During the late phase of HIV type 1 (HIV-1) replication, newly synthesized retroviral Gag proteins are targeted to the plasma membrane of most hematopoietic cell types, where they colocalize at lipid rafts and assemble into immature virions. Membrane binding is mediated by the matrix (MA) domain of Gag, a 132-residue polypeptide containing an N-terminal myristyl group that can adopt sequestered and exposed conformations. Although exposure is known to promote membrane binding, the mechanism by which Gag is targeted to specific membranes has yet to be established . Here we show that PI(4,5)P 2 binds directly to HIV-1 MA, inducing a conformational change that triggers myristate exposure. Related phosphatidylinositides PI, PI(3)P, PI(4)P, PI(5)P, and PI(3,5)P 2 do not bind MA with significant affinity or trigger myristate exposure. Structural studies reveal that PI(4,5)P 2 adopts an ''extended lipid'' conformation, in which the inositol head group and 2-fatty acid chain bind to a hydrophobic cleft, and the 1-fatty acid and exposed myristyl group bracket a conserved basic surface patch previously implicated in membrane binding. Our findings indicate that PI(4,5)P 2 acts as both a trigger of the myristyl switch and a membrane anchor and suggest a potential mechanism for targeting Gag to membrane rafts. matrix protein ͉ membrane targeting ͉ NMR ͉ lipid rafts

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.

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 Ca 2؉ -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 (K d ) of ϳ2 M 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.Gag is the major structural protein encoded by HIV-1 and contains all of the viral elements required to drive virus assembly (1-3). HIV-1 Gag targeting to the plasma membrane (PM) 2 is critical for proper and efficient assembly to produce progeny virions (1, 3-9). During virus maturation, Gag is cleaved into myristoylated matrix (myr(ϩ)MA), capsid, and nucleocapsid proteins, inducing major morphological reorganization of the virus (1, 2, 4, 5, 10). In many cell types, HIV-1 Gag budding and assembly has been shown to occur predominantly on the PM (4 -9, 11-18). Gag binding to the PM is mediated by the MA domain and enhanced by multimerization. Proper assembly and efficient binding of Gag to the PM requires a myristyl (myr) group as a membrane anchor and a cluster of basic residues localized within the N-terminal domain to facilitate interactions with acidic phospholipids (1,2,19,20).Steady progress has been made in defining both the viral and cellular determinants of HIV-1 assembly and release (6). However, the trafficking pathway used by Gag to reach assembly sites in the infected cell is poorly understood. Studies by Freed, Ono, and co-workers (21-23) demonstrated that the ultimate localization of HIV-1 Gag at virus assembly sites is dependent on phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P 2 ), a cellular factor localized at the inner leaflet of the PM (24 -26). Our structural studies revealed that PI(4,5)P 2 binds directly to HIV-1 MA, inducing a conformational change that triggers myr exposure (27). In addition to PI(4,5)P 2 ...

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)P 2 ). Exposure of the MA myristyl (myr) group is triggered by PI(4,5)P 2 binding and is enhanced by factors that promote protein self-association. In the studies reported herein, 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 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. KeywordsHuman immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2); myristyl (myr); matrix (MA); nuclear magnetic resonance (NMR); His-Glu salt bridge Corresponding Author: Jamil S. Saad, Ph.D., 845 19 th Street South, Birmingham, AL 35294;; saad@uab.edu. SUPPORTING INFORMATION AVAILABLE 2D 1 H-15 N HSQC NMR spectra of myr(−) and myr(+)MA as a function of pH (Figs. S1 and S2); 3D 13 C-edited/ 12 C-double-halffiltered NOE spectrum of myr(+)MA at pH 5.5 (Fig. S3); sedimentation equilibrium profiles for myr(+)MA at different pH values (Fig. S4); plots of chemical shift changes vs. pH used to determine the pK a value of His89 for myr(−)MA (Fig. S5); sedimentation equilibrium and velocity profiles for WT CA and various MACA mutants as a function of pH ( Fig. S6-S9); 2D NMR HSQC spectra (Fig. S10), sedimentation velocity profiles ( Fig. S11) and CD spectra (Fig. S12) for MA H89G mutants; 2D HSQC spectra and sedimentation velocity profiles of MA E12A mutants (Fig. S13); sedimentation velocity profiles for myr(+)MA-E12A/W184A/ M185A as a function of pH (Fig. S14); structural representation of salt bridge formation in the HIV-1 MA structures (Fig. S15); and, SDS-PAGE showing the effect of His89 and E12 mutations on virus production (Fig. S16). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 20...

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)P). 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)P, CaM, and membrane mimetics (nanodisc) bind to Akt(PHD). We show that PI(3,4,5)P 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)P-embedded membrane nanodisc to Akt(PHD) with a 10-fold tighter affinity than PI(3,4,5)P 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)P on the nanodisc surface. No detectable binding has been observed between Akt(PHD) and PI(3,4,5)P-free nanodiscs, demonstrating that PI(3,4,5)P 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.

No abstract

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.

For most retroviruses, including HIV-1, binding of the Gag polyprotein to the plasma membrane (PM) is mediated by interactions between Gag's N-terminal myristoylated matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2) in the PM. The Gag protein of avian sarcoma virus (ASV) lacks the N-myristoylation signal but contains structural domains having functions similar to those of HIV-1 Gag. The molecular mechanism by which ASV Gag binds to the PM is incompletely understood. Here, we employed NMR techniques to elucidate the molecular determinants of the membrane-binding domain of ASV MA (MA 87) to lipids and liposomes. We report that MA 87 binds to the polar head of phosphoinositides such as PI(4,5)P 2. We found that MA 87 binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups, indicating that the MA 87-IP binding is governed by charge-charge interactions. Using a sensitive NMRbased liposome-binding assay, we show that binding of MA 87 to liposomes is enhanced by incorporation of PI(4,5)P 2 and phosphatidylserine. We also show that membrane binding is mediated by a basic surface formed by Lys-6, Lys-13, Lys-23, and Lys-24. Substitution of these residues to glutamate abolished binding of MA 87 to both IPs and liposomes. In an accompanying paper, we further report that mutation of these lysine residues diminishes Gag assembly on the PM and inhibits ASV particle release. These findings provide a molecular basis for ASV Gag binding to the inner leaflet of the PM and advance our understanding of the basic mechanisms of retroviral assembly. Retroviral genomes encode a polyprotein called Gag, which contains all of the structural elements required for virus assembly. Subsequent to their synthesis, Gag proteins for most retroviruses are targeted to the plasma membrane (PM) 2 for assem-This work was supported by National Institutes of Health Grant 5R01 GM117837 (to J. S. S.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Tables S1 and S2 and Figs. S1-S9. The atomic coordinates and structure factors (codes 6CCJ, 6CE5, 6CUS, 6CV8, and 6CW4) have been deposited in the Protein Data Bank (http://wwpdb.org/). The chemical shift data reported in this paper have been deposited in the Biological Magnetic Resonance Bank with the accession codes 27396 and 30404.