CRYSTALLIZATION NOTE Preparation and Crystallization of a Complex between Human Adenovirus Serotype 2 Proteinase and Its 11-Amino-Acid Cofactor pVIc WILLIAM J. MCGRATH,JIANZHONG DING,ROBERT M. SWEET, AND WALTER F. MANGEL 1 Biology Department, Brookhaven National Laboratory, Upton, New York 11973 Received December 1, 1995, and in revised form March 22, 1996 Crystals have been obtained of the recombinant human adenovirus serotype 2 proteinase (AVP) in a complex with its 11-amino-acid cofactor pVIc. AVP– pVIc complexes were formed by the incubation of AVP with a 1.2-fold molar excess of pVIc prior to the crystallization trials. Diffraction-quality crystals were obtained at 18°C by the vapor-diffusion method with 5.6 mg/ml AVP–pVIc in 1.4 M sodium acetate and 0.1 M Hepes, pH 7.5. Diffraction data (99% complete to 2.6 Å resolution with R merge of 0.077) were collected from native crystals at room tempera- ture at beamline X12-C at the National Synchrotron Light Source. The crystals belong to space group P6 1 with unit cell dimensions a 5 b 5 114.2 Å, c 5 50.1 Å; a 5b5 90°, g5 120°. The unit cell dimensions and likely mass of the molecular species in the crystals were consistent with there being one 25 000-Da complex (1:1) per asymmetric unit. Additionally, one heavy-atom derivative, obtained by the soaking of preformed crystals, was isomorphous to the native crystal. Diffraction data obtained on these crystals were 95% complete to 3.0 Å resolution with an R merge of 0.076. Difference-Patterson analysis indicates three heavy atom sites in the derivative asymmetric unit. r 1996 Academic Press, Inc. INTRODUCTION Many animal and plant viruses contain a gene for a proteinase whose correct expression is absolutely required for the synthesis of infectious virus (Kraus- slich and Wimmer, 1988). Because of this and be- cause virus-coded proteinases are highly specific enzymes, they are appealing targets for antiviral therapy. The human adenovirus proteinase is re- quired to process 6 of the 12 major polypeptides from which adenovirus virions are assembled. Weber (1976) isolated a temperature-sensitive mutant H2ts1 (ts1) of human adenovirus serotype 2 (Ad2) that lacks proteinase activity at the nonpermissive tem- perature. Virions of ts1 assemble at the nonpermis- sive temperature but contain precursor proteins in place of the mature components present in wild-type virus. Such immature virions attach to cells but fail to initiate a productive infection (Hannan et al., 1983; Mirza and Weber, 1980). The mutation in ts1 was identified as a single base-pair change in a 204-codon open reading frame (L3 23 kDa) at the 38 end of the L3 family of late messages (Yeh-Kai et al., 1983). The L3 23K gene has been cloned and ex- pressed in Escherichia coli, and the resultant protein has been purified (Anderson, 1990; Mangel et al., 1995). Recombinant human adenovirus serotype 2 pro- teinase (AVP) has little activity compared to that in disrupted virions. This prompted a search for cofac- tors; two were discovered. One was the 11-amino- acid peptide from the carboxy terminus of the virion precursor protein pVI, pVIc (Mangel et al., 1993; Webster et al., 1993). It stimulates AVP activity 350-fold (Mangel et al., 1995). The other cofactor is the viral DNA, which in the presence of pVIc stimu- lates AVP activity 6000-fold (Mangel et al., 1993). The requirement of two cofactors for proteinase activity is rare; for one to be DNA is unprecedented. AVP is a difficult proteinase to classify.Analysis of the AVP sequence of 12 different adenovirus sero- types reveals no homology with any proteins (Ran- court et al., 1994). Inhibitor profiles imply it may be a serine (Bhatti and Weber, 1979; Chatterjee and Flint, 1987; Tremblay et al., 1983) or cysteine protein- ase (Grierson et al., 1994; Rancourt et al., 1994; Tihanyi et al., 1993; Weber and Tihanyi, 1994; Webster et al., 1993). AVP exhibits a very selective 1 To whom correspondence and reprint requests should be addressed at Biology Department, Brookhaven National Labora- tory, Upton, NY 11973. Fax: (516) 344-3407. JOURNAL OF STRUCTURAL BIOLOGY 117, 77–79 (1996) ARTICLE NO. 0072 77 1047-8477/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.