Novel Insights into Structure and Function of Factor XIIIa-Inhibitor

Article pubs.acs.org/jmc

Novel Insights into Structure and Function of Factor XIIIa-Inhibitor Tridegin Miriam Böhm,† Charlotte A. Baü ml,† Kornelia Hardes,‡ Torsten Steinmetzer,‡ Dirk Roeser,§ Yvonne Schaub,§ Manuel E. Than,§ Arijit Biswas,∥ and Diana Imhof*,† †

Pharmaceutical Chemistry I, Institute of Pharmacy, University of Bonn, Brühler Str. 7, 53119 Bonn, Germany Department of Pharmacy, Institute of Pharmaceutical Chemistry, Philipps University of Marburg, Marbacher Weg 6, 35032 Marburg, Germany § Protein Crystallography Group, Leibniz Institute for Age ResearchFritz Lipmann Institute (FLI), Beutenbergstr. 11, 07745 Jena, Germany ∥ Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany ‡

S Supporting Information *

ABSTRACT: The inhibition of the final step in blood coagulation, the factor XIIIa (FXIIIa) catalyzed cross-linking of fibrin monomers, is currently still a challenge in medicinal chemistry. We report synthesis, recombinant expression, disulfide connectivity, and biological activity of tridegin, the sole existing peptide representative displaying inhibitory activity on FXIIIa. Inhibition of the enzyme by this 66-mer cysteine-rich peptide is mediated by its C-terminal sequence, while the N-terminal part comprises structural information and contributes to inhibitor binding. Either of the production strategies examined leads to the formation of different disulfide-bridged isomers indicating the requirement of the correct fold for inhibitory activity. Molecular modeling and docking studies confirm disulfide bond isomer preference with respect to binding to FXIIIa, in turn, the knowledge of the enzyme−inhibitor interactions might bring about comprehensive ideas for the design of a suitable lead structure for addressing FXIIIa.

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INTRODUCTION Factor XIIIa (FXIIIa) is a transglutaminase (EC 2.3.2.13) that catalyzes the final step of blood coagulation, i.e., the covalent cross-linking of fibrin chains, and protects the fibrin clot from proteolytic degradation. FXIIIa is involved in a variety of other physiological functions including wound healing, angiogenesis, cardiac protection,1 and inflammation.2 While the plasmatic form (pFXIII) circulates in the blood as an A2B2 heterotetramer (with A being the protransglutaminase subunits and B being carrier subunits) there is also a cellular form (cFXIII), which is a dimer of A-subunits. Activation of pFXIII is achieved by either thrombin-catalyzed removal of an activation peptide in the presence of physiological Ca2+ concentrations or by addition of strongly increased Ca2+ concentrations. Cellular FXIIIa can be activated without cleavage at moderate calcium concentrations.3,4 Its involvement in hemostasis makes FXIIIa in general an interesting pharmaceutical target for both anticoagulative and thrombolytic therapy as well as a diagnostic marker. Also other indications concerning the FXIII concentration and/or activation point to FXIIIa being a risk factor for various pathophysiological conditions. This can be exemplified with the © 2014 American Chemical Society

known polymorphism (Val34Leu) of FXIIIa, which has been shown to be associated with an enhanced activation rate by thrombin,5 or high levels of FXIIIa representing an increased risk for myocardial infarction in women.6 In addition, various studies revealed an involvement of increased levels and/or an elevated activity of FXIIIa in cancer, in particular acute promyelocytic leukemia,7 hematogenous tumors,8 brain tumors,9 and nonsmall cell lung cancer.10 Specificity for FXIIIa is of major importance since other transglutaminases have a variety of different and important functions. Tissue transglutaminase (TGase2), the most extensively investigated transglutaminase, for example, is expressed ubiquitously and may also be localized extracellularly,11 which is also the case for FXIIIa. TGase 2 is involved in a variety of functions, including differentiation, migration, and cell death of various cell types as well as, e.g., matrix stabilization and remodeling.11,12 Therefore, from a medical point of view, a FXIIIa inhibitor that does not discriminate at Received: July 15, 2014 Published: November 21, 2014 10355

dx.doi.org/10.1021/jm501058g | J. Med. Chem. 2014, 57, 10355−10365

Journal of Medicinal Chemistry

Article

Table 1. Peptide Sequences and Length (Number of Amino Acids) sequencea

length

K LLPCKEWHQGIPNPRCWCGADLECAQDQYCAFIPQCRPRSELIKPMDDIYQRPVEFPNLPLKPRE 66 oxidized 1a oxidized 1a dimer K01LLPSKEWHQGIPNPRSWSGADLESAQDQYSAFIPQSRPRSELIKPMDDIYQRPVEFPNLPLKPRE 66 A32FIPQCRPRSELIKPMDDIYQRPVEFPNLPLKPRE66 D22LECAQDQYCAFIPQCRPRSELIKPMDDIYQRPVEFPNLPLKPRE66 C17WCGADLECAQDQYCAFIPQCRPRSELIKPMDDIYQRPVEFPNLPLKPRE66 I12PNPRCWCGADLECAQDQYCAFIPQCRPRSELIKPMDDIYQRPVEFPNLPLKPRE66 E07WHQGIPNPRCWCGADLECAQDQYCAFIPQCRPRSELIKPMDDIYQRPVEFPNLPLKPRE66 K01LLPCKEWHQGIPNPRCWCGADLECAQDQYCAFIPQC37 R38PRSELIKPMDDIYQRPVEFPNLPLKPRE66 1b with N-terminal carboxyfluoresceine 8 with N-terminal carboxyfluoresceine 9 with N-terminal carboxyfluoresceine

66 66 66 66 35 45 50 55 60 37 29 66 37 29

peptide 1a 1b 1c 2 3 4 5 6 7 8 9 cf-1b cf-8 cf-9 a

01

All peptides were prepared as C-terminal amides.

least to some extent against TGase2 would pose a risk for side effects. Thus far, however, only few specific inhibitors of FXIIIa have been described. These include nonpeptidic inhibitors, such as thioimidazoles13 or alutacenoic acids,14 as well as peptide derived inhibitors like 6-diazo-5-oxo-norleucine (DON) derivatives,15 chloromethyl ketones,16 or Michael acceptors.17 Recently, one of the latter has been used to stabilize and crystallize the calcium-activated form of FXIIIa.18 This allows detailed insights into the active site geometry of the enzyme. Most of these inhibitors, however, do not discriminate well between FXIIIa and the homologous TGase2. For example, within a series of acrylamide-derived inhibitors against the TGase2, several potent FXIIIa inhibitors have been identified.19 Tridegin, a pure peptidic inhibitor isolated from the giant amazon leech Haementeria ghilianii, has been shown to inhibit FXIIIa with high potency, while the inhibition of guinea pig tissue transglutaminase was much lower according to this report.20 In our previous work21 we started to investigate structure−function relationships of this peptide inhibitor by narrowing down the region that most likely interacts with the active site of FXIIIa to a C-terminal sequence around Gln52. We demonstrated that a sequence of only 18 amino acids (L43−L60) retained an IC50 value that was only about 8-fold higher than that of the full length sequence. Further truncation of the sequence, however, resulted in a dramatic decrease of inhibitory potency as did the exchange of Gln52 to glutamate, alanine, or ornithine. Furthermore, concerning the truncated Cterminal segments of the peptide we demonstrated that the Gln52 side chain, if present, is deamidated or linked to a small amine. Nonetheless, questions regarding tridegin still remained. These included, for example: (i) What is the function of the multiply disulfide-bridged N-terminal region of the peptide and is this function depending on the presence of these disulfide bonds? (ii) Which disulfide connectivity is formed in different ways of production? (iii) Is dimerization influencing inhibitory potency? (iv) How do different parts of the tridegin molecule interact with FXIII or FXIIIa in terms of binding affinity? These questions were thus addressed in the present study. Focus was laid on a detailed analysis of disulfide bridge formation depending on the production strategy using MS/MS analysis as well as functional assays to characterize the whole molecule and individual parts. Finally, computational studies were

performed to increase our knowledge about inhibitor−protein interactions.

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RESULTS AND DISCUSSION Design and Synthesis of Peptides. To further assess structure−activity relationships of tridegin and FXIIIa, we synthesized a series of tridegin-derived peptides (Tables 1 and S1, Supporting Information). Peptide 1 represents the sequence of tridegin (lead structure) as described earlier20,21 and was prepared as the linear, reduced precursor (1a) and the completely oxidized peptide 1b. Furthermore, from the oxidized form a dimerized peptide (1c) was purified and subjected to testing of its inhibitory activity because earlier findings of our group suggested an increased inhibitory potential of dimers.21 Peptide 2, a derivative of 1, in which all cysteine residues were exchanged by serine, was synthesized to investigate the influence of the disulfide bonds and the resulting peptide conformation on the activity of the inhibitor. Peptides 3 to 7 comprise a series of tridegin-derived peptides lacking different numbers of amino acids in the N-terminus. This was done as a reasonable follow-up experiment to our previous studies,21 where we already evaluated different truncated versions of the C-terminus of tridegin. Peptide 8 contains only the N-terminal 37 amino acids of tridegin (thereby including all six cysteine residues) and was prepared to assist in the elucidation of the cysteine connectivity of tridegin and the general role of the N-terminus for the inhibitory function. Peptide 9 represents the corresponding Cterminal part (29 amino acids), i.e., the tridegin sequence was split into two parts: peptides 8 and 9. In addition, peptides 1b, 8, and 9 were prepared as Nterminally carboxyfluoresceine-labeled derivatives (denoted cf1b, cf-8, and cf-9) for binding studies. Structural Analysis. Tridegin is a large oligopeptide that can also be regarded as a miniprotein, and therefore, it is likely to form a defined secondary structure. The most prominent structural feature is the intramolecular linkage of the six cysteine residues resulting in three disulfide bridges. Such constrained peptides generally tend to form highly restrained structural folds as can be found in peptides containing a cysteine knot, such as several known neurotoxins and cyclotides.22,23 Yet, the cysteine residues in tridegin are all concentrated in the N-terminal part rendering the C-terminus potentially flexible. This is very similar to the natural leech10356



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Figure 1. Chymotryptic digest of tridegin (1b) was analyzed by HPLC both before and after reduction with DTT (A). From the HPLC of the nonreduced sample, fractions were collected and subjected to MS analysis, again before (B) and after (C) reduction. From the nonreduced sample, peptide fragments containing disulfide bonds were identified, and their identity was confirmed by subsequent tandem mass spectrometry (D).

concentration by ultrafiltration was only possible to ∼0.7−1.0 mg/mL, suggesting that either (i) the peptide does not fold into one defined three-dimensional structure under our experimental conditions (and hence shows limited solubility due to misfolding and aggregation resulting from intermolecular contacts of protein sections that would not interact in their native state) or (ii) the monomeric tridegin preparation is folded into a defined structure, but shows limited solubility as a

derived thrombin inhibitor hirudin that also contains the six cysteine residues in the N-terminal domain and possesses a flexible C-terminal tail.24 To understand the structure and inhibitory properties of tridegin, we set out to crystallize it and to perform X-ray structural analysis. We first analyzed pure tridegin and found that the synthetic oxidized peptide consisted of monomeric, dimeric, and higher molecular weight species in aqueous buffers. Unfortunately, 10357



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contained a mixture of different disulfide bond isomers. Three (out of 15 possible forms) of them were clearly identified: (i) isomer A with the connection C5−C17, C19−C25, and C31− C37; (ii) isomer B with C5−C37, C19−C25, and C17−C31 linked; and finally (iii) isomer C containing links between C5− C31, C17−C37, and C19−C25. All conformational variants share one common disulfide bridge, i.e., the bond between C19 and C25, and at the same time, the three possible disulfide bond isomers containing this bridge are present. To assess whether the C-terminal part of the peptide has an influence on the disulfide formation, we synthesized a shorter tridegin variant 8. This peptide contains only the 37 N-terminal residues including all cysteines of the tridegin sequence. The analysis of disulfide connectivity was performed as aforementioned, except that the preseparation on HPLC could be omitted due to the lower complexity of the digest. LC−MS analysis of the digest resulted in fragments that supported the same three disulfide bond isomers detected for the full-length tridegin. Therefore, we concluded that the presence of the Cterminal part of tridegin does not significantly influence the oxidation result. During the oxidation reaction of both, 1b and 8, however, partly oxidized species were formed, which did not vanish after longer reaction times or even in the presence of atmospheric oxygen. For the less complex peptide 8, this side product of the oxidation reaction could be analyzed. Iodoacetamide derivatization and MS analysis of the partly oxidized species revealed that it contains two thiol groups. The derivatized peptide was then subjected to CID sequencing, which showed that the modified residues are found at positions C19 and C37 (Figure S2, Supporting Information). Interestingly, this means that the bridge between C19 and C25 is not formed here. This, in turn, seems to push the peptide into an irreversibly misfolded state. Activity Assays. In previous experiments we investigated which part of tridegin conveys most of its inhibitory potential. We could show that the C-terminal part of tridegin interacts with the active center of FXIIIa. Furthermore, we demonstrated that the isolated C-terminal segment of tridegin is a suitable glutamine-containing substrate of FXIIIa, which loses most of its inhibitory potency when transformed into the glutamate containing product by FXIIIa.21 In contrast, this effect is not observed with the full-length oxidized tridegin (1b). In the present report, we studied the influence of the Nterminal (cysteine-rich) part in more detail. Tridegin-derived peptides were synthesized, all of which contain the C-terminal part of the inhibitor and vary in length, sequence, or oxidation state of the N-terminal part (Table 1). For these peptides, IC50 values were determined (Figure 2). The IC50 values of the different truncated 35- to 60-mer peptides (3−7) weakly varied between 1.7 and 3.0 μM. Considering the fact that all of them were linear, cysteine-containing peptides prone to spontaneous oxidation, one can assume that the elongation of the N-terminal part did not result in major improvement in IC50 values. This is in accordance with our earlier findings, where we showed that a 22-mer peptide (S41−L62) lacking any disulfide bridge is sufficient to reach an IC50 value of about 1.9 μM.21 The oxidized full-length tridegin 1b, however, shows an even lower IC50 of about 0.5 μM. Full length variants of the inhibitor, which are either linear (1a) or contain serine residues instead of cysteine (2) also show similar IC50 values of 1.5 and 2.1 μM, respectively. Peptide 2 was tested in addition to the peptide 1a to exclude effects from spontaneous oxidation of cysteine residues, which might occur under the assay conditions.

genuine property. Crystallization experiments employing isolated tridegin yielded, unfortunately, no peptide crystals. We thus had to conclude that our tridegin preparation does not crystallize readily in isolation. We next investigated whether tridegin forms a stable complex with FXIII, possibly converting its target transglutaminase into the active conformation. Earlier observations of Arkona et al. suggested that tridegin might also bind to the inactive form of the enzyme.21 This interaction was thus examined in a gel filtration experiment (Figure S1, Supporting Information), yet revealed no indication for this specific complex formation. Cocrystallization experiments with monomeric tridegin and FXIII resulted in various crystals that could be grouped to ∼10 conditions under which well-defined protein crystals appeared, amenable to crystallographic data collection (Table S2, Supporting Information). These crystals clearly contained the structurally already known, inactive FXIII, but in no case additional electron density that could be attributed to tridegin was observed. This led to the conclusion that either the binding efficiency between tridegin and FXIII/FXIIIa is not sufficient to form a stable complex or purity and identity of the tridegin component need to be re-evaluated. Therefore, a closer look was taken to reveal which fact applies. In order to address the latter hypothesis, the elucidation of the disulfide connectivity in the tridegin monomer was of major importance. Therefore, after enzymatic digestion the resulting peptide fragments were analyzed by HPLC and subsequent MS and MS/MS. The HPLC profile of the tridegin 1b digest in oxidized and subsequently reduced states already showed differences in a variety of peaks. As the first step, fractions from the nonreduced digest (Figure 1A) were collected. Before and after the reduction by DTT the digest fragments were further analyzed by LC−MS and MS/MS. Comparison of the reduced and nonreduced fractions indicated disulfide linked fragments and, with the help of MS/MS analysis, the masses of these fragments could be attributed to distinct disulfide-linked sequences (Figure 1B−D). From the analysis of all disulfide-containing fragments (Table 2), it became clear that the oxidized tridegin monomer 1b Table 2. Fragments Found after Chymotryptic Digest of Tridegina expected mass [M + H]+

measured massb [M + H]+

connection

645.217

645.203

C17−C31

836.366

836.362

C17−C37

C GADLEC A

779.270

779.270

C19−C25

C19GADLEC25 AQDQY

1313.482

1313.480

C19−C25

C W C AFIPQ

983.412

983.399

C17−C31

C3AF IPQC37RPR

1206.588

1206.589

C31−C37

fragment C17W C31AF 17

37

C W C RPR 19

17

25

31

5

37

1544.819

1544.817

C5−C37

5

17

KLLPC KEW C W

1321.644

1321.628

C5−C17

KLLPC5KEW C31AF

1353.670

1353.673

C5−C31

KLLPC KEW C RPR

a

Unspecific cleavage (other than after W, F, Y, M, and L) occurred only marginally. Cleavage after R might be due to contamination with trypsin. bIf the peptide was detected in a higher charged state, [M + H]+ was calculated from this peak. 10358



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Figure 2. IC50 values of peptide variants of tridegin investigated in this study using 80 μM of the chromogenic substrate H-Tyr-Glu(pNA)-Val-LysVal-Ile-Gly-NH2 in the assay. Peptides 3−7 are linear, i.e., not oxidized; ox, oxidized.

Figure 3. (A) Synthesis of fluorogenic-substrates: (a) 1 equiv of Cbz-diaminobutane·HCl, 1 equiv of BOP, 2.5 equiv of DIEA, and DMF, 15 min at 0 °C, 4 h at room temp; (b) 10% Pd/C and H2 at ambient pressure in 90% acetic acid, room temp; (c) TFA, 1 h at room temp; (d) 0.95 equiv of CbzGlu-OBzl, 1 equiv of BOP, 2.5 equiv of DIEA, and DMF, 15 min at 0 °C, overnight at room temp; (e) 1 equiv of Fmoc-OSu in DMF; (f) standard Fmoc-SPPS on Rink-amide resin, followed by deprotection with TFA/Tis/H2O (95/2.5/2.5, v/v/v) and preparative HPLC. (B) Kinetic characterization of the two novel fluorogenic FXIIIa substrates 14 (●) and 15 (○).

Considering this, both peptides show comparable IC50 values, which are in turn similar to the values obtained for peptides 3− 7. Therefore, the final 3- to 4-fold decrease in IC50 of the fulllength tridegin 1b can be attributed to the structural change introduced when the peptide is oxidized. In previous studies we already proposed that the N-terminal part alone does not inhibit FXIIIa. Yet, the peptide segment used for this analysis (K01−Q36) was not fully oxidized because it did lack one cysteine residue in position 37.21 To strengthen this finding, we repeated the experiment with the oxidized N-terminal part of tridegin containing all six cysteine residues (8). Again, the peptide did not show any inhibitory action on FXIIIa, confirming the previous findings. The C-terminal counterpart of 8 was also analyzed and showed an IC50 value of 2.2 μM for peptide 9, which is also in accordance with what we already determined for peptides of similar length.21 Further attention was also given to the substrate-like behavior of isolated C-terminal parts of tridegin, as mentioned earlier. Peptides 1a, 1b, and 2−7 were incubated with FXIIIa and a chromogenic substrate (H-Tyr-Glu(pNA)-Val-Lys-Val-

Ile-Gly-NH2) for up to 1 h. All of the shortened peptides (3−7) showed total or partial loss of their inhibitory potency over time, while full-length peptides 1a, 1b, and 2 were nearly stable (Figure S3, Supporting Information). Moreover, inhibitory potency of the oxidized tridegin 1b was also measured with a novel fluorogenic FRET substrate (15) (Figure 3A). The synthesis of this hexapeptide substrate and of its elongated heptapeptide derivative 14 is shown in Figure 3A. Compared to previously described FRET substrates of FXIIIa, in which the quencher is attached to the Glu side chain, these new substrates carry the fluorophore 2-aminobenzoic acid at that position. This enables a convenient substrate synthesis by incorporation of the quencher 3-nitrotyrosine using standard Fmoc synthesis (for detailed synthesis and characterization see Supporting Information). Kinetic measurements provided a Km of 1.43 μM and kcat/Km values of 38232 s−1 M−1 for substrate 14. For the shorter analogue 15, a Km of 3.63 μM and a kcat/Km value of 38811 s−1 M−1 were determined, respectively (Figure 3B). Although Km was slightly lower for 14, substrate 15 was used for further measurements with tridegin due to its higher 10359



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Figure 4. IC50 determination of 1b. Measurements were performed with (A) 80 μM of the chromogenic substrate in the presence of 0.164 μM FXIIIa (IC50 = 0.68 μM) and with (B) 20 μM of the fluorogenic substrate 15 and 0.082 μM FXIIIa (IC50 = 0.30 μM).

monitored by fluorescence.25 To perform these measurements, fluorescence-labeling of one binding partner, i.e., cf-1b, cf-8, and cf-9, was required. In addition, to check whether the labeling strongly interferes with the FXIIIa-tridegin-interaction, we tested the IC50 value of cf-1b and found it to be 5.2 μM, which is about 10-fold higher than for the nonlabeled peptide 1b. However, as the peptide still showed inhibition of FXIIIa we used it for binding studies, keeping in mind that binding affinity will be weaker and the determined Kd values will thus be increased. For all labeled peptides binding to FXIIIa, the nonactivated A-subunit (FXIII-A and the B-subunit (FXIII-B) were analyzed. Neither peptide bound to the inactive A- or the B-subunit. In continuation of the aforementioned discussion, this may be the reason for the absence of the respective cocrystals. In contrast, all peptides (including the labeled analogue of the noninhibitory peptide 8) bound to FXIIIa. The determined Kd values were 95%, which was determined by analytical

strongly, it merely makes a moderate contribution to the overall inhibitory potency of tridegin. A recombinant variant of 1b proved to be less potent, whereas a synthetic dimer (1c) showed increased potency. IC50 values were determined both with a chromogenic and a novel fluorogenic method. Molecular modeling of the three folded isomers and subsequent docking studies to FXIIIa revealed different binding modes for all of them, with one molecule showing no binding to the active site. With these findings we were able to characterize a natural FXIIIa inhibitor, which might be a useful tool for further studies on the enzyme in vitro and in vivo and as a lead structure for perspective inhibitor design.

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EXPERIMENTAL SECTION

Materials. Inorganic salts and buffer components were obtained from Roth (Karlsruhe, Germany) or Sigma-Aldrich (St. Louis, USA). Fmoc-amino acids, coupling reagents, and resins for solid phase peptide synthesis were purchased from Orpegen Peptide Chemicals (Heidelberg, Germany), IRIS Biotech (Marktredwitz, Germany), and Intavis Bioanalytical Instruments (Cologne, Germany), respectively. Carboxyfluoresceine was purchased from Merck Millipore (Darmstadt, Germany). Fibrogammin was kindly supplied by CSL Behring. Recombinant FXIII-A, FXIII-B, and FXIIIa were purchased from Zedira (Darmstadt, Germany). 10362



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temperature using a Fluoroskan Ascent plate reader (Thermo Fisher Scientific, Vantaa, Finland) with λex = 320 and λem = 405 nm. Each well contained 175 μL of 50 mM Tris-HCl, pH 7.5, in 0.9% (w/v) NaCl, 50 μL of substrate dissolved at different concentrations in water, and 25 μL of activated Fibrogammin solution. The concentration of the A subunits was approximately 0.082 μM in the assay (total volume 250 μL), assuming that both subunits are activated. Microscale Thermophoresis Experiments. Microscale thermophoresis (MST) measurements were performed on a Monolith NT 115 instrument from Nanotemper (Munich, Germany). Tween-20 was added up to a final concentration of 0.1% to a freshly prepared solution of 2 μM carboxyfluoresceine-labeled peptide in buffer (50 mM Tris-HCl pH 7.5, 0.9% w/v NaCl). The peptide solution was then mixed with an equal volume of protein (FXIIIa, FXIII-A, or FXIII-B) solution (in the same buffer) of varying concentrations (3.1 μM to 0.1 nM), and 15 different protein concentrations were prepared per measurement. After 20 min of incubation, the solutions were transferred into a glass capillary, and thermophoresis was measured three times per capillary. The data were evaluated using NT analysis 1.2 and OriginPro 8G. Recombinant Expression. The recombinant expression of tridegin in Escherichia coli was performed according to Arkona et al.21 In brief, a synthetic cDNA sequence optimized for expression in E. coli was purchased from Entelechon (Regensburg, Germany). The sequence already contained the MscI and BamHI restriction sites necessary for cloning into the pET22b(+) vector (Novagen). The ligation fragment was excised from the vector in which it was provided by the manufacturer and ligated into pET22b(+). Ligation was performed in frame with the pelB leader sequence present in pET22b(+), resulting in expression of a tridegin variant carrying the N-terminal pelB leader sequence responsible for periplasmic localization. The vector was transformed into competent BL21(DE3) cells (Novagen). The bacteria were grown in M9 medium up to an optical density (OD) of ca. 0.4−0.8 at 600 nm. After induction with 1 mM IPTG, growth was allowed to continue overnight. The cells were harvested by centrifugation. Preparation of the periplasmic fraction was performed by a protocol adapted from the PeriPreps periplasting kit (Biozym Scientific, Germany). In short, 1 g of wet cell pellet was resuspended in 4 mL of periplasting buffer (200 mM Tris-HCl, pH 7.5, 20% sucrose, 1 mM EDTA, and 30 U/μL lysozyme). The mixture was incubated at room temperature for 5 min, then 6 mL of cold (4 °C) pure water was added, and incubation was continued on ice for 10 min. After centrifugation, the supernatant containing the periplasmic fraction was isolated. Additionally, the medium was harvested and freeze-dried. SDS-PAGE analysis revealed that although tridegin was present in the periplasmic fraction, the major amount of peptide had leaked into the medium; therefore, purification of tridegin was done from the medium fraction. Purification of Recombinant Tridegin. The protein from the periplasmic fraction was precipitated with 60% (NH4)2SO4 for 2 h at 4 °C and centrifuged. Then, the pellet was redissolved in a small volume of phosphate buffer (50 mM, pH 6.3, 150 mM NaCl). By addition of the same volume of 0.1% TFA in acetonitrile the majority of larger proteins was precipitated and the supernatant containing tridegin was isolated. After lyophilization, this supernatant was separated by analytical HPLC as described earlier. The lyophilized medium was redissolved in 1/30 of the original volume of water. Nonsoluble particles were removed by centrifugation. Tridegin was then purified from the medium by semipreparative HPLC as described above. Molecular Modeling and Docking Studies. The tridegin structure was modeled on the I-TASSER threading server (http:// zhanglab.ccmb.med.umich.edu/I-TASSER/; accessed on 18 April 2014). Three different models were generated for three commonly occurring folded isoforms of tridegin as obtained from the experimental data. Each isoform was constrained by the “S−γ” atom residue of individual cysteines participating in the disulfide bond to a distance of 2 Å. The models generated were inspected for the presence of correct disulfide bonds pertaining to individual isoforms. The disulfide bonds that were not correctly generated were manually

HPLC on a Shimadzu LC-10AT chromatograph (Duisburg, Germany) equipped with a Vydac 218TP54 column (C18, 5 μm particle size, 300 Å pore size, 4.6 × 250 mm). Gradient elution was achieved with 0.1% TFA in H2O (eluent A) and 0.1% TFA in acetonitrile (eluent B) at a flow rate of 1 mL/min. The elution gradient was chosen depending on peptide properties. Oxidized peptides were produced by self-folding of linear precursors in a buffer solution. A peptide solution (0.01 mM final concentration) was prepared using 40% isopropanol and 60% buffer (0.1 M Tris, 1 mM EDTA, pH 8.7) containing 1 mM oxidized glutathione (GSSG) and 2 mM reduced glutathione (GSH). The reaction was allowed to take place under argon atmosphere at 4 °C for 24 h. After freezedrying, the reaction product was purified by semipreparative HPLC as described above. Peptide Characterization. Peptides were characterized by analytical HPLC (see above), thin layer chromatography (TLC) gelelectrophoresis (for peptides larger than 7000 Da), amino acid analysis, and mass spectrometry. Gel electrophoresis (SDS-PAGE) was performed on tricine gels according to Schägger and Jagow.26 The resolving gel contained 18% acrylamide/bis(acrylamide) with 5% cross-linking, while the stacking contained 5% acrylamide/bis(acrylamide) with 3.3% cross-linking. The gels were fixed in 5% glutaraldehyde solution for 30 min and stained with colloidal Coomassie solution according to Candiano et al.27 For amino acid analysis, samples were hydrolyzed in 6 N HCl at 110 °C for 24 h, dried in a vacuum concentrator, redissolved, and analyzed on an LC 3000 system from Eppendorf-Biotronik (Hamburg, Germany). From these analyses, sample concentration and/or peptide content of the lyophilized powder was determined. Both matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry were applied for peptide characterization. MALDI mass spectra were acquired on an autoflex II instrument (Bruker Daltonics, Bremen, Germany). ESI mass spectra were measured on a micrOTOF-Q III device (Bruker Daltonics, Bremen, Germany). Determination of Disulfide Connectivity. Elucidation of disulfide connectivity in fully oxidized peptides was achieved by using a digestion protocol followed by MS and MS/MS analysis. A peptide solution containing approximately 1 mg/mL peptide was prepared in 50 mM sodium phosphate buffer pH 6. Then chymotrypsin (Boehringer Mannheim GmbH, 0.1 mg/mL in 1 mM HCl) was added to a final concentration of 16.7 μg/mL. Samples were incubated at 37 °C for 1.5 h and subsequently analyzed by analytical HPLC. In the case of 1b, fractions from HPLC were collected, lyophilized, and analyzed by MS and MS/MS individually. Subsequently, these fractions were fully reduced by the addition of DTT to a final concentration of 10 mM at pH > 7 and successive incubation for at least 30 min at RT. Mass spectrometric analysis was then repeated with the reduced samples, and results were compared to those of the nonreduced samples. All MS analyses were carried out on the aforementioned ESI system coupled to a DionexUltiMate 3000 LC (Thermo Scientific). The LC was equipped with an EC 100/2 NUCLEOSHELL RP18 column (C18, 100 × 2 mm, 2.7 μm particle size, 90 Å pore size). Gradient elution was carried out with 0.1% acetic acid in water (eluent A) and 0.1% acetic acid in acetonitrile (eluent B) at a flow rate of 0.3 mL/min. The gradient was 0% eluent B to 60% eluent B in 12 min. Peptide fragmentation was performed by collision induced decay (CID), and spectra were evaluated with BioTools version 3.2 (Bruker). Enzyme Activity Assay. The measurements of enzyme activity were performed with chromogenic and fluorogenic substrates. The activation of FXIII (Fibrogammin, CSL Behring Marburg, Germany) and the chromogenic measurements with the substrate H-TyrGlu(pNA)-Val-Lys-Val-Ile-Gly-NH2 were performed as described previously.21,28 The fluorogenic assays were done with the newly developed FRET substrates H-Tyr(3-NO2)-Glu(NH-(CH2)4-NH-Abz)-Val-Lys-Val-IleNH2 (15) and H-Tyr(3-NO2)-Glu(NH-(CH2)4-NH-Abz)-Val-LysVal-Ile-GlyNH2 (14).16 Fluorescence readings were performed in black 96 well plates (Nunc, Langenselbold, Germany) at room 10363



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imposed on YASARA version 13.3.26.29 The model was then energyminimized for the structure to adjust to natural disulfide bond lengths. Therefore, each model was strictly constrained by the order of disulfide bonds occurring in them. Subsequently all models were refined by running them on a short solvated MD (molecular dynamic) simulation protocol of 500 ps. The YAMBER force field (YASARA modified version of AMBER) was imposed for the MD simulation. The model with the energetic minimum in the simulation trajectory was chosen as the final model. Blind docking of each of the tridegin models was done on the recently released activated FXIII structure (PDB ID: 4kty).18 The activated FXIII structure was optimized first by removing the small molecule peptide like inhibitor (ID: PRD_001125). Blind docking was performed on the Z-dock docking server (http://zdock.umassmed.edu/; accessed on 28 May 2014). The best dock was inspected for the proximity of one of the putative five reactive glutamine residues to the central catalytic triad Cys314 residue as well as other residues of the catalytic triad (i.e., His373 and Asp396) and residues surrounding the catalytic triad that influence it (i.e., Trp279, Asp343, and Glu401). Trp279 is assumed to form an oxyanion hole that most likely stabilizes substrate-bound enzyme intermediates, while Asp343 and Glu401 direct the substrate to the catalytic site.

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German Heart Research Foundation (to M.B.) and the German Research Foundation (DFG) within priority program SPP1911 (to D.I.). The Bruker micrOTOF-Q instrument was funded by the University of Bonn, the Ministry of Innovation, Science and Research of North-Rhine Westfalia and the DFG.

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ABBREVIATIONS USED CID, collision induced decay; DIEA, N,N-diisopropylethylamine; DMF, dimethylformamide; ESI, electrospray ionization; FRET, Förster resonance energy transfer; FXIIIa, factor XIIIa; HPLC, high performance liquid chromatography; MALDI, matrix-assisted laser desorption/ionization; TLC, thin layer chromatography; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PyBOP, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis protocols for FRET-substrates, description of crystallographic procedures, analytical data of all peptides (Table S1), information about the protein crystals (Table S2), additional information on modeled tridegin isomers (Tables S3 and S4), gel filtration chromatogram of tridegin and FXIII (Figure S1), MS/MS spectrum of peptide 8 (Figure S2), OD traces of the chromogenic FXIIIa assay (Figure S3), analytical data of recombinant tridegin (Figure S4), an additional docking image (Figure S5), and the TGase activity assay (Figure S7). This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

(1) Muszbek, L.; Bereczky, Z.; Bagoly, Z.; Komáromi, I.; Katona, É. Factor XIII: A coagulation factor with multiple plasmatic and cellular functions. Physiol. Rev. 2011, 91, 931−972. (2) Ichinose, A. Factor XIII is a key molecule at the intersection of coagulation and fibrinolysis as well as inflammation and infection control. Int. J. Hematol. 2012, 95, 362−370. (3) Komáromi, I.; Bagoly, Z.; Muszbek, L. Factor XIII: Novel structural and functional aspects. J. Thromb. Haemostasis 2011, 9, 9− 20. (4) Kristiansen, G. K.; Andersen, M. D. Reversible activation of cellular factor XIII by calcium. J. Biol. Chem. 2011, 286, 9833−9839. (5) Ariëns, R. A.; Philippou, H.; Nagaswami, C.; Weisel, J. W.; Lane, D. A.; Grant, P. J. The factor XIII V34L polymorphism accelerates thrombin activation of factor XIII and affects cross-linked fibrin structure. Blood 2000, 96, 988−995. (6) Bereczky, Z.; Balogh, E.; Katona, E.; Czuriga, I.; Edes, I.; Muszbek, L. Elevated factor XIII level and the risk of myocardial infarction in women. Haematologica 2007, 92, 287−288. (7) Simon, A.; Bagoly, Z.; Hevessy, Z.; Csáthy, L.; Katona, E.; Vereb, G.; Ujfalusi, A.; Szerafin, L.; Muszbek, L.; Kappelmayer, J. Expression of coagulation factor XIII subunit A in acute promyelocytic leukemia. Cytometry, Part B 2012, 82, 209−216. (8) Palumbo, J. S.; Barney, K. A.; Blevins, E. A.; Shaw, M. A.; Mishra, A.; Flick, M. J.; Kombrinck, K. W.; Talmage, K. E.; Souri, M.; Ichinose, a.; Degen, J. L. Factor XIII transglutaminase supports hematogenous tumor cell metastasis through a mechanism dependent on natural killer cell function. J. Thromb. Haemostasis 2008, 6, 812−819. (9) Bárdos, H.; Molnár, P.; Csécsei, G.; Adány, R. Fibrin deposition in primary and metastatic human brain tumours. Blood Coagulation Fibrinolysis 1996, 7, 536−548. (10) Lee, S. H.; Suh, I. B.; Lee, E. J.; Hur, G. Y.; Lee, S. Y.; Lee, S. Y.; Shin, C.; Shim, J. J.; In, K. H.; Kang, K. H.; Yoo, S. H.; Kim, J. H. Relationships of coagulation factor XIII activity with cell-type and stage of non-small cell lung cancer. Yonsei Med. J. 2013, 54, 1394− 1399. (11) Gundemir, S.; Colak, G.; Tucholski, J.; Johnson, G. V. W. Transglutaminase 2: a molecular Swiss army knife. Biochim. Biophys. Acta 2012, 1823, 406−419. (12) Griffin, M.; Casadio, R.; Bergamini, C. M. Transglutaminases: Nature’s biological glues. Biochem. J. 2002, 368, 377−396. (13) Freund, K. F.; Doshi, K. P.; Gaul, S. L.; Claremon, D. A.; Remy, D. C.; Baldwin, J. J.; Pitzenberger, S. M.; Stern, A. M. Transglutaminase inhibition by 2-[(2-oxopropyl)thio]imidazolium derivatives: mechanism of factor XIIIa inactivation. Biochemistry 1994, 33, 10109−10119. (14) Kogen, H.; Kiho, T.; Tago, K.; Miyamoto, S.; Fujioka, T.; Otsuka, N.; Suzuki-Konagai, K.; Ogita, T. Alutacenoic acids A and B, rare naturally occurring cyclopropenone derivatives isolated from fungi: Potent non-peptide factor XIIIa inhibitors. J. Am. Chem. Soc. 2000, 122, 1842−1843.

AUTHOR INFORMATION

Corresponding Author

*Phone: +49-(0)228-7360258. E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. M.B. synthesized and characterized peptides 1−7, 9, cf-1b, and cf-9. M.B. performed all MS measurements and assigned disulfide connectivity of 1b. C.B. synthesized and characterized peptides 8 and cf-8, assigned disulfide connectivity of 8, and did recombinant expression of tridegin. M.B. and C.B. performed MST measurements. T.S. and K.H. designed and synthesized the used chromogenic and fluorogenic substrates. Enzyme kinetic measurements were performed by K.H., M.B., and C.B. Molecular modeling and docking were carried out by A.B. M.T, D.R., and Y.S. did crystallization experiments and X-ray analysis of crystals. D.I. designed experiments. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to C. Arkona (Free University of Berlin) for useful scientific discussions, to M. Engeser (University of Bonn) for access to the MALDI instrument, and to M. Famulok (University of Bonn) and NanoTemper (Munich) for access to the MST device. This work was supported by the 10364



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(15) Sabo, T. M.; Brasher, P. B.; Maurer, M. C. Perturbations in factor XIII resulting from activation and inhibition examined by solution based methods and detected by MALDI-TOF MS. Biochemistry 2007, 46, 10089−10101. (16) Hardes, K.; Zouhir Hammamy, M.; Steinmetzer, T. Synthesis and characterization of novel fluorogenic substrates of coagulation factor XIII-A. Anal. Biochem. 2013, 442, 223−230. (17) Heil, A.; Weber, J.; Büchold, C.; Pasternack, R.; Hils, M. Differences in the inhibition of coagulation factor XIII-A from animal species revealed by Michael Acceptor- and thioimidazol based blockers. Thromb. Res. 2013, 131, e214−222. (18) Stieler, M.; Weber, J.; Hils, M.; Kolb, P.; Heine, A.; Büchold, C.; Pasternack, R.; Klebe, G. Structure of active coagulation factor XIII triggered by calcium binding: basis for the design of next-generation anticoagulants. Angew. Chem., Int. Ed. 2013, 52, 11930−11934. (19) Prime, M. E.; Andersen, O. A.; Barker, J. J.; Brooks, M. A.; Cheng, R. K. Y.; Toogood-Johnson, I.; Courtney, S. M.; Brookfield, F. A.; Yarnold, C. J.; Marston, R. W.; Johnson, P. D.; Johnsen, S. F.; Palfrey, J. J.; Vaidya, D.; Erfan, S.; Ichihara, O.; Felicetti, B.; Palan, S.; Pedret-Dunn, A.; Schaertl, S.; Sternberger, I.; Ebneth, A.; Scheel, A.; Winkler, D.; Toledo-Sherman, L.; Beconi, M.; Macdonald, D.; MuñozSanjuan, I.; Dominguez, C.; Wityak, J. Discovery and structure-activity relationship of potent and selective covalent inhibitors of transglutaminase 2 for Huntington’s disease. J. Med. Chem. 2012, 55, 1021− 1046. (20) Finney, S.; Seale, L.; Sawyer, R. T.; Wallis, R. B. Tridegin, a new peptidic inhibitor of factor XIIIa, from the blood-sucking leech Haementeria ghilianii. Biochem. J. 1997, 324, 797−805. (21) Böhm, M.; Kühl, T.; Hardes, K.; Coch, R.; Arkona, C.; Schlott, B.; Steinmetzer, T.; Imhof, D. Synthesis and functional characterization of tridegin and its analogues: inhibitors and substrates of factor XIIIa. ChemMedChem 2012, 7, 326−333. (22) Daly, N. L.; Craik, D. J. Structural studies of conotoxins. IUBMB Life 2009, 61, 144−150. (23) Daly, N. L.; Rosengren, K. J.; Craik, D. J. Discovery, structure and biological activities of cyclotides. Adv. Drug Delivery Rev. 2009, 61, 918−930. (24) Grütter, M. G.; Priestle, J. P.; Rahuel, J.; Grossenbacher, H.; Bode, W.; Hofsteenge, J.; Stone, S. R. Crystal structure of the thrombin-hirudin complex: A novel mode of serine protease inhibition. EMBO J. 1990, 9, 2361−2365. (25) Baaske, P.; Wienken, C. J.; Reineck, P.; Duhr, S.; Braun, D. Optical thermophoresis for quantifying the buffer dependence of aptamer binding. Angew. Chem., Int. Ed. 2010, 49, 2238−2241. (26) Schägger, H.; von Jagow, G. Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 1987, 166, 368−379. (27) Candiano, G.; Bruschi, M.; Musante, L.; Santucci, L.; Ghiggeri, G. M.; Carnemolla, B.; Orecchia, P.; Zardi, L.; Righetti, P. G. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 2004, 25, 1327−1333. (28) Hardes, K.; Becker, G. L.; Hammamy, M. Z.; Steinmetzer, T. Design, synthesis, and characterization of chromogenic substrates of coagulation factor XIIIa. Anal. Biochem. 2012, 428, 73−80. (29) Krieger, E.; Darden, T.; Nabuurs, S. B.; Finkelstein, A.; Vriend, G. Making optimal use of empirical energy functions: Force-field parameterization in crystal space. Proteins: Struct., Funct., Bioinf. 2004, 57, 678−683.

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Novel Insights into Structure and Function of Factor XIIIa-Inhibitor

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