CALCIUM-FREE CALMODULIN IS A SUBSTRATE OF PROTEASES FROM HUMAN IMMUNODEFICIENCY VIRUS-1 AND VIRUS-2

Calcium-free calmodulin-(CaM) is rapidly hydrolyzed by proteases from both human immunodeficiency viruses (HIV) 1 and 2. Kinetic analysis reveals a sequential order of cleavage by both proteases which initiates in regions of the molecule known from X-ray crystallographic analysis of Ca2+/CaM to be associated with calcium binding. Although HIV-1 and HIV-2 proteases hydrolyze two bonds in common, the initial site of cleavage required for subsequent events differs in each case. The first bond hydrolyzed by the HIV-1 protease is the Asn-Tyr linkage in the sequence, -N-I-D-G-D-G-Q-V-N-Y-E-E-, found in the fourth calcium binding loop. In contrast, it is an Ala-Ala bond in the third calcium loop, -D-K-D-G-N-G-Y-I-S-A-A-E-, that is first hydrolyzed by the HIV-2 enzyme, followed in short order by cleavage of the same Asn-Tyr linkage described above. Thereafter, both enzyme proceed to hydrolyze additional peptide bonds, some in common, some not. Considerable evidence exists that inhibitors are bound to the protease in an extended conformation and yet all of the cleavages we observed occur within, or at the beginning of helices in Ca2+/CaM, regions that also appear to be insufficiently exposed for protease binding. Molecular modeling studies indicate that CaM in solution must adopt a conformation in which the first cleavage site observed for each enzyme is unshielded and extended, and that subsequent cleavages involve further unwinding of helices. The conclusion that the conformation of CaM is different from that of Ca2+/CaM is supported by the observation that Ca2+/CaM is resistant to hydrolysis by either enzyme. As well as demonstrating conformational differences between CaM and Ca2+/CaM, these studies provide further evidence that the two highly homologous human retroviral proteases may be distinguished enzymologically in terms of differential substrate specificities. In addition, some new and unpredicted sequences have been identified that undergo cleavage by these enzymes. Finally, the fact that an abundant, ubiquitous, and biologically important cellular protein is broken down by the HIV proteases could be physiologically relevant to HIV infection if the viral enzyme ever displays activity within the host cell.