A Novel Cross-linked RNase A Dimer With Enhanced Enzymatic Properties Brigitte L. Simons, 1,2 Harvey Kaplan, 2 Sylvie M. Fournier, 1 Terry Cyr, 1 and Mary Alice Hefford 1 * 1 Centre for Biologics Research, Biologics and Genetics Therapies Directorate, Health Canada, Ottawa, Ontario, Canada, K1A 0L2 2 Department of Chemistry, University of Ottawa, D’Iorio Hall, 10, Marie-Curie, Ottawa, Ontario, Canada, K1N 6N5 ABSTRACT A new cross-linked ribonuclease A (RNase A) dimer composed of monomeric units cova- lently linked by a single amide bond between the side-chains of Lys 66 and Glu 9 is described. The dimer was prepared in the absence of water by incubating a lyophilized preparation of RNase, sealed under vac- uum, in an oven at 858C. It was determined that the in vacuo procedure does not induce any significant conformational changes to the overall structure of RNase A, yet the amide cross-link has an increased acid lability, indicating that it is exposed and confor- mationally strained. Examination of X-ray crystallo- graphic structures indicates that Lys 66 and Glu 9 are not close enough for the in vacuo dimer to adopt any of the known domain-swapped conformations. Therefore, the in vacuo RNase A dimer appears to be a novel dimeric structure. The in vacuo RNase A dimer also exhibits a twofold increase in activity over monomeric RNase A on a per monomer basis. This doubling of enzymatic activity was shown using dsRNA and ssRNA as substrates. In addition to this enhanced ability to degrade RNA, the dimer is not inhibited by the cellular ribonuclease inhibitor pro- tein (cRI). Proteins 2007;66:183–195. V V C 2006 Wiley- Liss, Inc. Key words: ribonuclease A; in vacuo; enzymatic activity; covalent cross-linking; dimer INTRODUCTION Bovine pancreatic ribonuclease A (RNase A) is one of the most extensively studied and characterized proteins. It, like most other members of the RNase superfamily, is a monomeric protein; however, in 1962, Crestfield et al. 1 observed that stable, dimeric forms of bovine pancreatic RNase A could be formed by lyophilization from a concen- trated solution of 50% acetic acid. In 1967, he and his col- leagues demonstrated that these noncovalent RNase A dimers were two monomeric units that had exchanged their N-terminal segments, thus forming a domain- swapped conformation 2 similar to that observed many years later in bovine seminal ribonuclease (BS-RNase), 3 a uniquely homodimeric ribonuclease. More recently, many groups have undertaken the characterization of noncova- lent RNase A dimers generated by Crestfield’s method. 4–7 Rather than a single dimeric RNase A species, recent work had shown that RNase A dimers can exist in two distinctly different conformers: the N-terminal a-helix-swapped dimer and the C-terminal b-strand-swapped dimer. 8,9 The N-terminal a-helix (residues 1–15)-swapped conformer can be separated from the more dominant C-terminal b-strand (residues 116–124)-swapped conformer by cation exchange chromatography. 5,9–11 Both the N- and C-terminal domain- swapped dimers show increased catalytic activity toward dsRNA substrates. 5,10 The observation that the dimeric forms of RNase are cy- totoxic 12 has been of considerable interest as they may have applications as a therapeutic agent in preventing some types of cell proliferation (see Refs. 13 and 14, and references cited therein). BS-RNase and onconase, an am- phibian RNase, are also highly cytotoxic, and both have been shown to retain activity in the presence of the cellular inhibitor, cytosolic ribonuclease inhibitor (cRI). 15 This abil- ity to evade inactivation by cRI has been linked to the cyto- toxic and antitumor activities 16 of BS-RNase, and onconase and, by extension, to other homologues of RNase A. 17 In so- lution, BS-RNase forms an equal mixture of two distinct conformers, 17,18 both of which are composed of two identi- cal monomers linked by two disulfide bonds. In one of the two dimeric conformers, the two monomeric subunits appear to fold independently of one another. The other di- meric conformer, however, is assembled by the exchange of the N-terminal a-helix between subunits, a process that is commonly termed 3D domain swapping. 19 The noncovalent dimers first identified by Crestfield are also domain- swapped dimers. However, it has recently been demon- strated that cRI strongly binds to and inhibits noncovalent, domain-swapped RNase dimers. 20 It therefore appears that either some other property of the RNase dimers is responsi- ble for the cytotoxicity or, as argued by Sica et al., 21 a prop- erly formed dimeric structure can evade cRI but must be stable in the reducing environment of the cytosol to do so. Grant sponsor: National Sciences and Engineering Research Council (NSERC). *Correspondence to: Mary Alice Hefford, Centre for Biologics Research, Biologics and Genetics Therapies Directorate, AL: 2201C, Health Canada, Ottawa, ON, Canada, K1A 0L2. E-mail: mary_ hefford@hc-sc.gc.ca Received 27 January 2006; Revised 13 April 2006; Accepted 30 May 2006 Published online 16 October 2006 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/prot.21144 V V C 2006 Government of Canada. Exclusive worldwide publication rights in the article have been transferred to Wiley-Liss, Inc. PROTEINS: Structure, Function, and Bioinformatics 66:183–195 (2007)