 1hy7 - 1jk3 - 1fbl MMP-8 1fbl - 1hfc MMP-9 1eak MMP-10 1hy7 MMP-11 1hv5 MMP-12 1fbl - 1hy7 - 830c MMP-13 1cvx MMP-14 830c - 1jk3 - 1cxv MMP-15 1buv MMP-16 IF1 and IF2 1buv MMP-17 1hv5 - 830c - 1cxv MMP-19 IF1 and IF2 1hfc - 1fbl - 1hv5 MMP-20 830c - 1eak - 1i76 MMP-21 and Simil21 1hv5 - 1hy7 MMP-23 A and B 1hv5 - 1fbl - 1i76 MMP-24 1buv MMP-25 1hv5 - 1 mmq MMP-26 1jk3 - 830c - 1hy7 MMP-27 1i76 - 1hy7 - 830c MMP-28 1fbl - 1hfc - 1hy7 MMP-like1 1hv5 - 1 mmq hemopexin domains MMP-1 1fbl MMP-3 1fbl MMP-8 1fbl MMP-10 1fbl MMP-12 1fbl MMP-13 1fbl MMP-15 1gen MMP-16 IF1 1gen MMP-17 1gen MMP-25 1gen-1pex MMP-27 1fbl mature MMPs MMP-1 1fbl MMP-3 1fbl MMP-8 1fbl MMP-9 1gxd MMP-10 1fbl MMP-12 1fbl MMP-13 1fbl MMP-15 1gxd MMP-16 IF1 1gxd MMP-17 1gxd MMP-25 1gxd MMP-27 1fbl

% of identity

91 67-65 65-62-62 57-56-55 68- 67 64 86 95 61-60-60 93 55-53-53 71 69 49-47-45 51-50-49 61-60-59 45-40 47-47-44 72 55-51 54-52-51 61-61-60 52-50-49 43-42 85 52 57 53 51 49 39 42 42 39-35 50 88 56 62 51 55 53 52 39 42 37 39 53

a The percentage of sequence identity between the templates used and the sequence modeled is also shown.

activated, with the exception of MMP2 (Ala) and including MMP26, but is substituted in the other MMPs. Conversely, at position 459, trans-membrane, GPI-anchored and some furinactivated MMPs (MMP11, which is similar to GPI-anchored MMPs (Figures 1-3), and MMP28) have an aromatic residue, whereas other MMPs do not. Interestingly, the next residue, Tyr460, is conserved in all MMPs but MMP23. Structure Modeling: Hemopexin Domains. Satisfactory modeling of the structures of hemopexin domains was possible only for 12 MMPs out of 21 (Table 2), because for the other 9, the distance between the available templates and the sequence to be modeled was too high (that is, the sequence identity was below 30%). Analogously to the case of models for the catalytic domains, the analysis of PROCHECK and PROSA results for these models show that their quality is analogous to the quality of the experimental structures (see Table 3 and Table S1 of the 26


Table 3. PROCHECK Parameters (percentage of residues in the core, allowed, generously allowed and disallowed regions of the Ramachandran plot)50 and PROSA Z-score51 for the Various Structural Models Built in This Work MMP

core (%)

MMP1 MMP2 MMP3 MMP7 MMP8 MMP9 MMP10 MMP11 MMP12 MMP13 MMP14 MMP15 MMP16 MMP17 MMP19 MMP20 MMP21 MMP23 MMP24 MMP25 MMP6 MMP27 MMP28 MMP like1

87.9 78.3 93.8 86.0 88.8 89.6 89.5 88.4 89.0 92.3 87.9 88.1 78.5 80.4 78.6 86.8 93.1 82.4 76.8 75.9 80.4 82.2 89.2 83.6

MMP1 MMP3 MMP10 MMP12 MMP13 MMP15 MMP16IF1 MMP17 MMP25 MMP27

81.6 87.3 78.0 82.6 82.9 93.4 89.8 85.5 85.7 83.9

MMP1 MMP3 MMP8 MMP9 MMP10 MMP12 MMP13 MMP15 MMP16 MMP17 MMP25 MMP27

87.9 87.1 87.0 87.1 85.4 88.1 87.5 89.2 71.0 85.2 78.7 87.4

allowed (%)

generous (%)

catalytic domains 9.8 2.3 11.3 7.0 4.7 1.6 11.0 2.9 8.8 1.6 7.4 0.7 8.7 1.4 5.8 5.1 9.6 0.7 4.6 1.5 10.6 0.8 10.5 1.4 16.0 4.2 15.2 3.6 14.3 4.3 7.8 2.3 3.8 2.3 12.5 1.5 15.5 3.5 19.1 4.3 12.3 5.1 14.8 1.5 6.9 2.3 11.7 1.6 hemopexin domains 13.3 1.9 10.1 1.3 16.4 1.3 14.3 1.9 13.3 1.3 4.0 2.0 8.0 0.7 11.8 2.6 11.6 0.7 13.0 1.9 mature MMPs 10.5 0.6 10.3 1.6 9.8 1.9 11.3 1.7 10.2 2.5 9.1 1.3 10.6 1.3 8.1 2.1 21.0 5.9 10.8 3.0 12.7 6.0 9.4 2.6

disallowed (%)

Z-score

0.0 3.5 0.0 0.0 0.8 2.2 0.4 0.7 0.7 1.5 0.8 0.0 1.4 0.7 2.9 3.1 0.8 3.7 4.2 0.7 2.2 1.5 1.5 3.1

-9.21 -8.55 -7.12 -5.34 -9.01 -10.85 -7.73 -7.30 -7.88 -7.64 -7.78 -7.54 -7.86 -7.41 -7.09 -9.43 -6.58 -5.26 -8.27 -7.86 -6.39 -7.32 -7.09 -5.46

3.2 1.3 4.4 1.2 2.5 0.7 1.5 0.0 2.0 1.2

-7.33 -7.66 -7.31 -8.03 -8.32 -7.89 -7.30 -7.59 -7.99 -7.11

1.0 0.9 1.3 0.0 1.9 1.6 0.6 0.6 2.2 0.9 2.5 0.6

-11.37 -7.91 -8.87 -11.15 -9.72 -8.10 -7.85 -7.57 -6.05 -7.35 -6.50 -7.40

Supporting Information). All models feature the common fourbladed propeller fold, as expected. The alignment of the hemopexin domains shows a significant differentiation between trans-membrane MMPs and other MMPs, with entire stretches of sequence conserved in the former but different from the corresponding residues in the latter. A significant differentiation is also observed between the two gelatinases (MMP2 and MMP9), which can also be appreciated from their separation in two different branches of the phylogenetic trees of hemopexin domains (Figure 3). This contrasts quite remarkably with the substantial sequence similarity of their catalytic domains. Some interesting positions in the general alignment are as follows: 598 where MMP2 and MMP9 have a Gly, trans-membrane MMPs have a Tyr, and other MMPs most commonly have Asp/Glu; 607 where nonfurin activated MMPs have a Pro, and all other MMPs, except

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Structural Comparison of MMPs Table 4. Backbone RMSD (Å) between Structural Models of the Catalytic Domain and the Corresponding Experimental Structuresa MMP

no restraints

with restraints for the zinc(II) ions

with restraints for all metal ions

MMP-1 MMP-2 MMP-3 MMP-7 MMP-8 MMP-9 MMP-12 MMP-13 MMP-14

0.49 0.75 0.93 1.39 1.20 0.89 0.87* 0.59* 2.48

0.45* 0.71* 0.82* 1.38* 1.26 0.86* 0.90 0.70 1.73*

0.45* 0.71* 0.92 1.39 1.16* 0.90 0.89 0.70 1.80

a For each MMP three different models were generated, using a different approach to model the metal sites. The symbol / highlights which of the three models features the lowest RMSD value from the experimental structure.

Figure 6. Electrostatic surfaces of MMPs (based on models containing both the catalytic and the hemopexin domains, and having good energetics and geometric parameters). Top: face containing the catalytic zinc ion (green); Bottom: rotated by 180° around the x axis. The orientation is the same as in Figure 5.

Figure 5. Location of characteristic residues (black spheres) onto the surface of MMP1. The ligands of the catalytic zinc ion are also labeled. Top: face containing the catalytic zinc ion (grey sphere); Bottom: rotated by 180° around the x axis.

MMP21 and MMP23, do not; 627 which features a charge reversal between non furin-activated MMPs and MMP21 with a positive charge, and all others with a negative charge; 692 where non furin-activated MMPs and gelatinases have a hydrophobic amino acid, while the others mostly have Lys/ Arg. Structure Modeling: Mature Proteins. For 12 human MMPs, it was also possible to build a model of the mature protein (excluding transmembrane regions). As shown in Table 3, for

these models the quality indicated by PROSA and PROCHECK is somewhat lower than what obtained for the two experimental structures available (Table S1), even though still in the range of good values. This may be due to the additional difficulty of properly modeling the region connecting the two domains (see however below). The subsequent analyses refer to the 10 model structures with Z-score lower than -7.0 (i.e., MMP16 and MMP25 were discarded) plus the experimental structure of human MMP2. Figure 6 shows the electrostatic surface of several mature MMPs. The catalytic zinc ion is shown in green, and it is immediately apparent that it is largely solvent accessible in all MMPs. Accessibility to the zinc ion is indeed a requirement for its enzymatic activity. In all MMPs, the zinc ion is located within an elongated cavity in the catalytic domain largely negatively charged (Figure 6, top panel). The substrate to be cleaved is bound by MMPs in this cavity. The two protein regions defining the cavity have different electrostatic properties: the upper region in Figure 6 (top panel) is in all cases also strongly negatively charged, whereas the lower region is more variable and tends to be less negative with small hydrophobic patches exposed. Overall, the surface properties of the catalytic domains of the MMPs are strikingly similar in Journal of Proteome Research • Vol. 3, No. 1, 2004 27

research articles the surroundings of the active site, with only minor variations, both in terms of charge and shape. On the other hand, the opposite face of the catalytic domain (Figure 6, bottom panel) is much more variable. Given the broad similarity between substrate binding capabilities by the various MMPs, the above observations suggest that the protein face where the zinc lies is, expectedly, most important with respect to interaction with the substrate. The surface of the region connecting the catalytic and the hemopexin domains, as well as that of the hemopexin domain itself is much more variable (Figure 6). The hemopexin domain should serve an ancillary role in helping substrate binding at the catalytic site, besides contributing to regulation through modulation of protein-protein interactions. The seven nonfurin regulated MMPs in Figure 6 (MMPs 13, 8, 1, 27, 10, 3, 12) appeared to be strictly related in phylogenetic trees. When viewed from the face of the catalytic zinc ion (Figure 6, top panel), the shape of the surface of the hemopexin domains is quite similar in these seven, whereas electrostatic properties are different to some extent for MMP8, MMP12 and MMP13. The hemopexin surface features an elongated cavity, more shallow than that containing the catalytic zinc, which in the top panel of Figure 6 is essentially horizontal, mostly negatively charged, contoured by positive regions (but for MMP13) variously distributed in the various MMPs. The point of intersection between the cavity of the catalytic domain and that of the hemopexin domain is located within the linker region, and corresponds to a (mostly) nonpolar patch. Analogous surface features can also be observed for MMP2, where it is known that strong substrate binding is promoted by the fibronectin repeats, with the functional role of the hemopexin domain being relatively unclear.21,54 In the other gelatinase, MMP9, the isolate hemopexin domain has been recently shown to be capable to bind gelatin.23 MMP15 and MMP17 are instead substantially different from this general picture, in that the latter has a linker region strongly positively charged, rather than essentially nonpolar, whereas MMP15 features a less defined mostly nonpolar cavity on the hemopexin domain. Interestingly, the linker region is reported to play an important role in collagen binding by MMP14 (also called MT1-MMP),22 and is known to affect collagenolytic activity in MMP8.55 The opposite face of the hemopexin domain (Figure 6, bottom panel) is more shallow, without pockets. For the non furin-regulated MMPs, this face features a large negative patch extending from the linker regions to the center of the face. This face is characterized for MMP27 and MMP13 by the additional presence of two large positive patches, whereas in the other four MMPs the negative patch is surrounded by essentially neutral regions. An analogous pattern is observed for MMP2, as well as, if the extra residues in the linker region are not taken into account, for MMP17 and for MMP15. Inter-Domain Contacts. The availability of several structures of mature MMPs allowed us to analyze in detail and compare inter-domain contacts, which determine the energetics associated with the reciprocal orientation of the catalytic and hemopexin domains. Figure 7 shows the value of the total area of the inter-domain interface for MMPs considered, as well as its separation into the various contributions. The average total area of the interface is 2200 ( 250 Å2, with MMP1 having the smallest area (1850 Å2) and MMP17 the largest area (2740 Å2). As for individual contributions, it appears that all three possible inter-domain (catalytic-bridge, catalytic-hemopexin and bridge-hemopexin) contacts contribute similarly to the total 28


Andreini et al.

Figure 7. Area (Å2) of the interface between domains for various MMPs. Note that the values for MMP2 were calculated on the experimental structure. The total area is shown, as well as the contributions from the contacts between individual domains: catalytic (Cat), hemopexin (HX), and the bridging region (Bridge).

area. The bridge region is defined as that between the two other domains, whose boundaries have been defined according to SMART. The hemopexin-bridge interaction is the smallest among the three (averaging 440 ( 70 Å2), whereas the catalyticbridge, and catalytic-hemopexin interfaces are more similar in size with an area, respectively, of 850 ( 200 Å2 and 1000 ( 250 Å2). The two most significant exceptions are MMPs 15 and 17, with a somewhat larger differentiation, possibly also due to the different size of the bridge region. The size of the interface area observed here is in the range of average recognition surfaces in protein-protein complexes.56 In keeping with the similarity of the interfacial area discussed above, the overall energetics of inter-domain interactions is quite similar for all MMPs (with a standard deviation of ( 35%). The electrostatic and van der Waals contributions to the energetics of inter-domain interactions is comparable for all MMPs in Figure 7, essentially within the same indetermination (( 35%). The energetic contribution of inter-domain hydrogen bonds is essentially negligible. There is no significant correlation between individual contributions to the inter-domain interaction and the overall energetics. Interestingly, the contribution to the energetics of hemopexin-bridge, catalyticbridge, and catalytic-hemopexin interactions are more similar than the interface areas, likely as a consequence of the shorter distance between the hemopexin domain and the bridge. Overall, the similar energetics of inter-domain interactions in different MMPs suggests that the plasticity of the inter-domain interface should be similar in the proteins considered. A further level of detail in the present analysis is given by the inspection of specific amino acid contacts at the domain interfaces. The most common contacts between residues at the interface of the different domains involve both polar and nonpolar amino acids. Quite surprisingly, there is little conservation of these residues among the various MMPs considered, with the nonpolar ones being marginally more conserved. Residues 452-453 in the catalytic domain make contacts to both the bridge and the hemopexin domains, whereas residue 527 in the bridge makes contacts to both the catalytic and the hemopexin domains. Thus, the above-mentioned three residues glue together the three MMP domains. Despite this important role, all three residues are somewhat variable among different MMP sequences, with nonconservative substitutions.

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Overall, these data indicate that there are no, or very few, specific interactions that are functionally important over the entire MMP family, but rather the key factors are the overall size of the interface and the number of contacts. Note that the lack of highly specific contacts and the fact that the interfacial area is in the average range for proteinprotein interactions indicate that the relative orientation of the domains may have a certain degree of flexibility. Thus, although it appears unlikely that in solution such orientation is strongly different from what observed in the present models, some minor variations such as relative rotation centered on the contact region can occur. Indeed it is also possible that interaction with the substrate or with inhibitors actually brings about some inter-domain rearrangements as part of the physiological mechanism of action of MMPs. In this respect, it is noteworthy that comparison of the structure of free35 and TIMP-2 bound38 MMP2 shows that the reciprocal orientation of the catalytic and hemopexin domains experiences only a minor variation upon TIMP-2 binding. Substrate Binding. The binding of physiological substrates such as collagen or gelatin to the MMPs, involving both the catalytic and the hemopexin domains, may take place by contacting both domains from the same side or, alternatively, by contacting each of them on different/opposite sides (i.e., with collagen sort of hugging the MMP). Little experimental information is available on this matter. In the first hypothesis, for the two domains to contact the substrate simultaneously, it is necessary either that the substrate bends appropriately, or that some inter-domain rearrangement takes place, which should be allowed by the flexible linker. This hypothesis would lead to the speculation that the similar surface shape of the non furin-regulated MMPs indicates a common mode of contributing to the interaction with the physiological substrates such as collagen or gelatin, that is different for MMP15 and MMP17. In the alternative hypothesis of collagen hugging MMPs to contact the two domains from different sides, it would be tempting to suggest that the common surface properties observed for essentially all MMPs in the bottom panel of Figure 6 are the consequence of its central negatively charged region being crucial for interaction with the substrate. In all cases, the subtle variation in surface charges observed at and around the various regions discussed above can be physiologically important to tune the substrate affinity and/or specificity of each MMP, or to tune its interaction with other proteins. Note that the above description of possible binding modes assumes limited inter-domain rearrangement in the substrate-bound form with respect to our models, which reflect the substratefree state. Even within the group of non furin-regulated MMPs, the features of the protein surface do not provide substantial hints on the possible molecular determinants for the fact that only some of them (MMP1, MMP8, MMP13) are capable of binding native collagen, whereas the others mostly bind denatured collagen or gelatin.2,25 Indeed, there is also no significant differentiation between these sequences in the phylogenetic tree of hemopexin domains, even though the MMPs that bind native collagen are closer to one another in this tree than in the tree of the catalytic domains. However, MMP1 and MMP13 are further to one another than with respect to MMP12, which does not even bind gelatin. It is to be noted that MMP12 has a less negative electrostatic potential at the hemopexin face in the top panel of Figure 6 than the other MMPs. The role of the hemopexin domain with respect to catalytic activity is quite variable. For MMP1 deletion of the

hemopexin domain removes its ability to degrade native type I collagen.57,58 The isolate hemopexin domain of MMP2 instead does not bind collagen54 but it does appear to have a role in catalysis.21 Indeed, truncated form of MMP259 and MMP960 lacking the hemopexin domain show a catalytic activity similar to that of the full-length enzymes. The hemopexin domain of MMP13 binds collagen, but its presence is not necessary for catalytic activity toward a number of native collagen types.61 In MMP8, the hemopexin domain has been shown to be important for substrate specificity, but not for catalytic activity.62 These data suggest that the role of the hemopexin domain and importance for catalytic activity may be significantly different in different MMPs, as well as for different collagen types. Inspection of hemopexin domain shape and surface properties, as well as of their sequence alignment does not provide clear-cut indications as to what modulates their role. At this point, it is interesting to reconsider the location onto the MMP surface (Figure 5) of the residues identified in sequence alignments as characteristics for differentiation between different MMPs classes. It appears that there are a few distinct clusters of such residues, some of which are at and around the catalytic zinc cavity and the putative contact regions. Because these residues were identified based on their conservation within specific classes of MMPs (as defined based on phylogenetic trees), together with their replacement in different classes, it can be proposed that the clusters identified in Figure 5 correspond to key regions in tuning the affinity toward the different substrates. Most of these amino acid substitutions differentiate trans-membrane and membraneanchored MMPs from all other MMPs’. For instance, most of the residues (204, 223, 229, 459) around the catalytic zinc cleft highlighted in yellow in the top panel of Figure 5 are consistently replaced by aromatic side chains in trans-membrane MMPs. Residues 229 and 459 are replaced by aromatics also in GPI-anchored MMPs. It is noteworthy that these substitutions do not alter significantly the electrostatic properties around the zinc ion (Figure 6). Interestingly, residue 457 is an aromatic only in non furin-regulated MMPs and MMP2, indicating that there are some compensating replacements. On the hemopexin domain, 598 and 777 are aromatics in transmembrane MMPs, where, in addition, residue 683 is a Trp. In the same systems, Pro607 is replaced by Lys/Arg thus introducing a positive charge. The two residues 627 and 628 are particularly interesting as they lie close to the center of the hemopexin face. Residue 628 has a negatively charged side chain in most non furin-regulated MMPs, whereas it loses its charge in the other MMPs (but MMP9, where there is a Lys at this position). Residue 627 on the other hand is positively charged in non furin-regulated MMPs, and is negatively charged in most other MMPs, with, again, the exception of MMP9 where it is not charged. On the opposite face of the hemopexin domain, residue 581 is mostly negatively charged in nonfurin regulated and trans-membrane MMPs, whereas it is replaced by Ala or Gly in the other MMPs.

Conclusions The availability of the complete human genome sequence has allowed us to examine the whole family of human MMPs both in terms of phylogenetic trees and of 3D structural features. The available experimental information on the structures has been significantly expanded by structural modeling, shedding further light on the role of the two largest domains and on their relationship. The main findings of the work can Journal of Proteome Research • Vol. 3, No. 1, 2004 29

research articles be summarized as follows: (i) The phylogenetic trees indicate that the evolution of the individual domains of MMPs is correlated. This is a further proof of the involvement of both catalytic and hemopexin domains in substrate recognition. (ii) The analysis of the surface properties of the domains show that there is a high degree of conservation in the catalytic domain around the active site cleft. The hemopexin domain and linker regions are instead more variable, with only some regions conserved. (iii) The variability of the surface shape and electrostatics of the hemopexin domain, where present, is most likely a significant factor in modulating substrate specificity. (iv) The total interfacial area is quite constant, as well as, with the exceptions of MMPs 15 and 17, the different contributions to it. Correspondingly, also the inter-domain interaction energies are similar for all MMPs. The analysis of residues making inter-domain contacts indicates the absence of conserved individual contacts, and suggests that the functionally important features of the interaction are probably in the number of contacts and the size of the area of the interface. Electrostatic and van der Waals interactions make comparable contributions to the inter-domain interaction energy. The latter is high enough to keep the two domains close to one another, but likely allows some degree of relative inter-domain motion that may be relevant for the function.

Acknowledgment. We acknowledge financial support from the Ministry of the University and Research, MIUR (projects FIRB-RBNE01TTJW and FIRB-RBNE01ZL3R) the Ministry of Health (project "Terapie innovative antitumorali basate sul blocco dell’invasione cellulare dell’angiogenesi" in collaboration with the Istituto Superiore di Sanita`), and from Ente Cassa di risparmio di Firenze, project “Development of methodologies of industrial interest in pharma-biotechnology”. Supporting Information Available: A figure depicting the multiple sequence alignment including all mature proteins, showing the residue numbering used in the present work, a table with PROCHECK parameters and PROSA Z-score for the templates used to build the structural models, and a table with computed inter-domain interaction energies. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Borkakoti, N. Prog. Biophys. Mol. Biol. 1998, 70, 73-94. (2) Sternlicht, M. D.; Werb, Z. Annu. Rev. Cell Dev. Biol. 2001, 17, 463-516. (3) Docherty, A. J.; O’Connell, J.; Crabbe, T.; Angal, S.; Murphy, G. Trends Biotechnol. 1992, 10, 200-207. (4) Hembry, R. M.; Murphy, G. J. Rheumatol. 1992, 19, 61-64. (5) Peress, N.; Perillo, E.; Zucker, S. J. Neuriphatol. Exp. Neurol. 1995, 54, 16-22. (6) Wernicke, D.; Seyfert, C.; Hinzmann, B.; Gromnica-Ihle, E. J. Rheumatol. 1996, 23, 590-595. (7) Shapiro, S. D. Curr. Opin. Cell Biol. 1998, 10, 602-608. (8) Egeblad, M.; Werb, Z. Nat. Rev. Cancer 2002, 2, 161-174. (9) Visse, R.; Nagase, H. Circ. Res. 2003, 92, 827-839. (10) King, M. K.; Coker, M. L.; Goldberg, A.; McElmurray, J. H.; Gunasighe, H. R.; Mukherjee, R.; Zile, M. R.; O’Neill, T. P.; Spinale, F. G. Circ. Res. 2003, 92, 177-185. (11) Daja, M. M.; Niu, X.; Zhao, Z.; Brown, J. M.; Russell, P. J. Prostate Cancer Prostatic Dis. 2003, 6, 15-26. (12) Jung, S. S.; Zhang, W. B.; Van Nostrand: W. E. J. Neurochem. 2003, 85, 1208-1215. (13) Lorenzl, S.; Albers, D. S.; Relkin, N.; Ngyuen, T.; Hilgenberg, S. L.; Chirichigno, J.; Cudkowicz, M. E.; Beal, M. F. Neurochem. Int. 2003, 43, 191-196. (14) McCawley, L. J.; Matrisian, L. M. Curr. Opin. Chem. Biol. 2001, 13, 534-540.

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Bioinformatic Comparison of Structures and Homology-Models of

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