Journal of Crystal Growth 122 (1992) 293—297 ~ CRYSTAL North-Holland GROWTH Systematic investigation of crystallization parameters for protein—nucleic acid complexes: application to an active truncated form of Escherichia coli topoisomerase I Patricia C. Weber The DuPont Merck Pharmaceutical Company, P.O. Box 80228, Wilmington, Delaware 19880-0228, USA Chang-Xi Zhu and Yuk-Ching Tse-Dinh Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA An experimental strategy to systematically screen possible crystallization parameters for protein: oligonucleotide complexes is described and successfully used in the cocrystallization of a truncated form of Escherichia coli topoisomerase I with an oligo(thymidylic acid) polymer of 8 bases. Crystals are orthorhombic (a = 65.9 A, b = 74.0 A, and c = 140.2 A) and diffract to at least 3 A resolution. 1. Introduction ber of possible solution parameters [7—91. In the case of protein—oligonucleotide complexes, fac- DNA topoisomerases have been demonstrated tors specific to the oligonucleotide such as length, to be targets of antineoplastic and antibacterial sequence, and the presence of blunt-ended frag- agents. Recently, camptothecin has been shown ments, may also need to be screened. For exam- to promote cancer cell death through its interac- tion with type I DNA topoisomerase during DNA replication [1,21. Structural studies of these pro- teins could provide important information to aid in the design of new anticancer agents or antibi- otics. For this reason, we initiated crystallization studies of Escherichia coli topoisomerase I, a single chain, multidomain protein (fig. 1) of 864 amino acids and 97,400 MW [3]. Despite little overall amino acid sequence homology, the E. coli and human topoisomerases I show some con- servation in active site structure, including a simi- lar utilization of tyrosine as the catalytic nude- Fig. 1. Schematic Illustration of E. coli topoisomerase I ophile [4]. The catalytic activity of the E. coli Domains. The intact protein of 864 amino acids consists of enzyme is retained in an 85,000 MW fragment three domains. The amino terminal domain, on the left, (top85) that lacks the carboxy terminal domain, contains the active site. Three zinc-binding regions separate but includes the active site [5] and three zinc- the amino and carboxy terminal domains. Limited proteolytic binding domains [61. digestion results in a carboxy terminal domain that tightly binds single-stranded DNA. The truncated, active protein The search for crystallization conditions of a (top85) crystallized here includes the amino terminal and specific protein can involve scanning a large num- zinc-binding domains. 0022-0248/92/$05.00 © 1992 — Elsevier Science Publishers B.V. All rights reserved