Serpins fold into a native metastable state and utilize a complex conformational change to inhibit target proteases. An undesirable result of this conformational flexibility is that most inhibitory serpins are heat sensitive, forming inactive polymers at elevated temperatures. However, the prokaryote serpin, thermopin, from Thermobifida fusca is able to function in a heated environment. We have determined the 1.8 Å x-ray crystal structure of thermopin in the native, inhibitory conformation. A structural comparison with the previously determined 1.5 Å structure of cleaved thermopin provides detailed insight into the complex mechanism of conformational change in serpins. Flexibility in the shutter region and electrostatic interactions at the top of the A -sheet (the breach) involving the C-terminal tail, a unique structural feature of thermopin, are postulated to be important for controlling inhibitory activity and triggering conformational change, respectively, in the native state. Here we have discussed the structural basis of how this serpin reconciles the thermodynamic instability necessary for function with the stability required to withstand elevated temperatures.Serpins (peptidase inhibitor family I4) are the largest superfamily of protease inhibitors with over 800 members identified to date (1-4). A recent authoritative classification of known peptidase inhibitors reveals that serpins are the only inhibitor family present in all three superkingdoms of life (Eukarya, Bacteria, and Archaea) as well as certain viruses (1). The majority of serpins are found in multicellular eukaryotes and inhibit serine or (more rarely) cysteine proteases. Inhibitory serpins are unusual molecules that fold into a native metastable state and utilize a complex conformational change to achieve protease inhibition (4 -6). Upon docking with a target protease, a flexible exposed region termed the reactive center loop (RCL) 1 is cleaved and the region N-terminal to the site of proteolysis (termed P1-P15) 2 inserts into the center of the large A -sheet, forming an additional -strand and causing a large conformational change (termed the Stressed (S) to Relaxed (R) transition) throughout the molecule (8 -10). Following cleavage, and throughout the S to R transition, the protease remains covalently attached to the serpin via an acyl bond between the side chain of the active site serine (or cysteine) and the carbonyl oxygen of the P1 residue in the RCL. The x-ray crystal structure of antitrypsin in complex with trypsin revealed that the enzyme is translocated 75 Å to the base of the inhibitor where it is trapped in a distorted, inactive conformation (11-14). The events following successful protease inhibition are irreversible, and serpins are thus termed suicide inhibitors. The size and conformational flexibility of serpins allow for an exquisite degree of functional control, and many serpins require specific co-factors to effectively inhibit target enzymes. For example, the thrombin/factor Xa inhibitor antithrombin circulates in a rel...