AP-2 alpha/sigma2 complex bound to HIV-1 Nef

Emma Mairson '17


I. Introduction

HIV-1 has a small genome that encodes several proteins including the accessory protein, Nef. Nef is considered a good drug target to inhibit the pathogenic effects of HIV-1. While it is not necessary for infection, the expression of Nef increases the effectiveness of HIV replication and disease progression. A primary function of Nef is downregulation the CD4 receptor on the surface of infected cells. CD4 is a transmembrane co-receptor that is used in the host-immune response and interaction with other cells. By downregulating CD4, Nef allows infected cells to evade immune response and prevents against superinfection, reinfection of an already infected cell, which increases probability of cell apoptosis.

II. Structure Overview

Nef is a small polymorphic protein, about 200-215 amino acids in length. Its N-terminus is myristoylated, meaning that myristic acid is attached to an N-terminal glycine residue. Nef has a folded core, flexible N-terminal, C-terminal, and central flexible loop . In the crystal structure the Nef/AP2 complex is displayed as a tetramer. However, in order to understand the residue interactions between the Nef and AP2, it is possible to focus on only one portion of the tetramer.

The ability of Nef to downregulate CD4 depends on three factors. The first is Nef myristoylation. When Nef is myristoylated, it can form a complex with the AP-2 complex, which goes on to interact with the cytosolic tail of the CD4 receptor. This tripartite interaction leads to a quick internalization of CD4, which is then delivered to lysosomes. The second category of factors important in Nef-mediated CD4 downregulation are specific residues in the flexible loop, including the (Leu 164 & Leu 165). The dileucine motif has hydrophobic interactions with residues in sigma 2. The third important factor is the (Asp 174 & Asp 175). Asp 174 and Asp 175 are involved in core-to-loop intracomplex interactions. For Asp 174 it is a hydrogen bond while for Asp 175 it is a salt bridge. The dileucine and diacidic motifs, as well as other residues in the Nef loop, are crucial in Nef:AP-2 binding.

Adaptor protein 2 (AP-2) is a heterotetramer composed of alpha, beta-2, mu, and sigma-2 subunits. However, Nef specifically interacts with the alpha-sigma-2 hemicomplex. In the alpha subunit, Lys 298 and Arg 341 are by the dileucine motif and are required for Nef binding and CD4 downregulation.

III. Nef

Except for the central loop, the Nef core contains five alpha helices, and a five-stranded beta sheet. The central loop consists of two additional helices, H4 and H5. It is the central loop that does the most interaction with the alpha and sigma-2 subunits.

A. H4 helix

The (Leu 164 & Leu 165) anchors the central loop into a pocket of sigma-2. The pocket is made up of hydrophobic residues. Nef Glu 160 also binds to basic residues in alpha (Arg 21) and sigma-2 (Arg 15). Glu154 also interacts with a basic patch in sigma-2 (Arg 61 and Arg 10). Interestingly, the conformation of H4 is specified by its interaction with alpha-sigma-2 and not other parts of Nef. This is because H4 has little to no interaction with the Nef core.

B. H5 and C-terminal

The H5 and C-terminal segment are sandwiched between alpha-sigma2 and the Nef core. H5 situates right against the beta sheet of the Nef core and allows the hydrophobic core loop to properly orient the dileucine motif. The C-terminal part of the loop has tight turns that insert charged and hydrophobic side chains into complementary interactions with both alpha and sigma-2. These tight turns are anchored at the ends by H5 and by the strand beta-5 of the core. The anchoring occurs through (1) ahydrogen bond between nef Asp 174 and a main-chain amide of (Asp 174 & Gln 104) and (2) a salt-bridge between Nef Asp175 of the loop and Nef Arg 134 of the core beta sheet.

C. H3

Gln 104 and Asp 108 all bind to a basic area on alpha (Lys 298, Lys 299), through hydrogen bonding and salt bridges, respectively. While the central loop interactions are most significant, the Nef core interactions involving H5, H3, and the C-terminal are also important.

IV. References

Ren, X., Park, S. Y., Bonifacino, J. S., and Hurley, J. H. (2014). How HIV-1 Nef hijacks the AP-2 clathrin adaptor to downregulate CD4. Elife, 3, e01754.

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