Calmodulin Disrupts the Structure of the HIV-1 MA Protein

Abstract

The MA protein from HIV-1 is a small, multifunctional protein responsible for regulating various stages of the viral replication cycle. To achieve its diverse tasks MA interacts with host cell proteins and it has been reported that one of these is the ubiquitous calcium -sensing calmodulin (CaM) which is up-regulated upon HIV-1 infection. The nature of the CaM-MA interaction has been the subject of structural studies using peptides based on the MA sequence that have led to conflicting conclusions. The results presented here show that CaM binds intact MA with 1:1 stoichiometry in a Ca 2+ -dependent manner and that the complex adopts a highly extended conformation in solution as revealed by small-angle X-ray scattering. Alterations in tryptophan fluorescence suggest that the two tryptophans at the N-terminus of MA mediate the CaM interaction. Major chemical shift changes occur in the NMR spectrum of MA upon complex formation, while chemical shift changes in the CaM spectrum are quite modest and are assigned to residues within the target-protein binding hydrophobic clefts of CaM. The NMR data indicate that CaM binds MA via its N-and C-terminal lobes and induces a dramatic conformational change involving a significant loss of secondary and tertiary structure within MA. Circular dichroism experiments suggest that MA looses ~20% of its α-helical content upon CaM binding. Thus CaM binding is expected to impact upon the accessibility of interaction sites within MA that are involved in its various functions.

Solution Structure of Calmodulin Bound to the Binding Domain of the HIV-1 Matrix Protein

“…Tryptophan fluorescence data additionally suggested that interactions are possibly mediated by insertion of Trp 16 and Trp 36 in hydrophobic pockets of CaM (57). Although these findings agree with most of our previous (59,61) and current data, the structure of CaM⅐MA- (8 -43) clearly shows that Trp 36 is exposed to solvent and is not involved in any interaction with CaM; the orientation of the helix positions the indole group of Trp 36 away from the binding pocket (Fig.…”

“…In vivo and in vitro studies revealed that CaM interacts with additional HIV-1 proteins like Nef, Tat, and gp160 (27,34,37,52,53,56). Small-angle x-ray scattering studies have provided a global picture of CaM bound to full-length MA (57) or to short peptides derived from the MA protein (58). Our laboratory (59) and others (57,60) have shown that CaM binds directly to the MA protein in a calcium-dependent manner.…”

“…Small-angle x-ray scattering studies have provided a global picture of CaM bound to full-length MA (57) or to short peptides derived from the MA protein (58). Our laboratory (59) and others (57,60) have shown that CaM binds directly to the MA protein in a calcium-dependent manner. We have shown that binding of CaM induces a conformational change in MA, triggering myr exposure (59).…”

Structural and Biophysical Characterization of the Interactions between the Death Domain of Fas Receptor and Calmodulin

“…This unfolding event also releases helix ␣6 and make it accessible to CaM. The ability of CaM to disrupt protein targets has been recently observed in studies of the interactions between CaM and the human immunodeficiency virus type-1 matrix protein (37,45,64). CaM-induced unfolding of protein targets is not unusual.…”

The Motif of Human Cardiac Myosin-binding Protein C Is Required for Its Ca2+-dependent Interaction with Calmodulin

“…The final expressed recombinant protein included an in-frame N-terminal polyhistidine tag that was not removed due to the proteolytic sensitivity of the C1C2EEE protein. CaM and 15 N-labeled CaM were expressed in E. coli Rosetta 2 cells and purified as described previously (42). Stock solutions of C1C2 and C1C2EEE were stored in a buffer containing 25 mM Tris, 350 mM NaCl, and 2 mM TCEP, pH 7.0; stock solutions of the motif were stored in buffer containing 20 mM Tris, 50 mM NaCl, 2 mM TCEP, pH 6.8; and stock solutions of CaM were stored in a buffer containing 50 mM MOPS, 150 mM NaCl, and 10 mM CaCl 2 , pH 7.4.…”