Entropic switch regulates myristate exposure in the HIV-1 matrix protein

Abstract

The myristoylated matrix protein (myr-MA) of HIV functions as a regulator of intracellular localization, targeting the Gag precursor polyprotein to lipid rafts in the plasma membrane during virus assembly and dissociating from the membrane during infectivity for nuclear targeting of the preintegration complex. Membrane release is triggered by proteolytic cleavage of Gag, and it has, until now, been believed that proteolysis induces a conformational change in myr-MA that sequesters the myristyl group. NMR studies reported here reveal that myr-MA adopts myr-exposed [myr(e)] and -sequestered [myr(s)] states, as anticipated. Unexpectedly, the tertiary structures of the protein in both states are very similar, with the sequestered myristyl group occupying a cavity that requires only minor conformational adjustments for insertion. In addition, myristate exposure is coupled with trimerization, with the myristyl group sequestered in the monomer and exposed in the trimer (K(assoc) = 2.5 +/- 0.6 x 10(8) M(-2)). The equilibrium constant is shifted approximately 20-fold toward the trimeric, myristate-exposed species in a Gag-like construct that includes the capsid domain, indicating that exposure is enhanced by Gag subdomains that promote self-association. Our findings indicate that the HIV-1 myristyl switch is regulated not by mechanically induced conformational changes, as observed for other myristyl switches, but instead by entropic modulation of a preexisting equilibrium.

Fig. 1.

(A) Overlay of expanded portions of the 2D [¹H–¹⁵N]HSQC spectra obtained for myr-MA [25 μM (black), 100 μM (blue), 400 μM (cyan), and 800 μM (green)] and myr(–)-MA (500 μM, red). A subset of myr-MA signals differ significantly from those of myr(–)-MA at low concentration due to sequestration of the myristyl group but shift progressively toward the frequencies of myr(–)-MA as the concentration is increased (denoted by arrows), consistent with an equilibrium shift toward a myristate-exposed species at higher concentrations. (B) Representative sedimentation equilibrium data obtained for myr-MA and myr(–)-MA at 20°C. Data for myr-MA fit to a momomer–trimer equilibrium with association constant (K_assoc) of 2.5 ± 0.6 × 10⁸ M^–2 (theoretical curve shown in green), whereas the myr(–)-MA data fit best to a homogeneous, monomeric species (red). Higher-order myr-MA oligomers are formed at concentrations >100 μMat20°C. At 35°C, where optimal NMR data were obtained, K_assoc for myr-MA shifts to 4.0 ± 0.8 × 10⁷ M^–2.

Fig. 2.

(A) Stereoview of 20 superposed NMR structures determined for myr(s)-MA (backbone atoms of Gly-2–Ala-120 in blue and carbon atoms of myr 1 in red). For comparison, representative NMR (magenta) and x-ray (green) structures of myr(–)-MA are also shown. (B) Semitransparent surface representation of myr(s)-MA showing the partial penetration of the myristyl group (red spheres). (C) Ribbon drawing of myr(s)-MA showing side chains (green) that exhibit [¹H–¹H]NOEs with the myristyl group (red). The orange segment is fully disordered in myr(–)-MA, and the blue segment undergoes a minor conformational adjustment (reflected by chemical shift changes) upon insertion of the myristyl group.

Fig. 3.

Representation of the myr(s)-MA[myr(e)-MA]₃ equilibrium showing the experimentally determined NMR structure of myr(s)-MA and a proposed model for the [myr(e)-MA]₃ trimer. The model was generated by superpositioning three identical copies of a representative NMR structure of myr-MA [generated by using only myr(–)-MA NMR restraints] onto the coordinates of the trimeric myr(–)-MA x-ray structure. The model is consistent with the NMR chemical shift changes observed for myr-MA at protein concentrations that favor self-association. Lysine residues that are important for efficient virus replication (67) and have been proposed to interact with the membrane surface (25) are shown in blue. The intermolecular myr–myr interactions may be disrupted upon binding to membranes.

Fig. 4.

(A) Sedimentation equilibrium data obtained for myr-MA–CA (3.3 μM, 20°C) and best-fit theoretical curve (cyan) for a monomer–trimer equilibrium (K_assoc = 4.6 ± 1.1 × 10⁹ M^–2). (B) Portions of 2D [¹H–¹⁵N]HSQC spectra obtained for myr-MA–CA (50 μM; pink dashed lines), myr(–)-MA (500 μM, red), and concentration-dependent spectra obtained for myr-MA (see Fig. 1 caption for color definitions). The data obtained for myr-MA–CA follow trends observed for myr-MA. (C) Schematic representation of the monomer–trimer and myr(s)–myr(e) equilibrium (myr, MA, and CA are shown as black lines and green and red spheres, respectively). The CA domain promotes self-association and thereby shifts the equilibrium toward the myr(e) state. (D) Other Gag domains (yellow spheres, p2; blue spheres, NC) that promote assembly and membrane binding are expected to also shift the equilibrium toward the myr(e) state. Proteolytic cleavage of the MA–CA junction during the early phase of replication eliminates these effects, allowing the equilibrium to shift toward the myr(s) state.