Crystal Structure of a Truncated Mutant of Glucose- Fructose Oxidoreductase Shows that an N-terminal Arm Controls Tetramer Formation J. Shaun Lott 1,2 , Dirk Halbig 3 , Heather M. Baker 1,2 , Michael J. Hardman 2 Georg A. Sprenger 3 and Edward N. Baker 1,2,4 * 1 School of Biological Sciences, University of Auckland Auckland, New Zealand 2 Institute of Molecular Biosciences, Massey University Palmerston North, New Zealand 3 Institut fu Èr Biotechnologie, 1 der Forschungszentrum Ju Èlich GmbH, D-52425, Ju Èlich Germany 4 Department of Chemistry University of Auckland Auckland, New Zealand N-terminal or C-terminal arms that extend from folded protein domains can play a critical role in quaternary structure and other intermolecular associations and/or in controlling biological activity. We have tested the role of an extended N-terminal arm in the structure and function of a periplasmic enzyme glucose-fructose oxidoreductase (GFOR) from Zymo- monas mobilis. We have determined the crystal structure of the NAD  complex of a truncated form of the enzyme, GFORÁ, in which the ®rst 22 residues of the N-terminal arm of the mature protein have been deleted. The structure, re®ned at 2.7 A Ê resolution (R cryst  24.1%, R free  28.4%), shows that the truncated form of the enzyme forms a dimer and implies that the N-terminal arm is essential for tetramer formation by wild-type GFOR. Truncation of the N-terminal arm also greatlyincreasesthesolventexposureofthecofactor;sinceGFORactivity is dependent on retention of the cofactor during the catalytic cycle we conclude that the absence of GFOR activity in this mutant results from dissociation of the cofactor. The N-terminal arm thus determines the quaternary structure and the retention of the cofactor for GFOR activity and during translocation into the periplasm. The structure of GFORÁ also shows how an additional mutation, Ser64Asp, converts the strict NADP  speci®city of wild-type GFOR to a dual NADP  /NAD  speci®city. # 1998AcademicPress Keywords: Glucose-fructoseoxidoreductase;oligomerisation;N-terminal arm;cofactorbinding;crystalstructure *Corresponding author Introduction Protein oligomerisation provides a mechanism by which biological activity can be controlled. Thus, changes in the relationships between the subunitsofanoligomericenzymeprovideforallo- steric regulation of activity (Fersht, 1977). Within oligomeric proteins, however, the extent and strength of the association between protein sub- units can vary greatly. An analysis of protein dimers,forexample,showedthatthepercentageof surface area that is buried on dimerisation ranged from6.5%to29.4%(Jones&Thornton,1995),and it is reasonable to assume that dimer stability var- ies correspondingly. Likewise, in higher oligomers itisoftenthecasethatoneinterfaceismoreexten- sive than a second; many tetramers are best described as a dimer of dimers, because the primary interface that forms the dimer is much more extensive than the secondary interfaces that generate the tetramer. This can give rise to dimer- tetramer equilibria that may also regulate activity. Indeed, there is no real distinction, in composition or interactions, between the interfaces that are formedbetweenproteinsthatonlyassociatetransi- ently, as in signalling pathways, and those that are involved in the permanent association of oligomers. In recent years it has become clear that exten- sions of the polypeptide, beyond the con®nes of a foldedproteindomainorsubunit,canplayavery important role in oligomerisation. This may E-mailaddressofthecorrespondingauthor: ted.baker@auckland.ac.nz Abbreviationsused:GFOR,glucose-fructose oxidoreductase;GFORÁ,GFORS64Dmutantwith residues1-22deleted. doi:10.1006/jmbi.1998.4245 available online at http://www.idealibrary.com on J. Mol. Biol. (1998) 304, 575±584 0022-2836/00/040575±10$35.00/0 # 1998AcademicPress