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NMR Evidence for an Unusual Peptide Bond at the N-Extein–Intein Junction

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NMR Evidence for an Unusual Peptide Bond at the N-Extein–Intein Junction Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein–intein junction Alessandra Romanelli*†, Alexander Shekhtman†‡, David Cowburn‡, and Tom W. Muir*§ *Laboratory of Synthetic Protein Chemistry, The Rockefeller University, 1230 York Avenue, New York, NY 10021; and ‡New York Structure Biology Center, New York, NY 10027 Edited by Rowena G. Matthews, University of Michigan, Ann Arbor, MI, and approved March 9, 2004 (received for review October 13, 2003) Protein splicing is a posttranslational autocatalytic process in which (or O 3 N) acyl shift. This is known to be a spontaneous chemical an intervening sequence, termed an intein, is removed from a host rearrangement (10) and presumably does not require the intein. protein, the extein. Although we have a reasonable picture of the Although we have a reasonable overview of the various steps in basic chemical steps in protein splicing, our knowledge of how protein splicing, the mechanistic details of autocatalysis remain these are catalyzed and regulated is less well developed. In the poorly understood. There have been several high-resolution crystal current study, a combination of NMR spectroscopy and segmental structures of inteins (11–16) and related proteins (5, 7, 17). These isotopic labeling has been used to study the structure of an active structures have shed some light on the mechanism of the first step protein splicing precursor, corresponding to an N-extein fusion of in protein splicing, the N 3 S (or N 3 O) acyl shift. As illustrated ؊ 1 the Mxe GyrA intein. The JNC؅ coupling constant for the ( 1) in Fig. 1B, the highly conserved block B His and Thr residues are scissile peptide bond at the N-extein–intein junction was found to located close to the scissile (Ϫ1) amide bond in all of the intein be Ϸ12 Hz, which indicates that this amide is highly polarized, structures determined to date. The imidazole side chain of the perhaps because of nonplanarity. Additional mutagenesis and conserved histidine (His-75 in Mycobacterium xenopi DNA gyrase NMR studies indicate that conserved box B histidine residue is A, Mxe GyrA) seems to be positioned to participate in general essential for catalysis of the first step of splicing and for maintain- acid͞base type catalysis, whereas the threonine appears to be part ing the (؊1) scissile bond in its unusual conformation. Overall, of a oxyanion hole that would stabilize the presumed tetrahedral these studies support the ‘‘ground-state destabilization’’ model as oxythiazolidine intermediate (Fig. 1B). part of the mechanism of catalysis. Perhaps the most remarkable feature of these structural studies relates to the conformation of the scissile (Ϫ1) peptide bond itself. rotein splicing is an autocatalytic process in which an internal In cases where N-extein residues were actually present in the Pintein domain excises itself from a host protein, the extein. The crystallized constructs, the (Ϫ1) amide has been found in a variety first intein was discovered in 1990 as a spacer within the Saccha- of conformations ranging from normal trans (15, 16) to distorted romyces cerevisiae vacuolar ATPase gene (1, 2). Since then, Ͼ100 trans (13) to a cis configuration (12). This has led to the hypothesis putative members of this protein domain family have been identi- that the scissile (Ϫ1) peptide bond is activated toward nucleophilic fied from all three branches of life (3). In addition, a growing attack by distortion away from the most stable trans configuration, number of proteins have been shown to contain autoprocessing the strain being imposed by specific tertiary interactions involving domains that are orthologous to inteins at the structural and͞or conserved intein residues (Fig. 1B). Although intriguing, there are mechanistic levels (e.g., refs. 4–7). There are three broad categories two major caveats to this ‘‘ground-state destablization’’ model; first, of inteins: (i) Maxi-inteins, the largest class, contain a homing an unusual (Ϫ1) amide conformation is not found in all N-extein– endonuclease domain inserted into the core intein sequence (Fig. intein crystal structures (15, 16), and second, all of the structures 1A). This endonuclease domain is not required for splicing activity solved to date involved inteins inactivated through mutation of and can be deleted (8). (ii) Mini-inteins lack a homing endonuclease conserved residues. For example, in the Mxe GyrA intein structure, domain and are consequently much smaller in size. (iii) Trans- Cys-1 was mutated to a serine. Moreover, the construct contained splicing inteins lack a peptide linkage between the N- and C- only a single N-extein residue and consequently the positively terminal halves of the domain. Thus, the intein is split in two and charged ␣-amino group was very close to the active site of the intein. splicing only occurs on reconstitution of the fragments. All three Conceivably, these mutations͞alterations could have affected the classes of inteins are characterized by several conserved sequence (Ϫ1) amide conformation. motifs (blocks A, B, F, and G) containing the critical residues for Clearly, it would be desirable to obtain a crystal structure of an catalyzing the splicing reaction (Fig. 1A). Indeed, with one excep- active N-extein–intein precursor. This has proven difficult because tion (noted below), there are no sequence requirements in the of the short half-life of splicing precursors, on the order of a few extein in order for splicing to take place, and so intein activity is hours (18, 19). Indeed, an active S. cerevisiae vacuolar ATPase (Sce promiscuous with respect to the extein. VMA) intein has been shown to catalyze protein splicing within the The mechanism of protein splicing has been studied extensively crystalline lattice (15). Thus, there is no information currently by using biochemical approaches (Fig. 1A) (9). The first step in the available regarding the conformation of the scissile (Ϫ1) amide 3 3 standard protein splicing mechanism involves an N S(orN bond in an active N-extein–intein precursor. In the present study, we O) acyl shift in which the N-extein unit is transferred to the set out to address this issue by using a combination of solution NMR BIOCHEMISTRY sidechain SH or OH group of a Cys͞Ser residue, located at the immediate N terminus of the intein. Next, the entire N-extein unit is transferred to a second conserved Cys͞Ser͞Thr residue at the This paper was submitted directly (Track II) to the PNAS office. intein–C-extein boundary (ϩ1 position within the C-extein) in a Abbreviations: Sce VMA, Saccharomyces cerevisiae vacuolar ATPase; RP-HPLC, reverse- transesterification step. The resulting branched intermediate is then phase HPLC; Gdm⅐Cl, guanidinium chloride; HSQC, heteronuclear single quantum correla- resolved through a cyclization reaction involving a conserved tion; Mxe GyrA, Mycobacterium xenopi DNA gyrase A. asparagine residue at the C terminus of the intein. The intein is thus †A.R. and A.S. contributed equally to this work. excised as a C-terminal succinimide derivative. In the final step, an §To whom correspondence should be addressed. E-mail: [email protected]. amide bond is formed between the two exteins following an S 3 N © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0306616101 PNAS ͉ April 27, 2004 ͉ vol. 101 ͉ no. 17 ͉ 6397–6402 Downloaded by guest on September 23, 2021 Fig. 1. The mechanism of protein splicing. (A)(Upper) Schematic illustrating the conserved regions within the intein domain family. The white bar represents the intein primary structure with conserved blocks A, B, F, and G indicated by colored boxes. The homing endonuclease domain found in the majority of inteins is also indicated. Conserved residues implicated in the splicing mechanism are shown below the bar. (Lower) The basic protein splicing mechanism. Note that a small number of inteins contain an Ala as the first residue, rather than Cys or Ser. These inteins are thought to directly use the ϩ1 nucleophile within the C-extein in the first step (40). (B)(Upper) Ribbon representation of the Mxe GyrA crystal structure with the active site residues indicated in ball and stick (12). (Lower) Diagram of the N-terminal splice junction of the Mxe GyrA intein. The scissile (Ϫ1) amide is highlighted in red, and purple lines indicate possible hydrogen bond interactions to this amide with corresponding distances in Å. spectroscopy and segmental isotopic labeling. Specifically, NMR Importantly, this mutation does not affect the N-terminal splicing was used to study the structure of the (Ϫ1) amide in an active reaction (22), which is the focus of this study, and for clarity is protein splicing precursor, prepared by semisynthesis, correspond- referred to as the wild-type sequence hereafter. For construction of ing to an N-extein fusion of the Mxe GyrA intein. the plasmid pTrcHis-Xa-GyrAwt, which encodes a factor Xa cleavage sequence between a poly(His) tag and the intein, the appropriate Materials and Methods PCR product was cloned into the pTrcHisA vector (Invitrogen) by Peptide Synthesis. The peptide, H-AAMR[13CЈ]F-SR, was synthe- using BamHI and HindIII restriction sites. This expression plasmid sized on a 3-mercaptopropionamide-4-methylbenzhydrylamine was used as a template for site-directed mutagenesis with the (MBHA) resin (20) by using the in situ neutralization͞ QuikChange site-directed mutagenesis kit (Stratagene). This re- 2-(hydroxybenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexa- sulted in the generation of the following expression vectors: fluorophosphate (HBTU) activation protocol for Boc-SPPS (21). pTrcHis-Xa-GyrAT72V; pTrcHis-Xa-GyrAN74A; pTrcHis-Xa- After chain assembly, the peptide ␣-thioester was cleaved off the GyrAH75A; pTrcHis-Xa-GyrAT72VN74A; pTrcHis-Xa-GyrAT72VH75A; resin with anhydrous HF containing 4% vol͞vol p-cresol (1 h, 4°C) and pTrcHis-Xa-GyrAN74AH75A.
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