Screening Using Polymorphs for the Crystallization ... - ACS Publications

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Screening Using Polymorphs for the Crystallization of Protein− Ligand Complexes Published as part of the Crystal Growth & Design virtual special issue on the 14th International Conference on the Crystallization of Biological Macromolecules (ICCBM14). Laura Vera,† Claudia Antoni,†,‡ Laurent Devel,† Bertrand Czarny,† Evelyn Cassar-Lajeunesse,† Armando Rossello,‡ Vincent Dive,† and Enrico A. Stura*,† †

CEA, iBiTec-S, Service d’Ingénierie Moléculaire des Protéines (SIMOPRO), Gif-sur-Yvette, F-91191, France Dipartimento di Farmacia, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy

‡

S Supporting Information *

ABSTRACT: An efficient crystallization screening method is important in drug design to yield high resolution crystallographic structures of protein−ligand complexes to understand inhibitor selectivity and potency for various members of an enzyme family. The strategy starts with a single condition for each protein−ligand complex, and more trials encompassing all polymorph crystallization conditions are included later, eventually defaulting to a more extensive screening for difficult cases. The polymorph screening approach relies on an intrinsic positive feedback mechanism. New polymorphs are constantly discovered since certain ligands favor variant lattices. The new best diffracting polymorph is selected for single-conditions testing, ensuring that as more forms are discovered, the resolution of the structures obtained improves. Continual optimization of the conditions for all crystal forms yields new solutions that become increasingly effective in protein−ligand crystallization trials. More polymorphs imply more lattices suitable to accommodate ligands of greater diversity. Wider seeding opportunities combined with optimized enzyme-specific crystallization conditions improves the outcome and accelerates the screening process so that a conventional full-range crystallization screening is only rarely needed. Having tested this approach with a large repertoire of 100 ligands and 4 enzymes, we expect the method to perform equally well on similar drug-discovery projects.

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INTRODUCTION In humans, the matrix metalloproteinase (MMP) family is composed by 23 closely related proteins that share a catalytic zinc ion and the typical metzincin fold.1 Since a variety of pathological states correlate with abnormal activity of these enzymes, synthetic compounds have been developed to bring these enzymes under control.2,3 The need for selectivity is evidenced from clinical administration of broad-spectrum inhibitors that has resulted in severe adverse effects.4 Improved selectivity has been achieved by moving away from ligands with the powerful hydroxamate zinc-chelating group in favor of inhibitors with phosphinic,5 carboxylic acids,6 and pyrimidine trione7 chelating groups or without any chelating group, as in the case of MMP-13.8 Examples of each of these inhibitor classes can be found among the 100 or so compounds we are using in our structural comparative studies. The number of MMP catalytic domains that we can currently produce is limited to MMP-8, MMP-9, MMP-12, and MMP-13. For MMP-13, we have reported the structure of the full form of inactive enzyme (E223A) in complex with peptides.9 Inactive forms, where the active site glutamate is replaced by an alanine, are important to study © 2013 American Chemical Society

enzyme−substrate interactions. Selective inhibitors that inhibit strongly one member of the family can be 3 orders of magnitude less effective on other members. To reduce proteolytic self-degradation during crystal growth, when studying low potency inhibitors, a less active enzyme, with a glutamine instead of a catalytic glutamate, is chosen. The combinatorial possibilities of enzyme constructs and ligands is so vast that a strategy is needed to reduce its complexity. For each of the enzymes in our study, sequence, crystallization conditions, and a number of structures have been reported.7,8,10−12 The details of the constructs used in prior studies were taken into account to select the most suitable forms for crystallization, and the reported crystallization conditions were used to guide the initial screens. This approach did not work reliably. Although MMP-9 had been crystallized previously (PDB code: 2OVZ7), its crystallization proved problematic even with the same sequence, a related inhibitor, and identical methodology. In the context of our drug-design Received: October 19, 2012 Revised: January 18, 2013 Published: March 5, 2013 1878

dx.doi.org/10.1021/cg301537n | Cryst. Growth Des. 2013, 13, 1878−1888

Crystal Growth & Design

Article

Figure 1. Flow-chart showing the steps in single drop screening for all MMPs in this study. The success rate for the various experiments is given as a percentage from 88 cocrystallized MMP-12 complexes. Seeding is extremely important since spontaneous nucleation occurs in only a small number of trials. The crystallization conditions are selected to improve the chances of obtaining large crystals suitable for X-ray diffraction experiments from single drop screening (including streak-seeding). Because certain inhibitor increase the nucleation probability, in 5% of experiments we get spontaneous nucleation and crystals suitable for X-ray data collection. The self-nucleated crystals belong often most frequently to the P21212 polymorph. The space group most frequently reported in the PDB by other groups is for C2 polymorph (Table 1). The success rate (resolution better than 2 Å) for streak seeded single drops is 12%. With 6 drop bracketing, involving pH and precipitant concentration variations, the success rate improves to 75%. The 2% rate for the emergence of new polymorphs with MMP-12 is a low figure. Most MMP-12 crystallizations have been carried out before the application of the polymorph-based screening with the six-drop bracketing strategy instead. The results obtained for the other MMPs suggest that new may polymorphs emerge with greater frequency. For MMP-9 we have solved only 25 complexes to date: the crystals have been grown in eight different space groups not reported in the PDB database by other groups. For MMP-8 with 42 complex structures solved to date (some from ligand soaking) we are working with 8 polymorphs. The latest project, MMP-13 with only 13 complexes, we are now working with 7 polymorphs.

project, the 4−12 weeks mentioned in original report7 for growing crystals was considered inappropriate. While searching for a new strategy to speed-up the process, we found that, for MMP-9, screening a large ensemble of inhibitors with a limited number of conditions was more effective than screening one complex with numerous crystallization conditions. The fastest nucleation was obtained with MMP-9 in complex with a bifunctional inhibitor.13 Microcrystals nucleated reliably but were difficult to improve. Streak seeding14 other MMP-9 complexes with seeds from these microcrystals yielded cocrystals that could be easily optimized. Extensive streak seeding yielded many polymorphs useful for further screening. The strategy developed for MMP-9 was applied to all other MMP-inhibitor complexes, with excellent success yielding crystals for almost all inhibitors in the sub-micromolar range. Crystallization of complexes with poor affinity inhibitors remain problematic. Extremely hydrophobic ligands also need special consideration for their solubilization in aqueous solutions.15 In materials science, polymorphism indicates the existence of a solid material in more than one form or crystal structure. Here we show that polymorphism is extremely common in protein crystallization. Understanding the implications of

extensive polymorphism in protein crystals can be exploited to devise new screening strategies as reported here.

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EXPERIMENTAL PROCEDURES

Constructs. The synthetic gene encoding the “mini-catalytic domain” of MMP-9 comprising residues Met109−Gly215 and Gln391− Gly444, without the additional fibronectin domains, was cloned into a pET-14b vector for expression under the PT7 promoter (Novagen). Synthetic genes encoding the catalytic domain of the metalloelastase (cdMMP-12) Met98−Lys266, the truncated pro-collagenase-3 (proMMP-13) Leu20−Asp272, and the truncated pro-collagenase-2 (proMMP-8) Phe21−Gly262 were obtained from Geneart (Geneart-AG, Germany). These genes were inserted into the pET24a vector for expression under the PT7 promoter, between the NdeI and BamHI sites. All plasmids were propagated in the Escherichia coli strain XL1Blue at 37 °C. Mutagenesis. Mutagenesis of inactive cdMMP-12 E219Q (primer 5′-tttctgaccgcggtgcatcagattggccatag-cct-3′) or inactive cdMMP-9 E402Q (primer 5′-cgtggcggcgcatcagttcggcca-3′) were performed using a site-directed mutagenesis kit from Stratagene (Heidelberg, Germany). The construct was verified by DNA sequencing using the ABI PRISM 310 Genetic analyzer (Applied Biosystem). Expression and Refolding. Recombinant catalytic domain of the various MMPs were expressed in E. coli cells BL21 (DE3 star) carrying the MMP catalytic domain encoding plasmids. Bacteria were grown in 1879



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LB medium containing ampicillin (50 μg/mL) for MMP-9 and kanamycin (50 μg/mL) for the other MMPs at 37 °C. At an absorbance of 0.6 (λ = 600 nm), protein expression was induced with 0.5 mM isopropyl-β-thiogalactopyranoside (IPTG). Five hours after induction the cells were harvested by centrifugation at 5000g for 30 min at 4 °C. The pellet was resuspended in buffer A (5 mM benzamidinochloride, 5 mM β-mercaptoethanol, 100 mM Tris-HCl, pH 8.5) and incubated with lysozyme for 1 h at 4 °C. The suspension was then passed through a cell disruption system (Constant Systems Ltd., Daventry Northants, England) and centrifuged for 30 min at 8000g at 4 °C. The pellet was washed three times with buffer B (2 M urea, 5 mM β-mercaptoethanol, 100 mM Tris-HCl, pH 8.5) and then dissolved in buffer C (8 M urea, 100 mM Tris-HCl, pH 8.5). After centrifugation, the concentration of supernatant was determined by the Bradford assay and adjusted in buffer C to 100−200 μg/mL. The solution was dialyzed for 3 h against 10 vol of buffer D (100 mM NaCl, 10 mM CaCl2, 0.1 mM ZnCl2, 3 M urea, 50 mM Tris-HCl, pH 7.5) and then overnight against 10 vol of buffer D1 (50 mM NaCl, 10 mM CaCl2, 0.1 mM ZnCl2, 50 mM Tris-HCl, pH 7.5) and finally 3 h against 10 volumes of buffer E (50 mM NaCl, 10 mM CaCl2, 50 mM Tris-HCl, pH 7.5) at 4 °C. The resulting dialysis of protein was centrifuged at 12000g for 40 min to eliminate precipitates. Purification of MMP Catalytic Domains. The refolded hMMP-9 wild-type catalytic domain and its active site glutamine mutant were purified to homogeneity over affinity chromatography with sepharosecoupled hydroxamate inhibitor (Pro-Leu-Gly-NHOH).16 The enzymes were eluted from the affinity column with a buffer consisting of 20 mM Tris buffer pH 11.0 into a tube with 1 M Tris buffer pH 7.0. Fractions containing the protein were pooled and concentrated for crystallization. The human MMP-8 catalytic domain was refolded as a pro-form and then treated with 1 mM 4-aminophenylmercuric acetate (APMA) for 2 h at 37 °C, to yield an active catalytic domain consisting of residues Gly84 to Gly262. The concentrated cdMMP-8 was finally purified on a HiPrep 16/60 Sephacryl S-100 HR column (GE Healthcare, France) and concentrated for crystallization. The human MMP-13 pro-form of the catalytic domain was activated directly during the refolding without autolysis to produce the catalytic domain comprising residues Tyr104 to Asp272. The concentrated cdMMP-13 was then purified by a HiPrep 16/60 Sephacryl S-100 HR column (GE Healthcare, France) and concentrated for crystallization. The human MMP-12 catalytic domain and catalytic site mutants (E219Q and E219A) were used directly after refolding without a purification step. To prevent self-degradation 1−10 mM acetohydroxamic acid (AHA; PDB code HAE) was added to the proteinases during the concentration step using an Amicon stirred ultrafiltration cell with a 3500 Da molecular weight cutoff ultrafiltration regenerated cellulose disk membrane (Millipore, Billerica, MA, USA). Characterization. Refolded proteins were analyzed by SDS-PAGE and were found to migrate as a single band. Protein molecular weights were determined by ESI-MS (Bruker esquire HCT 8000), and the presence of the mutation was verified by MALDI-TOF (Applied Biosystem, MALDI-TOF-TOF 4800) after a tryptic digestion. Protein concentrations were determined by the Bradford assay, using a BioRad reagent, and verified by OD at 278 nm. The activity of MMPs was determined by titration with a specific inhibitor, and using a fluorimetric substrate (Mca-R-P-K-P-V-E-Nval-W-R-K(Dnp)-NH2). Crystallization Experiments. Crystallization experiments were typically carried out in CrysChem sitting drop vapor diffusion plates with protein drops of 1 and 1 μL precipitant solution. The vapor diffusion trays are stored in a cooled incubator at 20 °C. Initial screening carried out on each of MMP-8, MMP-9, MMP-12, and MMP-13 was based on a review of previously published crystallization conditions7,8,10−12 analyzed in a systematic manner using the principles of reverse screening17 but selecting conditions from the standard Stura Screens (Molecular Dimensions, Ltd.).18 Typically, 7 μL of protein−ligand sample is mixed, 1 μL is used for single crystallization condition screening, the remaining volume is used for further screening or for optimization. Crystals were systematically optimized by streak seeding.14

MMP-9 Screening and Crystallization. Initial screening with MMP-9 was carried out with the MMP-12 selective phosphinic inhibitor RXP470.1 (Protein Database (PDB) ligand code R47; ligand 2D chemical structures can be obtained from the PDB and found in Supporting Information)5 whose affinity for MMP-9 is only 1.76 μM. Although RXP470.1 shares many similarities with AM-409 (PDB entry OVZ; ligand code 5MR), crystallized with MMP-9 and having an affinity for the enzyme of 0.020 μM, no crystals were observed within one week. Once it was realized that it would not have been possible to carry out the inhibitor screening project with crystals that appear after 4−12 weeks,7 a more extensive screening was carried out with the same inhibitor without success. Since lack of success was probably due to the poor affinity of RXP470.1 for MMP-9,19 in later tests related analogues with higher affinity were used. The new strategy consisted of a 6-condition screening on a large ensemble of MMP-9-inhibitor complexes. Various strategies are used to select the six screening or optimization conditions. In the “reverse screening recombination” approach, the six screening conditions are a combination of two working solutions17 A and B mixed in different ratios in a linear fashion (i.e., A, 80%A/20%B, 60%A/40%B ... 20%A/80%B, B). An example of two such solutions is N8WS4 (17.5% PEG 20000, 125 mM NaCl, 100 mM MES, pH 5.5) and N9WS3 (45% MPEG 5000, 200 mM imidazole piperidine, pH 8.5). This allows screening pH values between 5.5 and 8.5. An alternative approach used more often for optimization than for screening involves bracketing around previously successful conditions (3 different pHs; higher and lower PEG concentrations). Given the coarseness of the limited screen, most complexes did not give any crystals except for a hydroxamic acid-based bifunctional ligand, MP24 (PDB ligand code 0Y3), consisting of two inhibitors linked together with a spacer.13 By bracketing around the determined crystallization conditions and by using streak seeding,14 several other MMP-9 complexes were also crystallized. Largely, the inhibitors had a chemical substructure (see Supporting Information for 2D chemical structures) related to the monomeric constituents of the bifunctional inhibitor. Because seeding can be followed by an epitaxial jump,20 where related lattices are selected instead of duplicating with fidelity that of the original seeds, new polymorphs were often generated. These MMP-9 polymorphs were used in further seeding experiments. A minor modification to the screening strategy was used to get crystallization conditions for the MMP-9 complexes with 470B (PDB ligand code R4B; Supporting Information; affinity for MMP-9: 0.055 μM) and 470C (affinity for MMP-9 0.340 μM; PDB ligand code R4C; Supporting Information) two analogues of RXP470.1 (R47).19 After this complex failed to crystallize using the approach used so far for the other ligand complexes seeded from the MP24 initial cocrystals, a different “reverse screening recombination” was used. Since R4C and 5MR7 are both phosphinic inhibitors the screening conditions were chosen by mixing the crystallization conditions for PDB entry OVZ7 (3.0 M NaCl, 0.1 M Tris, pH 8.0) with N8WS4. Crystals nucleated spontaneously at the lower pH values. A new working solution, N9WS11 (3.5 M NaCl, 100 mM MES, pH 5.5) was mixed. With this solution nucleation of 470C cocrystals was instantaneous, and the rapidity of nucleation allowed for fast optimization of the crystallization conditions. The rate of nucleation was later slowed down by reducing the precipitant concentration, varying the concentration of NaCl, and adding Li2SO4. The pH range was screened with the linear buffer combination PCTP (Na propionate, Na cacodylate, Bis-Tris-propane).21 The final optimized conditions were 1.9 M NaCl, 10% PEG 20 K, 100 mM PCTP (Na propionate, Na cacodylate, Bis-Tris-propane 80% at pH 4; 20% at pH 9.5), 40 mM Li2SO4. Streak seeding from these crystals into drops of another other phosphinic inhibitor (R4B; Supporting Information) complex gave also a second C2221 polymorph (Table 2). The best crystals were obtained from 1.9 M NaCl, 100 mM PCTP (80% pH 4.0/20% pH 9.5), 40 mM Li2SO4, 10% PEG 20,000 without seeding. Flash Cooling. Crystals are soaked for short or long periods in selected mixtures of cryoprotectant compounds15 to optimize the resolution to which they diffract. Crystals are retrieved from the cryoprotectant solution with a cryo-loop and plunged in liquid 1880



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Table 1. Space Groups Reported for MMP-12 Catalytic Domainsa MMP12 polymorphism space group C2 P21212 P21212 P212121 P212121 P21 P21 P31 P6322 I222 P4132

cell dimensions (Å) 51 60 54 69 63 38 67.8 61.5 33.5 46.9 61.8 112.6 42 63 113.2 47.4 106.5 65.9 39.7 60.6 58.4 123.8 123.8 69.7 123.2 123.2 168.2 67.4 87.2 169.2 125 125 125

angles (deg) 90 115 90 90 90 90 90 90 90 90 90 90 90

90 90 90 90 90 90 90 90 95 90 103.1 90 90 120 90 120 90 90 90 90

Z 4 4 4 8 8 8 4 18 24 16 24

PBD codes 11

b

12

1JK3, 1Y93, 2OXZ,A1 2OXW,A1 2OXU,A1 2HU6,A2 3EHX,A3 3EHY,A3 3F15,A4 3F16,A4 3F17,A4 3F18,A4 3F19,A4 3F1A,A4 3LK8,A4 3LKA,A5 3N2U,A6 3N2V,A6 3NX7,A6 3RTS,A7 c32 A7 A7

3RTT, 4GUY, 4I03 1RMZ,12 3LIK,6 3LIL,6 3LIR,6 3LJG,6 3TS4,6 3TSK,6 4EFS,6 4GR0,c19 4GR3,c19 4GQLc19 4GR8c,e19 4H30c30 4H84c30 4H49c30 3UVC, TBDd27 1OS2,eA8 1OS9eA8 1UTZeA9 1JIZeA10 1UTTeA9

highest resolution (Å) 1.03 1.15 1.31 1.51 1.43 2.16 1.80 1.85 2.50 2.60 2.20

a

The constructs have minor variations at the amino and carboxy termini and the mutation F171D. bA1−A10 refer to additional references available in Supporting Information. cThis study; contributions from our laboratory are in bold. dTBD: not yet deposited; cell parameters reported in Vera et al. 2011.27 eMMP12 with sequence or terminus variations.

Figure 2. Comparison of the packing of molecules of MMP-12 in four crystal lattices. (a) The P21212 MMP-12 polymorph is most commonly observed crystal form for self-nucleated crystals in our laboratory and the most common form used for the crystallization of peptide based inhibitors and for seeding of other ligands. The packing can accommodate a certain amount of self-degradation at the N-terminus. Such degradation results in a translation along a major crystal packing direction (compare 470C: PDB entry 4GR8 in green and 470A: PDB entry 4GR3 in gray). The two lattices are sufficiently similar to allow seeds of one form to induce nucleation of the other lattice. In this packing the ligands are well separated from each other. (b) In the C2 form, the phenyl rings of two symmetry related batimastat (PDB entry 1JK3; ligand code BAT) molecules are in van der Waals contact. Such interaction is not required to obtain crystals in this crystal form since even small inhibitors like acetohydroxamic acid (AHA) (PDB code 1Y93)12 can adopt this packing. The ease with which this small weak binding inhibitor can be replaced by other ligands make this form suitable for soaking experiments. It is well represented in the PDB (Table 1). The packing in the C2 and P21212 MMP-12 polymorphs are not related. (c) The MMP-12 packing in the P212121 space group observed for the complex with EN238 (PDB entry 4H84; ligand code Y38) is unrelated to either the C2 or the P21212 polymorphs. The ligand moieties are shown in yellow ball and stick representation with MMP-12 in a different orientation from that for (a) and (b) to better show the phenyl-isopropyl ligand−ligand interactions. (d) Another polymorph is obtained with a bifunctional inhibitor, LC20 (PDB entry 4H30; ligand code OZD). The space group and similarity in cell parameters between 4H30 and 4H84 is deceptive since these pack differently starting with a distinct homodimeric association. This reinforces the idea that coupling two inhibitors with a linker is an effective strategy for the creation of new polymorphs. Screening Strategy. Screening is carried out initially using a single reservoir conditions with all the ligands for which complex crystal structures were needed. The ligand is first added to the protein solution in a 1:1 stoichiometric ratio for monofunctional high affinity ligands and a 1:2 ratio for bifunctional ligands. Excess ligand is used for lower affinity inhibitors. The ligand-protein drop is dispensed in the vapor diffusion well and reservoir solution is layered on top without mixing. The crystallization conditions chosen for the single test correspond to the best diffracting crystals obtained in previous studies as are the seeds used for streak seeding.14 The seeding procedure is carried out within 10 min after setting-up the drops, just before the sitting drop reservoir is sealed. The crystals from which the seeds are picked-up are seldom older than three months. A few large crystals

nitrogen in ESRF pucks which are then stored in a Dewar of liquid nitrogen. Data Collection. More 70 inhibitors have been crystallized in complex with one of the four MMPs and found to be suitable for data collection. About 1000 samples were tested at synchrotron facilities, namely, at the ESRF (beamlines ID29, ID14-1, ID14-2, ID14-3, ID144, ID23-1, ID23-2, and BM30) in Grenoble and at the Soleil storage ring on beamline Proxima-1 in Saclay. Data processing was carried out using the automated system available at the synchrotron facilities or on the laboratory computers using either XDS22 or MOSFLM.23 Molecular replacement was carried out using MOLREP24 and refinement with with REFMAC25 or phenix.refine.26 1881



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Table 2. Space Groups Reported for MMP-9 Catalytic Domainsa MMP9 polymorphism space group P41212 P21 P21 P1 P21 P21212 P212121 C2221 C2221

cell dimensions (Å) 56.0 56.0 262.6 44.1 48.7 67.9 40.2 97.4 45.7e 39.9 98.9 47.1 45 49 136 74 99 46 34 57 172 68.5 69 85 57 76 94

e

angles (deg) 90.0 90.0 90.0 90.0 102.6 90.0 90.0 112.0 90.0 90.03 111.95 89.98 90.0 96.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0

PBD codesb

Z 16 4 4 4 4 8 8 8 8

A11

A11

7

7

highest resolution (Å) 7

7

1GKC, 1GKD, 2OVX, 2OVZ, 2OW0, 2OW1, 2OW2 4H1Qc30 4H2Ec30 4H3Xc30 4H82c30 TBDd 4HMAc30 4JIJc TBD (ligand R4C)d TBD (ligand R4B)d

7

2.00 1.59 1.76 1.90 1.76 1.94 1.70 1.97 2.43

a

The variation in the constructs are at the amino terminus. The catalytic Glu is often mutated to Gln to prevent autolysis. bA11 refers to an additional reference available in Supporting Information. cThis study; contributions from our laboratory are in bold. dTBD: not yet deposited; cell parameters may vary with refinement. eCell parameters correspond to the first PDB entry in the list.

Figure 3. Comparison of the packing of polymorphs of MMP-9. (a) The packing of two complexes of MMP-9 with two hydroxamate-based bifunctional inhibitors MP24 (PDB entry 4H2E; ligand code 0Y3) and LC29 (PDB entry 4H82; ligand code L29) in space groups P21 and P1, respectively. Although the lattice is essentially identical, crystals of LC29 in P1 diffract to 1.9 Å, while those grown in space group P21 for either MP24 or LC29 diffract to only 2.9 Å. (b) The packing in the P41212 space group (PDB entry 2OVZ; ligand code 5MR) does not match any crystal contact in the P21 and P1 space groups. A conserved interaction between MMP-9 molecules can be found with crystals grown in the C2221 space group with a similar phosphinic inhibitor (PDB ligand code R4B; Supporting Information). The ligands do not mediate the conserved interaction. (c) The homodimeric assembly is conserved between MMP-9 bound to bifunctional ligands both in a 1:1 and 1:2 association (comparison of PDB entries 4H82 and 4H1Q). Other lattice contacts are not duplicated. (d) Better conservation of MMP-9 lattice interactions is observed for the complex with the monofunctional ligand CC27 (PDB entry 4H3X; ligand code 10B). Two homodimers pack in a conservative manner, but a large translation differentiates a third packing interaction conserved in panel a). all new crystallization drops set up in the multicondition screen. In addition, 25 μL NaCl at 5 M is added to all reservoirs with clear drops. The seeding of these drops is repeated with seeds of the different polymorphs at appropriate time intervals that may vary from 6 h to 1 week. The success rate from the second screening and NaCl reservoir addition approaches 70−80% for ligands with good affinity. Ligands of lower affinity that do not provide sufficient protection from proteolytic self-degradation of the enzyme are tested with lower activity mutants

suitable for X-ray data collection are obtained from this initial screen (Figure 1). A reservoir refinement step based on reverse screening17 is carried out to improve the size and appearance of all complexes for which a line has been obtained as a result of streak seeding. For those ligands that have not responded to streak seeding under the single crystallization condition, a second screening is carried out consisting of individual crystallization conditions for all polymorphs available for the enzyme being crystallized. The seeding procedure is carried out using the same methodology as previously used for the single condition for 1882



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(E → Q) or inactive mutants (E → A; substrates and weakly inhibitory mini-proteins). Rescue Strategy. Further screening is carried with different PEGs, buffers, and salts for all those ligand-MMP complexes that are considered to be “promising” based on their precipitation pattern. This labeling as “promising” is arbitrary and the success rate for the further screening is less than 10%. The rescue becomes really effective as new polymorphs are discovered. The most successful polymorphs are often obtained with better affinity ligands related in chemical space. Identification of New Polymorphs. New potential polymorphs are obtained by spontaneous nucleation or via epitaxial jumps20 using ligands with special properties such as bifunctional ligands or inhibitors with substituents that cannot be accommodated in existing lattices. Since protein-precipitant drops are systematically streak seeded,14 all crystals found growing outside the seed line are potentially unrelated to the seeds. At times new polymorphs can be identified because their crystal habit is different from that of the seed crystals, but in most cases it is ascertained from X-ray diffraction studies.

determination for our complexes that we have reported (PDB codes: 3LIK, 3LJG, 3LIR, 3LIL, 3TS4, 3TSK, 4EFS)6 and have recently deposited in the protein database (PDB codes: 4GQL, 4GR0, 4GR3, and 4GR8)19 belong the P21212 space group. This polymorph is most frequently obtained by spontaneous nucleation and the crystal form used for the first seeding trial (Figure 2). Only a minority of inhibitors tested do not respond to seeding with the P21212 polymorph but grow spontaneously or only respond to seeding with the monoclinic C2 polymorph. Recently, two new polymorphs have been obtained in space group P212121 with cell parameters a = 46.9 Å; b = 61.8 Å; c = 112.6 Å (PDB entry: 4H30) and a = 42.0 Å; b = 63.0 Å; c = 113.2 Å (PDB entry: 4H84) corresponding to a bifunctionalized inhibitor and the same inhibitor with just the linker extension. These will be included in future seeding trials. Another polymorph of MMP-12 with the phosphinic inhibitor 553 in space group P21 with cell parameters a = 39.5 Å; b = 70.8 Å; c = 58.1 Å; and β = 103.7° was characterized in other studies.27 A characteristic of the C2 polymorph and of these new crystal forms is the role of the ligand in the formation of crystal contacts (Figure 2b−d). The ligand importance in the discovery of the polymorphs does not preclude their use in seeding trials with dissimilar ligands. MMP-9 Polymorphism. Various known MMP-9 crystal forms are reported in Table 2 and the 2D chemical structures are in Supporing Information. The two bifunctional inhibitor complexes with MMP-9: MP24 (PDB entry: 4H2E; ligand code 0Y3) and LC29 (PDB entry: 4H82; ligand code L29) adopt similar packing in space groups P21 and P1 respectively (Figure 3a). The change in space group from P21 to P1 is probably needed to compensate for slight variation in the crystal contacts. Although the two MMP-9 constructs are not identical, most features of the packing are conserved. The MP24 complex has been obtained with the wild-type sequence with four amino acids shorter at the N-terminus. The construct used for the LC29 complex involves an MMP-9 which is less active and therefore less susceptible to self-degradation since the catalytic glutamate is mutated to glutamine. The P21 complex between MP24 and MMP-9 is characterized by one bifunctional inhibitor bringing together two MMP-9 molecules. Seeds from crystals from this complex are at the origin of successive seeding experiments with MMP-9 resulting in the various polymorphs obtained. These first crystals diffracted to 2.9 Å resolution, while those for the MMP-9 complex with LC29 in space group P1 diffracted to 1.9 Å. Attributing the higher resolution to an improved IC50 (7-fold better for LC29 versus MP24 for MMP-9) would not take into consideration the power of crystallization as a purification method. Logically, the improved affinity would result in a more homogeneous sample at a 1:1 protein−ligand stoichiometry. Unfortunately, this does not always translate into improved diffraction. When the same MMP-9 in complex with LC29 was crystallized in space group P21, with cell parameters a = 39.8 Å; b = 98.4 Å; c = 47.1 Å; β = 112.2°, these crystals diffracted to only 2.84 Å. In this case, higher resolution data was obtained by breaking the symmetry generated by weak protein−protein interactions. The molecules previously crystallographically related maintain the packing, and the symmetry becomes noncrystallographic with a minor rearrangement of the unit cell. The lattice adjustment observed here is less spectacular than the space group change from trigonal to monoclinic and a change obtained for a complex involving a small GTPase, but the improvement in resolution is comparable (3 to 2 Å for the GTPase).28 A larger

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RESULTS Polymorphism and Isomorphism. True polymorphism refers to the existence of a solid material in more than one crystalline state. The crystal composition should be identical, something that is seldom true for protein crystals due to differences in solute molecules, more or less ordered, incorporated from the crystallization medium. In the case of protein−ligand complexes, any chemical difference in the ligand structure can be considered a breach of the requirement of identical composition even if such changes have no impact on crystal packing. Such strict definition of a polymorph would not be useful to report and analyze the results of this study. Crystals with similar cell parameters and angles even if not isomorphous for crystallographic uses such as data scaling or phasing are effectively the same crystal form for seeding purposes. Understanding the packing of the various complexes (Figures 2−5) is important when choosing the first polymorph to use for screening and seeding (Figure 1). With this working definition of polymorphism, even small differences in protein sequence may not represent a significant change in composition. Small sequence differences may change the predisposition for one lattice compared to another but does not imperatively force the protein to adopts a different packing, although it may be considered normal for it to do so. We have obtained crystals of MMP-12 in the P21212 space group with substantial variations in cell parameters between inhibitor complexes: RXP470.1 (PDB ligand code: R47; Supporting Information), 470A (R45) and 470B (R4B) (RXP470.1: 69.6 Å; 63.4 Å; 36.9 Å)19 with the uncleaved protein and the complex with 470C (R4C) with the shortened protein (67.8 Å; 61.5 Å; 33.5 Å). The termini are ordered in the lattice for both the longer version and the cleaved version of the protein. A translation differentiates that two forms although the packing remains essentially the same (Figure 2a). The RMSD for the MMP-12 in RXP470.1, 470A and 470B that does not exceed 0.14 Å and the maximum deviations in cell parameters is only 0.8 Å in a, 0.6 Å in b and 0.9 Å in c indicating that the crystals are isomorphous with different inhibitors. The complexes have all been grown from seeds derived from the RXP470.1 diffracting to 1.15 Å resolution. Crystals of the complex with 470C obtained with a protein that has been proteolytically degraded were seeded in the same way. MMP-12 Polymorphism. The various MMP-12 crystal forms are reported in Table 1 and the 2D chemical structures in Supporting Information. Most crystals used in structure 1883



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Table 3. Space Groups Reported for MMP-8 Catalytic Domainsa MMP8 polymorphism space group

cell dimensions (Å)

angles (deg)

Z

P212121

33.1 69.4 72.6

90.0 90.0 90.0

4

P212121 P212121 P212121 P212121 P21 P21 P21 P21 P1 I222 C2221 P41212

32.4 34.9 44.7 48.6 33.2 30.2 42.3 32.3 33.3 61.0 68.7 68.8

90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 77.7 90.0 90.0 90.0

52.7 61.3 80.8 49.2 68.5 68.0 69.4 69.0 47.1 69.2 86.6 68.8

67.3 68.5 108.1 68.7 78.3 69.7 52.7 32.8 61.3 88.5 68.0 136.2

90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 98.1 90.0 96.5 90.0 92.4 90.0 105.4 90.0 80.0 77.0 90.0 90.0 90.0 90.0 90.0 90.0

4 4 8 8 4 4 4 2 2 8 8 16

PBD codesb 3TT4,c6 1A85,A13 1A86,A13 1BZS,A14 1I73,A15 1I76,A15 1JH1,A16 1MMB,A17 1JAN,A18 1JAO,A19 1JAP,A19 1JAQ,A20 1ZS0A21 1ZP5,A22 1ZVXA21 1MNCA23 1KBCA24 4IYUc 2OY2A1 RXP470.1 TBD (ligand R47)c,d 3DNGA25 3DPEA25 2OY4A1 1JJ9A26 bifunctional inhibitor TBD (ligand L88)c,d 41WUc

highest resolution (Å) 1.20 1.80 2.10 1.80 1.50 1.50 1.56 2.00 1.60 1.70 2.00 1.88 1.16

a Variations in the constructs are at the amino and carboxy termini are noted. Catalytic domain constructs: N: (1A85, 1A86, 2OY2, 2OY4, 1JH1: 158 aa); G: (3TT4: 159 aa); M: (1JAP, 1JAO, 1JAQ, 1JJ9, 1I76, 1I73, 1ZP5, 1ZS0, 1ZVX, 1MMB, 3DPF, 3DNG, 3DPE: 163 aa); L: (1MNC: 163 aa); F: (1JAN, 1KBC: 164aa); FD: (1BZS: 165aa). bA1−A26 refer to additional references available in Supporting Information. cThis study; contributions from our laboratory are in bold. dTBD: not yet deposited; cell parameters may vary with refinement.

Figure 4. Comparison of the packing of polymorphs of MMP-8. (a) The dominant crystal form for most MMP-8 complexes is represented in the PDB entry 3TT4. Typically the crystals grow as long needles. Various packing elements found in this polymorph can be found in other crystal forms. PDB entry 1ZP5 differs by only a small translation. (b) PDB entries 1MNC and 2OY4 show greater variation with a major rearrangement of the layer that stacks perpendicularly on top of the dominant packing direction common among the various polymorphs. (c) Even when a long peptide is cocrystallized with MMP-8, the dominant packing direction is maintained and the MMP-8 molecules are simply translated perpendicularly to make space. (d) Even the change from P212121 to P41212 that requires a new interaction to mediate the 4-fold screw axis is accomplished with the preservation of this dominant packing mode.

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Table 4. Space Groups Reported for MMP-13 Catalytic Domainsa MMP13 polymorphism space group

cell dimensions (Å)

angles (deg)

Z

PBD codesb

P21212 C2

60.3 85.2 40.9 134.4 36.5 95.3

90.0 90.0 90.0 90.0 130.5 90.0

4 8

P21212 P21212 P21212 C2 P1 P21 P21 C2 C2 P32

120.9 96.5 36.6 133.2 72.4 36.4 81.1 108.2 36.0 75.1 106.2 38.7 36.3 70.0 69.1 35.9 135.3 69.4 41.6 60.0 75.0 135.4 35.8 139.4 161.8 72.0 138.1 96.5 96.5 67.5

90.0 90.0 90.0 90.0 92.4 90.0 90.0 90.0 90.0 90.0

3TVCc6 1XUC,A27 1XUD,A27 1XUR,A28 3ELM,A31 3I7G,A32 3I7I,A32 456C,A33 830C,A33 4A7B,A34 2YIG,A35 3ZXHA36 3KEJ,A30 3KEKA30 3KECA30 1YOUA29 4JA3c 3KRYA38 1ZTQA39 RXP470.1 TBD (ligand R47)c,d 2PJTA40 2OZRA37 2D1NA41

90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 94.2 104.9 104.9 90.0 91.8 90.0 108.9 90.0 124.6 90.0 90.0 120.0

8 8 8 8 4 8 4 16 32 6

highest resolution (Å) 2.34 1.60 1.97 2.05 2.30 2.18 1.90 2.00 1.56 2.80 2.30 2.37

a

Variations in the constructs are at the amino and carboxy termini are noted. Catalytic domain constructs: Y: (3KRY: 164 aa); A: 3LJZ, 3O2X: 164 aa); P: (1FM1, 1FLS, 1ZTQ, 2PJT: 165 aa); G: (2D1N: 166 aa); M: (2OW9, 2OZR: 170 aa); D: (3KEC, 3KEJ, 3KEK: 167 aa); E: (1YOU, 456C, 830C: 168 aa); YD: (3TVC, 4A7B: 169 aa); N: (1XUC, 1XUD, 1XUR, 2YIG, 3ELM, 3I7G, 3I7I, 3ZXH: 171 aa). bA27−A41 refer to additional references available in Supporting Information. cThis study; contributions from our laboratory are in bold. dTBD: not yet deposited; cell parameters may vary with refinement.

Figure 5. The polymorphism of MMP-13·ligand complexes can be separated in four groups: (a) The main group is represented by three polymorphs: PDB entry 1XUC in space group C2 with eight molecules in the unit cell (Z = 8; 2 mol/asym unit), the most frequent (Table 4), 3KRY in P1 (Z = 4) and 1ZTQ in space group P21 (4 mol/asym unit). (b) Three polymorphs populate the second group with PDB entry 3KEJ with (Z = 8; 2 mol/asym unit) in P21212, 1PJT in space group C2 (Z = 16; 4 mol/asym unit) for, and 2OZR again in space group C2 (Z = 32; 8 mol/asym unit). (c) The third group includes PDB entry 3KEC (Z = 8; 2 mol/asym unit) in P21212 and entry 2OW9 in C2 (Z = 8; 2 mol/asym unit). The superimposition of the lattices in each group is notable. (d) The fourth packing group consists of PDB entry 3TVC (ligand code E3P) in P21212 at 2.34 Å resolution and the complex with ligand 470.1 (PDB ligand code R47; coordinates to be deposited) in P21. No contact found in these structures match those found in any of the crystals forms previously reported (Table 4). The P21212 form has only a single molecule in the asymmetric unit, an advantage for faster structure determination. The resolution achieved with crystals of this polymorph has been extended to 1.9 Å with a different ligand. The P21 polymorph with two molecules in the asymmetric unit diffracts to 1.56 Å, a resolution comparable with the polymorphs of the main group. These polymorphs can be obtained from each other by streak-seeding. Given the partial conservation of the packing, an epitaxial jump is needed to achieve the transition between the two polymorphs. The superpositions for Figures 2−5 were done with pyMOL.34

jump20 with the sliding of one crystal contact (Figure 3d). The crystallization of MMP-9 in complex with ligand R4C, an RXP470.1-like phosphinic inhibitor (Supporting Information), yields a new polymorph in space group C2221 (Table 2). It is interesting to note that this polymorph shares crystal contacts with the tetragonal OVZ polymorph (Figure 3b). MMP-8 Polymorphism. The various MMP-8 crystal forms are reported in Table 3 and the 2D chemical structures in Supporting Information. The orthorhombic crystal form represented by protein database entry 3TT46 is the most common polymorph of MMP-8 independently of the construct (Table 3; Figure 4a). The crystals were obtained from 17.5% PEG 20000, 0.1 M MES, 0.125 M NaCl in the presence of a

gain was achieved for the MP24·MMP-9 complex, from 2.9 Å (PDB entry: 4H2E)29 to 1.59 Å, when the stoichiometry was changed from 1 MP24 bifunctional ligand for 2 MMP-9, to an excess of ligand (PDB entry: 4H1Q)29 (Figure 3c). The example shows how similar lattices can be used to improve the resolution. In the 2:1 MMP-9/ligand complex, the spacer forces two MMP-9 molecules together in a relationship that is less relaxed that with monofunctional inhibitors. The polymorph obtained for the MMP-9 complex with CC27 (monofunctional inhibitor portion of MP24; Supporting Information; PDB entry: 4H3X29) is comparable to the polymorph obtained with the bifunctional inhibitor. Illustrating the freedom gained without the spacer, the lattice changes through an epitaxial 1885



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crystallization of other MMP catalytic domains is less problematic, but it is always a good strategy to make use of potent inhibitors to improve seed quality and optimize the crystallization screening for the various polymorphs. The optimized protocol is then applied to the crystallization of complexes with weaker inhibitors. For seeding, a polymorph that is tolerant of protein degradation is preferred. In the case of MMP-12 in complex with 470C (PDB code 4GR819), the enzyme was proteolytically degraded but the crystals obtained still diffracted to 1.3 Å. The space group is preserved and the packing is closely related to crystals of the intact enzyme (PDB code 4GR3;19 Figure 2a) from which the seeds were derived. Although proteolysis shortens the enzyme by seven amino acids at the N-terminus and one residue at the C-terminus, the termini are well-defined in the electron density both in the truncated form as they are in the intact catalytic domain. The adaptability of the P21212 polymorph, illustrated by this example, is as important as the seeding step. Analysis of MMP-12 crystal structure depositions, which were probably carried out without seeding, shows that minor sequence variations often result in modified packing of MMP-12 inhibitor complexes in the lattice (Table 1). An important mutation in MMP-12 that makes the enzyme easier to crystallize is F171D. This mutation is present in our construct but not in PDB entry 1UTZ, space group P6322, nor in entry 1UTT, space group P4132. The length of the MMP-12 used to obtain these structures is only one residue longer at the N-terminus compared to ours. With the addition of another extra Cterminal amino acid in entry 1ROS results in space group H3. The addition of a C-terminal hexa-His in 2W0D results in crystals adopting the monoclinic P21 space group, while P1 is preferred for the same construct without the tag (PDB entries 2WO8, 2WO9, and 2WOA). The screening approach given in the flowchart (Figure 1) can be subject to modification depending on information, or assumptions, regarding the crystallization behaviors of particular ligands. The same MMP-12 construct used in our laboratory can also crystallize in space group C2. This is the most common polymorph found in the PDB (Table 1). It is unrelated to the P21212 crystal form and the crystals are slower to grow, but they can become very large and are often well ordered. The C2 crystals form might be the best choice to grow crystals for neutron crystallographic studies, should the need arise. The crystallographic 2-fold generates a homodimer which is stabilized in the case of the complex with batimastat (PDB ligand code BAT; Supporting Information) by interactions between symmetry-related phenyl groups from the ligand (Figure 2b). Stabilization interactions are not essential for the growth of crystals of this polymorph that are readily obtained with acetohydroxamic acid (PDB entry 1Y93, ligand code HAE),12 a ligand effective in preventing self-degradation when used in millimolar concentration but readily replaced by most inhibitors and for this reason is also a good choice for soaking experiments. Our first choice of crystallization conditions and seeding polymorph shifts from our customary P21212 to C2 for ligands expected to stabilize a batimastat-like homodimer. Homodimer Containing Polymorphs. The first MMP-9 inhibitor complex obtained in our laboratory was with the bifunctional inhibitor MP24 (ligand code 0Y3; PDB entry 4H2E; Table 2) which forces MMP-9 dimerization.30 All subsequent MMP-9 polymorphs have been generated by seeding with these crystals or using related crystallization conditions and show closely related homodimers (Figure 3).

slowly degraded substrate. The inhibitor was introduced by soaking. Crystals can be grown without this convoluted procedure, but for certain inhibitors for which the cocrystals grow as thin long needles this is the fastest option. Crystals of the various polymorphs grown in our laboratory are morphologically indistinguishable, with the exception of the P41212 crystal form (Figure 4). The morphology may reflect the crystal packing which in all these crystals grow fastest along the dominant packing direction. This packing interaction is also found in other orthorhombic polymorphs such as protein database entries 1ZP5 and 1MNC. Other packing contacts are also relatively well preserved except for a small translation in 1ZP5 (Figure 4a) and a more serious departure in 1MNC (Figure 4b). Surprisingly, the collagen-mimic peptide cocrystals in the P212121 space group were also obtained by streak seeding given that only the dominant packing direction is conserved. The tetragonal form appeared in the same drop (Figure 4), and seeds were collected and streak seeded in other drops to obtain crystals that diffracted to 1.16 Å. As the space group changes to P21 for the RXP470.1 (R47) cocrystals (Table 3), a dimer is present in the asymmetric unit. This noncrystallographic dimer in the P21 crystal form is generated by a 21 symmetry operation in the 3TT4-like polymorphs and in the P41212 and P212121 lattices, obtained from seeding with crystals of the 3TT4-like polymorph. The approach of using a bifunctional ligand (to be deposited; PDB ligand code L88) to obtain a new polymorph (Table 3) has given cocrystals in C2221 (Figure 4).30 MMP-13 Polymorphism. The various MMP-13 crystal forms are reported in Table 4 and the 2D chemical structures are in Supporting Information. The catalytic domain of MMP13 is a recent addition to our MMP repertoire and currently only 11 MMP-13 ligand complexes have been obtained. Eight polymorphs characterize these complexes among which the orthorhombic P21212 crystal form reported for an inhibitor consisting of a simple pseudo dipeptide with a P2′ glutamate chelating the catalytic zinc (PDB entry: 3TVC6) is particularly interesting for fast structure determination as there is only one molecule in the asymmetric unit (Table 4). The polymorphs previously discovered appear to cluster into three packing groups with identical contact patches but with changes in space group (Figure 5a−c) and without shared packing elements common to all groups. The polymorphs obtained so far for MMP-13 show a pattern similar to that obtained for the MMP12, MMP-9, and MMP-8, where some but not all packing interactions are shared by the polymorphs (Figure 5d).

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DISCUSSION Matrix metalloproteinases (MMPs) are important pharmaceutical targets that have been extensively studied by X-ray crystallography and for which various investigators have reported a large number of different crystal lattices (Tables 1−4) for constructs that are closely related. Polymorphism is quite common for these enzymes, but it has not been exploited until now to speed-up crystallization and structure determination. Growing crystals of wild-type enzyme complexes requires a fast and efficient screening protocol. Speed is more important with weak inhibitors than for potent ones to avoid proteolytic degradation. Previous crystallographic studies with MMP-9, using the P41212 polymorph (Table 2) that is slow to crystallize, were carried out exclusively with the low activity glutamine mutant, while we have been able to determine the crystal structures for PDB entries 4H3X and 4H2E with a fully active catalytic domain with an active site glutamate. The 1886



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second exploits the extensive sequence conservation among members of the MMP family. We have started looking at flexible linkers of different lengths that can impose distance constraints to allow access to certain polymorphs but not to others.30

The crystals of an unrelated polymorph obtained in space group C2221 (Table 2) for one RXP470.1-like phosphinic inhibitor show unrelated homodimeric associations (Figure 3b). One of these associations matches that found in the P41212 polymorph. The similarity in the ligands (codes 5MR7 for PDB entry 1OVZ and R4C not yet deposited; Supporing Information) does not ensure the conservation of the interface that places these ligands in close proximity. The difference between a tryptophan in 5MR compared and an alanine in R4C is significant. The conserved interface in the MMP-9 5MR and R4C crystals differs from most other homodimeric interfaces that are frequently influenced by the ligand. The second interface (Figure 3b) in 1OVZ, mediated by the tryptophan in the ligand, is an asymmetric crystal contact since it does not generate a 2-fold symmetric dimer. The situation found in the MMP-12 batimastat complex (Figure 2b; PDB entry 1JK311) with two inhibitors interacting across a 2-fold axis is not the typical situation. The symmetric dimers found in most other MMP-9 ligand complexes that we have obtained (Figure 3a,c,d) are mainly mediated by protein−protein interactions. The homodimer mediated by the phenyl containing inhibitor EN238 (PDB entry 4H84;28 Table 1; Figure 2c) is symmetric where the ligand interacts across the 2-fold with MMP-12 residues as is the case for the polymorph generated by a tryptophan in an MMP-12 complex reported in Vera et al., 201127 (Table 1). Exposed hydrophobic groups present in inhibitors appear to have a tendency to modify the manner in which protein−ligand complexes crystallize leading to new polymorphs. The tendency of exposed hydrophobic groups to be involved in lattice interactions is generally true in protein crystallization. A comparison of the lattices of Fab complexes with two tryptophan mutants of protein L: PpL-A66W (PDB entry: 1YMH32) and PpL-D55A-Y64W (PDB entry: 1MHH33) shows that in both cases the tryptophans mediate crystal interactions. The two polymorphs are related to each other, and analysis of the packing shows a lattice translation coherent with the tryptophan position being shifted by two positions along the protein sequence. This suggests that introducing of displacing hydrophobic residues not involved in pharmacological interactions along the chemical structure can lead to modification of crystal interactions and generate new polymorphs. However, bifunctional ligands are probably the most effective choice to modify enzyme homodimerization and consequently its tendency to crystallize.29,30 Stoichiometry and Affinity. Bifunctional ligands were instrumental in obtaining the initial MMP-9 crystals but not without problems. The relative low affinity of MP24, designed for MMP-213 rather than for MMP-9, resulted in crystals that diffracted at high resolution (1.5 Å; PDB code: 4H1Q)29 only when an excess of inhibitor was used in the optimization stage of the crystallization trials. With the correct stoichiometry to ensure that the bifunctional ligand binds to two enzymes simultaneously, larger crystals were obtained but the data extended to only 2.9 Å (PDB code: 4H2E)29 at best. Affinity is very important to obtain well ordered cocrystals with bifunctional ligands,30 but, because nucleation is generally much higher that that for monofunctional ligands, it is of lesser importance for the generation of polymorphs for crystal seeding purposes. Future Developments. Asymmetric bifunctional ligands and cross-seeding between MMPs are approaches that we have left unexplored. The first creates an asymmetric dimer which will have to pack differently from a symmetric homodimer, the

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CONCLUSION The soaking approach, with its advantages and disadvantages, is likely to remain the fastest way to obtain protein-inhibitor crystal structures to discern the binding mode of inhibitors.31 The screening method for protein−ligand complexes proposed here, with the use of seeding into a sitting drop immediately after setup using a single crystallization condition, is also highly effective. It allows for lattice readjustments and the creation of new polymorphs that can be used in future seeding experiments augmenting the arsenal of seeds available to stimulate the growth of new protein−ligand complexes in a process with positive feedback. These alternative approaches work well together. Obtaining the same ligand complex in more than one polymorph will allow a better understanding of whether ligand binding causes conformational changes in its target enzyme and the extent of these changes. Having seeds of a large number of polymorphs becomes an important asset when critical questions are asked regarding the influence of lattice contacts on the binding of the ligand. Nucleation is the biggest obstacle to crystallization, and seldom extra screening can overcome it, but seeding can, and with more polymorphs, a suitable one might be found.

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ASSOCIATED CONTENT

S Supporting Information *

Table of data on MMP12, MMP9, MMP8, MMP13 ligands and additional references. The structures whose lattices have been discussed in this paper: 4H2E, 4H1Q, 4H82, 4H30, 4H49, 4H84, 4H3X, 4H76, 4HMA, 4GQL, 4GR3, 4GR0, 4GR8 have been deposited in the Protein Database and associated with this publication. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the ESRF and SOLEIL for beam time and to their staff for assistance. ABBREVIATIONS MMP: matrix metalloproteinase; PTCP: linear buffer composed of sodium propionate, sodium cacodylate and Bis-Tris propane; RMSD: root-mean-square deviation; PDB: protein database; AHA: acetohydroxamic acid

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REFERENCES

(1) Tallant, C.; Marrero, A.; Gomis-Ruth, F. X. Matrix metalloproteinases: Fold and function of their catalytic domains. Biochim. Biophys. Acta 2010, 1803, 20−28. (2) Matter, H.; Schudok, M. Recent advances in the design of matrix metalloproteinase inhibitors. Curr. Opin. Drug Discovery Dev. 2004, 7, 513−535.

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