Abstract
The N-terminal matrix (MA) domain of the HIV-1 Gag protein is responsible for binding to the plasma membrane of host cells during viral assembly. The putative membrane-binding interface of MA was previously mapped by means of mutagenesis and analysis of its trimeric crystal structure. However, the orientation of MA on membranes has not been directly determined by experimental measurements. We present neutron reflectivity measurements that resolve the one-dimensional scattering length density profile of MA bound to a biomimetic of the native viral membrane. A molecular refinement procedure was developed using atomic structures of MA to determine the orientation of the protein on the membrane. The orientation defines a lipid-binding interface consistent with previous mutagenesis results. The MA protein maintains this orientation without the presence of a myristate group, driven only by electrostatic interactions. Furthermore, MA is found to penetrate the membrane headgroup region peripherally such that only the side chains of specific Lys and Arg residues interact with the surface. The results suggest that electrostatic interactions are sufficient to favorably orient MA on viral membrane mimics. The spatial determination of the membrane-bound protein demonstrates the ability of neutron reflectivity to discern orientation and penetration under physiologically relevant conditions.
Highlights
The association of peripheral membrane proteins to lipid bilayers is accomplished by a variety of molecular mechanisms, including the insertion of nonpolar components into the hydrophobic core of membranes, specific lipidbinding sites, and electrostatic interactions with charged membrane surfaces
A single anionic lipid membrane tethered to a gold surface was formed with surface coverage of >99% as determined by a slab model analysis of the reflectivity data (Fig. S2)
The initial question of whether ÀmyrMA without its hydrophobic anchor interacts with the charged tethered bilayer lipid membrane (tBLM) was immediately answered by the observation of a change in the measured reflectivity spectra
Summary
The association of peripheral membrane proteins to lipid bilayers is accomplished by a variety of molecular mechanisms, including the insertion of nonpolar components into the hydrophobic core of membranes, specific lipidbinding sites, and electrostatic interactions with charged membrane surfaces. The conformation and spatial orientation of these peripheral proteins with respect to the membrane surface are important for their function; it can be difficult to experimentally identify the membraneprotein interface from solution structures. Techniques such as electron spin resonance, x-ray reflectivity, fluorescence, and solid-state NMR have been developed to study such systems [1,2,3]. A bipartite mechanism is implicated in both membrane association and selectivity for the plasma membrane. This mechanism includes a hydrophobic myristate anchor that is cotransla-
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