Conformation of the Dileucine‐Based Sorting Motif in HIV‐1 Nef Revealed by Intermolecular Domain Assembly

Abstract

The human immunodeficiency virus 1 (HIV-1) Nef protein is a pathogenicity factor required for effective progression to AIDS, which modulates host cell signaling pathways and T-cell receptor internalization. We have determined the crystal structure of Nef, allele SF2, in complex with an engineered SH3 domain of human Hck showing unnaturally tight binding and inhibitory potential toward Nef. This complex provides the most complete Nef structure described today, and explains the structural basis of the high affinity of this interaction. Intriguingly, the 33-residue C-terminal flexible loop is resolved in the structure by its interactions with a highly conserved hydrophobic groove on the core domain of an adjacent Nef molecule. The loop mediates the interaction of Nef with the cellular adaptor protein machinery for the stimulated internalization of surface receptors. The endocytic dileucine-based sorting motif is exposed at the tip of the acidic loop, giving the myristoylated Nef protein a distinctly dipolar character. The intermolecular domain assembly of Nef provides insights into a possible regulation mechanism for cargo trafficking.

HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck

“…Note that SH3-HART Phe97 (F97; green spheres) occupies the position of I96 in the wild-type crystal structure. Recent crystallographic analysis of a related high affinity Nef-SH3 interface supports this model [29]. …”

“…Because NaPP1 binds to the Hck-TA active site in a specific location, it serves as a chemical probe for conformational changes that may occur in response to Nef binding. In addition to the gatekeeper mutation, we modified the SH3 domain to enhance interaction with Nef [29,30]. This modification enabled stable association of Hck with Nef in solution-based kinase assays, thus mimicking the stable association that is likely to occur between Hck and Nef at cellular membranes [17].…”

Interaction with the Src Homology (SH3-SH2) Region of the Src-family Kinase Hck Structures the HIV-1 Nef Dimer for Kinase Activation and Effector Recruitment

“…A precedent for intercomplex Nef⅐SH3 contacts comes from the work of Horenkamp et al (38), in which the x-ray crystal structure of the Nef core domain was determined in complex with an engineered high affinity Hck SH3 domain. This structure, referred to as Nef⅐Hck SH3 B6 , also crystallized as a dimer of complexes with the Nef C-terminal loop making contacts with a hydrophobic crevice adjacent to the SH3 RT-loop recognition site.…”

Link between Primate Lentiviral Coreceptor Usage and Nef Function

“…We found that residues I123 and L146 are located on two central α helices, which are located within the core domain of Nef (Figure 6A). These two helices are kept in place by the two gatekeeper residues F122 and Y145 (F94 and W117 in HIV- 1 Nef), which divide the intermediate groove into a recognition site for SH3 domains and a binding site for endocytic sorting motifs (Horenkamp et al, 2011). I123 and L146 oppose each other on these two helices at a minimal distance of 4.2 Å but are not directly surface accessible (Figures 6B and 6C).…”