Extended Data Fig. 3: Purification of the CD4-gp120-CCR5 complex.
From: Structural basis of coreceptor recognition by HIV-1 envelope spike
a, Schematic of expression constructs for HIV-1 gp120, human CCR5 and CD4. Segments of gp120 are designated as follows: C1–C5, conserved regions 1–5; V1–V5, variable regions 1–5; and His-tag, a six-histidine tag. Tree-like symbols represent glycans. Abbreviations used for segments of CCR5 are: N, N terminus; TM1–TM7, transmembrane helices 1–7; ECL1–ECL3, extracellular loops 1–3; ICL1–ICL3, intracellular loops 1–3; and CT, cytoplasmic tail. For CD4, the following abbreviations are used: D1–D4, immunoglobulin (Ig) domains 1–4; and strep tag, a purification tag. The transmembrane segment (TM) and cytoplasmic tail (CT) in grey are truncated in the expression construct. b, Unmodified human CCR5 in complex with HIV-1 gp120 and four-domain CD4 was purified by the following steps. (1) Complex formation: HIV-1 gp120 (light blue) and strep-tagged, four-domain CD4 (green) were incubated with CCR5 (magenta)-expressing cells to allow formation of the CD4–gp120–CCR5 complex on cell surfaces. (2) Strep-tag purification: the CCR5 complex and some of the CD4–gp120 complex were captured to strep-tactin resin via the strep-tagged CD4 (strep tag in purple). They were eluted by d-desthiobiotin under mild conditions. (3) Negative selection by an anti-V3 antibody to remove the CD4–gp120 complex. The CCR5 complex was further purified by size-exclusion chromatography. c, The purified CD4–gp120–CCR5 complex was resolved by gel-filtration chromatography on a Superose 6 column in the presence of the detergent LMNG. The molecular-mass standards include thyoglobulin (670 kDa), ferritin (440 kDa), γ-globulin (158 kDa) and ovalbumin (44 kDa). The expected size of the CCR5 complex is ~310 kDa (120 kDa for gp120, 50 kDa for four-domain CD4, 40 kDa for CCR5 and ~100 kDa for LMNG micelle). Peak fractions were analysed by Coomassie-stained SDS–PAGE (lanes 1–3). Labelled bands were confirmed by western blot and protein sequencing. The experiment was repeated independently at least 15 times with similar results.
