Subcellular distribution of Calmodulin (CaM) in human immunodeficiency virus type-1 (HIV-1)-infected cells is distinct from that observed in uninfected cells. CaM has been shown to interact and co-localize with the HIV-1 Gag protein in infected cells. However, the precise molecular mechanism of this interaction is not known. Binding of Gag to CaM is dependent on calcium and is mediated by the N-terminal-myristoylated matrix (myr(+)MA) domain.Wehave recently shown that CaM binding induces a conformational change in the MA protein, triggering exposure of the myristate group. To unravel the molecular mechanism of CaM-MA interaction and to identify the minimal CaM binding domain of MA, we devised multiple approaches utilizing NMR, biochemical, and biophysical methods. Short peptides derived from the MA protein have been examined. Our data revealed that whereas peptides spanning residues 11-28 (MA-(11-28)) and 31-46 (MA-(31-46)) appear to bind preferentially to the C-terminal lobe of CaM, a peptide comprising residues 11-46 (MA-(11-46)) appears to engage both domains of CaM. Limited proteolysis data conducted on the MA-CaM complex yielded a MA peptide (residues 8-43) that is protected by CaM and resistant to proteolysis. MA-(8-43) binds to CaM with a very high affinity (dissociation constant = 25 nM) and in a manner that is similar to that observed for the full-length MA protein. The present findings provide new insights on howMAinteracts withCaMthat may ultimately help in identification of the functional role of CaM-Gag interactions in the HIV replication cycle. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.