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The Thioesterase Domain from a Nonribosomal Peptide Synthetase As a Cyclization Catalyst for Integrin Binding Peptides

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The Thioesterase Domain from a Nonribosomal Peptide Synthetase As a Cyclization Catalyst for Integrin Binding Peptides The thioesterase domain from a nonribosomal peptide synthetase as a cyclization catalyst for integrin binding peptides Rahul M. Kohli*, Junichi Takagi†, and Christopher T. Walsh*‡ *Department of Biological Chemistry and Molecular Pharmacology, and †The Center for Blood Research, Department of Pathology, Harvard Medical School, Boston, MA 02115 Contributed by Christopher T. Walsh, December 13, 2001 Nonribosomal peptide synthetases responsible for the production amide bonds in the linear precursor, suggested that the TycC TE of macrocyclic compounds often use their C-terminal thioesterase could serve as a permissive macrocyclization catalyst (Fig. 1A) (TE) domain for enzymatic cyclization of a linear precursor. The (6–8). The elements of the substrate that appeared necessary for excised TE domain from the nonribosomal peptide synthetase cyclization included the potential for preorganization of the responsible for the production of the cyclic decapeptide tyrocidine substrate in an antiparallel ␤-sheet, as well as side-chain require- A, TycC TE, retains autonomous ability to catalyze head-to-tail ments for an N-terminal D-phenylalanine and a penultimate macrocyclization of a linear peptide thioester with the native ornithine. The sum of our site-specific alterations have led us to sequence of tyrocidine A and can additionally cyclize peptide propose a model for the minimal substrate requirements for analogs that incorporate limited alterations in the peptide se- cyclization and raised the question of the effect on cyclization of quence. Here we show that TycC TE can catalyze macrocyclization simultaneous alterations in potentially substitutable positions of peptide substrates that are dramatically different from the (Fig. 1B) (8). native tyrocidine linear precursor. Several peptide thioesters that We now address the ability of TycC TE to catalyze the retain a limited number of elements of the native peptide sequence cyclization of peptide thioesters with different therapeutic po- are shown to be substrates for TycC TE. These peptides were tential, switching from antibiotic endpoints to peptide inhibitors designed to integrate an Arg-Gly-Asp sequence that confers po- of integrin receptors. The molecules studied, as they differ tential activity in the inhibition of ligand binding by integrin dramatically from the natural tyrocidine substrate, allow for receptors. Although enzymatic hydrolysis of the peptide thioester examination of the effects of aggregate changes in the substrate substrates is preferred over cyclization, TycC TE can be used on a on TycC TE catalysis of cyclization. preparative scale to generate both linear and cyclic peptide prod- Integrins are ␣͞␤ heterodimeric receptors that mediate nu- ucts for functional characterization. The products are shown to be merous cell–cell and cell–matrix interactions (9, 10). Many inhibitors of ligand binding by integrin receptors, with cyclization natural ligands for integrins contain an Arg-Gly-Asp (RGD) ␣ and N -methylation being important contributors to the nanomo- sequence that is believed to be important for recognition by the BIOCHEMISTRY lar potency of the best inhibitors of fibrinogen binding to ␣IIb␤3 integrins, including fibrinogen (ligand for ␣IIb␤3 and ␣v␤3), integrin. This study provides evidence for TycC TE as a versatile vitronectin (ligand for ␣v␤3), and fibronectin (ligand for ␣5␤1) macrocyclization catalyst and raises the prospect of using TE (11). Peptides containing the RGD motif are able to inhibit catalysis for the generation of diverse macrocyclic peptide libraries receptor-ligand binding and therefore are potentially useful that can be probed for novel biological function. therapeutic agents. With the introduction of a conformational constraint, peptides cyclized by means of a disulfide linkage have acrocyclic structure is a desirable feature for biologically been shown to have a higher affinity and increased specificity Mor pharmaceutically active compounds. Macrocyclization when compared with their linear counterparts (12, 13). Speci- stabilizes products from degradation and decreases the confor- ficity also is linked to the structural presentation of the RGD mational flexibility of a molecule compared with its linear analog motif, as presentation on a ␤ type IIЈ turn gives ␣IIb␤3 speci- by constraining it to a biologically active conformation (1). In the ficity, whereas presentation on a ␤ type I turn gives specificity for biosynthesis of macrocyclic natural products, such as the mac- the integrin ␣v␤3 (14, 15). rolactam antibiotic tyrocidine, the macrolactone antifungal In this work, we explore the ability of TycC TE to catalyze fengycin, and the macrolactone siderophore enterobactin by cyclization of rationally designed peptide thioesters and evaluate nonribosomal peptide synthetases (NRPSs), a C-terminal thio- the integrin inhibition activity of the macrolactam enzymatic esterase (TE) domain introduces this constraint by catalyzing products. The utility of the excised TE domain for the synthesis cyclization of a linear precursor (2, 3). Analogous cyclases in of macrocyclic molecules with novel bioactivity is revealed by our polyketide synthetase assemblies generate erythromycin and characterization of the cyclization of these integrin binding ketolide antibiotics as well as other bioactive macrolactones peptides. (4, 5). We have recently shown that several 28- to 35-kDa C-terminal Materials and Methods TE domains excised from megadalton NRPS subunit proteins Substrate Synthesis. Peptide N-acetylcysteamine thioester retain autonomous macrocyclization activity with peptide thio- (SNAC) substrates were synthesized by solid-phase peptide ester substrates that mimic their natural protein-bound peptide phosphopantetheinyl substrates (6, 7). As a representative ex- ample, TycC TE, the C-terminal TE domain from the synthetase Abbreviations: NRPS, nonribosomal peptide synthetase; TE, thioesterase; RGD, Arg-Gly- Asp; SNAC, N-acetylcysteamine thioester; MALDI-TOF MS, matrix-assisted laser desorption responsible for the biosynthesis of tyrocidine, macrocyclizes a ionization–time of flight MS. soluble decapeptide thioester with the native peptide sequence ‡To whom correspondence should be addressed. E-mail: christopher࿝walsh@hms. of the antibiotic tyrocidine A (Fig. 1A). We have investigated the harvard.edu. substrate specificity of TycC TE by site-specific alterations in the The publication costs of this article were defrayed in part by page charge payment. This soluble substrate. Substitution of single residues of the peptide article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. sequence by alanine, as well as targeted substitution of ester for §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.251668398 PNAS ͉ February 5, 2002 ͉ vol. 99 ͉ no. 3 ͉ 1247–1252 Downloaded by guest on September 25, 2021 Fig. 1. (A) The natural reaction of the C-terminal TE domain from the NRPS responsible for production of the cyclic decapeptide antibiotic tyrocidine A is shown (Left). The terminal peptidyl carrier protein (PCP) is shown with its phosphopantetheine arm represented by SH. A linear decapeptide is transferred to the active-site serine (OH) of the TE domain, followed by head-to-tail cyclization giving the product tyrocidine A. (Right) The experimental design for studying the isolated TE domain. The protein-bound substrate is replaced by a decapeptide SNAC (TLP), which is cyclized by the isolated TE domain, TycC TE. The portions of the substrate that have been shown to tolerate substitution without leading to a more than 3-fold drop in the rate of cyclization are shown in gray (6, 8).(B) The proposed model for the minimal substrate, based on examination of substrates with site-specific alterations (6–8). The curved line represents a variable length linker. R represents variable side chains, X represents NH2 or OH, and LG represents a leaving group. synthesis followed by solution-phase thioester formation. Pep- Medical Institute͞Harvard Medical School. For each cyclic tide synthesis was carried out on a Perseptive Biosystems 9050 product, multiple fragment ions were found that contained the synthesizer (0.3 mmol scale) by using 2-chlorotrityl resin deri- D-Phe-1 to C-terminal Leu linkage expected from head-to-tail vatized with Leu (Novabiochem). 9-Fluorenylmethoxycarbonyl cyclization. For cyclo-IT1, see observed peptides in Fig. 3. For ␣ (Fmoc)-N -Me-Arg(Mtr) was purchased from Bachem. All cyclo-IT2, detected peptide fragments included the sequence other amino acids and coupling reagents were obtained from LF observed 261.0 (calculated 261.2), LFA 332.1 (332.2), Novabiochem. Couplings were carried out with Fmoc-protected and LFAAR 559.3 (559.3). For cyclo-IT3, detected fragments amino acids, except for the N-terminal D-Phe, which was Boc- included the a-ion of LF 233.1 (233.2) and the b-ion of protected. Side-chain protecting groups were: Boc for Orn and LF(NMe)RGD 603.4 (603.3). For kinetic analysis of substrates, ␣ Trp, Mtr for N -Me-Arg, Pbf for Arg, and t-Bu for Asp. For all peptide thioester concentration was varied, and product forma- ␣ couplings not involving N -Me-Arg, 30-min single coupling using tion at 1 min was quantified and fitted to the Michaelis–Menten diisopropylcarbodiimide (DIPCDI)͞1-hydroxybenzotriazole kinetic model. (HOBt) chemistry was conducted. Coupling of Fmoc-N␣-Me- Arg(Mtr) to the growing peptide was carried out with DIPCDI͞ Preparative Scale Generation of Linear Peptide Acid and Cyclic Pep-
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