Elena
Decaneto‡
ab,
Tatiana
Vasilevskaya‡
c,
Yuri
Kutin‡
a,
Hideaki
Ogata‡
a,
Moran
Grossman
b,
Irit
Sagi
d,
Martina
Havenith
b,
Wolfgang
Lubitz
a,
Walter
Thiel
c and
Nicholas
Cox
*ae
aMax Planck Institute for Chemical Energy Conversion, Stiftstraße. 34-36, D-45470, Mülheim an der Ruhr, Germany. E-mail: nicholas.cox@cec.mpg.de
bDepartment of Physical Chemistry II, Ruhr-Universität Bochum, 44780 Bochum, Germany
cMax-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, D-45470, Mülheim an der Ruhr, Germany
dDepartment of Biological Regulation, The Weizmann Institute of Science, Rehovot, Israel
eResearch School of Chemistry, Australian National University, Canberra, ACT 2601, Australia. E-mail: nick.cox@anu.edu.au
First published on 14th September 2017
Matrix metalloproteinases (MMP) are an important family of proteases which catalyze the degradation of extracellular matrix components. While the mechanism of peptide cleavage is well established, the process of enzyme regeneration, which represents the rate limiting step of the catalytic cycle, remains unresolved. This step involves the loss of the newly formed N-terminus (amine) and C-terminus (carboxylate) protein fragments from the site of catalysis coupled with the inclusion of one or more solvent waters. Here we report a novel crystal structure of membrane type I MMP (MT1-MMP or MMP-14), which includes a small peptide bound at the catalytic Zn site via its C-terminus. This structure models the initial product state formed immediately after peptide cleavage but before the final proton transfer to the bound amine; the amine is not present in our system and as such proton transfer cannot occur. Modeling of the protein, including earlier structural data of Bertini and coworkers [I. Bertini, et al., Angew. Chem., Int. Ed., 2006, 45, 7952–7955], suggests that the C-terminus of the peptide is positioned to form an H-bond network to the amine site, which is mediated by a single oxygen of the functionally important Glu240 residue, facilitating efficient proton transfer. Additional quantum chemical calculations complemented with magneto-optical and magnetic resonance spectroscopies clarify the role of two additional, non-catalytic first coordination sphere waters identified in the crystal structure. One of these auxiliary waters acts to stabilize key intermediates of the reaction, while the second is proposed to facilitate C-fragment release, triggered by protonation of the amine. Together these results complete the enzymatic cycle of MMPs and provide new design criteria for inhibitors with improved efficacy.
The MMP family of proteins has been extensively studied. The catalytic Zn2+ ion is located near the protein surface, with the cleft above the metal site allowing binding of substrate peptides. In its resting state the Zn ion is coordinated by three His residues (His239, His243, His249 for MT1-MMP) and at least one water molecule which serves as the nucleophile during substrate hydrolysis.7,8 Mechanistic studies at close-to-neutral pH revealed that this water molecule is strongly polarized forming a bridge between the conserved glutamate base (Glu240) and the Lewis acidic Zn.9–11
A four-step catalytic mechanism of peptide hydrolysis has been proposed for MMPs.7,12–14 Upon peptide docking (Fig. 1a, ES, 1) the reaction starts with the nucleophilic attack of the carbonyl carbon of the scissile bond by the catalytic water molecule. The side chain of Glu240 acts as a proton shuttle, transferring a proton of the glutamate-polarized water to the nitrogen atom of the scissile amide forming a metal penta-coordinated intermediate complex (ES‡, 2). The deprotonated water nucleophile then attacks the C–O bond of the peptide leading to cleavage of the C–N bond (3). This step is coupled to or followed by the transfer of the second water-derived proton to the amine group via the glutamate base. Theoretical modeling suggests that a second auxiliary solvent water molecule acts as an electrophile, stabilizing the newly formed carboxylate product.7 The last step corresponds to the release of the carboxylate product and the re-uptake of a water molecule. (Fig. 1a, 4).
Fig. 1 (a) Proposed catalytic mechanism of peptide hydrolysis of MMPs; (b) schematic illustration of the binding features of an acetohydroxamic acid (AHA) molecule to the catalytic zinc of MMPs. |
Crystallographic characterization of the intermediates of the reaction cycle has thus far been elusive. Bertini et al.15 have reported structures of two different MMPs where substrate was present, capturing the cleaved peptide fragments in the catalytic pocket. These are the earliest intermediates that can be characterized suggesting that the catalytic steps (Fig. 1a, 2, 3) are fast and the rate-limiting step instead represents product release (Fig. 1a, 4). This is in agreement with steady-state and pre-steady-state kinetics data from the tumor necrosis factor-a converting enzyme (TACE),16 another member of the MMP family. In addition, computational studies suggest that the loss of the bidentate C-terminus fragment from the Zn2+ during (Fig. 1a, 4) is the rate limiting step with an estimated barrier of 20 kcal mol−1;13 however this intermediate has yet to be characterized. The large barrier is derived from the negatively charged C-fragment coordinating the positive Zn2+ ion in a bidentate fashion. It is also not clear in what order the peptide fragments are released. The study by Bertini et al.15 characterized an intermediate with both C- and N-fragment in the catalytic pocket and a second intermediate with only the N-fragment suggesting, surprisingly, that C-fragment dissociation occurs before N-fragment dissociation. This has led to a concerted mechanism being proposed involving partial carboxylate detachment to initiate N-fragment release, coupled with solvent water insertion.
Pinpointing the protein structural elements that foster this progression has been challenging. Point mutations are found to strongly affect the rate of catalysis,17 particularly when replacing the glutamate base;18 however, as the relevant mutated residues are implicated in both the hydrolysis and product release steps, their precise role in enzyme reactivation is unclear. The role of auxiliary/solvent water in product release is also not resolved, nor is there good experimental evidence for auxiliary water molecule(s) invoked in theoretical treatments of the catalytic cycle. This is mainly due to the difficulty of crystallizing the protein in its native form owing to autocatalysis. As a consequence, MMPs are typically crystallized in the presence of various inhibitors including: regulator proteins (TIMPs),19,20 peptides and small synthetic molecules, such as hydroxamic acids, e.g. acetohydroxamic acid21 (AHA, Fig. 1b) with the latter chelating the catalytic zinc ion displacing the water nucleophile.22 To some extent the AHA-bound form of the enzyme models the ES‡ intermediate, at least in terms of the coordination of the Zn ion. However, as these inhibitors are small, they can be removed after crystallization by extensive washing to at least allow the solvation of the Zn ion in its resting state to be examined as achieved in the study of Bertini.15 Using this approach, both MMP-8 and MMP-12 have been characterized leading to two different descriptions of the resting state solvation sphere. In MMP-8 only one water ligand is resolved with full occupancy, while two further water molecules have only 20% occupancy. If the latter are neglected, the resulting geometry is a flattened tetrahedron. In contrast, the active site of MMP-12 contains three coordinating water molecules leading to an almost regular octahedral geometry of the Zn site.15 For MT1-MMP, the system used in this study, only an X-ray structure with a TIMP has been reported.19,20 The uninhibited coordination number has instead been inferred from stopped-flow X-ray absorption (XAS), which suggested that the metal is tetracoordinated, i.e. it only has one water ligand, the nucleophilic water.23
Regardless of the precise number of coordinating water molecules in the resting state, it is the presence of solvent in the ‘activated’ states of catalysis which is crucial to address function. Recent THz absorbance spectroscopy suggests that solvent water dynamics plays a crucial role for the enzymatic activity and protein recognition.23 Here we report the crystal structure of the catalytic domain of human MT1-MMP with a small peptide bound at its catalytic site, modeling the first product state after peptide cleavage. This novel structure identifies two auxiliary water molecules whose role in the enzymatic reaction was analyzed using optical, magnetic resonance and computational techniques. Together these data provide new information on the catalytic mechanism of MMPs and the importance of the immediate solvation sphere in directing catalysis.
The state-averaged complete active space self-consistent field (SA-CASSCF) method was used for calculation of on-site zero-field splitting (ZFS) parameters and of d–d transitions in Co(II) coordination complexes. The active space in the SA-CASSCF calculations included seven electrons and five 3d-based molecular orbitals. 10 (18) roots were considered for the quartet (doublet) state. Dynamic correlation effects were included by using second-order N-electron valence perturbation theory (NEVPT2).55,56 The ZORA Hamiltonian was employed in all these calculations, and the ZORA-recontracted version of the def2-TZVP basis set was used for all atoms. The spin–orbit coupling was represented by the spin–orbit mean field operator.57
Fig. 2 Superposition of the structures of the obtained MT1-MMP (blue) and the enzyme inhibited by TIMP-1 (red, PDB: 3MA2)20 and by TIMP-2 (green, PDB: 1BUV).19 The protein inhibitor TIMPs are not shown for clarity. Zn ions (catalytic Zn1 and structural Zn2) are colored in gold; Ca ions (Ca1 and Ca2) in cyan. |
The approximately spherical supercomplex contains four metal sites. Two sites contain zinc, whereas the other two sites contain calcium. The catalytic zinc is bound (Fig. 3) in the central part of the active-site cleft coordinated by the imidazole Nε2 nitrogens of His239, His243 and His249.7,12 As no inhibitor was included in the crystallization, the remaining ligands of the catalytic site should be solvent derived. This however was not the case. Additional electron density was found close to the active site pocket which could be fitted with a sequence of at least three histidines. This additional density is suspected to be associated with the His-tag engineered protein to allow purification (ESI,† S6). This His-tag was inserted into the flexible “hinge” region which links the catalytic domain to a C-terminal hemopexin domain.61In vitro maturation of the protein construct leads to autoproteolysis of the hinge region, which is not necessary for catalytic function.26 We suspect that the inadvertent presence of the residual His-tag in crystal preparations is required to stabilize the enzyme during the crystallization process as no His-tag free population was observed. We note that there is no evidence for the His-tag associating with the complex in solution and that the Zn/His-tag complex is not an intermediate of the hydrolysis reaction. Nevertheless, the structure obtained for the catalytic Zn site is novel. While it shares features with earlier inhibited structures using small synthetic molecules and peptides, it is the first to resolve multiple water interactions with the putative C-terminus product fragment, which computational modeling has predicted for the first product intermediate of the reaction cycle. A complete list of all reported MMP structures with a carboxylate bound at the catalytic Zn site is given in the ESI,† S6, see Table S2 and Fig. S13.
Fig. 3 Crystal structure of the catalytic zinc ion (Zn1) of MMP-MT1 with a small peptide (His-tag, yellow stick) bound as a bi-dentate ligand via its C-terminus (PDB entry 5H0U). The Zn has three protein derived ligands: His239, His243 and His249 and distant water ligands: a water in front of the page (Wf) and a water behind the page (Wb). |
The interaction of the His-tag with the protein cleft is shown in the ESI,† S6. The MMPs active site is characterized by three unprimed pockets (S1 to S3) and three primed pockets (S1′ to S3′) which drive the binding of the substrates. The primed pockets interact with the N-terminus fragment, while the C-terminus fragment instead interacts with the unprimed pockets. The interaction of the C-terminus fragment of the His-tag was found in the unprimed pockets (S1 and S2).
The two additional Zn sites are described in the ESI:† a structural zinc coordinated by the imidazole Nε2 nitrogens of His186 and His201, the Nδ1 nitrogen of His214, and one carboxylate oxygen of Asp188; and a third adventitious Zn binding site that was found on the surface of the protein exposed to the solvent and corresponds to a crystallization artifact. It is bound via the carboxylate oxygen of Asp248 and stabilized by one of the histidine residues from the His-tag (ESI,† S5 and S6).
(1) a hydrogen bond between the α oxygen of the carboxylate and the transiently protonated Glu240 residue;
(2) a hydrogen bond between the β oxygen of the carboxylate and a near-by auxiliary water molecule, which acts as an electrophile.
Both of these features have been seen in crystal structures of MMPs;62–64 in our structure the carboxylate C-terminus of the His-tag is in H-bonding distance to one oxygen of the Glu240 (2.6 Å) and an auxiliary water molecule, termed throughout this study as water in front (Wf) as it is located on the front face of the Zn ion in the projection where the three His residues are located below the Zn and the Glu240 sits behind the Zn on the left hand side (Fig. 3 and 4a). A survey of all structures reveals that the presence of Wf is correlated with the asymmetric bidentate carboxylate ligand motif (Zn–O carboxylate distances of ≈2.0 and ≈2.5 Å). In the complete set of MMP structures with an exogenous carboxylate ligand, the carboxylate coordination mode is plastic, binding as a symmetric bidentate ligand all the way to a monodentate ligand, see ESI,† S6, Table S2.
The more novel feature of our crystal structure is an additional auxiliary water molecule, termed throughout this study as water back (Wb) as it is located on the back face of the Zn ion. It interacts with both the Zn2+ ion and the 2nd coordination sphere proline residue. Such an auxiliary water molecule has only been observed once before, in a structure of MMP3 with a bound peptide.65 This earlier structure, however, did not contain Wf, nor did it stabilize a bidentate motif for the carboxylate. It instead more closely resembled the 1st product state that could be trapped in the study of Bertini, in which the carboxylate has already partially detached from the Zn2+ ion (mono-dentate). Wb in our structure is located such that it provides H-bonds to the α-oxygen of the C-terminus, the same as Wf. Owing to its location near Glu240, it represents the best candidate to displace/replace the bound C-fragment. We hypothesize that this process is triggered upon proton transfer from the Glu240 to the amine, breaking the H-bond between the Glu240 and C-terminus. A small rearrangement of Wb can then act to recover the H-bond network between Glu240 and the C-fragment, stabilizing an intermediate containing a monodentate carboxylate ligand (see Section 4). Within this interpretation, the MMP3 structure65 described above would then be representative of a later timepoint in this progression.
Lastly, we note that the position of the C-terminus/Glu240 couple in our structure also provides insight into how the C-fragment and N-fragment of the hydrolysis reaction likely interact immediately after peptide cleavage. The existing crystal structure of Bertini et al. locates the position of N-fragment (IAG) after the hydrolysis reaction. In our structure additional electron density is seen in approximately the same region, which we assigned to two cryoprotectant (glycerol) molecules. If instead this density is fitted to the IAG molecule, which is approximately the same size, the position of the amine head group in the 1st product state can be proposed. The nitrogen of the amine is within H-bonding distance of both the C-terminus (Oα) and the Glu240 (both 2.7 Å). We also note that in this model the Glu240 interacts with both the N-terminus and C-terminus fragments via a single oxygen (Fig. 4b). This is the same orientation for the Glu240 as seen in the earlier Bertini structures and in other MMPs with bound carboxylates (ESI,† S5 and S6). This is an important deviation from theoretical models of the first product state, which have instead assumed that the C- and N-fragments interact via a bidentate Glu240 (Fig. 4c) as is proposed for the gem-diol intermediate state. This result then implies that for the last proton transfer event, involving proton shuttling between the transiently protonated Glu240 and the amine, only requires one oxygen (the protonated one) of the Glu240, as recently suggested by Vasilevskaya et al.,13 making good chemical sense in so far that the proton transfer distance is significantly decreased using this motif.
Fig. 4 (a) Modeled position of a putative N-fragment associated with the catalytic site (steroview). The position of the N-fragment (IAG) was reoptimized from the structure(s) of Bertini et al.15 in the location of cryoprotectant molecules seen in our structure. (b and c) Possible proton transfer intermediates, with model b consistent with our crystallographic data. |
Importantly, for MMP homologues (e.g. MMP-1 and MMP-12) Zn/Co replacement does not change the coordination number/geometry of the active site.69,70 The protocol for replacing the “spectroscopically silent” Zn2+ ions in MT1-MMP with Co2+ is described in the ESI,† S3. The modified enzyme is fully active as demonstrated with a standard assay developed for MMPs based on a FRET mechanism.24,25 In this assay, the consumption of a fluorogenic collagen-like peptide leads to an increase of fluorescence at 400 nm allowing the reaction kinetics to be monitored. Interestingly the cobalt enzyme has a lower substrate turnover frequency of approximately 20% and a 2 fold increase of the Michaelis Menten constant KM, (36 ± 15 vs. 85 ± 8 μM, Fig. 5A and B). The increase in KM can be interpreted in a number of ways. As the rate limiting step of the reaction is the loss of the two peptide fragments from the protein cavity, the simplest enzymatic model requires four states (see Fig. 1): (i) free enzyme (E) + substrate (S); (ii) the enzyme–substrate complex (ES); (iii) the enzyme–product complex (EI); and (iv) free enzyme + product(s). Such a scenario can still be rationalized within the Michaelis Menten model, but KM includes a pre-factor (KM′) which takes into account the product dissociation step.
(1) |
(2) |
(3) |
Fig. 6 (a) Electronic absorption spectra of Co2+ substituted MT1-MMP. The dashed line represents an estimate of the scattering background due to protein aggregation. (b) Corresponding low temperature MCD spectrum (1.8 K, 7 T). (c) With AHA inhibitor. The red dashed lines represent a fitting of the spectra assuming eight bands modeled in terms of a Gaussian profile. Individual bands are colored green (catalytic), blue (structural) and grey (uncertain). See also ESI,† S7. |
To refine this picture, low temperature MCD of Co2+-substituted MT1-MMP was performed. These measurements identify multiple spin-allowed d–d transitions consistent with two or more protein-bound Co2+ ions (Fig. 6b). The profile could be modeled in terms of eight transitions. The addition of the inhibitor AHA was used to assign these transitions to either the catalytic site or the structural site; with the latter not being influenced by the binding of AHA (Fig. 6c). Bands 1–4 are all modified by the inhibitor and are thus assigned to the catalytic site: band 1 (17200) band 2 (18000) are significantly diminished or completely lost upon inhibitor binding whereas band 3 (20000) decreases in intensity by 50% and is shifted to the red (500 cm−1), and band 4 doubles in intensity (its position is not well defined in the non-inhibited form). Bands 5–8 are unchanged and thus assigned to the structural site (see ESI,† S7). It is noted that the structural site should also have some contribution in the 17000–21000 cm−1 region and thus the fitted bands should only be assigned dominantly to one species or the other. The electronic transitions observed represent the highest spin-allowed d–d transition of Co2+: i.e. transitions between electronic states derived from the ground state 4F and excited state 4P terms.95 For the free ion these levels are separated by 16800 cm−1 (15B), with the crystal/ligand field (10 Dq < 5000 cm−1) tuning their exact positions (Fig. 7).95 The bands assigned to the catalytic site in its uninhibited form best resemble those seen for 5 coordinate Co2+ complexes. Such complexes typically resolve two MCD features of negative ellipticity, derived from the splitting of the 4P term, here split by ≈2500 cm−1, supporting the assignment of bands 1–2 to 4A2′(F) → 4A2′(P) and band 3 to 4A2′(F)→ 4E′′(P).
The induction of MCD signals as a function of the temperature and applied field provides further support for this assignment.66,96,97 The MCD C-term activity of Co2+ complexes is dependent on the ordering and separation of the two Kramers doublets that describe its ground magnetic spin state. In the instance where the separation is large, only one Kramers state is populated at cryogenic temperatures (0–20 K) and thus the induction of the MCD signal – described by its magnetization curve – is temperature invariant (the magnetization curves overlay). In contrast, if the separation is small, the relative populations of the two Kramers states will significantly vary as a function of temperature (<100 cm−1) and as such its magnetization curves will vary with temperature; a phenomenon called ‘nesting’. The separation between the two Kramers states is defined by the zero-field splitting of the complex, which is defined by the parameter D. For Co2+ complexes the zero-field splitting comes about due to spin–orbit coupling (SOC) and its magnitude broadly correlates with the coordination number.66,96,97 For tetrahedral complexes, the singlet ground electronic state (4A2) is not strongly affected by SOC, whereas for octahedral complexes, the orbitally degenerate ground electronic state will split (to 1st order) due to the SOC mechanism; 5 coordinate complexes fall in between, with the SOC mechanism operating to 2nd order.97 This sets up a hierarchy of D values which Co2+-substituted enzymes typically follow: D is small (or negative) for tetrahedral complexes, moderate for 5 coordinate complexes and large for octahedral complexes, although there is significant overlap within these three ligand fields.66,96,97
Magnetization curves for these two bands (2 and 3) assigned to the catalytic state show nesting behavior consistent with a fine structure splitting value of approximately D = +25 cm−1, and thus consistent with a 5 and 4 coordinate Co2+ complex. We note that band 1 has a non-physical magnetization curve presumably due to the overlap with another unresolved feature (i.e. structural site). It is less clear what band 4 represents. This band does not display the same field and temperature dependence lacking the nesting behavior of bands 2 and 3, suggesting a large, positive fine structure value (D > +100 cm−1). This simple, weak feature may represent a second population of the catalytic site in which it adopts an octahedral coordination with a 4T1g(F) → 4T1g(P) transition. The presence of both these signals would then suggest that the resting state solvation of the Co2+ substituted enzyme is best described in terms of a dynamic 1st coordination sphere with two or three water molecules. Whether both the front and back waters can bind independently of each other in a 5 coordinate geometry, or if instead there is a preferred solvation complex in the 5-coordinate form cannot be deduced from this data.
Upon binding of the inhibitor, the spectrum collapses into a doublet centered at 19624 cm−1, although a shoulder may be present at 18000 cm−1, which could be an underlying structural site signal. This feature shows no nesting behavior (D > 130 cm−1), suggesting that the catalytic site with the inhibitor bound is best described in terms of an octahedral coordination. Both bands are assigned to a single complex, with the splitting due to a lowering of perfect octahedral symmetry. This requires the inhibited active site to have one water ligand. It is not clear whether the additional water binds on the front or back face of the complex (Fig. 8).
Subsequently pulse double resonance EPR experiments71 were performed to directly assess the number of water ligands that bind at the catalytic Co2+ ion and to examine whether these are displaced by AHA. Davies 1H-ENDOR measurements show that there is at least one auxiliary water ligand in both the inhibited and non-inhibited state, with hyperfine coupling (Adip) of about 3 MHz typical for aqueous transition metal complexes.99,100 It has a ‘Pake-like’ pattern indicative of a dipolar-coupled species (sitting perpendicular to the principal axis of the D tensor) that is lost upon deuteration. Both properties are only consistent with the presence of one or more water molecules in the 1st coordination sphere. These results immediately imply that the AHA form of the cofactor is 6 coordinate, with three His ligands, two ligands from the inhibitor and 1 auxiliary water, in agreement with MCD data. A second, more strongly coupled water, is resolved by 2H-ESEEM/HYSCORE (see Fig. 10B). The observed hyperfine coupling of 1.5 MHz for 2H corresponds to Aobs ≈ 9.8 MHz for 1H due to the difference in the gyromagnetic ratios. We reason that the signal is too broad to be readily observed in the corresponding 1H-ENDOR experiment. Indeed we encountered a similar situation for the Mn/Fe cofactor of the R2lox protein.101 The hyperfine coupling of this 1st coordination sphere water is approximately the same as observed earlier for the Zn finger protein Transcription Factor IIIA98 and is assigned to the catalytic water. These results imply that the cofactor must be at least 5 coordinate in the non-inhibited state, with three His ligands, the polarized/catalytic water and at least 1 auxiliary water. We find that the 1.5 MHz signal is lost upon AHA binding (see Fig. 10C), replaced by a smaller coupling of 1.1 MHz, presumably the OH of the AHA. As the 1.5 MHz coupling is completely abolished, AHA is bound to all catalytic sites at this concentration. One further observation can be made from this dataset. A non-exchangeable proton signal with a splitting of 3.4 MHz is also resolved in the 1H-ENDOR spectrum of the non-inhibited sample; this signal is not lost upon deuteration. It is likely derived from the non-exchangeable proton(s) of the histidine ligands. This signal is however lost upon binding of AHA, suggesting that the ligand field of the catalytic Co2+ changes upon binding of the inhibitor. This final result supports the notion that the non-inhibited state is different from the inhibited state, i.e. 5-coordinate, in agreement with MCD data. However as with the MCD measurements, EPR results do not show how the auxiliary water(s) binds in either the non-inhibited or inhibited state.
The validity of these models was tested by comparing the calculated D value for each model with experimental values determined from MCD measurements (Table 1). As expected, the tetrahedral models (C, D) yield D values of the wrong sign and magnitude and can thus be discarded. One 5-coordinate model (E), in which Wn directly interacts with Glu240 yields a positive intermediate D value (+33 cm−1) in agreement with the dominant species observed by MCD. The other 5-coordinate models in which an additional water is located between Wn and Glu240 (F and G) yield negative D values and are thus not supported. Finally, the 6-coordinate models (H, I) yield large positive D values, larger than 80 cm−1. These models could therefore explain the second species that may be present in the MCD data. We note that other calculated magnetic parameters (e.g.59Co and 14N hyperfine constants) were not discriminatory in terms of coordination/solvation number assignment.
CASSCF | NEVPT2 | |||
---|---|---|---|---|
D | E/D | D | E/D | |
C | +11.8 | 0.27 | +10.0 | 0.28 |
D | −16.8 | 0.10 | −14.2 | 0.11 |
E | +39.1 | 0.01 | +33.4 | 0.04 |
F | −20.3 | 0.30 | ±16.2 | 0.33 |
G | −27.3 | 0.12 | −23.3 | 0.10 |
H | +119.2 | 0.07 | +110.5 | 0.10 |
I | +96.1 | 0.19 | +84.0 | 0.21 |
J | −22.2 | 0.29 | −18.9 | 0.28 |
K | +89.2 | 0.29 | ±83.2 | 0.33 |
Exp. (native) | D = +24.5 cm−1; E/D = 0.10 | |||
Exp. (inhibited) | D > +100 cm−1; E/D = 0.13 |
We considered two further computational models (J, K) to investigate how auxiliary water interacts with the cofactor in the presence of bound AHA: model J does not include an additional first coordination sphere (auxiliary) water, whereas model K does. The hydroxamate anion (CONHO−) binds bidentately to the catalytic Zn2+ ion leading to a distorted trigonal-bipyramidal geometry around the metal, and it also forms a hydrogen bond with the carboxylate group of Glu240. The –NH– group forms another hydrogen bond with the carbonyl oxygen of Ala200, and van der Waals interactions and hydrophobic contacts play a critical role in stabilizing the inhibitor-enzyme complex.102 In the QM/MM optimized geometry for model K, water binds only to the front face (Wf). Comparison between computed and experimental MCD parameters shows that, as expected, model J is inconsistent with MCD data yielding a small negative D value. In contrast, structure K is consistent with the experimentally observed D value, i.e. >100 cm−1, derived from the MCD measurements and is in agreement with ENDOR results that showed the AHA complex has one first coordination sphere water. Structure K represents a genuine octahedral complex, with a water ligand stabilizing the bidentate ligation of the AHA. Thus all magneto-optical and magnetic resonance data presented above can be interpreted as follows:
(1) the solvation of the non-inhibited state is such that the Co/Zn ion is 5-coordinate (with only the Wb auxiliary water bound) which may be in equilibrium with a 6-coordinate population (with both auxiliary waters bound);
(2) the solvation of the inhibited state is such that the Co/Zn ion has one auxiliary water bound (Wf) rendering the complex 6-coordinate.
Finally it should be emphasized that QM/MM geometry optimizations with Co2+ instead of Zn2+ converged to qualitatively identical structures with a maximum absolute difference in bond length of 0.18 Å (ESI,† S9).
Fig. 12 Complete reaction cycle for MMPs including the sequence of steps involved in product release and the role of auxiliary/solvent water in this process. Structures (a, resting), (g and h) represent intermediates trapped in the Bertini study.15 Structures (b) and (c) show the peptide binding and cleavage steps. Structure (d) represents the 1st product state of the reaction following peptide cleavage, developed in this study. Structures (e) and (f) show two possible resolvation pathways, with only (e) leading to C-fragment loss. |
We favour a resting state structure that has at least one additional, non-catalytic water molecule (Wb, Fig. 12a). A third water ligand (Wf) may also associate with the Zn2+ rendering the site octahedral. The dynamic nature of the solvation sphere – under physiological conditions – is not surprising and has literature precedent.8,23 Importantly, we find that Wb more readily associates with the metal site than Wf. In all 5-coordinate DFT models the second solvating water is Wb rather than Wf, presumably because Wb forms an H-bond to the second sphere Pro259 residue, whereas no such interaction with the protein is available for Wf. Both waters are displaced by the bulky substrate, which is found to coordinate in a bidentate fashion leading to the gem-diol intermediate (Fig. 12b and c). Following the cleavage of the peptide backbone the first predicted intermediate contains a newly formed C-terminus of one product fragment coordinating the Zn2+ as an asymmetric bidentate ligand (Fig. 12d). The bound carboxylate is stabilized by two interactions: (i) an H-bond between the α-oxygen of the carboxylate and transiently protonated Glu240; and (ii) an H-bond to the β-oxygen of the carboxylate and an auxiliary water. Both these structural elements are found in the crystal structure we report here, with the auxiliary water bound on the front face (Wf). Our structure deviates from the original computational proposal7 in two important ways:
(1) The position of the Glu240 prohibits it from forming a bidentate H-bonding network to the amine product, as is proposed in the gem-diol intermediate.
Instead, we can model a monodentate H-bonding bridge between the two protein fragments. We speculate that, for this step in the cycle, an H-bonding interaction involving only the transiently protonated oxygen of the Glu240 would assist the subsequent proton transfer to the amine fragment as the tunneling distance is significantly smaller.
(2) The structure includes a second water (Wb) which is also within H-bonding distance to the α-oxygen of the carboxylate, further participating in stabilizing the asymmetric carboxylate binding motif.
We suspect that this water represents the water used as the nucleophile (Wn) in the next reaction cycle as it is uniquely placed to displace the α-oxygen of the product carboxylate. We speculate that the trigger for this event is the proton movement to the amine. Upon proton transfer, Wb moves within the coordination sphere of the Zn2+ (approx. 2 Å) to replace the lost H-bonding network between the α-oxygen of the product and the Glu240. Simultaneously it may also gain an H-bond to the amine. At this point the water can displace the α-oxygen forming the first experimentally characterized intermediate of the reaction, a mono-dentate 5-coordinate complex reported by Bertini and coworkers (Fig. 12g). This structure also exhibits a favourable H-bonding network between both product fragments facilitated by the incoming/relocated Wb and the Glu240. The mono-dentate C-terminus fragment is then released from the Zn2+ site, perhaps in competition with solvent, i.e. water binding on the back face, as seen in our resting state models (Fig. 12h). We note though that such a water molecule was not observed in the Bertini structure.15 Nevertheless, carboxylate loss would strengthen the Zn2+ water (Wn) interaction, shortening the Wn–Zn2+ bond and disrupting its interaction with the amine. The amine, which at this point only weakly associates with the Glu240 (via H-bonding) is then also released.
The above sequence suggests that the C-fragment loss can be short-circuited slowing enzymatic turnover. Following proton transfer to the amine destabilizing the Zn-carboxylate complex, the solvation sphere can respond in more than one way. Instead of Wb moving to restore the H-bonding network involving the Glu240 as described above (Fig. 12e), Wf could instead directly ligate the Zn2+ ion rendering the complex 6-coordinate, thus preventing the release of the carboxylate (Fig. 12f). This is indeed what we observe for the Co enzyme inhibited with AHA – an additional water binds on the front face rendering the complex 6-coordinate. Presumably both re-solvation pathways are in equilibrium, however we suspect that Wf binding is more likely to occur for the Co2+ enzyme as opposed to the native Zn2+ form, owing to the higher coordination number preference of Co2+. This simple rationale would then explain the slower turnover of the Co2+ enzyme. Thus, the solvation and inhibition of the cofactor via small synthetic molecules are likely interrelated providing another crucial element needed for understanding the affinity of inhibitors of MMPs and their rational design. The strategy would be to ensure that the inhibitor mimics substrate/solvent interactions, either by replicating 1st solvation sphere interactions, or by the inclusion of functional groups which can replace the 1st solvation shell and its H-bonding to the protein surface. It is less clear how solvent/substrate interactions can be exploited toward selective inhibition of MMPs. This forms ongoing work of our laboratory.
Footnotes |
† Electronic supplementary information (ESI) available: (S1) Expression and purification of the active catalytic domain of MT1-MMP; (S2) molar extinction coefficient determination for wild type MT1-MMP; (S3) preparation of apo-MT1-MMP (metal-free material)/preparation of Co2+-MT1-MMP; (S4) inhibition of Co2+-MT1-MMP by acetohydroxamic acid (AHA); (S5) X-ray data-collection statistics for MT1-MMP; (S6) additional crystallographic figures. Comparison of the X-ray structure of MT1-MMP to literature MMP structures; (S7) analysis of Co2+ EPR and MCD spectra from wild type and inhibited Co2+-MT1-MMP; (S8) QM/MM geometry optimizations of structural models A-K; (S9) comparison of computed key distances in Co2+ and Zn2+ QM/MM models; (S10) optimized QM/MM geometries (QM part); (S11) optimized model geometries used for EPR parameter calculations; (S12) calibration of computed cobalt isotropic hyperfine coupling constants; (S13) references. See DOI: 10.1039/c7cp05572b |
‡ ED (biochemistry), TV (computations), YK (EPR) and HO (crystallography) made complementary and equal contributions to the manuscript. |
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