Refined NMR structure of alpha-sarcin by 15N-1H residual dipolar couplings

(15)N-(1)H residual dipolar couplings (RDC) have been used as additional restraints to refine the solution structure of the ribotoxin alpha-sarcin. The RDC values were obtained by partial alignment of alpha-sarcin in the binary mixture of n-dodecyl hexa(ethylene glycol)/hexanol. A total of 131 RDCs were measured and 106 were introduced in the final steps of the calculation protocol following the main calculation based on nuclear Overhauser enhancements and torsion angle restraints. A homogeneous family of 81 conformers was obtained. The resulting average pairwise root-mean-square deviation corresponding to the superposition of the 20 best structures is 0.69+/-0.12 A for the backbone and 1.29+/-0.14 A for all heavy atoms. The new structural features derived from the refined structure, compared with the non-refined structure of alpha-sarcin, consist of new hydrogen bonds and a better definition of the backbone conformation. In particular, the loop segment spanning Gly 60 to Lys 70 shows a single conformation, corresponding to the most populated family of conformers observed in the unrefined structure. The information derived from the analysis of the refined structure and the comparison with the homologous protein restrictocin could help in establishing further structure-function relationships concerning alpha-sarcin which can be reasonably extrapolated to other members of the ribotoxin family.