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Solution Structure of Hirsutellin a – New Insights Into the Active Site And

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Solution Structure of Hirsutellin a – New Insights Into the Active Site And Solution structure of hirsutellin A – new insights into the active site and interacting interfaces of ribotoxins Aldino Viegas1, Elias Herrero-Gala´ n2, Mercedes On˜ aderra2, Anjos L. Macedo1 and Marta Bruix3 1 REQUIMTE-CQFB, Departemento de Quimica, Faculdade de Cieˆ ncias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 2 Departemento de Bioquı´mica y Biologı´a Molecular I, Facultad de Quı´mica, Universidad Complutense, Madrid, Spain 3 Departemento de Espectroscopı´a y Estructura Molecular, Instituto de Quı´mica Fı´sica ‘Rocasolano’, Consejo Superior de Investigaciones Cientificas, Madrid, Spain Keywords Hirsutellin (HtA) is intermediate in size between other ribotoxins and less cytotoxic protein; NMR; ribonucleases; specific microbial RNases, and thus offers a unique chance to determine RNase T1; structure; a-sarcin the minimal structural requirements for activities unique to ribotoxins. Here, we have determined the structure of HtA by NMR methods. The Correspondence M. Bruix, Departamento de Espectroscopı´a structure consists of one a-helix, a helical turn and seven b-strands that y Estructura Molecular, Instituto de Quı´mica form an N-terminal hairpin and an anti-parallel b-sheet, with a characteris- Fı´sica ‘Rocasolano’, Serrano 119, 28006 tic a + b fold and a highly positive charged surface. Compared to its Madrid, Spain larger homolog a-sarcin, the N-terminal hairpin is shorter and less posi- Fax ⁄ Tel: +34 91 561 94 00 tively charged. The secondary structure elements are connected by large E-mail: [email protected] loops with root mean square deviation (rmsd) values > 1 A˚, suggesting some degree of intrinsically dynamic behavior. The active site architecture Database Structural data has been submitted to the of HtA is unique among ribotoxins. Compared to a-sarcin, HtA has an Protein Data Bank and BioMagResBank aspartate group, D40, replacing a tyrosine, and the aromatic ring of F126, databases under the accession numbers located in the leucine ‘environment’ close to the catalytic H113 in a similar 2kaa and 16018, respectively arrangement to that found in RNase T1. This unique active site structure is discussed in terms of its novel electrostatic interactions to understand the (Received 28 November 2008, revised 20 efficient cytotoxic activity of HtA. The contributions of the N-terminal January 2009, accepted 16 February 2009) hairpin, loop 2 and loop 5 with regard to protein functionality, protein– doi:10.1111/j.1742-4658.2009.06970.x protein and protein–lipid interactions, are also discussed. The truncation and reduced charge of the N-terminal hairpin in HtA may be compensated for by the extension and new orientation of its loop 5. This novel orienta- tion of loop 5 re-establishes a positive charge on the side of the molecule that has been shown to be important for intermolecular interactions in ribotoxins. Ribotoxins are a family of toxic extracellular fungal 400 bp fragment, known as the a fragment, and blocks RNases that display specific ribonucleolytic activity protein synthesis, leading to cell death by apoptosis against a single phosphodiester bond in the sarcin ⁄ ricin [6]. Several ribotoxins have been isolated (clavin [7], loop of the ribosomal RNA [1–4]. This bond (G4325– c-sarcin [8], gigantin [9] and Aspf 1 [10]), with a-sarcin A4324 in the 28S subunit) is located at an evolution- [11–13] (from Aspergillus giganteus) and restrictocin arily conserved site with important roles in ribosome [14,15] (from A. restrictus) being the best characterized. function, namely elongation factor 1-dependent bind- The sequence identity between a-sarcin and restrictocin ing of aminoacyl-tRNA and elongation factor 2-cata- is 85%, and they share a basic pI and common tertiary lyzed GTP hydrolysis and translocation [5]. Cleavage structure [4,13,14]. They fold into an a + b structure of this phosphodiester bond results in release of a with a central five-stranded antiparallel b-sheet and an Abbreviation HtA, hirsutellin A. FEBS Journal 276 (2009) 2381–2390 ª 2009 The Authors Journal compilation ª 2009 FEBS 2381 Solution structure of hirsutellin A A. Viegas et al. a-helix. They are highly twisted in the right-handed fungal HtA was obtained and 2D 1H-NMR methodol- sense, creating a convex face against which the a-helix ogy [22] was used to determine the three-dimensional is packed. In addition, the N-terminal residues form a structure of HtA in aqueous solution. Our results show b-hairpin that may be considered as two consecutive that the structure is well determined (pairwise minor b-hairpins connected by a hinge region. Further- rmsd = 0.98 A˚for all backbone atoms), and the glo- more, the nature and location of the catalytic residues bal fold is similar to that reported for cytotoxins. as well as the enzymatic mechanism (they are cyclizing However, differences can be found in the conformation RNases) are also conserved [16–18]. For these reasons, of loops, the b-hairpin and the relative position of the ribotoxins may be considered to belong to the larger catalytic residues in the active site. The results family of fungal ⁄ microbial secreted RNases, usually obtained will be discussed and compared with those represented by the nontoxic ribonuclease T1 [19]. The reported for other members of the fungal extracellular main structural differences between ribotoxins and RNase family. nontoxic RNases are the length and arrangement of the loops and the N-terminal b-hairpin, which are Results believed to be responsible for ribotoxin cytotoxicity. Hirsutellin A (HtA) is a 130-residue extracellular Assignment protein produced by the invertebrate fungal pathogen Hirsutella thompsonii. This protein displays biological The 1H assignments for the backbone and side chains properties similar to those of the a-sarcin family [4,20]. are nearly complete. The observed conformational Sequence alignment with microbial RNases and ribo- chemical shifts for alpha and amide protons, calculated toxins revealed a significant similarity even though the as dHtA–dRC (Fig. 1), resemble those reported for sequence identity between HtA and other ribotoxins is a-sarcin [11]; this suggests that the global fold and 3D marginal, only about 25%. This is lower than structure that are characteristic of the ribotoxin family the sequence identity observed among all other known are present in HtA. Analysis of these assignments pro- ribotoxins, which is always above 60%. It is suggested vides some interesting clues concerning HtA structure. that the common structural core is conserved in HtA, First, several protons show d values below 0 ppm. One with the most significant differences being the length of these shielded nuclei is a gamma proton of P68 with of the loops connecting the a-helical and b-sheet a chemical shift of )0.32 ppm. Tellingly, the gamma regions and the N-terminal hairpin. protons of the structurally related P98 in a-sarcin also A recent study characterized HtA and evaluated its have low d values ()0.83 and )0.31 ppm). Second, the ribotoxin characteristics [4]. It showed conclusively that labile OH protons of S38, Y70, T92, T112 and Y98 HtA is a member of the a-sarcin ⁄ restrictocin ribotoxin exchange slowly enough with the water molecules to family. Furthermore, far-UV CD analysis confirmed be observable in the NMR spectra, and consequently the predominance of b-structure predicted by the their resonances could be assigned. All these NMR sequence similarity between HtA and a-sarcin. The data clearly indicate that HtA has a compact fold with N-terminal b-hairpin characteristic of ribotoxins is a tightly structured core. shorter in HtA than in a-sarcin, but this structural motif is still present. The active site residues and cata- Disulfide bonds and structure determination lytic mechanism also appear to be conserved. The puta- tive loop 3 in HtA possesses a net positive charge and The disulfide pairings of HtA were previously pre- hydrophilic properties that are thought to be responsi- dicted from sequence alignment with other members of ble for interacting with the sarcin ⁄ ricin loop, providing the ribotoxin family. Here, we have found experimen- HtA with specific ribonuclease activity [4,13,15]. With tal evidence by searching for Hb–Hb and Ha–Hb NOEs regard to its interaction with lipid vesicles, HtA and between cysteines. At least one intercysteine NOE a-sarcin show a significant difference: a-sarcin pro- could be found for C6–C129 and for C57–C108. The motes the aggregation of lipid vesicles but HtA does long-range NOEs between residues surrounding the not. Both proteins change the permeability of mem- cysteines confirm the cysteine pairing defined here. branes but HtA is more efficient. These differences are This pattern agrees with the arrangement present in thought to be related to dissimilarities in loop 2 and other ribotoxins, and is fully compatible with the the N-terminal b-hairpin, which have been proposed to distance restraints discussed below. be specifically involved in vesicle aggregation [21]. After seven cycles of NOE assignment and structure In order to better understand the structural require- calculation by cyana ⁄ candid, a set of 20 structures that ments for the specific activities of these proteins, satisfy the experimental constraints was obtained. The 2382 FEBS Journal 276 (2009) 2381–2390 ª 2009 The Authors Journal compilation ª 2009 FEBS A. Viegas et al. Solution structure of hirsutellin A 1 1 Fig. 1. Ha and HN conformational shifts (dobserved–drandom coil) in ppm for HtA at pH 4.1 and 298 K. The amino acid sequence and the elements of secondary structure are shown; b-strands are represented by arrows and the a-helix by a spiral. coordinates of these 20 conformers have been deposited Table 1B. Calculation statistics. in the Research Collaboratory for Structural Bioinfor- Mean Minimum Maximum matics Protein Data Bank under accession number 2kaa.
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