CRYSTAL STRUCTURE OF FOOT-AND-MOUTH DISEASE VIRUS LEADER PROTEASE

Alba Guarné¹, José Tormo¹, Regina Kirchweger^2,3, Doris Pfistermueller², Tim Skern² & Ignacio Fita¹

¹Centre d'Investigació i Desenvolupament (CSIC). Jordi Girona, 18-26. 08034-Barcelona (Spain). ²Institute of Biochemistry, Medical Faculty, University of Vienna. A-1030 Vienna (Austria). ^{3(present address)}Sloan Kettering Memorial Cancer Center, East 68^th St. New York (EEUU).

Leader protease from Foot-and-Mouth Disease Virus (FMDV) cleaves itself from the growing polyprotein and inhibits the translation of the host cell RNA capped messengers by the cleavage of the eucaryotic initiation factor (eIF4G). We have recently elucidated the three-dimensional structure of the leader protease. In order to obtain suitable crystals we have worked with two forms of the leader protease: the normal length inactive mutant C23A (residues 1 to 173) and the deletion inactive mutant C23A (residues 1 to 167). Crystals of the deletion inactive mutant C23A (1-167) belongs to the space group C222₁, with unit cell parameters a=49.6, b=129.0, c=124.7. The structure was determined by multiple isomorphous replacement techniques. Initial phases were calculated at 3.0Å (using SHARP), and were significantly improved using density modification procedures. Crystals of the normal length inactive mutant C23A (1-173) belongs to space group P2₁2₁2₁, with unit cell dimensions of a=65.1, b=100.4, c=276.0Å; and contains eight molecules in the asymmetric unit. Initial phases were obtained by molecular replacement using the shortest form structure as a model. The crystallographic refinement was performed using data between 2.0-3.0Å yielding crystallographic R-factors of 23.5% and 26.1%, with corresponding R-free of 28.8% and 29.8%, for the shortest and normal length forms respectively. Leader protease structure reveals the overall fold of papain-like enzymes in spite of the low sequence similarity shared with this family of cysteine-proteases. The leader protease has a globular core (residues 1-155) and a flexible region exposed to the solvent (residues 156-173). Crystal packing of the normal length structure reveals a helicoidal disposition of the eight molecules present in the asymmetric unit, where neighbouring molecules are related by local binary axis, exchanging of the neighbouring carboxy-terminal extensions and the intermolecular disulphide bridges found in the crystal packing of the shortest form, as well. The three-dimensional structure of the leader protease allows to establish the molecular basis of substrate binding recognition and the P' requirements for the eIF4G cleavage.