Crystal structure of the peptidyl-cysteine decarboxylase EpiD complexed with a pentapeptide substrate

Article1 December 2000free access Crystal structure of the peptidyl-cysteine decarboxylase EpiD complexed with a pentapeptide substrate Michael Blaesse Michael Blaesse Abteilung für Strukturforschung, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany Search for more papers by this author Thomas Kupke Thomas Kupke Lehrstuhl für Mikrobielle Genetik, Universität Tübingen, Auf der Morgenstelle 15, Verfügungsgebäude, 72076 Tübingen, Germany Search for more papers by this author Robert Huber Robert Huber Abteilung für Strukturforschung, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany Search for more papers by this author Stefan Steinbacher Corresponding Author Stefan Steinbacher Abteilung für Strukturforschung, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany Search for more papers by this author Michael Blaesse Michael Blaesse Abteilung für Strukturforschung, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany Search for more papers by this author Thomas Kupke Thomas Kupke Lehrstuhl für Mikrobielle Genetik, Universität Tübingen, Auf der Morgenstelle 15, Verfügungsgebäude, 72076 Tübingen, Germany Search for more papers by this author Robert Huber Robert Huber Abteilung für Strukturforschung, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany Search for more papers by this author Stefan Steinbacher Corresponding Author Stefan Steinbacher Abteilung für Strukturforschung, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany Search for more papers by this author Author Information Michael Blaesse1, Thomas Kupke2, Robert Huber1 and Stefan Steinbacher 1 1Abteilung für Strukturforschung, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany 2Lehrstuhl für Mikrobielle Genetik, Universität Tübingen, Auf der Morgenstelle 15, Verfügungsgebäude, 72076 Tübingen, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:6299-6310https://doi.org/10.1093/emboj/19.23.6299 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Epidermin from Staphylococcus epidermidis Tü3298 is an antimicrobial peptide of the lantibiotic family that contains, amongst other unusual amino acids, S-[(Z)- 2-aminovinyl]-D-cysteine. This residue is introduced by post-translational modification of the ribosomally synthesized precursor EpiA. Modification starts with the oxidative decarboxylation of its C-terminal cysteine by the flavoprotein EpiD generating a reactive (Z)-enethiol intermediate. We have determined the crystal structures of EpiD and EpiD H67N in complex with the substrate pentapeptide DSYTC at 2.5 Å resolution. Rossmann-type monomers build up a dodecamer of 23 point symmetry with trimers disposed at the vertices of a tetrahedron. Oligomer formation is essential for binding of flavin mononucleotide and substrate, which is buried by an otherwise disordered substrate recognition clamp. A pocket for the tyrosine residue of the substrate peptide is formed by an induced fit mechanism. The substrate contacts flavin mononucleotide only via Cys-Sγ, suggesting its oxidation as the initial step. A thioaldehyde intermediate could undergo spontaneous decarboxylation. The unusual substrate recognition mode and the type of chemical reaction performed provide insight into a novel family of flavoproteins. Introduction Antimicrobial peptides represent a universal defence mechanism that is found in multicellular organisms as a key component of their innate immunity and in bacteria as a tool to antagonize bacterial competitors (Jack et al., 1995; Lehrer and Ganz, 1999). Gram-positive bacteria synthesize mostly peptides of ∼20–40 amino acids that are quite similar to eukaryotic defence peptides. The structural diversity observed among these peptides overcomes restrictions by the genetic code by post-translational modifications involving reactions like acylation, heterocyclic ring formation, glycosylation, lipoylation and cyclization of the peptide backbone or formation of thio-ether bridges. Epidermin is a tetracyclic peptide produced and secreted by Staphylococcus epidermidis Tü3298 (Schnell et al., 1988) (Figure 1A). It is a member of the lantibiotic family, a group of mostly plasmid-encoded, ribosomally synthesized and post-translationally modified antimicrobial peptides that are secreted by and mainly act against Gram-positive bacteria. Lantibiotics interact with lipid-bound peptidoglycan precursors and form transient, potential-dependent pores in cytoplasmic membranes of bacteria (Brötz et al., 1998; Breukink et al., 1999). They are characterized by the presence of the thio-ether amino acids lanthionine and 3-methyllanthionine (lanthionine-containing antibiotic peptides) and additionally contain α,β-didehydroamino acids (Sahl and Bierbaum, 1998). The biosynthesis of epidermin from the precursor peptide EpiA involves a series of post-translational modification reactions (Figure 1). In particular, the formation of the unsaturated amino acid S-[(Z)-2-aminovinyl]-D-cysteine (Allgaier et al., 1986) from Ser19 and the C-terminal cysteine residue Cys22 of EpiA has been studied in detail. The reaction involves addition of a (Z)-enethiol group to the didehydroalanine residue generated from Ser19 by dehydration. Figure 1.(A) Biosynthesis of epidermin starting from the 52-amino-acid precursor EpiA. All serine (Ser3, 16 and 19) and threonine (Thr8 and 14) residues of the propeptide are converted to didehydroalanine and didehydrobutyrine residues respectively. Cysteine thiols are stereo-selectively added, which yields two meso-lanthionine (Ala-S-Ala) and one (2S,3S,6R)-3-methyllanthionine (Abu-S-Ala) ring system. S-[(Z)-2-aminovinyl]-D-cysteine is shaded in grey. The didehydroaminobutyric acid residue (Abu) generated from Thr14 remains present in the mature peptide. The propeptide is cleaved off by the protease EpiP. (B) Chemical reaction performed by EpiD. The C-terminal Cys22 is converted to the reactive (Z)-enethiol structure present in mature epidermin (shaded in grey). Download figure Download PowerPoint Flavoprotein EpiD catalyses the formation of the reactive (Z)-enethiol group by oxidative decarboxylation of the C-terminal cysteine residue (Kupke et al., 1994; Kupke and Götz, 1997) (Figure 1B). The structure of the EpiD reaction product has been confirmed by NMR spectroscopy (Kempter et al., 1996). The flavin cofactor that has been identified as non-covalently bound flavin mononucleotide (FMN) is concomitantly reduced to FMNH2. EpiD is essential for epidermin biosynthesis, as a naturally occurring mutation of the epiD gene of Staphylococcus epidermidis, Tü3298/EMS11, is no longer able to produce epidermin. The underlying point mutation Gly93Asp in EpiD turned out to impair FMN binding (Kupke et al., 1992). The substrate specificity of EpiD has been investigated by mass spectrometry applying single peptides or peptide libraries that varied the C-terminal sequence of EpiA (SFNSYCC) (Kupke et al., 1994, 1995). Substrate recognition requires peptides of at least about four to five amino acids in length with a C-terminal consensus sequence of [V/I/L/(M)F/Y/W]-[A/S/V/T/C/(I/L)]-C. The last but second tyrosine can be replaced by a large hydrophobic residue and the exchange of the penultimate cysteine by any small residue does not influence the reaction rates significantly. However, the C-terminal cysteine residue turned out to be essential for the decarboxylation reaction as peptides with serine or homocysteine at the C-terminus are not decarboxylated. The same is true for both the free carboxylate group and the free thiol group of the terminal cysteine as neither the amide SFNSYCC-NH2 nor the thiol-ethylether SFNSYCC(SEt) is decarboxylated. EpiD belongs to a ubiquitous enzyme superfamily whose members are found from bacteria to man and appear to have quite distinct functions (Espinosa-Ruiz et al., 1999; Kupke et al., 2000). Its closest relatives, MrsD from Bacillus sp. HIL-Y85/54728 (Altena et al., 2000) and MutD from Streptococcus mutans (Qi et al., 1999), are, like EpiD, involved in the biosynthesis of lantibiotics, mersacidin and mutacin III, respectively. The Dfp proteins implicated in DNA and panthothenate metabolism (Spitzer and Weiss, 1985; Spitzer et al., 1988) have an N-terminal flavin-binding domain homologous to EpiD and occur in eubacteria and archaea. In vitro Escherichia coli Dfp promotes the decarboxylation of the terminal cysteine moiety of (R)-4′-phospho-N-pantothenoyl-cysteine to (R)-4′-phosphopantetheine in coenzyme A biosynthesis (Kupke et al., 2000). Another group of flavoproteins, however, with unknown molecular targets, includes SIS2/HAL3 from Saccharomyces cerevisiae (Ferrando et al., 1995) and AtHal3 from Arabidopsis thaliana (Espinosa-Ruiz et al., 1999). The recently determined crystal structure of AtHal3 revealed a trimer composed of flavodoxin-like monomers (Albert et al., 2000). SIS2/HAL3 was reported to influence cell cycle regulation and salt and osmotic tolerance, and AtHal3 is also related to salt and osmotic tolerance and plant growth. We have determined the crystal structure of EpiD, a functionally well characterized member of this novel family of flavoproteins, to gain insight into its structure and molecular mechanism. In particular, the substrate complex of EpiD offers insight into the mode of substrate binding by this novel family of proteins and explains the substrate specificity of EpiD involved in an unusual post-translational modification. Results and discussion Structure determination Recombinant EpiD was crystallized in two related monoclinic crystal forms in space group C2 with a dodecamer of tetrahedral symmetry in the asymmetric unit (Table I). The structure was solved by single isomorphous replacement (SIR) and 12-fold non-crystallographic symmetry averaging at 4.3 Å resolution. The density allowed fitting of a polyalanine model, which was used to solve the second crystal form with data to 2.5 Å resolution by Patterson search methods. The inactive mutant H67N was co-crystallized with the substrate-like peptide DSYTC in the cubic space group I2(1)3 with four monomers in the asymmetric unit. In this crystal form, with data to 2.6 Å resolution, the dodecamer is generated by a crystallographic 3-fold axis. The model includes residues 1–173 and one molecule of FMN for each monomer. The C-terminal residues 174–181 of each monomer are disordered in all crystal forms, whereas residues 148–157, which function as a substrate recognition clamp, are disordered in the crystal structure of substrate-free EpiD or contribute to crystal packing but become completely ordered in EpiD-H67N complexed with the peptide DSYTC. The structures were refined to the following crystallographic R-factor values: Rwork 22.1%, Rfree 25.5%; and Rwork 20.9%, Rfree 22.6%, using data to 2.5 and 2.6 Å resolution, respectively (Table II). Table 1. Data collection and phasing statistics Data sets NATI1 NATI2 AuCl3 DSYTC Data collection space group C2 C2 C2 I2(1)3 asymmetric monomers 12 12 12 4 Vm (Å3/Da) 2.9 3.1 3.1 5.5 limiting resolution (Å) 2.5 3.7 4.3 2.57 last shell (Å) 2.50–2.56 3.71–3.91 4.30–4.53 2.68–2.57 unique reflections 81 936 28 987 14 935 56 352 mean redundancy 2.6 (2.4) 2.1 (1.8) 1.9 (1.8) 2.7 (2.1) completeness (%) 87.1 (73.3) 92.4 (82.1) 75.0 (57.5) 93.2 (59.4) mean I/σI 10.2 (2.8) 5.8 (2.4) 2.9 (2.1) 12.2 (2.0) Rsym (%) 5.8 (24.8) 10.2 (27.3) 14.4 (32.2) 6.8 (34.9) Phasing Riso (%) 25.9 – number of sites 12 RCullis 0.76 – phasing power 1.43 – NATI1 (a = 164.7 Å, b = 110.0 Å, c = 152.9 Å; β = 90.4°), native data set used for refinement; NATI2 (a = 176.5 Å, b = 110.5 Å, c = 154.2 Å; β = 94.3°), native data set used for SIR phasing and initial model building. AuCl3 (a = 175.0 Å, b = 110.3 Å, c = 153.9 Å; β = 94.3°), DSYTC (a = b = c = 223.55 Å) pentapeptide complex. Rsym = Σ|I(h)i − |/Σ Riso = Σ|FPH − FP|/ΣFP RCullis = (r.m.s. lack of closure)/(r.m.s. isomorphous difference). Phases were calculated in the resolution range 15.0–4.3 Å and had a mean figure of merit of 0.3. Cyclic 12-fold averaging resulted in a back-transformation R-factor of Rback = 18.1%. Table 2. Refinement statistics EpiD EpiD-H67N-DSYTC Resolution range (Å) 20.0–2.5 20.0–2.6 Reflections in working set 77 526 53 471 Reflections in test set 4087 (5%) 2881 (5%) Rcryst (%) 22.1 20.9 Rfree (%) 25.5 22.6 Protein atoms (non-H) 15 994 5608 Solvent atoms (non-H) 416 136 Cofactor atoms (non-H) 372 124 Peptide atoms (non-H) – 160 Average B-factor (Å2) 37.3 49.5 R.m.s. ΔB (Å2) 1.5 1.7 Deviations from ideality (r.m.s.) bond lengths (Å) 0.008 0.008 bond angles (°) 1.29 1.29 Subunit and dodecamer structure EpiD forms dodecamers with 23 point symmetry from 181 amino acid residue monomers with trimers disposed on the vertices of a tetrahedron (Figure 2). The oligomeric state of the protein in solution was confirmed by size exclusion chromatography (Kupke et al., 2000). The particle has an outer diameter of ∼110 Å and encloses a cavity of ∼35 Å in diameter. Trimers appear as compact trigonal prisms with ∼65 Å in side length and a height of ∼35 Å. In the dodecamer a total of 28.3% of the monomer surface is shielded mainly by trimer contacts. The trimer interface buries a surface of 1864 Å2 per monomer, which represents 22.4% of its total surface (68% hydrophilic and 32% hydrophobic). In contrast, the dimer interface buries only 475 Å2 per monomer, which represents 5.9% of its total surface (69% hydrophilic and 31% hydrophobic). In AtHal3, the trimers represent the biological units (Albert et al., 2000) reflecting the small interaction surface of the dimer contact. Figure 2.Monomer, trimer and dodecamer structure of EpiD. (A) Monomer structure. β-S1 to S6 in yellow, α-helices in dark red and 310-helices in light red. The substrate peptide (in green) is embraced by the substrate binding clamp (S7 and S8 in blue). (B) View along the 3-fold axis. The FMN cofactor is buried in the centre of the trimer interface. The top rim near the trimer axis is formed by α-helix H8. (C) View along the 2-fold axis. The substrates are shown in light yellow. The distance between the two active sites is ∼31 Å (Sγ to Sγ) indicated by a white arrow. Download figure Download PowerPoint Each monomer consists of a single domain with a Rossmann-type fold (Rossmann et al., 1974), which is composed of a central parallel β-sheet of six strands (S1–S6) arranged in the topology 3-2-1-4-5-6. This results in a 2-fold topological symmetry including S3, S2 and S1 in the N-terminal half and S4, S5 and S6 in the C-terminal half. The central β-sheet is flanked by a total of nine α-helices (H1–H9), generating a three-layer αβα protein (Figures 2A and 3). In the trimer, the β-sheet of each monomer runs parallel to the 3-fold axis (Figure 2B), exposing α-helices H1 to H8 to the solvent. Figure 3.Secondary structure of EpiD and alignment with representative homologues MrsD, MutD, Arabidopsis thaliana AtHal3a and Saccharomyces cerevisiae SIS2. Residues involved in dodecamer formation are in green (dimer contacts) and orange (trimer contacts), FMN-binding residues in yellow and residues contacting the substrate are in dark blue. The substrate recognition clamp is shaded in light blue. The PASANT and PXMNXXMW motifs are boxed. The strictly conserved active site base His67 is shown in red. Alignment with AtHal3a is structure based (Albert et al., 2000). The largest deviations occur at H3, contributing to the dimer contact in EpiD and between S3 and H4 (boxed region). The latter substitutes for this dimer contact in AtHal3 and might contribute to substrate binding. Pro143 (168) and Met162 (183) have conserved positions, indicating a substrate recognition loop shorter by four residues in AtHal3 compared with EpiD. Download figure Download PowerPoint In the dodecamer, each monomer contacts two neighbours in the trimer and one in the dimer. Trimer contacts are formed by α-helices H5, H7 and the N-terminal part of α-helix H8, which pack against the N-terminus of α-helix H5, the C-terminal end of H6 and the stretch connecting both helices (Figure 2B). The N-terminus of α-helix H4 contributes indirectly to trimer contacts via the FMN cofactor and directly by side-chain contacts to α-helix H7. Therefore, the FMN cofactor significantly contributes to the contacts within the trimer at the centre of its side face. The dimethylbenzene ring of the isoalloxazine moiety is bound to a hydrophobic patch, whereas the ribityl chain is surrounded by a hydrophilic cavity that opens into the interior of the particle. The trimers in the dodecamer are related by dyads (Figure 2C). The dimer interface involves α-helix H1, the 310-helices H2 and H3 and the connecting coil region. The C-terminal end of the 310-helix H2 contacts the 310-helix H3 of the neighbouring molecules, whereas α-helix H3 contacts α-helix H2 according to the 2-fold symmetry. The interface is dominated by hydrophilic residues and harbours five water molecules. A cluster of hydrophobic residues mainly at the surface contributes either to FMN binding or to the specificity pocket for the large hydrophobic side chain of the substrate peptide. The outer surface at the resulting dimer interface has the shape of a flat groove with FMN cofactors bound at both ends. Residues in this groove contribute to substrate binding. Sequence motifs involved in a novel mode of FMN binding The FMN cofactor is well buried at the centre of the trimer side face contacting three subunits (Figures 2, 3 and 4). However, it can be clearly assigned to one subunit that provides the majority of interactions. There, the isoalloxazine ring is located at the C-terminal end of the β-strands perpendicular to the plane of the central β-sheet. The loop regions connecting the β-strands and α-helices S1/H1, S2/H2, S4/H5 and S5/H7 contribute to its binding. Two of these loop regions, S4/H5 and S5/H7, have been recognized as sequence motifs that characterize this novel family of flavoproteins allowing the identification of members of the family (Figure 3) (Kupke et al., 2000). The region S4/H5 is constituted by the most conserved PASANT motif (residues 81–86) that supports the pyrimidine portion of the isoalloxazine ring by a hydrogen bond of the backbone amide of Ala84 to the oxygen O3. Ser83, Asn85 and Thr86 bind the phosphate group of FMN by their hydrophilic side-chain atoms. Pro81 and Ala82 are not in direct contact with FMN, but Pro81 is present in a cis conformation, resulting in a sharp turn of the chain after β-strand S4. This geometry seems necessary, as the mutant Pro81Ala is no longer able to bind FMN (Kupke et al., 2000). The region S5/H7 formed by the sequence motif PXMNXXMW (residues 114–121) has only a few contacts to the pyrimidine portion of the FMN cofactor. The backbone carbonyl oxygen of Asn115 contacts nitrogen N3 (2.8 Å) of the isoalloxazine and its side-chain carboxamide forms polar interactions with the oxygen O4 at a distance of 3.5 Å. Met120 is located above the pyrimidine system at a distance of 4.2 Å from C2, and is part of a conserved hydrophobic cluster, together with Pro114, Met116 and Trp121. The conserved Asn117 is involved in substrate binding, whereas Trp121 supports Pro143 on the outside of the molecule at the beginning of the substrate binding clamp. Figure 4.FMN binding. (A) The cofactor is buried between two molecules in the trimer (gold and light green). The phosphoribityl moiety is buried ina cavity towards the interior of the particle. The PASANT and PXMNXXMW motifs contribute to contacts with the pyrimidine and phosphoribityl moiety. Leu51 of a neighbouring trimer (dark green) contacts one edge of the dimethylbenzene ring. (B) Schematic representation of FMN contacts. Download figure Download PowerPoint In the trimer, a neighbouring subunit contributes residues 64–67 preceding α-helix H4 to contacts with the isoalloxazine ring, including the active site base His67. The phosphoribityl group is exclusively anchored by residues of one molecule in a hydrophilic depression opening onto the interior of the dodecamer. A neighbouring trimer contributes only a single contact to the C7-methyl group of FMN with the side chain Cδ1 of Leu51. Some recurrent features of diverse flavin-binding proteins involved in dehydrogenation reactions have been reported (Fraaije and Mattevi, 2000). The oxidized isoalloxazine is essentially planar in most cases, which is also true for EpiD, but both the si or re side can be exposed to the substrate. In EpiD the re side faces the substrate. The N1-C2=O2 locus of the pyrimidine part is generally found in contact with a positively charged residue, the N-terminus of an α-helix or bound by a cluster of peptide nitrogens. In EpiD, O2 is hydrogen bonded to the backbone amide of Ala84 (2.8 Å) of α-helix H5. However, no other positively charged atoms are near to N1 that could stabilize the negative charge evolving upon reduction of FMN. Nϵ of His67 is 4.6 Å and O3 of ribityl 4.1 Å away from N1. However, a water molecule that is also found in AtHal3 (Albert et al., 2000) bridges N1 (3.2 Å) and O2 (3.0 Å) of the isoalloxazine ring and O2 (2.8 Å) of the ribityl chain. The hydrophilic environment might be important in stabilizing the negative charge evolving on N1 upon reduction of FMN. An important feature modulating the redox potential of FMN is a hydrogen-bond donor close to N5, the atom directly involved in the oxidation reaction. In EpiD, the backbone amide of Ile13 is located at a distance of 3.2 Å in the plane trough N5 and N10, well within the range 2.8–3.3 Å usually observed (Fraaije and Mattevi, 2000). The angle of 126° between N10, N5 and the amide nitrogen falls within the reported range of 116−170°. N3 is hydrogen bonded to the carbonyl group of Asn115 (2.8 Å). Nϵ of His67 is located on the re side 3.5 Å above N10, with an angle of 86° between N5, N10 and Nϵ, and is 4.5 Å away from N5. EpiD shares the Rossmann-type fold with flavodoxin-like proteins (Smith et al., 1983; Fukuyama et al., 1990; Rao et al., 1992) as shown by a survey of 81 crystal structures of FMN-binding proteins in the PDB (Bernstein et al., 1977) with SCOP (Murzin et al., 1995). This yields 34 enzymes that represent 17 unique proteins. Although EpiD and flavodoxin have the gross binding region with respect to the folding architecture in common, the orientation of the FMN cofactor is clearly different (Figure 5). Flavodoxins expose the si side to the solvent whereas EpiD has the re side exposed. The most prominent difference is the location of the phosphoribityl chains, which are located on opposing sides of the central β-sheet. This novel mode of interaction between FMN and a Rossmann-type fold can be expected to be representative for this family of flavoproteins as it is associated with characteristic sequence motifs (Kupke et al., 2000). This has been corroborated by the structure of AtHal3, which shows an essentially identical binding mode (Albert et al., 2000). Only the dimer contact to Leu51 in EpiD is replaced by a hydrophobic contact to Trp81 and the dimethylbenzene ring is additionally supported by Trp78. Figure 5.Superposition of EpiD (colours as in Figure 2A) and flavodoxin (purple) as a representative of FMN-binding proteins. Structural equivalents to S3 and H3 of EpiD are absent in flavodoxin. The FMN cofactors bind to topologically similar positions, although with different orientation of the ribityl moiety and exposing different sides (EpiD, re-side; flavodoxin, si-side). Download figure Download PowerPoint Substrate recognition clamp and specificity of binding To address substrate binding, the inactive mutant H67N of EpiD was crystallized in the presence of the pentapeptide DSYTC, which shows higher solubility compared with the natural C-terminus of EpiA, NSYCC or SYCC; the latter being the shortest substrate of EpiD. The penultimate cysteine was exchanged for threonine to exclude intramolecular disulfide bridge formation. These changes do not affect substrate recognition (Kupke et al., 1995). To prevent conversion of the substrate, the mutant EpiD-His67Asn was chosen, in which the active site base His67, located above the isoalloxazine, is replaced by Asn, resulting in a complete loss of enzymatic activity (Kupke et al., 2000). The substrate pentapeptide will be numbered according to the EpiA sequence, i.e. P-Asp18 to P-Cys22. The pentapeptide substrate is bound along the channel that connects the 2-fold axis and the FMN cofactor in an extended conformation, well defined by electron density (Figure 6A). There, it is embraced by a substrate recognition clamp comprising residues Pro143 to Met162. The binding clamp forms a highly twisted antiparallel β-sheet (S7 and S8) with Ser152, Ser153 and Gly154 in the turn region. The binding clamp and substrate together form a three-stranded β-sheet with the peptide running parallel to S7. The remaining edge of the substrate β-strand faces α-helix H1 and the S5/H7 loop (PXMNXXMW motif) (Figure 6B). The clamp adopts a right-handed double-helical tertiary structure that results in a turn of almost 90° at residues Lys147 and Asn158. The first residue of the clamp, Pro143, is fixed by hydrophobic contact to Trp121 of the PXMNXXMW motif, whereas the last, Met162, contacts Pro81 of the PASANT motif. At the C-terminal end it is anchored to the protein surface by α-helix H9. As the substrate recognition clamps of three out of four crystallographically independent monomers are engaged in crystal contacts, their precise geometry differs slightly, indicating their adaptability to the substrate in line with a rather broad specificity. However, the cysteinyl residues at the active site and all residues that are ordered in both the apo and complex structures are in virtually identical positions. Figure 6.Substrate binding. (A) 2Fo − Fc electron density contoured at 1σ for the substrate pentapeptide is shown in green, that of the substrate recognition clamp in blue. (B) The substrate peptide (P-Ser19 to P-Cys22) forms a regular parallel β-sheet with β-strand S7 of the substrate clamp (Phe149 to Ile151) and backbone H-bonds to the protein surface on the opposite side to Asn14 (carbonyl and amide of P-Tyr20) and Asn117 (amide and carboxylate of P-Cys22). (C) Surface representation of the binding pocket for P-Tyr20 coloured according to the H-bonding properties of the contributing protein residues (grey, hydrophobic; red, H-bond donors; blue, H-bond acceptors). Download figure Download PowerPoint Residues of two additional subunits contribute to substrate binding. Ile68 next to the active site base His67 from a neighbour in the trimer helps to position the substrate cysteine P-Cys22 at the flavin by a hydrophobic contact to its methylene group. A neighbour related by the 2-fold symmetry contributes only to the binding pocket that accommodates the side chain of the last but second P-Tyr20. This pocket consists of two parts, one constituted by static residues from the protein surface and the other by Phe149 and Ile151 of the mobile binding clamp (Figure 6C). Binding of the large last but second residue therefore reflects a dynamic process involving an induced fit mechanism. The floor of the overall hydrophobic pocket is rather hydrophilic, including Asn17, Asn19 and His20. The front of the pocket is closed by the backbone of the N-terminal aspartate residue P-Asp18 of the substrate-like peptide. The position of the latter is fixed by the hydrogen bond of its carboxylate group to the hydroxyl group of Tyr156 from the specificity loop. The structure provides a straightforward explanation of the previously determined substrate requirements of EpiD (Kupke et al., 1995). The observed binding site extends from the FMN cofactor to the 2-fold axis of the particle, which accommodates approximately five amino acids in an extended conformation. Shorter peptides will not form essential interactions with the binding clamp and longer peptides will probably not make additional contacts as they extend beyond the 2-fold axis of the particle. More specific interactions occur between P-Tyr20 and the protein where large hydrophobic residues are required, whereas the penultimate P-Cys21 can be replaced by any residue of comparable size. Larger residues will result in a steric conflict with Asn159 of the binding clamp that crosses the substrate between the side chains of P-Ser19 and P-Thr21. Binding of substrates to flavoproteins is usually observed at a site located underneath the protein surface, where the access is gated by mobile loops, side chains or flexible domains, resulting in the exclusion of solvent. Alternatively, an exact match to a narrow active-site channel can be utilized as a strategy to expel solvent (Fraaije and Mattevi, 2000). In EpiD, the substrate binding clamp does not contribute to solvent exclusion at the active site but only to binding of the substrate. Solvent at the active site is excluded by the complementarity between P-Cys22 and non-flexible residues on the protein surface. Therefore, EpiD combines both strategies of substrate binding. With the exception of the side chain of P-Tyr20, all contacts and active site residues reside within the trimer. Obvio