AN UNUSUAL ROUTE TO THERMOSTABILITY IN PYROPHOSPHATASES Adrian Goldman*, Tiina Salminen*, Alexei Teplyakov[[daggerdbl]], Barry Cooperman+ and Reijo Lahti*. *University of Turku, Turku, Finland; [[daggerdbl]]European Molecular Biology Laboratory, Hamburg, Germany; +University of Pennsylvania, Philadelphia, USA.

Rigid-body motions of entire monomers to produce tighter oligomers may be yet another way in which proteins can be made thermophilic. By comparing the structures of Thermus thermophilus and E. coli PPases (T- and E-PPases) we showed that the chief determinant of the increased thermostability of T-PPase appears to be a change in the oligomerisation and oligomeric interfaces of these hexameric enzymes. Their sequences are 47% identical and the monomer structures superimpose extremely well (rmsd of 1 Å per Ca). However, the hexamers superimpose poorly (rmsd of 2.2 Å per Ca) because of a complex rigid-body motion: with respect to the E-PPase hexamer, each T-PPase monomer in the hexamer is skewed by about 1 Å in the xy plane, is 0.3 Å closer to the centre of the hexamer in the z-direction, and is rotated by about 7deg. about its centre of gravity. The rigid-body translation accounts for most of the difference between the hexamer and monomer superpositions. In T-PPase the hexamer is packed more tightly than in E-PPase: the amount of surface area buried upon oligomerisation increases by 16%, in particular at one of the trimer-trimer interfaces. Three small loops involved in oligomerisation adopt different conformations. Consequently, the number of hydrogen-bonding and ionic interactions almost doubles, which allows the tighter-packing to occur. The new interactions interlock across all the oligomeric interfaces in the hexamer.

Bacterial PPases, only 170 residues long, contain large active sites (~15 active site residues; ~12 Å across) and have little in the way of a hydrophobic core. Much of the protein, therefore, does multiple duty: for instance, one helix contributes to the hydrophobic core of the protein, provides active-site residues and is a key part of the trimer-trimer interfaces. (Thermo)stability and activity appear to be intimately linked. Although these enzymes are not allosteric, solution data on E-PPase variant proteins show that oligomerisation stabilises the conformation of the enzyme that binds substrate and vice versa. The D₃ bacterial inorganic pyrophosphatases seem to be a minimalist protein design.