Neutron reflectometry study of the conformation of HIV Nef bound to lipid membranes

Abstract

Nef is an HIV-1 accessory protein that directly contributes to AIDS progression. Nef is myristoylated on the N-terminus, associates with membranes, and may undergo a transition from a solution conformation to a membrane-associated conformation. It has been hypothesized that conformational rearrangement enables membrane-associated Nef to interact with cellular proteins. Despite its medical relevance, to our knowledge there is no direct information about the conformation of membrane-bound Nef. In this work, we used neutron reflection to reveal what we believe are the first details of the conformation of membrane-bound Nef. The conformation of Nef was probed upon binding to Langmuir monolayers through the interaction of an N-terminal His tag with a synthetic metal-chelating lipid, which models one of the possible limiting cases for myr-Nef. The data indicate that residues are inserted into the lipid headgroups during interaction, and that the core domain lies directly against the lipid headgroups, with a thickness of ∼40 A. Binding of Nef through the N-terminal His tag apparently facilitates insertion of residues, as no insertion occurred upon binding of Nef through weak electrostatic interactions in the absence of the specific interaction through the His tag.

Figure 1

Illustration of the experimental system. Neutrons strike the opposite face of the lipid monolayer from where Nef interacts. The dimensions of Nef from the most extended form of the Geyer and Peterlin model (17) are shown at the left, along with the dimensions of the two lipids. For the purpose of size comparison, a model of a highly compact form of Nef is shown on the right. This figure is meant to illustrate the experimental setup and not necessarily to show the true conformation of Nef at the membrane.

Figure 2

(a) NR data for a 65% d-DSIDA/Cu²⁺ + 35% d-DPPC monolayer alone (○) and with bound 6His-d-Nef (■) adsorbed from solution at 0.5 μM. Adsorption was arrested after 2.0 h by exchanging the subphase with pure Tris buffer. (b) SLD profiles corresponding to the data in a, with uncertainty limits determined from a Monte Carlo resampling procedure (41). The profiles for the lipids alone are denoted by a cyan/blue/pink color scheme, and the profiles with d-Nef are denoted by a black/red/yellow color scheme.

Figure 3

Change in area upon adsorption of 6His-d-Nef to a monolayer of 35% DPPC + 65% DSIDA/Cu²⁺ with no reducing agent present (●) and with 5 mM DTT in the buffer (□). In each case, time 0 corresponds to the time at which adsorption was first detected in the reflectivity data.

Figure 4

(a) The NR curve resulting from the extended form of Nef in Fig. 1 (dashed line) along with the experimental data and best fit (solid line with symbols). (b) SLD profile calculated for the extended form of Nef smeared to 10 Å resolution along with the best-fit profile.

Figure 5

Heavy atom distributions for subpopulations of simulated conformations binned according to the distance between the center of the globular domain and the center of the 6His tag. The center of the His tag is at 0 Å.

Figure 6

(a) Four conformations yielding good agreement with the experimental data from the ensemble of simulated structures. These conformations are representative of the small fraction of solutions that could be found to match the experimental data. The core domain is shown in blue, the flexible regions in green, and the rigid regions, including the His tag, in yellow. (b) The dashed line shows the calculated NR curve resulting from the conformation shown at upper left in a along with the experimental data and the best fit. (c) Calculated SLD profile from the conformation in a compared with the best-fit profile. The calculated profile was smeared to 10 Å resolution. The other conformations in a show comparable agreement with the experimental data. The difference in the calculated and experimental reflectivity curves at q_z = 0.04 Å⁻¹ in b is due to the presence of a weak tail in the experimental profile.