Ruba H. Ghanam

Structure of the myristylated human immunodeficiency virus type 2 matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in membrane targeting.

During the late phase of retroviral replication, newly synthesized Gag proteins are targeted to the plasma membrane (PM), where they assemble and bud to form immature virus particles. Membrane targeting by human immunodeficiency virus type 1 (HIV-1) Gag is mediated by the PM marker molecule phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)], which is capable of binding to the matrix (MA) domain of Gag in an extended lipid conformation and of triggering myristate exposure. Here, we show that, as observed previously for HIV-1 MA, the myristyl group of HIV-2 MA is partially sequestered within a narrow hydrophobic tunnel formed by side chains of helices 1, 2, 3, and 5. However, the myristate of HIV-2 MA is more tightly sequestered than that of the HIV-1 protein and does not exhibit concentration-dependent exposure. Soluble PI(4,5)P(2) analogs containing truncated acyl chains bind HIV-2 MA and induce minor long-range structural changes but do not trigger myristate exposure. Despite these differences, the site of HIV-2 assembly in vivo can be manipulated by enzymes that regulate PI(4,5)P(2) localization. Our findings indicate that HIV-1 and HIV-2 are both targeted to the PM for assembly via a PI(4,5)P(2)-dependent mechanism, despite differences in the sensitivity of the MA myristyl switch, and suggest a potential mechanism that may contribute to the poor replication kinetics of HIV-2.

Myristate Exposure in the Human Immunodeficiency Virus Type 1 Matrix Protein Is Modulated by pH

Human immunodeficiency virus type 1 (HIV-1) encodes a polypeptide called Gag that is capable of forming virus-like particles (VLPs) in vitro in the absence of other cellular or viral constituents. During the late phase of HIV-1 infection, Gag polyproteins are transported to the plasma membrane (PM) for assembly. A combination of in vivo, in vitro, and structural studies have shown that Gag targeting and assembly on the PM are mediated by specific interactions between the myristoylated matrix [myr(+)MA] domain of Gag and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Exposure of the MA myristyl (myr) group is triggered by PI(4,5)P2 binding and is enhanced by factors that promote protein self-association. In the studies reported here, we demonstrate that myr exposure in MA is modulated by pH. Our data show that deprotonation of the His89 imidazole ring in myr(+)MA destabilizes the salt bridge formed between His89(Hδ2) and Glu12(COO-), leading to tight sequestration of the myr group and a shift in the equilibrium from trimer to monomer. Furthermore, we show that oligomerization of a Gag-like construct containing matrix-capsid is also pH-dependent. Disruption of the His−Glu salt bridge by single-amino acid substitutions greatly altered the myr-sequestered−myr-exposed equilibrium. In vivo intracellular localization data revealed that the H89G mutation retargets Gag to intracellular compartments and severely inhibits virus production. Our findings reveal that the MA domain acts as a “pH sensor” in vitro, suggesting that the effect of pH on HIV-1 Gag targeting and binding to the PM warrants investigation.

Binding of Calmodulin to the HIV-1 Matrix Protein Triggers Myristate Exposure

Steady progress has been made in defining both the viral and cellular determinants of retroviral assembly and release. Although it is widely accepted that targeting of the Gag polypeptide to the plasma membrane is critical for proper assembly of HIV-1, the intracellular interactions and trafficking of Gag to its assembly sites in the infected cell are poorly understood. HIV-1 Gag was shown to interact and co-localize with calmodulin (CaM), a ubiquitous and highly conserved Ca2+-binding protein expressed in all eukaryotic cells, and is implicated in a variety of cellular functions. Binding of HIV-1 Gag to CaM is dependent on calcium and is mediated by the N-terminally myristoylated matrix (myr(+)MA) domain. Herein, we demonstrate that CaM binds to myr(+)MA with a dissociation constant (Kd) of ∼2 μm and 1:1 stoichiometry. Strikingly, our data revealed that CaM binding to MA induces the extrusion of the myr group. However, in contrast to all known examples of CaM-binding myristoylated proteins, our data show that the myr group is exposed to solvent and not involved in CaM binding. The interactions between CaM and myr(+)MA are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. As revealed by NMR data, both CaM and MA appear to engage substantial regions and/or undergo significant conformational changes upon binding. We believe that our findings will provide new insights on how Gag may interact with CaM during the HIV replication cycle.

Role of the HIV-1 Matrix Protein in Gag Intracellular Trafficking and Targeting to the Plasma Membrane for Virus Assembly

Human immunodeficiency virus type-1 (HIV-1) encodes a polypeptide called Gag that is able to form virus-like particles in vitro in the absence of any cellular or viral constituents. During the late phase of the HIV-1 infection, Gag polyproteins are transported to the plasma membrane (PM) for assembly. In the past two decades, in vivo, in vitro, and structural studies have shown that Gag trafficking and targeting to the PM are orchestrated events that are dependent on multiple factors including cellular proteins and specific membrane lipids. The matrix (MA) domain of Gag has been the focus of these studies as it appears to be engaged in multiple intracellular interactions that are suggested to be critical for virus assembly and replication. The interaction between Gag and the PM is perhaps the most understood. It is now established that the ultimate localization of Gag on punctate sites on the PM is mediated by specific interactions between the MA domain of Gag and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], a minor lipid localized on the inner leaflet of the PM. Structure-based studies revealed that binding of PI(4,5)P2 to MA induces minor conformational changes, leading to exposure of the myristyl (myr) group. Exposure of the myr group is also triggered by binding of calmodulin, enhanced by factors that promote protein self-association like the capsid domain of Gag, and is modulated by pH. Despite the steady progress in defining both the viral and cellular determinants of retroviral assembly and release, Gag’s intracellular interactions and trafficking to its assembly sites in the infected cell are poorly understood. In this review, we summarize the current understanding of the structural and functional role of MA in HIV replication.

NMR, Biophysical, and Biochemical Studies Reveal the Minimal Calmodulin Binding Domain of the HIV-1 Matrix Protein

Background: Calmodulin interacts with the HIV-1 Gag protein in infected cells. Results: The minimal calmodulin binding domain of Gag has been identified. Conclusion: Residues 8–43 of MA are important for CaM binding. Significance: Our findings may help in identification of the functional role of CaM-Gag interactions in the HIV replication cycle. 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. We have 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 how MA interacts with CaM that may ultimately help in identification of the functional role of CaM-Gag interactions in the HIV replication cycle.

Mutations that mimic phosphorylation of the HIV‐1 matrix protein do not perturb the myristyl switch

Recent studies indicate that the matrix domain (MA) of the HIV‐1 Gag polyprotein directs Gag to the plasma membrane for virus assembly via a phosphatidylinositol‐4,5‐bisphosphate (PIP2)–dependent myristyl switch mechanism. MA also has been reported to direct nuclear trafficking via nuclear import and export functions, and some studies suggest that nuclear targeting may be regulated by MA phosphorylation (although this proposal remains controversial). We have prepared and studied a series of HIV‐1 MA mutants containing Ser‐to‐Asp substitutions designed to mimic phosphorylation, including substitutions in regions of the protein involved in protein–protein interactions and known to influence the myristyl switch (S6D, S9D, S67D, S72D, S6D/S9D, and S67D/S72D). We were particularly interested in substitutions at residue 6, since conservative mutations adjacent to this site strongly perturb the myristyl switch equilibrium, and this site had not been genetically tested due to its involvement in post‐translational myristylation. Our studies reveal that none of these mutations, including S6D, influences the PIP2‐ or concentration‐dependent myristyl switch equilibrium. In addition, all of the mutants bind liposomes with affinities that are only slightly reduced in comparison with the native protein. In contrast, the myristylated mutants bind liposomes with substantially greater affinity than that of the native, unmyristylated protein. These findings support the hypothesis that phosphorylation is unlikely to significantly influence membrane‐mediated intracellular trafficking.

Structural and Biophysical Characterization of the Interactions Between Calmodulin and the Pleckstrin Homology Domain of Akt

Background: Akt translocation to the plasma membrane (PM) is modulated by calmodulin (CaM). Results: CaM forms a tight complex with the PHD of Akt, which represents a novel class of CaM-binding domains. Conclusion: The binding mode of CaM to Akt(PHD) suggests a synergistic role in membrane binding and subsequent activation. Significance: Characterization of Akt-CaM is critical for understanding Akt activation. The translocation of Akt, a serine/threonine kinase, to the plasma membrane is a critical step in the Akt activation pathway. It is established that membrane binding of Akt is mediated by direct interactions between its pleckstrin homology domain (PHD) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). There is now evidence that Akt activation in many breast cancer cells is also modulated by the calcium-binding protein, calmodulin (CaM). Upon EGF stimulation of breast cancer cells, CaM co-localizes with Akt at the plasma membrane to enhance activation. However, the molecular details of Akt(PHD) interaction with CaM are not known. In this study, we employed NMR, biochemical, and biophysical techniques to characterize CaM binding to Akt(PHD). Our data show that CaM forms a tight complex with the PHD of Akt (dissociation constant = 100 nm). The interaction between CaM and Akt(PHD) is enthalpically driven, and the affinity is greatly dependent on salt concentration, indicating that electrostatic interactions are important for binding. The CaM-binding interface in Akt(PHD) was mapped to two loops adjacent to the PI(3,4,5)P3 binding site, which represents a rare CaM-binding motif and suggests a synergistic relationship between CaM and PI(3,4,5)P3 upon Akt activation. Elucidation of the mechanism by which Akt interacts with CaM will help in understanding the activation mechanism, which may provide insights for new potential targets to control the pathophysiological processes of cell survival.

The Interplay between Calmodulin and Membrane Interactions with the Pleckstrin Homology Domain of Akt.

The Akt protein, a serine/threonine kinase, plays important roles in cell survival, apoptosis, and oncogenes. Akt is translocated to the plasma membrane for activation. Akt-membrane binding is mediated by direct interactions between its pleckstrin homology domain (PHD) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). It has been shown that Akt activation in breast cancer cells is modulated by calmodulin (CaM). However, the molecular mechanism of the interplay between CaM and membrane binding is not established. Here, we employed nuclear magnetic resonance (NMR) and biochemical and biophysical techniques to characterize how PI(3,4,5)P3, CaM, and membrane mimetics (nanodisc) bind to Akt(PHD). We show that PI(3,4,5)P3 binding to Akt(PHD) displaces the C-terminal lobe of CaM but not the weakly binding N-terminal lobe. However, binding of a PI(3,4,5)P3-embedded membrane nanodisc to Akt(PHD) with a 103-fold tighter affinity than PI(3,4,5)P3 is able to completely displace CaM. We also show that Akt(PHD) binds to both layers of the nanodisc, indicating proper incorporation of PI(3,4,5)P3 on the nanodisc surface. No detectable binding has been observed between Akt(PHD) and PI(3,4,5)P3-free nanodiscs, demonstrating that PI(3,4,5)P3 is required for membrane binding, CaM displacement, and Akt activation. Using pancreatic cancer cells, we demonstrate that inhibition of Akt-CaM binding attenuated Akt activation. Our findings support a model by which CaM binds to Akt to facilitate its translocation to the membrane. Elucidation of the molecular details of the interplay between membrane and CaM binding to Akt may help in the development of potential targets to control the pathophysiological processes of cell survival.