Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly

Abstract

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. Recent studies have shown that phosphatidylinositol (PI) 4,5-bisphosphate [PI(4,5)P(2)], a factor that regulates localization of cellular proteins to the plasma membrane, also regulates Gag localization and assembly. 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.

Fig. 1.

Representative NMR data and structure of HIV-1 myrMA bound to di-C₄-PI(4,5)P₂. (a) Overlay of 2D ¹H-¹⁵N HSQC spectra upon titration with di-C₄-PI(4,5)P₂ [50 μM, 35°C; di-C₄-PI(4,5)P₂:MA = 0:1 (black), 1:1 (red), 2:1 (gray), 4:1 (magenta), 8:1 (green), 16:1 (blue)]. (b) ¹⁵N NMR chemical-shift titration data, which fit to 1:1 binding isotherms (K_d = 150 ± 30 μM). (c) Representative ¹³C-edited/¹²C-double-half-filtered NOE data showing unambiguously assigned intermolecular NOEs. (d) Stereoview showing the best-fit backbone superposition of the 20 refined structures calculated for the myr(e)MA:di-C₄-PI(4,5)P₂ complex. Structural statistics are summarized in Table 1, which is published as supporting information on the PNAS web site.

Fig. 2.

Structure of the di-C₈-PI(4,5)P₂:myr(−)MA complex. (a) Interactions between di-C₈-PI(4,5)P₂ (sticks) and myr(−)MA (colored according to electrostatic surface potential) showing the 2′-fatty acid extending in a hydrophobic cleft and the inositol ring packing against a basic patch of the protein. (b) The structure is rotated ≈90° relative to a, and the C₈ acyl chains are shown in space-filling format.

Fig. 3.

Structure of the di-C₈-PI(4,5)P₂:myr(−)MA complex showing the electrostatic interactions implicated in binding.

Fig. 4.

Comparison of the myrMA structures before (gray) and after (slate) binding to di-C₄-PI(4,5)P₂. (a) Superposition showing that di-C₄-PI(4,5)P₂ binding induces only minor structural changes in the loop connecting helices I and II and much larger changes in the structure and orientation of helix I. (b) View of the N terminus of myr(s)MA showing the orientation of helix I and relative location of Glu-12 associated with sequestration of the myristyl group. (c) View of the N-terminal portion of the di-C₄-PI(4,5)P₂:myr(e)MA complex showing the packing and hydrogen-bonding interactions that stabilize the myr(e) conformation.

Fig. 5.

Membrane-binding model predicted from the structural studies. (a) Trimer model constructed by superpositioning three identical copies of myr(e)MA:di-C₄-PI(4,5)P₂ onto the coordinates of the trimeric of myr(−)MA x-ray structure (38) and substituting the 1′- and 2′-C₈ acyl chains by C₁₈ and C₂₀ acyl chains, respectively. The myristyl group and residues that contact the phosphatidylinositide (yellow, with red phosphates) are colored green and blue, respectively. (b) The exposed 1′-fatty acids and myristyl groups project from a highly basic surface (Arg and Lys sidechains shown in blue) in a manner expected to synergistically promote membrane binding. The lipid model is from ref. .