The highly refined solution structure of the cytotoxic ribonuclease alpha-sarcin reveals the structural requirements for substrate recognition and ribonucleolytic activity

alpha-Sarcin selectively cleaves a single phosphodiester bond in a universa lly conserved sequence of the major rRNA, that inactivates the ribosome. Th e elucidation of the three-dimensional solution structure of this 150 resid ue enzyme is a crucial step towards understanding alpha-sarcin's conformati onal stability, ribonucleolytic activity, and its exceptionally high level of specificity. Here, the solution structure has been determined on the bas is of 2658 conformationally relevant distances restraints (including stereo especific assignments) and 119 torsional angular restraints, by nuclear mag netic resonance spectroscopy methods. A total of 60 converged structures ha ve been computed using the program DYANA. The 47 best DYANA structures, fol lowing restrained energy minimization by GROMOS, represent the solution str ucture of alpha-sarcin. The resulting average pairwise root-mean-square-dev iation is 0.86 Angstrom for backbone atoms and 1.47 A for all heavy atoms. When the more variable regions are excluded from the analysis, the pairwise root-mean-square deviation drops to 0.50 Angstrom and 1.00 Angstrom, for b ackbone and heavy atoms, respectively. The alpha-sarcin structure is simila r to that reported for restrictocin, although some differences are clearly evident, especially in the loop regions. The average rmsd between the struc turally aligned backbones of the 47 final alpha-sarcin structures and the c rystal structure of restrictocin is 1.46 Angstrom. On the basis of a dockin g model constructed with alpha-sarcin solution structure and the crystal st ructure of a 29-nt RNA containing the sarcin/ricin domain, the regions in t he protein that could interact specifically with the substrate have been id entified. The structural elements that account for the specificity of RNA r ecognition are located in two separate regions of the protein. One is compo sed by residues 51 to 55 and loop 5, and the other region, located more tha n 11 A away in the structure, is the positively charged segment formed by r esidues 110 to 114. (C) 2000 Academic Press.