CRYSTAL-STRUCTURE OF A PYRIMIDINE DIMER-SPECIFIC EXCISION-REPAIR ENZYME FROM BACTERIOPHAGE-T4 - REFINEMENT AT 1.45 ANGSTROM AND X-RAY-ANALYSIS OF THE 3 ACTIVE-SITE MUTANTS

Crystallographic study of bacteriophage T4 endonuclease V, which is in volved in the initial step of the pyrimidine dimer-specific excision r epair pathway, has been carried out with respect to the wild-type and three different mutant enzymes. This enzyme catalyzes the cleavage of the N-glycosyl bond at the 5'-side of the pyrimidine dimer, and subseq uently incises the phosphodiester bond at the apyrimidinic site throug h a beta-elimination reaction. The structure of the wild-type enzyme r efined at 1.45 Angstrom resolution reveals the detailed molecular arch itecture. The enzyme is composed of a single compact domain classified as an all-alpha structure. The molecule is stabilized mainly by three hydrophobic cores, two of which include many aromatic side-chain inte ractions. The structure has a unique folding motif, where the amino-te rminal segment penetrates between two major a-helices and prevents the ir direct contact, and it is incompatible with the close-packing categ ory of helices for protein folding. The concave surface, covered with many positive charges, implies an interface for DNA binding. The glyco sylase catalytic center, which comprises Glu23 and the surrounding bas ic residues Arg3, Arg22 and Arg26, lie in this basic surface. The crys tal structures of the three active-site mutants, in which Glu23 was re placed by Gln(E23Q) and Asp (E23D), respectively, and Arg3 by Gln (R3Q ), have been determined at atomic resolution. The backbone structures of the E23Q and R3Q mutants were almost identical with that of the wil d-type, while the E23D mutation induces a small, but significant, chan ge in the backbone structure, such as an increase of the central kink of the H1 helix at Pro25. In the catalytic center of the glycosylase, however, these three mutations do not generate notable movements of pr otein atoms, except for significant shifts of some bound water molecul es. Thus, the structural differences between the wild-type and each mu tant are confined to the remarkably small region around their replaced chemical groups. Combined with the biochemical studies and the differ ence circular dichroism measurements, these results allow us to conclu de that the negatively charged carboxyl group of Glu23 is essential fo r the cleavage of the N-glycosyl bond, and that the positively charged guanidino group of Arg3 is crucial to bind the substrate, a DNA duple x containing a pyrimidine dimer. The amino terminal alpha-amino group is located at a position approximately 4.4 Angstrom away from the carb oxyl group of Glu23. These structural features are generally consisten t with the reaction scheme proposed by Dodson and co-workers.