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. 2018 Nov 7;7:e37688. doi: 10.7554/eLife.37688

Figure 2. Reintroduction of V5 loop NLGSs does not hinder VRC01GL binding to the 426c core.

(A–D) BLI curves and the corresponding equilibrium dissociation constants for VRC01GL IgG binding to the S278A/T462A/T465A (A), S278A/T462A (B), S278A/T465A (C), and S278A (D) 426c core constructs lacking either one or several glycans in the V5 and D loops. The concentrations of 426c core injected and the color key is indicated on each panel. Fitted curves are colored as black dotted lines. The vertical dashed lines indicate the transition between association and dissociation phases. (E) Ribbon diagram of the 426c core†-VRC01GL complex crystal structure. gp120 is colored blue, VRC01GL Fab is colored yellow (heavy chain: dark yellow; light chain: light yellow), and resolved gp120 glycans are shown in surface representation and colored green. (F) Close-up view of the gp120 Asn460 contacts with the backbone carbonyl and amide groups of the light chain VRC01GL residue Ile02. (G) Close-up view of the (GlcNAc)1 at position Asn463 of gp120. Oligosaccharides are labeled by the corresponding Asn residue they are linked to. Hydrogen bonds are represented as dashed lines. (H–I) Semi-quantitative LC-MS/MS analysis of VRC01GL-based IP experiments depicting the relative signal intensities for identified Asn460 (H) and Asn463 (I) glycoforms in unbound (blue), first binding event (red), and second binding event (yellow) fractions. The ‘unbound’ material indicates 426c core glycoforms that did not bind VRC01GL well following three binding steps. The ‘first’ binding event corresponds to 426c core elution fractions following collection of the sample flow-through and three rigorous wash cycles. The ‘second’ binding event follows a rebinding of the aforementioned flow-through, performing three additional washes, and eluting any residual bound material from the VRC01GL affinity column and collecting this fraction. Colored dots associated with their corresponding histogram bars represent individual values extracted from each experimental replicate, with the bar itself representing the experimental mean signal fraction.

Figure 2.

Figure 2—figure supplement 1. Representative LC-MS/MS glycan identifications of 426c core constructs.

Figure 2—figure supplement 1.

The top-left ribbon diagram corresponds to the sample analyzed. The LC-MS/MS fragmentation pattern is indicated in the top-right inset. A graphical depiction of the Asn276 residue (dotted white circle) and its associated identified glycan (blue: N-acetyl glucosamine, green: mannose) are represented on the spectrum. Green peak labels correspond to precursor peptides with/without LC-MS/MS fragmentation occurring within the glycan. Red/orange peak labels represent identified x, y, and z fragments. Blue/teal peak labels highlight identified a, b, and c fragments. A) Schematic of possible LC-MS/MS peptide fragmentation patterns. The peptide N- and C-termini are labeled. Possible fragmentation positions are denoted as grey text, with x, y, and z fragments represented as red/orange labels and blue/teal fragments representing a, b, and c fragments. Modified R-groups are denoted in red text. (B–C) Representative LC-MS/MS spectra of detected V5 glycosylation profile of the 426c core†-VRC01GL (GnTI-/--expressed) (B) and a ligand-free 426c core containing a glycan at position Asn460 (GnTI-/--expressed) (C). D–J) Various additional representative LC-MS/MS spectra of an Asn276 glycopeptide containing a (GlcNAc)2-(Man)5 oligosaccharide from ligand-free 426c core (HEK293F-expressed) (D), an Asn276 glycopeptide of the 426c core†-VRC01GL complex with a detectable (GlcNAc)2-(Man)4 sugar (GnTI-/--expressed) (E), an Asn276 peptide from the 426c core†-VRC01GL complex that is unglycosylated at position Asn276 (GnTI-/--expressed) (F), a 426c core Asn276 glycopeptide containing a (GlcNAc)2-(Man)5 oligosaccharide (GnTI-/--expressed) (G), a 426c S278T core Asn276 glycopeptide containing a (GlcNAc)2-(Man)5 oligosaccharide (GnTI-/--expressed) (H), and an Asn276 peptide of a 426c S278T core that is unglycosylated at position Asn276 (GnTI-/--expressed) (I).