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 (Kassoc ‫؍‬ 2.5 ؎ 0.6 ؋ 10 8 M ؊2 ). The equilibrium constant is shifted Ϸ20-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.

A myristoyl switch regulates membrane binding of HIV-1 Gag

“…One would therefore predict that addition of the CA domain, which contains additional multimerization signals, should promote myristate exposure at lower protein concentrations than myrMA. This is exactly what Tang et al (11) found. The monomer 7 trimer equilibrium constant is 20 times higher for myrMA-CA than for myrMA.…”

Point Mutations in the HIV-1 Matrix Protein Turn Off the Myristyl Switch

“…NOEs between the myristate group and the side chains of Val-7 and Leu-8 were also observed for the WT myrMA protein. 27 These NMR observations indicate that mutation of Arg-7 and Leu-8 does not greatly alter the conformation of the loop and that residues 7 and 8 play an important role into the stabilization of the myr(s) form.…”

“…In addition, the relative NOE intensities and cross-peak patterns matched the data obtained for the WT myr(s)MA protein. 27,31 The main differences between the structures are localized within the disordered loop formed by residues Gly-2 -Ser-9. For V7R, the side chain of Arg-7 is poised to make a salt bridge with the side chain of Glu-52, and the mythelene groups of Arg-7 contribute to the hydrophobic interactions with the methylene groups of the myristate ( Figure 6).…”

“…In contrast, for the wild type (WT) myrMA protein, a subset of signals shift progressively toward the frequencies observed for myr(-)MA upon increasing the protein concentration due to a concomitant shift in the monomer-trimer equilibrium toward the trimeric species and exposure of the myristyl group. 27 The absence of concentration-dependent shifts for V7R, L8A, and L8I indicates that these mutants exist in a unique conformation over the range of concentrations tested. Sedimentation equilibrium (SE) data obtained for V7R, L8A and L8I are similar in appearance to SE data obtained for myr(-) MA, and confirm that the myrMA mutants remain monomeric at concentrations as high as 1 mM.…”

HIV-1 Gag: Flipped out for PI(4,5)P 2

“…A decade ago, it was proposed that the interaction of the MA domain of Pr55 Gag with membrane is regulated by a myristyl switch (12), and a substantial amount of evidence supports this model (13)(14)(15). Most recently, structural studies from Summers and colleagues (16) demonstrated that the myristate is sequestered when MA is in a monomeric form, which binds membrane poorly. In contrast, when downstream sequences in CA that promote oligomerization are added to MA, the myristate becomes exposed, and membrane binding is substantially increased.…”