Proc. Natl. Acad. Sci. USA Vol. 94, pp. 2874–2879, April 1997 Biochemistry Crystal structure of varicella-zoster virus protease XIAYANG QIU*, CHERYL A. JANSON†,JEFFREY S. CULP†,SUSAN B. RICHARDSON‡,CHRISTINE DEBOUCK‡, WARD W. SMITH*, AND SHERIN S. ABDEL-MEGUID*§ Departments of *Macromolecular Sciences, †Protein Biochemistry, and ‡Molecular Genetics, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406 Communicated by William N. Lipscomb, Harvard University, Cambridge, MA, January 27, 1997 (received for review October 28, 1996) ABSTRACT Varicella-zoster virus (VZV), an a-herpes vi- quence for chymotrypsin-like and G-T-S-MyA for subtilisin- rus, is the causative agent of chickenpox, shingles, and posther- like proteases. All human herpes virus proteases cleave a petic neuralgia. The three-dimensional crystal structure of the peptide bond between an alanine and a serine (6). Differences serine protease from VZV has been determined at 3.0-Å resolu- in substrate specificity exist. For example, HSV-1 protease tion. The VZV protease is essential for the life cycle of the virus cannot cleave the protein substrate of CMV protease, but the and is a potential target for therapeutic intervention. The CMV protease can cleave that of HSV-1 protease (7). structure reveals an overall fold that is similar to that recently Recently the crystal structure of CMV protease has been reported for the serine protease from cytomegalovirus (CMV), a reported (8–11). The structure reveals a new fold that has not herpes virus of the b subfamily. The VZV protease structure been reported for any other serine protease, and an active site provides further evidence to support the finding that herpes virus consisting of a novel catalytic triad in which the third member proteases have a fold and active site distinct from other serine of the triad is a histidine instead of an aspartic acid. The proteases. The VZV protease catalytic triad consists of a serine structure also suggests a catalytic tetrad composed of a serine, and two histidines. The distal histidine is proposed to properly two histidines, and an aspartic acid. The limited sequence orient the proximal histidine. The identification of an a-helical homology with CMV protease precluded detailed modeling of segment in the VZV protease that was mostly disordered in the the VZV protease structure. Here we report the crystal CMV protease provides a better definition of the postulated structure of the serine protease from VZV, the first structure active site cavity and reveals an elastase-like S* region. Struc- of the serine protease from an a-herpes virus. Comparison of tural differences between the VZV and CMV proteases also the VZV and CMV protease structures should facilitate better suggest potential differences in their oligomerization states. understanding of the substrate specificity and catalytic mech- anism of herpes virus proteases, and provide a structural basis Members of the human herpes virus family are responsible for for the rational design of antiviral agents. a variety of diseases from subclinical infections to fatal diseases in the immunocompromised or immunosuppressed. The fam- MATERIALS AND METHODS ily is divided into three subfamilies designated a, b, and g. The a subfamily includes herpes simplex viruses 1 and 2 (HSV-1 Crystallization and Data Acquisition. VZV protease contains and HSV-2) and varicella-zoster virus (VZV); the b subfamily 605 residues, of which residues 1–236 (protease domain) have full includes cytomegalovirus (CMV) and human herpes viruses 6 catalytic activity. The molecule used in this study contains only and 7; and the g subfamily includes Epstein–Barr virus and amino acids 10–236, in which the cysteine at position 10 of the human herpes virus 8. Viruses of the a subfamily are among authentic sequence (12) has been replaced by a methionine for those causing serious diseases. HSV-1 is the virus responsible Escherichia coli expression. These changes do not alter the for herpes labialis (cold sores), whereas HSV-2 causes genital protease activity. The VZV protease was purified from E. coli herpes. VZV is a neurotropic a-herpes virus responsible for cells using Ni-NTA-agarose (Qiagen, Chatsworth, CA) and Su- chickenpox, shingles, and postherpetic neuralgia: primary ex- perdex 75 (Pharmacia) chromatographies (J.S.C., unpublished posure to the virus results in chickenpox, reactivation of the data). A small portion of the final VZV protease product is 717 virus after a period of latency gives rise to shingles, and daltons less than expected as detected by matrix-associated laser postherpetic neuralgia is probably the result of nerve damage desorption ionization mass spectrometry, corresponding to a six during the active replication phase of shingles (1). amino acid deletion from the C terminal. Crystals were grown by An essential step in herpes virus assembly (2) is the pro- the method of vapor diffusion in hanging drops using 0.1 M teolytic processing of an assemblin protein designated ICP35 phosphate buffer (pH 6.2) containing 2.5 M NaCl as precipitant in HSV-1 (3). Processing of the assemblin protein is catalyzed and 10 mgyml protein mixed 1:1 with the precipitant in the drop. by a virally encoded serine protease that contains the assem- The symmetry of the diffraction was consistent with that of the blin protein at its C terminus (3). This protease catalyzes its hexagonal space group P6422 (or P6222) having a 5 b 5 90.0 Å own cleavage to produce an N-terminal domain having full and c 5 117.4 Å. There is one molecule per asymmetric unit, with 3 catalytic activity (4, 5). Herpes protease domains show signif- aVMof 3.1 Å yDa and about 60% solvent. Diffraction data were icant sequence homology within each subfamily, but only very collected with a Siemens multiwire area detector mounted on a limited homology between different subfamilies (Fig. 1, Table Siemens rotating-anode x-ray generator producing graphite- 1). For example, the VZV protease shows 50% identity to monochromated CuKa radiation and processed with the program HSV-1 and HSV-2 proteases, but only 26% to CMV protease. XENGEN (13). The native data are 90% complete to 3.0 Å with an There is little sequence homology to other known proteins, Rmerge (S I 2^I&yS^I&) of 0.07. including the absence of the conserved G-X-SyC-G-G se- Abbreviations: HSV-1, herpes simplex virus type 1; HSV-2, herpes The publication costs of this article were defrayed in part by page charge simplex virus type 2; VZV, varicella-zoster virus; CMV, human payment. This article must therefore be hereby marked ‘‘advertisement’’ in cytomegalovirus; I site, inactivation site. accordance with 18 U.S.C. §1734 solely to indicate this fact. Data deposition: The atomic coordinates reported in this paper have been deposited in the Protein Data Bank, Chemistry Department, Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA Brookhaven National Laboratory, Upton, NY 11973, accession no. 0027-8424y97y942874-6$2.00y0 1VZV. PNAS is available online at http:yywww.pnas.org. §To whom reprint requests should be addressed. 2874 Downloaded by guest on September 28, 2021 Biochemistry: Qiu et al. Proc. Natl. Acad. Sci. USA 94 (1997) 2875 FIG. 1. The structure-assisted alignment of human herpes virus proteases. The secondary structure elements of CMV and VZV protease are underlined and labeled. Helical (AA-A7) regions (blue), strands (B1-B7) (red), and the conserved catalytic triad (green). CMV numbering is used. Heavy Atom Derivatives. To identify heavy atom derivatives tion, obtained using 8.0–4.0 Å data, was the highest peak (u1 5 36 different compounds were screened, each at several differ- 103.88, u2 5 12.58, u3 5 271.88). It was 25s above the mean and ent concentrations. The presence of the slightest amount of 1s higher than the second highest peak. Translation searches mercury destroyed the crystals. Two poor derivatives finally were carried out in the two possible space groups P6222 and were obtained by soaking a crystal in 1 mM potassium gold P6422, the latter giving a better solution (T 5 27.0 Å, 13.9 Å, cyanide for 1 day, and another in 2 mM trimethyllead acetate 19.6 Å) with a 5s in peak height and 52.6% in R-factor for data for 3 days. The gold data set was 81% complete to 4.5 Å with in the 8.0–3.0 Å range. After rigid body refinement the an Rmerge of 0.14 and Riso (S uFPH 2 FPuySuFHou) of 0.19, while R-factor was reduced to 50.6%. Examination of crystal packing the lead was 92% complete to 4.2 Å with an Rmerge of 0.20 and revealed a tight dimer interface similar to that observed in the Riso of 0.17. The gold binding site was identified by difference CMV protease structure (8). Using the calculated phases from Patterson methods using programs from CCP4 (14). The lead the molecular replacement solution, the gold heavy atom site, at a position similar to the gold, could be seen only by position was found to be identical to that found from the difference Fourier methods. The phasing power and RCullis (S difference Patterson, further confirming the correctness of the uFHo 2 FHcuySuFHou) of the gold derivative were 1.2 and 0.79 molecular replacement solution. However, the resulting map and those of the lead derivative were 1.0 and 0.82, respectively. was still uninterpretable. The combined figure of merit was 0.37, and the resulting Structure Solution and Refinement. The crystal structure of electron density map was not interpretable. VZV protease was solved by combining phases from the two Molecular Replacement. After determining the CMV pro- heavy atom derivatives and the molecular replacement solution.