ANALYTICAL APPROACH dues of the cleavage product (the y peptide). A proposal for the active site for the autocatalytic cleavage has been made by using the sequence compari son described and by analyzing the dis tribution of potentially important resi dues in the vicinity of the cleavage site. Important residues for the proteolysis mechanism should be conserved among the four sequences, given that all nodaviruses demonstrate the cleavage. These data and the three-dimensional structure of BBV have helped us to identify several residues probably in volved in the cleavage mechanism (Cys 69, Asp 75, Tyr 176, and Lys 192). One of these residues (Asp 75) is very close to the cleavage site (Figure 4). The role of cleavage in the matura tion of the particle is not clear. Capsid structures with Τ = 3 symmetry dis play two types of twofold joints (Figure 3). Although the subunits involved in the molecular contacts are identical, the nature of these contacts is differ ent. Those occurring at the quasi-two fold axes have direct interactions be tween the subunits (Figure 3f). Con tacts occurring at the icosahedral two fold axes are mediated by a polypep tide lying in a groove just inside the surface of the particle (Figure 3e). The cleavage may permit the full insertion of the polypeptide into the groove.
Crystallographic studies of other mem bers of the nodavirus family (FHV and NOV) are in progress. In these studies, the known three-dimensional structure of BBV is used to generate a first set of low-resolution phases; noncrystallographic symmetry techniques are then used to generate an accurate high-reso lution electron density map. At the same time, more detailed studies of the cleavage kinetics, the pH optimum of the cleavage, and the relation between cleavage and infectivity are under way (24). Finally, specific residues probably involved in the cleavage are being al tered by molecular genetics methods to test their role in catalysis (25).
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Future directions This work is the first step in under standing the detailed chemistry of a relatively simple biological process.
This work was possible because we had access to X-ray synchrotron sources for data collection (at LURE, Orsay, France, and at CHESS, Cornell University, Ithaca, NY) and to a supercomputer (the Cyber 205 at Purdue University) for all of our calculations. We thank Paul Kaesberg, Roland Rueckert, and their colleagues at the Institute for Molecular Vi rology, University of Wisconsin—Madison for re sults prior to publication and helpful discussions. We thank Tom Gallagher and Roland Rueckert for permission to use Figure 1. We thank Sharon Fateley for help in preparing this manuscript.
References (1) J o h n s o n , J. E . In Crystallography in North America; M c L a c h l a n , D., Jr.; Glusker, J . P . , E d s . ; A m e r i c a n Crystallo g r a p h i c Association: L a n s i n g , M I , 1983; pp. 410-14. (2) R o s s m a n n , M . G.; J o h n s o n , J. E . Ann. Rev. Biochem. 1 9 8 9 , 5 8 , 5 3 3 - 7 3 . (3) L o n g w o r t h , J. F.; Archibald, R. D. TV. Z. J. Zool. 1 9 7 5 , 2 , 233-36.
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Figure 4. Stereoview of the region near the cleavage site in the capsid subunit. Colored spheres 1 Â in radius have been superimposed on each atom. Asparagine 363 is the carboxy terminus of protein β and alanine 364 (8 Â from 363) is the amino terminus of protein 7 . Residue 75 (OD2 75) in β is an aspartic acid that is hydrogen bonded to 363 (Asn) and may play a role in the cleavage reac tion.
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1348 A ·• ANALYTICAL CHEMISTRY, CHEMISTRY, VOL. 61, NO. 23, DECEMBER 1, 1989
