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• W2007343995 abstract "The crystal structure of the bidomain PCP-C from modules 5 and 6 of the nonribosomal tyrocidine synthetase TycC was determined at 1.8 Å resolution. The bidomain structure reveals a V-shaped condensation domain, the canyon-like active site groove of which is associated with the preceding peptidyl carrier protein (PCP) domain at its donor side. The relative arrangement of the PCP and the peptide bond-forming condensation (C) domain places the active sites ∼50 Å apart. Accordingly, this PCP-C structure represents a conformational state prior to peptide transfer from the donor-PCP to the acceptor-PCP domain, implying the existence of additional states of PCP-C domain interaction during catalysis. Additionally, PCP-C exerts a mode of cyclization activity that mimics peptide bond formation catalyzed by C domains. Based on mutational data and pK value analysis of active site residues, it is suggested that nonribosomal peptide bond formation depends on electrostatic interactions rather than on general acid/base catalysis. The crystal structure of the bidomain PCP-C from modules 5 and 6 of the nonribosomal tyrocidine synthetase TycC was determined at 1.8 Å resolution. The bidomain structure reveals a V-shaped condensation domain, the canyon-like active site groove of which is associated with the preceding peptidyl carrier protein (PCP) domain at its donor side. The relative arrangement of the PCP and the peptide bond-forming condensation (C) domain places the active sites ∼50 Å apart. Accordingly, this PCP-C structure represents a conformational state prior to peptide transfer from the donor-PCP to the acceptor-PCP domain, implying the existence of additional states of PCP-C domain interaction during catalysis. Additionally, PCP-C exerts a mode of cyclization activity that mimics peptide bond formation catalyzed by C domains. Based on mutational data and pK value analysis of active site residues, it is suggested that nonribosomal peptide bond formation depends on electrostatic interactions rather than on general acid/base catalysis. Nonribosomal peptide synthetases (NRPSs) are very large multidomain enzymes (0.15–1.5 MDa) that catalyze simple chemical reactions in a repetitive, assembly line-like manner to produce a broad variety of secondary peptidic metabolites, including many antimicrobial agents and siderophores (Walsh, 2004Walsh C.T. Polyketide and nonribosomal peptide antibiotics: modularity and versatility.Science. 2004; 303: 1805-1810Crossref PubMed Scopus (450) Google Scholar, Finking and Marahiel, 2004Finking R. Marahiel M.A. Biosynthesis of nonribosomal peptides.Annu. Rev. Microbiol. 2004; 58: 453-488Crossref PubMed Scopus (587) Google Scholar). These synthetases can be described as a linear arrangement of modules, which are responsible for the specific incorporation and often modification of one building block into the peptidic product (Sieber and Marahiel, 2005Sieber S.A. Marahiel M.A. Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics.Chem. Rev. 2005; 105: 715-738Crossref PubMed Scopus (437) Google Scholar). As the order of NRPS modules mostly reflects the sequence of amino acids in the peptidic product (Figure 1), the reprogramming of NRPS by reshuffling their constituent modules and manipulation of their specificities offers a promising route for the creation of novel bioactive compounds (Nguyen et al., 2006Nguyen K.T. Ritz D. Gu J.-Q. Alexander D. Chu M. Miao V. Brian P. Combinatorial biosynthesis of novel antibiotics related to daptomycin.Proc. Natl. Acad. Sci. USA. 2006; 103: 17462-17467Crossref PubMed Scopus (201) Google Scholar, Stachelhaus et al., 1995Stachelhaus T. Schneider A. Marahiel M.A. Rational design of peptide antibiotics by targeted replacement of bacterial and fungal domains.Science. 1995; 269: 69-72Crossref PubMed Scopus (250) Google Scholar). At least three different domains are necessary to build up an NRPS module that elongates peptide intermediates: the adenylation (A) domain, the peptidyl carrier protein (PCP) domain, and the condensation (C) domain. The A domain (∼550 aa) is responsible for the recognition and activation of the building block by adenylation, which in turn is then covalently attached as a thioester to the prosthetic 4′-phospho-pantetheine (Ppan) group of a subsequent PCP domain (∼80 aa). After loading of the PCP domains of two neighboring modules with their cognate substrates, the C domain (∼450 aa), located in between, catalyzes the condensation (i.e., peptide bond formation) between the two substrates. This condensation reaction is strictly unidirectional, leading to a downstream-directed synthesis of the NRPS product (Figure 1). As the NRPS activity proceeds, the growing peptide chain is continuously translocated toward the most C-terminal module in the synthetase, where it is eventually cleaved off by hydrolysis, intramolecular cyclization, or reduction (Walsh, 2004Walsh C.T. Polyketide and nonribosomal peptide antibiotics: modularity and versatility.Science. 2004; 303: 1805-1810Crossref PubMed Scopus (450) Google Scholar). Even though X-ray and NMR structures of isolated A (Conti et al., 1997Conti E. Stachelhaus T. Marahiel M.A. Brick P. Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S.EMBO J. 1997; 16: 4174-4183Crossref PubMed Scopus (554) Google Scholar, May et al., 2002May J.J. Kessler N. Marahiel M.A. Stubbs M.T. Crystal structure of DhbE, an archetype for aryl acid activating domains of modular nonribosomal peptide synthetases.Proc. Natl. Acad. Sci. USA. 2002; 99: 12120-12125Crossref PubMed Scopus (227) Google Scholar), PCP (Koglin et al., 2006Koglin A. Mofid M.R. Lohr F. Schafer B. Rogov V.V. Blum M.M. Mittag T. Marahiel M.A. Bernhard F. Dotsch V. Conformational switches modulate protein interactions in peptide antibiotic synthetases.Science. 2006; 312: 273-276Crossref PubMed Scopus (124) Google Scholar, Weber et al., 2000Weber T. Baumgartner R. Renner C. Marahiel M.A. Holak T.A. Solution structure of PCP, a prototype for the peptidyl carrier domains of modular peptide synthetases.Struct. Fold. Des. 2000; 8: 407-418Abstract Full Text Full Text PDF Scopus (157) Google Scholar), and C (Keating et al., 2002Keating T.A. Marshall C.G. Walsh C.T. Keating A.E. The structure of VibH represents nonribosomal peptide synthetase condensation, cyclization and epimerization domains.Nat. Struct. Biol. 2002; 9: 522-526PubMed Google Scholar) domains have been solved, little is known about their interactions. With the exception of the initiation module, each PCP domain interacts within an NRPS not only with the adenylation domain, but also with the preceding and following C domains, where it either accepts or donates the growing peptide intermediate (Lai et al., 2006bLai J.R. Koglin A. Walsh C.T. Carrier protein structure and recognition in polyketide and nonribosomal peptide biosynthesis.Biochemistry. 2006; 45: 14869-14879Crossref PubMed Scopus (67) Google Scholar). NMR titration experiments revealed profound structural plasticity of the PCP domain that changes its tertiary structure dependent on the domain type with which it needs to interact during different steps of NRPS action (Koglin et al., 2006Koglin A. Mofid M.R. Lohr F. Schafer B. Rogov V.V. Blum M.M. Mittag T. Marahiel M.A. Bernhard F. Dotsch V. Conformational switches modulate protein interactions in peptide antibiotic synthetases.Science. 2006; 312: 273-276Crossref PubMed Scopus (124) Google Scholar). Furthermore, the prosthetic Ppan group merely spans ∼20 Å, by far too little to simultaneously reach the upstream and downstream C domains. Accordingly, not only the domains have to be located so that their distance relative to each other is minimized, as exemplified by the fatty acid synthases (Maier et al., 2006Maier T. Jenni S. Ban N. Architecture of mammalian fatty acid synthase at 4.5 Å resolution.Science. 2006; 311: 1258-1262Crossref PubMed Scopus (282) Google Scholar, Jenni et al., 2006Jenni S. Leibundgut M. Maier T. Ban N. Architecture of a fungal fatty acid synthase at 5 Å resolution.Science. 2006; 311: 1263-1267Crossref PubMed Scopus (117) Google Scholar), but the PCP domain has to adapt its structure such that the prosthetic Ppan group reaches all the neighboring domains. As a consequence, any experimentally determined oligodomain structure might represent a distinct state of NRPS interdomain communication during nonribosomal peptide synthesis. The C domains share a highly conserved histidine motif (HHxxxDG, core 3) (Marahiel et al., 1997Marahiel M.A. Stachelhaus T. Mootz H.D. Modular peptide synthetases involved in non-ribosomal peptide synthesis.Chem. Rev. 1997; 97: 2651-2673Crossref PubMed Scopus (868) Google Scholar), which is essential for condensation activity (Stachelhaus et al., 1998Stachelhaus T. Mootz H.D. Bergendahl V. Marahiel M.A. Peptide bond formation in nonribosomal peptide biosynthesis.J. Biol. Chem. 1998; 273: 22773-22781Crossref PubMed Scopus (249) Google Scholar, Bergendahl et al., 2002Bergendahl V. Linne U. Marahiel M.A. Mutational analysis of the C-domain in nonribosomal peptide synthesis.Eur. J. Biochem. 2002; 269: 620-629Crossref PubMed Scopus (91) Google Scholar). One model suggests that the second histidine of this motif acts as a general base catalyst, restoring the nucleophilicity of the acceptor substrate by deprotonation of its α-ammonium group prior to the initial attack of the amino group onto the carboxyl group of the donor substrate (Bergendahl et al., 2002Bergendahl V. Linne U. Marahiel M.A. Mutational analysis of the C-domain in nonribosomal peptide synthesis.Eur. J. Biochem. 2002; 269: 620-629Crossref PubMed Scopus (91) Google Scholar). In this work, the first X-ray structure of a PCP-C bidomain is presented, which was excised from modules 5 and 6 of the tyrocidine synthetase TycC. Within the tyrocidine biosynthesis cluster and its three constituent NRPSs (TycA, TycB, and TycC), this C domain catalyzes the elongation of the nonapeptide DPhe-Pro-Phe-DPhe-Asn-Gln-Tyr-Val-Orn by L-Leu to the linear precursor of tyrocidine A (Figure 1). Although this reaction is not catalyzed in vitro by the isolated PCP-C bidomain, it was found to mediate peptide bond formation during the cyclization of synthetic peptides. To investigate the substrate acceptance of internal C domains, the recombinant PCP-C bidomain from modules 5 and 6 of the tyrocidine NRPS, TycC (Bacillus brevis, ATCC 8185), was produced in its apo-form (TycC5-6) (i.e., without the Ppan modification at S43). The upstream PCP domain was chemoenzymatically primed with various acceptor substrates, as shown for P1-coenzyme A (CoA) in Figure 2. For this chemoenzymatic reaction, the permissive phospho-pantetheine transferase Sfp was used to catalyze the transfer of synthetic peptidyl-Ppan arms onto the PCP domain with the corresponding CoA substrates (Belshaw et al., 1999Belshaw P.J. Walsh C.T. Stachelhaus T. Aminoacyl-CoAs as probes of condensation domain selectivity in nonribosomal peptide synthesis.Science. 1999; 284: 486-489Crossref PubMed Scopus (254) Google Scholar). Even though no elongated peptide product could be detected after incubation with potential acceptor substrate mimics, HPLC analysis of the priming reaction surprisingly revealed the formation of the cyclic hexapeptide, cP1 (Figure 2), in 20-fold excess over the background reaction. The head-to-tail connectivity of this product was delineated by MS-MS spectrometry (Figure 3). The use of an N-terminally acetylated variant of this hexapeptidyl-CoA substrate caused no product release—neither of the cyclic peptide, nor of the linear peptide released by hydrolysis of the PCP-bound thioester (data not shown). Furthermore, the peptidyl-Ppan modification of the isolated recombinant PCP domain of TycC5 alone led to no increased cyclization ratio compared to the background reaction. To demonstrate a direct role of the C domain in this cyclization reaction, mutants of the HHxxxDG motif (Marahiel et al., 1997Marahiel M.A. Stachelhaus T. Mootz H.D. Modular peptide synthetases involved in non-ribosomal peptide synthesis.Chem. Rev. 1997; 97: 2651-2673Crossref PubMed Scopus (868) Google Scholar) in PCP-C were prepared by site-directed mutagenesis. Since the second histidine of this motif had been suggested to be essential for catalysis (Bergendahl et al., 2002Bergendahl V. Linne U. Marahiel M.A. Mutational analysis of the C-domain in nonribosomal peptide synthesis.Eur. J. Biochem. 2002; 269: 620-629Crossref PubMed Scopus (91) Google Scholar), it was mutated to HAxxxDG and HVxxxDG, respectively. Under the same assay conditions, PCP-C mutants in position 224 showed neither peptide cyclization nor thioester hydrolysis, even though an Sfp-dependent consumption of substrate due to the priming of the PCP domain could be monitored by LC-MS (Figure 2). In contrast, the corresponding AHxxxDG mutant in which the first histidine of the HHxxxDG motif (H223) was replaced still displayed ∼28% of the wild-type activity. The use of six other peptidyl-substrates (P2–P7; Table 1), the cyclization activity of PCP-C was further explored. With lengths between 5 and 8 amino acids, the type and the absolute configuration of the terminal amino acids was altered in comparison to P1. The PCP-C bidomain only cyclized substrates that contained D-configured N termini (P1, P3, and P7). No hydrolysis product was cleaved off the enzyme, except in the case of the octapeptidyl substrate, P6. The C-terminal L-Gln cannot be exchanged to L-Ala in these reactions. Whenever a C domain-dependent cyclization was observed, autocatalytic formation of the cyclic product occurred with relative amounts of 5%–17%, as determined by peak integration of UV/VIS-recorded chromatograms.Table 1Substrates Used in Cyclization AssaysCoA SubstrateCyclization (Ratio)aMinus signs indicate that no cyclization product was observed.HydrolysisP1DPhe-Pro-Phe-DPhe-Asn-Gln++ (20:1)−P2LPhe-Pro-Phe-DPhe-Asn-Gln−−P3DAla-Pro-Phe-DPhe-Asn-Gln++ (7:1)−P4LAla-Pro-Phe-DPhe-Asn-Gln−−P5DPhe-Pro-Phe-DPhe-Asn-Ala−−P6DPhe-Pro-Phe-DPhe-Ala-Ala-Asn-Gln−++P7DPhe-Pro-Phe-Asn-Gln+ (n.d.)bn.d. indicates values not determined and refers to the ratio of cyclization to hydrolysis.−Hexapeptides with D-configured N termini and C-terminal glutamine are cyclized by PCP-C with ratios ranging from 7- to 20-fold in comparison with background reactions. Hydrolysis was only observed when the octapeptidic substrate P6 was used.a Minus signs indicate that no cyclization product was observed.b n.d. indicates values not determined and refers to the ratio of cyclization to hydrolysis. Open table in a new tab Hexapeptides with D-configured N termini and C-terminal glutamine are cyclized by PCP-C with ratios ranging from 7- to 20-fold in comparison with background reactions. Hydrolysis was only observed when the octapeptidic substrate P6 was used. Overall, it is shown here that the TycC6 condensation domain exhibits a novel in vitro cyclization activity on peptides presented at its donor site. The primary sequence of the substrate, P1, was derived from the first six amino acids of the decapeptide tyrocidine. The condensation domain tested, however, was from the 10th module (TycC6) of the tyrocidine NRPS cluster, where it elongates the nonapeptidyl substrate, DPhe-Pro-Phe-DPhe-Asn-Gln-Tyr-Val-Orn, by addition of L-Leu to the scaffold's C terminus. Synthetic CoA substrates with a C-terminal ornithine, however, lacked sufficient stability for in vitro assays, presumably caused by formation of a six-membered lactam due to an attack of the ornithine δ-amino group onto the CoA thioester. For intramolecular head-to-tail cyclization of P1, a proximal arrangement of both termini must be sterically adopted within the active site of the C domain so that intramolecular peptide bond formation can proceed. Considering the linear tyrocidine precursor, it is assumed that it tends to prefold by intramolecular hydrogen bonds due to its delicate primary sequence in the N-terminal part (Kuo and Gibbons, 1980Kuo M.-C. Gibbons W.A. Nuclear Overhauser effect and cross-relaxation rate determinations of dihedral and transannular interproton distances in the decapeptide tyrocidine A.Biophys. J. 1980; 32: 807-836Abstract Full Text PDF PubMed Scopus (46) Google Scholar). The alteration of D- and L-configured amino acids, as well as their hydrophobicity and steric strain, are believed to be important for such a prearrangement, which might also partially occur in the truncated peptidyl substrates assayed here. Taking our current understanding of the selectivity of C domains (Bergendahl et al., 2002Bergendahl V. Linne U. Marahiel M.A. Mutational analysis of the C-domain in nonribosomal peptide synthesis.Eur. J. Biochem. 2002; 269: 620-629Crossref PubMed Scopus (91) Google Scholar, Belshaw et al., 1999Belshaw P.J. Walsh C.T. Stachelhaus T. Aminoacyl-CoAs as probes of condensation domain selectivity in nonribosomal peptide synthesis.Science. 1999; 284: 486-489Crossref PubMed Scopus (254) Google Scholar, Clugston et al., 2003Clugston S.L. Sieber S.A. Marahiel M.A. Walsh C.T. Chirality of peptide bond-forming condensation domains in nonribosomal peptide synthetases: the C5 domain of tyrocidine synthetase is a (D)C(L) catalyst.Biochemistry. 2003; 42: 12095-12104Crossref PubMed Scopus (70) Google Scholar) into account, one might hence suspect that the intramolecular cyclization mimics the mode of intermolecular peptide bond formation catalyzed by condensation domains with their cognate substrates. One indication for this is given by the observation that the mutants of the second histidine in the HHxxxDG core motif (H224) showed no cyclization activity at all. The structure of the excised PCP-C bidomain was solved at 1.8 Å resolution by multiple anomalous diffraction (MAD) experiments from crystals of SeMet-labeled protein. Mass-spectrometric analysis of the SeMet-labeled TycC5-6 PCP-C bidomain showed that, as in the previous case of EntB (Drake et al., 2006Drake E.J. Nicolai D.A. Gulick A.M. Structure of the EntB multidomain nonribosomal peptide synthetase and functional analysis of its interaction with the EntE adenylation domain.Chem. Biol. 2006; 13: 409-419Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), the absence of iron in the culture medium induced transfer of a Ppan group onto S43 of the PCP domain (data not shown). This posttranslational modification initially prohibited growth of SeMet-labeled crystals and is apparently caused by the action of the endogenous phospho-pantetheinyl transferase, EntD, which is part of the Escherichia coli enterobactin biosynthesis cluster. Addition of 15 μM Fe(II) to the SeMet-labeling medium abolished this modification by suppressing the enterobactin biosynthesis cluster. After refinement, each asymmetric unit of the PCP-C bidomain crystals contains one polypeptide chain defined from R3 to M455 and from Q461 to L522, 294 water molecules, and two sulfate ions and dioxane molecules, which originate from the crystallization buffer. The PCP domain of module 5 of TycC (residues M1–T82) measures approximately 20 Å × 24 Å × 30 Å, the C domain of module 6 (residues V101-L522) ca. 65 Å × 50 Å × 40 Å. Both domains are connected via an 18-residue linker (A83-P100) running along the PCP-C domain interface (Figure 4A). The structure of the PCP domain closely corresponds to the A/H state (Koglin et al., 2006Koglin A. Mofid M.R. Lohr F. Schafer B. Rogov V.V. Blum M.M. Mittag T. Marahiel M.A. Bernhard F. Dotsch V. Conformational switches modulate protein interactions in peptide antibiotic synthetases.Science. 2006; 312: 273-276Crossref PubMed Scopus (124) Google Scholar). This state was found in the PCP domain of the third module of TycC as an intermediary conformational state that is present in both the Ppan-modified (holo [H]) and unmodified (apo [A]) PCP domain (Koglin et al., 2006Koglin A. Mofid M.R. Lohr F. Schafer B. Rogov V.V. Blum M.M. Mittag T. Marahiel M.A. Bernhard F. Dotsch V. Conformational switches modulate protein interactions in peptide antibiotic synthetases.Science. 2006; 312: 273-276Crossref PubMed Scopus (124) Google Scholar). A superposition with the A/H-like state of the PCP domain of module 3 of TycC (Weber et al., 2000Weber T. Baumgartner R. Renner C. Marahiel M.A. Holak T.A. Solution structure of PCP, a prototype for the peptidyl carrier domains of modular peptide synthetases.Struct. Fold. Des. 2000; 8: 407-418Abstract Full Text Full Text PDF Scopus (157) Google Scholar) gives an rmsd of 1.67 Å for 56 Cα positions. In the holo-form of PCP domains, the A/H state is in conformational equilibrium with the H state, which becomes increasingly stabilized by interactions with other holo-PCP-recognizing domains, like the editing thioesterase II (Koglin et al., 2006Koglin A. Mofid M.R. Lohr F. Schafer B. Rogov V.V. Blum M.M. Mittag T. Marahiel M.A. Bernhard F. Dotsch V. Conformational switches modulate protein interactions in peptide antibiotic synthetases.Science. 2006; 312: 273-276Crossref PubMed Scopus (124) Google Scholar). This H state diverges significantly from the conformation of the TycC5 PCP domain, as 78 Cα positions superimpose with an rmsd of only 5.5 Å, mostly along helix αI and αIV with a displacement of 2.3 Å for 18 Cα positions. Accordingly, the PCP-C domain interface observed in the crystal structure has to represent a state, where adoption of the A/H state for the PCP domain is compatible for loading with an aminoacyl residue by the preceding A domain. The only as-yet-known structure of a C domain is VibH from the vibriobactin synthesis cluster (Keating et al., 2002Keating T.A. Marshall C.G. Walsh C.T. Keating A.E. The structure of VibH represents nonribosomal peptide synthetase condensation, cyclization and epimerization domains.Nat. Struct. Biol. 2002; 9: 522-526PubMed Google Scholar), which recognizes in trans both the donor substrate, PCP-bound 2,3-dihydroxy-benzoic acid, and the acceptor substrate, norspermidine. Like the isolated VibH domain, the TycC6 condensation domain consists of two mainly separated and structurally similar subdomains, an N-terminal (V101–S268) and a C-terminal subdomain (A269–L522). Both subdomains are arranged in a V-shaped fashion and belong to the chloramphenicol-acetyltransferase (CAT) fold. There are only two major contact sites between these two CAT-like subdomains, thus giving rise to a large, canyon-like active site groove: one is mainly made at the floor of the active site canyon and comprises the loop, β8-β9 (T359–V374), with a short, internal 310 element and the conserved arginine R361 that stabilizes the loop conformation by H-bond interactions between its side chain and the peptide carbonyls of L366, E367, and I369. Besides a salt bridge between the side chains of D365 and R252, most H-bond interactions between the floor loop and the N-terminal subdomain are derived from the main chain, as this loop is made up of mostly hydrophobic residues. The second region is strand β11 that is donated from the C-terminal to the N-terminal subdomain, and thereby extends the four-stranded β sheet (β1-β6-β5-β4) of the latter. This extension (N438–F465) spans like a bridge over the active site canyon (Figures 4A and 7C), and appears to be rather flexible due to high thermal B-factors as compared with the remaining condensation domain. A superposition of the TycC6 C domain (residues 101–522) with VibH (PDB code: 1L5A ; pairwise sequence identity, 19%) shows structural similarity with an overall rmsd of 1.58 Å for 197 Cα-carbons (Figure 4B). Due to the fact that the arrangement of the two CAT-like subdomains is off-rotated by 12°, in the superposition only the C-terminal CAT-like subdomains are fitted properly, whereas the orientation of the N-terminal CAT-like subdomain of the TycC6 C domain as a whole—compared with the corresponding VibH N-terminal subdomain—is slightly distorted. The hinge-like region, around which this swiveling motion is centered, corresponds to S268 (VibH: S174) in the short connection between helices α5 and α6. Accordingly, structural comparisons of the corresponding subdomains were performed (Figure 4C), yielding a lower rmsd value of 1.35 Å for 149 Cα-carbons of the C-terminal CAT-like subdomains and an increased value of 1.70 Å for 116 Cα-carbons of the N-terminal CAT-like subdomains. Two sulfate ions are located in the vicinity of the two domains' active site residues, S43 and H224, respectively (Figure 4A). The sulfate ion next to the PCP domain forms salt bridges to the conserved residues, H42 (Nδ, 3.11 Å) and R45 (Nɛ, 2.92 Å; Nη, 2.89 Å), and is additionally stabilized by the dipole moment of helix αII, which points with its N terminus onto this sulfate. Obviously, this sulfate anion occupies the supposed position of the phosphate group of the Ppan arm, if the latter is esterified to S43 of the PCP domain. The sulfate ion in the C domain's active site forms a salt bridge to the catalytically active residue, H224 (Nɛ2, 2.87 Å), and an H bond to the amide nitrogen of G229 (2.68 Å). A structure-based sequence alignment between the VibH and TycC6 C domains and other NRPS C and epimerase domains (Figure 5A) indicates that, in the E domain, there are several sites distributed all over the protein where minor loop insertions and deletions occur. According to this alignment, there is no hint of an insertion unique to epimerase domains that may block the acceptor side of the substrate channel, as suggested previously (Keating et al., 2002Keating T.A. Marshall C.G. Walsh C.T. Keating A.E. The structure of VibH represents nonribosomal peptide synthetase condensation, cyclization and epimerization domains.Nat. Struct. Biol. 2002; 9: 522-526PubMed Google Scholar). There are several indications that the observed PCP-C domain arrangement is not the peptide bond-forming conformational state, where the Ppan-bound nonapeptide is passed from the donor side of the C domain's active site. First, the relative orientation of the PCP and C domains (Figure 4A) places the catalytic residues, S43 and H224, at a distance of 47 Å (Figure 5B), which is more than the Ppan arm can span (∼20 Å). Second, Lai et al., 2006aLai J.R. Fischbach M.A. Liu D.R. Walsh C.T. A protein interaction surface in nonribosomal peptide synthesis mapped by combinatorial mutagenesis and selection.Proc. Natl. Acad. Sci. USA. 2006; 103: 5314-5319Crossref PubMed Scopus (52) Google Scholar identified several residues of the EntB PCP domain from the enterobactin biosynthesis cluster that are crucial for productive interaction with the downstream C domain. Those residues of EntB, M249, F264, and A268, correspond to M47, L63, and F67 of the TycC5 PCP domain. As these residues are proximal to the Ppan attachment site, S43, they are far off-positioned from the surface of the TycC6 condensation domain (Figure 6A). (A) Overview of the PCP domain and its interactions with the C domain. The core motif commonly found in PCP domains is highlighted in yellow, and the linker is displayed in red. Residues corresponding to those identified to be crucial for interaction with the downstream C domain by Lai et al., 2006aLai J.R. Fischbach M.A. Liu D.R. Walsh C.T. A protein interaction surface in nonribosomal peptide synthesis mapped by combinatorial mutagenesis and selection.Proc. Natl. Acad. Sci. USA. 2006; 103: 5314-5319Crossref PubMed Scopus (52) Google Scholar are colored blue. (B) Detailed view of the 18 residue linker connecting the PCP and C domains and its interactions with the PCP domain and the C domain. (C) Superposition of the observed A/H state and a modeled H state of the TycC5 PCP domain. As E56 is translocated by 28 Å, at least this residue would be no longer involved in interactions with the downstream C domain. (D) Tentative model of a catalytically competent PCP-C domain interaction. The TycC5 PCP domain is shown in the modeled H state (blue, orientation as in Figure 6C; orange, orientation compatible with a positioning of the Ppan arm in the active site of the C domain). Nevertheless, there are several well-defined interactions found at the domains' interface. Besides the connection via the linker region PCP-C domain, interactions occur in two separated clusters, thereby occluding 1089 Å2 from bulk solvent access. In the first cluster (Figure 6A), H bonds are formed between the side chains of Y6 and K397 (2.67 Å) and between water molecule HOH88 and the side chains of Q29 (3.19 Å), Q394 (2.84 Å), K397 (2.70 Å), and the peptide group preceding G328 (3.08 Å), respectively. Interactions in the second cluster are mainly made by H bonds. Besides bridging water molecules, there are also direct interactions of residues of the PCP domain with those of the C domain. For instance, R16 forms H bonds to both G405 (2.60 Å) and D408 (2.83 Å), and E56 forms salt bridges to H406 (3.00 Å) and K273 (4.31 Å). Interactions of the linker comprise hydrophobic interactions formed by F88 with the residues W261 and F265 of the C domain. Furthermore, several residues of the linker are involved in an intricate H-bond network with both, the PCP and C domain (Figure 6B). Not only does N86 interact with T82 (3.15 Å) and the carboxylic group of I79 (3.18 Å), but its Oδ atom also forms an H bond with the amide nitrogen of F88 (2.91 Å). Likewise, the side chain of D257 interacts with the amide nitrogen of V93 (2.91 Å), whereas the indole amine function of W261 is H bonded to the amide oxygen of I90. Overall, it is possible that the domain arrangement observed here represents the domains' relative positions in which the PCP domain interacts with either the preceding A or C domain. For example, loading of the Ppan arm of the PCP domain with a cognate amino acid by action of the A domain may trigger an A/H→H transiti" @default.
• W2007343995 created "2016-06-24" @default.
• W2007343995 creator A5015160212 @default.
• W2007343995 creator A5033695434 @default.
• W2007343995 creator A5041775175 @default.
• W2007343995 creator A5064573004 @default.
• W2007343995 creator A5088666284 @default.
• W2007343995 date "2007-07-01" @default.
• W2007343995 modified "2023-10-11" @default.
• W2007343995 title "Structural and Functional Insights into a Peptide Bond-Forming Bidomain from a Nonribosomal Peptide Synthetase" @default.
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