Thus, the structure of HIV-1 Env has been studied extensively. gp120. Therefore, we identified the possibility of the involvement of a second gp120-CD4 interaction interface during viral entry, and also provided a reasonable explanation for the broad activity of neutralizing antibody ibalizumab. Initially synthesized as a heavily glycosylated gp160 precursor1, the human immunodeficiency virus type I (HIV-1) envelope glycoprotein (Env) is cleaved enzymatically by cellular proteases before binding to the cell surface as two MELK-8a hydrochloride non-covalently associated subunits: gp120 and gp41. Gp120 is the receptor binding subunit2 that interacts subsequently with the primary receptor CD4 and a co-receptor (either CCR5 or CXCR4) and undergoes a series of entry-related conformational changes. Gp41 is responsible for the fusion of the viral and cellular membranes3 by rearranging from a four-helix collar consisting of discontinuous helices containing N- and C-terminal heptad repeats to a thermodynamically stable six-helix bundle structure4,5,6,7. In addition to its critical role in the life cycle of HIV-1, Env is also the sole target on the surface of HIV-1 virions for antibody-mediated neutralization4,8. Thus, the structure of HIV-1 Env has been studied extensively. Since Kwong and colleagues reported the first MELK-8a hydrochloride crystal structure of a monomeric HIV-1 gp120 core nineteen years ago9, a wide variety of structures of gp120 and its outer domain have been determined using soluble two-domain CD4 and co-receptor mimics, as well as with numerous antibodies that target the CD4 binding site (CD4bs) or the outer domain of gp12010,11,12,13. Furthermore, some crystal structures of the unliganded gp120 core have been determined successfully, although it seems that the antibody alone or in combination with CD4 is essential for crystallization of gp120, by MELK-8a hydrochloride acting as stabilizing agents and crystallizing chaperones14. Recently, structures of pre-fusion full-length gp120 from an engineered, cleaved HIV-1 Env trimer, termed BG505 SOSIP.664 gp140, and a native, cleaved HIV-1 Env trimer referred to as EnvCT, have been resolved at relatively high resolution15,16. Furthermore, a gp140 trimer with a liganded CD4 complex has also been reported17. However, to the best of our knowledge, the structure of a binary complex of HIV-1 gp120 and soluble CD4 has not been reported. HIV-1 CRF07_BC is a recombinant form that mainly circulates in Northwest China18. To define precisely the conformational change of HIV-1 CRF07_BC gp120 induced only by the engagement of soluble CD4, ruling out an influence of co-receptor mimics, we solved the crystal structure of the HIV-1 gp120 core with an extended stem of variable loop MELK-8a hydrochloride 3 (V3)19,20, which is designated as coreV3e, in complex with the N-terminal two domains (D1D2) of CD49,21 (hereafter referred to as CD4D1D2). Compared with the crystal structure of pre-fusion gp120 in the soluble BG505 SOSIP.664 gp140 trimer, our CD4-bound gp120 coreV3e structure showed certain small differences22. Unexpectedly, a novel non-canonical binding interface between HIV-1 gp120 and CD4 was found, for the first time, based on crystal packing. Residual mutagenesis on this interface was harmful to Env-mediated cell-cell fusion and pseudotyped HIV-1 infection, indicating that this interface is important for HIV-1 fusion and entry. Moreover, based on the structural similarity with the ibalizumab-CD4 complex, the non-canonical interface may provide clues for the broad and potent antiviral activity of ibalizumab23. Results Overall structure of CRF07_BC gp120 coreV3e and CD4D1D2 We attempted to obtain crystals of the binary complex comprising HIV-1 gp120 and D1D2 of human CD4 using a series of gp120 crystallization variants, which contained different truncations of the first three variable loops MELK-8a hydrochloride (V1CV3), and deletions of the N and C termini. However, only the gp120 variant containing 10 additional Rabbit Polyclonal to NFAT5/TonEBP (phospho-Ser155) residues in the stem of the V3 loop could produce crystals of the complex. Monoclinic crystals of the gp120 core with an extended V3 stem (coreV3e) from HIV-1 CRF07_BC in complex with two-domain CD4 (Compact disc4D1D2) were attained successfully and employed for data collection. The complicated structure was resolved at 2.47?? (Desk S1; Fig. 1A). Open up in another window Amount 1 Overall framework of CRF07_BC gp120 coreV3e and Compact disc4D1D2 complicated.(A) The complicated of gp120 coreV3e/Compact disc4D1D2. Phe43 from Compact disc4, the vital residue in preserving the overall framework of CRF07_BC gp120 coreV3e and Compact disc4D1D2 complicated, was proven as blue sticks. The internal domain, external domain, and bridging sheet of gp120 had been provided as cyan, magenta, and yellowish ribbons, respectively. Compact disc4D1D2 is proven in green ribbon. (B) Aspect view from the gp120 coreV3e, shaded such as (A). The entire structure of the binary complex is comparable to reported counterparts in CD4-containing ternary structures previously. Needlessly to say, the gp120 primary includes three domains: the external domain, the internal domain,.