Author response: Germline VRC01 antibody recognition of a modified clade C HIV-1 envelope trimer and a glycosylated HIV-1 gp120 core (2018)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract VRC01 broadly neutralizing antibodies (bnAbs) target the CD4-binding site (CD4BS) of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env). Unlike mature antibodies, corresponding VRC01 germline precursors poorly bind to Env. Immunogen design has mostly relied on glycan removal from trimeric Env constructs and has had limited success in eliciting mature VRC01 bnAbs. To better understand elicitation of such bnAbs, we characterized the inferred germline precursor of VRC01 in complex with a modified trimeric 426c Env by cryo-electron microscopy and a 426c gp120 core by X-ray crystallography, biolayer interferometry, immunoprecipitation, and glycoproteomics. Our results show VRC01 germline antibodies interacted with a wild-type 426c core lacking variable loops 1–3 in the presence and absence of a glycan at position Asn276, with the latter form binding with higher affinity than the former. Interactions in the presence of an Asn276 oligosaccharide could be enhanced upon carbohydrate shortening, which should be considered for immunogen design. https://doi.org/10.7554/eLife.37688.001 Introduction Despite the tremendous impact of HIV-1 on human health, no efficacious HIV-1 vaccine currently exists. The HIV-1 envelope (Env) glycoprotein is a class-I fusion protein responsible for host attachment and fusion of the viral and cellular membranes (Dalgleish et al., 1984). Following expression, Env trimerizes and undergoes furin-mediated cleavage to yield non-covalent gp120-gp41 pre-fusion trimers anchored in the viral membrane (Haim et al., 2013). As the sole target of neutralizing antibodies, Env is the focus of intense interest for current vaccine design initiatives. However, HIV-1 Env relies on multiple mechanisms of immune evasion – including dense glycosylation, sequence variation, conformational masking, and presentation of decoy epitopes (Burton and Mascola, 2015; Cuevas et al., 2015; Jardine et al., 2015; Kwong et al., 2002; Wei et al., 2003; Zhou et al., 2017). For these reasons, development of an Env-based vaccine capable of eliciting broadly neutralizing antibodies (bnAbs) has proven challenging. The VRC01-class of bnAbs is of particular interest for HIV-1 vaccine development due to the exceptional potency and breadth of several of its well-characterized members (Huang et al., 2016; Zhou et al., 2015). These bnAbs derive from the VH1-2 variable heavy chain gene (Scheid et al., 2011; Wu et al., 2011), have been isolated from multiple HIV-1-infected patients (Zhou et al., 2013), and putative non-mutated precursors have been identified in naïve individuals (Jardine et al., 2016a). VRC01-class bnAbs are characterized by an unusually short five amino-acid light chain complementary-determining region (CDR) L3 loop (Zhou et al., 2015) and much higher levels of somatic hyper-mutation than antibodies targeting other pathogens (Wu et al., 2015). They bind the CD4-binding site (CD4BS) in a way reminiscent of the interactions formed with the viral receptor CD4, making extensive CDRH2-mediated contacts while also exhibiting multiple amino acid alterations in the CDRL1 loop relative to germline precursors (Wu et al., 2015; Zhou et al., 2013). Although N-linked glycosylation sites (NLGSs) that surround the CD4BS sterically limit recognition by bnAbs (Zhou et al., 2017), particularly those present at position Asn276 in Loop D and along the V5 loop, mature VRC01 bnAbs overcome this barrier and potently neutralize numerous HIV-1 viral clades (Zhou et al., 2017; Huang et al., 2016; Stewart-Jones et al., 2016; Wu et al., 2015). In contrast, the inferred germline precursors of VRC01-class bnAbs lack detectable binding to trimeric Env constructs harboring glycans at these locations (Jardine et al., 2013; McGuire et al., 2016; McGuire et al., 2013; Medina-Ramírez et al., 2017b; Stamatatos et al., 2017). Whereas most recombinant trimeric Env antigens do not bind germline precursors of VRC01-class bnAbs, a few recently designed constructs have been shown to bind and activate this specific class of B cell receptors (BCRs) (Jardine et al., 2013; McGuire et al., 2016; McGuire et al., 2013; Medina-Ramírez et al., 2017a). We previously engineered a trimeric HIV-1 Env protein able to bind most VRC01-class precursors (McGuire et al., 2013). This construct was a trimeric gp140 protein derived from the clade C 426c virus and lacked variable loops 1, 2, and 3, along with the putative NLGSs at positions Asn276 (loop D), Asn460, and Asn463 (V5 loop) (McGuire et al., 2016). Other constructs have also been engineered to engage the inferred precursors of VRC01-class bnAbs, all of which harbored mutations eliminating the NLGSs in loop D (at position Asn276) and in the V5 loop (Briney et al., 2016; Jardine et al., 2013; McGuire et al., 2016; McGuire et al., 2013; Medina-Ramírez et al., 2017a; Tian et al., 2016). Additionally, a gp120 core derived from the 01dG5 clade virus, which naturally lacks a glycan at position Asn276, was also shown to engage the inferred germline precursor of the VRC01 antibody (VRC01GL) (Wu et al., 2015). Although such glycan-depleted ‘germline-targeting’ immunogens activate B cells expressing germline VRC01-class BCRs in vivo (Briney et al., 2016; Dosenovic et al., 2015; Tian et al., 2016), they largely fail to elicit mature antibodies capable of bypassing the restrictions imposed by the glycan at position Asn276 (Zhou et al., 2017). However, a recent study demonstrated the successful elicitation of CD4BS-targeted antibodies, distinct from the VRC01 lineage, upon immunization of rabbits with an engineered clade C Env trimer (Dubrovskaya et al., 2017). To better understand the potential avenues of elicitation of VRC01-class bnAbs, we structurally characterized complexes between VRC01GL and two clade C Env constructs using a combination of cryo-electron microscopy (cryoEM) and X-ray crystallography. One of the constructs is a soluble trimeric 426c SOSIP with three NLGSs removed at positions Asn276, Asn460, and Asn463, and is based on our prior work (McGuire et al., 2016; McGuire et al., 2013). The second construct is a monomeric 426c core containing all wild-type NLGSs (including those at positions Asn276, Asn460, and Asn463), but lacks variable loops 1, 2, and 3. The 426c strain naturally lacks NLGSs surrounding the CD4BS at positions Asn234 and Asn362(363), which are present in other clades. Our structural analysis revealed that the absence of these glycans leads to a reduction of local oligosaccharide density in the vicinity of the NLGS at position Asn276. Integrating this data with biolayer interferometry (BLI) assays and glycoproteomics, we demonstrate here that VRC01GL could bind to a 426c core construct in the presence of all naturally occurring NLGSs surrounding the CD4BS, including the NLGS at position Asn276 and with its associated glycan. We also show the affinity of VRC01GL for the 426c core could be modulated by altering protein expression conditions to enrich for longer glycans, and also by shortening glycans via endoglycosidase treatment. These results suggest that priming of VRC01-class bnAbs may be possible using an HIV-1 gp120 derivative containing a glycan at position Asn276. Consequently, future epitope-based vaccine design strategies utilizing a 426c core preserving all NLGSs may be a promising route for guiding elicitation of VRC01-class bnAbs. Results CryoEM structure of VRC01GL in complex with a modified 426c HIV-1 SOSIP glycoprotein trimer Based on the known enhanced ability of VRC01GL (and related germline antibodies) to bind 426c constructs lacking putative NLGSs at positions Asn276, Asn460, and Asn463 (McGuire et al., 2013), and the lack of detectable binding to 426c DS-SOSIP (Figure 1A), we engineered a modified 426c DS-SOSIP trimer recapitulating the aforementioned glycan depletion mutations for structural analysis. This construct harbors the S278A, T462A and T465A mutations, abolishing the corresponding NLGSs and enabling binding to VRC01GL (Figure 1B, Figure 1—figure supplement 1). It also contains the SOSIP (Sanders et al., 2002) and the 201C-433C (DS) mutations (Kwon et al., 2015) and is a chimera of 426c gp120 and BG505 gp41 (Joyce et al., 2017). This glycan-depleted protein construct is henceforth referred to as 426c DS-SOSIP D3 (Figure 1—figure supplement 1). The VRC01GL construct comprises the germline VH gene reverted sequences of VH1-2*02, which includes CDRH1 and CDRH2 along with the mature CDRH3 of VRC01 and the germline VK3-11 with the mature CDRL3 of VRC01 (Figure 1—figure supplement 2). Initial complex formation was evaluated using negative-staining EM, which revealed sub-stoichiometric binding of VRC01GL Fab to 426c DS-SOSIP D3 (Figure 1—figure supplement 3A). The VRC01GL Fab appeared to have a much lower affinity for 426c DS-SOSIP D3, compared to the VRC01GL IgG (the latter bound with an apparent equilibrium dissociation constant of 43 nM (Figure 1B), neglecting the effect of avidity). We next attempted to enhance binding of VRC01GL Fab to 426c DS-SOSIP D3 by utilizing a mild glutaraldehyde cross-linking strategy. As expected, we observed significantly increased saturation of 426c DS-SOSIP D3 trimers by VRC01GL Fab, indicating covalent tethering following initial engagement of VRC01GL to the CD4BS was a suitable approach to enrich for and study VRC01GL-bound complexes (Figure 1—figure supplement 3A). We therefore engineered a disulfide bond between G459Cgp120 (426c DS-SOSIP D3†) and the VRC01GL heavy chain A60C (denoted as 426c DS-SOSIP D3†-VRC01GL) (Figure 1—figure supplement 1 and Figure 1—figure supplement 2). This strategy was previously employed to enhance binding of VRC01MAT to SOSIP trimers without altering interface contacts (Stewart-Jones et al., 2016). Using this method, we purified an enriched fraction of Fab-bound trimers and used this sample for structural characterization (Figure 1C, Figure 1—figure supplement 3A–D). This strategy led us to determine two cryoEM reconstructions of the 426c DS-SOSIP D3†-VRC01GL complex (Figure 1D–E, Figure 1—figure supplement 3E–H, Figure 1—figure supplement 4A–D, Figure 1—figure supplement 5): one with three bound Fabs at 3.8 Å resolution (Figure 1D, Table 1, Figure 1—figure supplement 3E, Figure 1—figure supplement 4A–B), and one with two bound Fabs at 4.8 Å resolution (Figure 1E, Figure 1—figure supplement 3H, Figure 1—figure supplement 4C–D). Figure 1 with 6 supplements see all Download asset Open asset Structural characterization of the 426c DS-SOSIP D3†-VRC01GL complex. (A–B) BLI binding data of immobilized VRC01GL IgGs binding to WT 426c DS-SOSIP (A) or 426c DS-SOSIP D3 trimers. The concentrations of 426c DS-SOSIP trimers injected are indicated on each panel. Fit curves are colored as black dotted lines. A KD could not be determined in (A) due to the weak responses observed. The vertical dotted lines indicate the transition between association and dissociation phases. (C) Size-exclusion chromatogram of the purified 426c DS-SOSIP D3†-VRC01GL complex used for cryoEM structure determination. The pooled fractions used for cryoEM are highlighted in light blue. (D) Two orthogonal views of the 3.8 Å cryoEM reconstruction sharpened with a B-factor of −250 Å2 whereas the glycan density is shown unsharpened. (E) Two orthogonal views of the asymmetric 4.8 Å reconstruction with two bound Fabs. (F) Surface representation of the 426c SOSIP trimer highlighting differences in glycosylation compared to the BG505 SOSIP. Glycans not present in 426c are colored light-gray and outlined. Glycans present in the 426c strain but removed by mutation from the 426c DS-SOSIP D3† construct are colored magenta and outlined. The gp120 surface buried at the interface with VRC01GL is indicated as a dotted outline and is colored yellow. (G) Comparison of the gp120 bridging sheet conformation when VRC01GL-class Fabs are bound to either 426c DS-SOSIP D3† trimer (Top-left) or a previously solved 426c gp120 core lacking selected NLGSs, such as the Asn276 NLGS (PDB: 5IGX) (Top-right). Comparisons of β20β21 loop conformations of each complex are shown below corresponding top panels. (H) Comparison of glycan density and position between VRC01GL-bound and VRC01GL-free protomers in the asymmetric cryoEM reconstruction shown in (E). (Top) Asn197 and Asn386 glycan density is stronger for protomers bound to VRC01GL Fab than for the gp120 protomer not bound to VRC01GL (Bottom). In panels D-H, gp120 protomers are shown in blue, gp41 in red, N-linked glycans in green and VRC01GL in dark and light yellow for the heavy and light chains, respectively. https://doi.org/10.7554/eLife.37688.002 Table 1 CryoEM data collection, refinement, and model validation statistics. https://doi.org/10.7554/eLife.37688.009 ParameterValueData CollectionNo. of Micrographs1993No. of Particles134,443Pixel size, Å1.36Defocus range, μM2.0–3.5Voltage, kV300Dose Rate, counts/pix/sec8Electron dose, e-/Å243RefinementResolution, Å3.8Map-sharpening B factor, Å2−230Model validation (3 Fab structure)Favored rotamers, %98.36%Poor rotamers, %0.30%Ramachandran outliers, %0.13%Clash Score0.99Molprobity score EM ringer score1.02 1.97 Similarly to what was reported for the B41 SOSIP trimer (Ozorowski et al., 2017), the 426c DS-SOSIP D3†-VRC01GL V1/V2 apex is closed under cryoEM conditions (Figure 1D,E,F) whereas it appears open in the conditions we used for negative-staining sample preparation (Ozorowski et al., 2017) (Figure 1—figure supplement 3B). We note that the closed 426c DS-SOSIP D3†-VRC01GL trimeric Env conformation observed in cryoEM lacks a formed gp120 bridging sheet, which is reminiscent of other closed SOSIP trimer structures (Figure 1G) (Julien et al., 2015; Lyumkis et al., 2013b; Pancera et al., 2014; Stewart-Jones et al., 2016). VRC01GL-class antibodies have recently been shown to also bind to core gp120 constructs in the presence of a bridging sheet (Scharf et al., 2016). Our data reveal that VRC01GL could also bind a prefusion closed conformation, which had previously only been reported for its mature counterpart, VRC01MAT (Stewart-Jones et al., 2016) (Figure 1G). Structural analysis of the region surrounding the CD4BS in 426c DS-SOSIP D3 Removal of CD4BS-surrounding carbohydrates has been shown to enhance binding of CD4BS-targeted germline VRC01-class antibodies and to increase the antigenicity of this region (McGuire et al., 2016; McGuire et al., 2013; Stamatatos et al., 2017; Zhou et al., 2017). Our structural analysis reveals that the 426c DS-SOSIP naturally lacks an NLGS at position Asn234 near the CD4BS, which is otherwise conserved in 80% of known circulating HIV-1 strains (Crooks et al., 2015). Instead, 426c features a glycan at Asn230 that is more remote from the VRC01 epitope than glycan Asn234 (Jardine et al., 2016b) (Figure 1F). The oligosaccharide at position Asn230 appears to be highly dynamic, since only the two proximal N-acetyl-glucosamine (GlcNAc) moieties are resolved in the reconstruction (Figure 1—figure supplement 6) and does not interact with VRC01GL or other glycans in the complex. Previous structural characterization of clades A and G SOSIP trimers established that glycans at positions Asn276 and Asn234 are in close proximity to each other and likely restrain each others’ conformational freedom (Jardine et al., 2016b; Stewart-Jones et al., 2016; Zhou et al., 2017). The absence of glycan Asn234 in 426c gp120 reduces local carbohydrate crowding near the CD4BS which could increase accessibility of this neutralization supersite (Stewart-Jones et al., 2016) and lead to altered local glycan processing (Behrens et al., 2018; Bonomelli et al., 2011). The 426c DS-SOSIP also lacks an NLGS at position Asn362(363), which is present in 42% of strains deposited in the HIV database (Gaschen et al., 2001) (Figure 1F). This oligosaccharide is located distally from the viral membrane side of the SOSIP trimer (Figure 1F) and is sandwiched between the VRC01MAT heavy chain and glycan Asn386 in the structure of VRC01MAT bound to the JR-FL SOSIP trimer (clade B) (Stewart-Jones et al., 2016). Analysis of the asymmetric 426c DS-SOSIP D3†-VRC01GL structure, comprising two Fabs, revealed that Fab-bound protomers feature slightly better-resolved density for glycan Asn386 than the free protomer when visualized at the same contour level (Figure 1H). These observations suggest that VRC01GL may stabilize the Asn386 glycan either through reduction of its conformational freedom and/or via direct interactions with the Fab framework region. The absence of glycan Asn362 or other topologically equivalent oligosaccharides in the 426c gp120 sequence likely contributes to increased accessibility of the CD4BS to VRC01GL-class bnAbs due to the close proximity of this glycan to the epitope (Stewart-Jones et al., 2016) (Figure 1F). Similarly to what is observed in available VRC01MAT/SOSIP complex structures (Stewart-Jones et al., 2016), glycan Asn197 density is also strongest when bound to VRC01GL, but appears weaker in the unbound protomer (Figure 1H), again indicating either Fab-induced stabilization or restriction of movement. The position of glycan Asn197 differs substantially between available structures of monomeric gp120 constructs bound to VRC01GL-class Fabs and the VRC01GL-bound SOSIP trimer reported here (Figure 1G) (Scharf et al., 2016). This variation in Asn197 positioning is guided by the formation of the gp120 bridging sheet in monomeric gp120, which would otherwise only form following CD4 receptor binding in the context of trimeric Env (Figure 1G) (Kwon et al., 2012; Zhou et al., 2010). This conformational difference includes the β20/β21 loop, whose orientation in the 426c DS-SOSIP D3†-VRC01GL complex differs relative to crystal structures of VRC01GL-class antibodies in complex with monomeric gp120 (Figure 1G) (Scharf et al., 2016). Although the β20/β21 loop is close to the VRC01 paratope, VRC01MAT was reported to have minimal preference in the conformation of the bridging sheet or β20/β21 region, as 87% of its contact surface area includes the conformationally invariant outer domain of gp120 (Zhou et al., 2010). Whether or not the conformation of the β20/β21 region directly impacts germline VRC01-class antibody binding affinities remains unclear. However, VRC01GL in complex with gp120 constructs lacking this domain have been determined (eOD-GT6 and eOD-GT8), demonstrating that these germline mAbs do not strictly require this region for CD4BS recognition when glycans surrounding the CD4BS are also removed (Jardine et al., 2013). Wild-type V5 loop NLGSs of the 426c core did not hinder binding to VRC01GL Fabs One of the mechanisms by which HIV-1 Env has evolved to avoid detection by the progenitors of VRC01-class bnAbs is by selection of V5 loop NLGSs (Huang et al., 2016; Li et al., 2011; Zhou et al., 2010) which sterically limit access to the CD4BS. The observation that VRC01GL may accommodate carbohydrates surrounding the CD4BS in our 426c DS-SOSIP D3†-VRC01GL cryoEM structure prompted us to assess the effect on binding of the two V5 loop putative NLGSs mutated in 426c DS-SOSIP D3†-VRC01GL. With 426c Env trimers, we previously found that VRC01GL binding could be detected following removal of glycan Asn276, and was further enhanced following removal of wild-type NLGSs at positions Asn460 and Asn463 (McGuire et al., 2013). We also demonstrated that removing the V1/V2, and V3 loops in gp140 Env trimers further increased binding of multiple VRC01-class germline antibodies relative to trimers only containing glycan-depleting mutations (McGuire et al., 2014; McGuire et al., 2016). However, the effects of V1/V2 and V3 loop deletion on VRC01GL binding to 426c core constructs in the presence of glycans remains unclear. Here we reintroduced the two NLGSs at positions Asn460 and Asn463 (in the V5 loop) and assessed their individual and cumulative effects on VRC01GL engagement of the 426c core construct comprised of gp120 residues 44 to 492, and lacking the V1/V2 and V3 variable loops (Kwon et al., 2012). Expanding on our prior work (McGuire et al., 2016), the following four 426c core glycan-deleted combinations were tested using HEK293F-expressed protein constructs: S278A/T462A/T465A, S278A/T465A, S278A/T462A, and S278A (Figure 1—figure supplement 1, Figure 2A–D, Supplementary file 1). Figure 2 with 1 supplement see all Download asset Open asset 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), binding and second binding The 426c core glycoforms that did not bind VRC01GL following three binding The binding to 426c core fractions following of the sample and three The binding a of the aforementioned three and bound from the VRC01GL affinity and this associated with their corresponding individual from each with the the signal Reintroduction of the NLGS at position Asn460 in the 426c core had no detectable impact on VRC01GL binding affinity the of a putative carbohydrate at position Asn460 with the bound VRC01GL Fab et al., (Figure Supplementary file 1). our work removing NLGSs at positions Asn460 and Asn463 via the mutations had only a impact on VRC01GL engagement to trimeric 426c constructs compared to the increase in binding observed following removal of the Asn276 NLGS (McGuire et al., 2016; McGuire et al., 2013). To better understand the of these observations with the monomeric 426c core we engineered a 426c core†-VRC01GL complex containing all wild-type NLGSs (426c We these using which lack and are to complex N-linked carbohydrates and We determined its crystal structure at Å resolution endoglycosidase to et al., 2012; and 2010) (Figure Table 2). Despite harboring an no glycan density could be resolved at position Asn460 in either of the two of the asymmetric Instead, the Asn460 side chain is to the backbone amide and carbonyl groups of the VRC01GL et al., In of this we detected only Asn460 when of this sample with to (Figure Figure supplement only levels of glycosylation were detected at Asn460 by LC-MS/MS analysis of 426c core the (Figure supplement Additionally, we VRC01GL-based experiments utilizing a VRC01GL affinity and the same 426c core Semi-quantitative LC-MS/MS of the 426c gp120 core from fractions that did not bind to VRC01GL and those that did revealed no difference in glycan of the Asn460 NLGS (Figure This indicated this is in 426c core and its impact on VRC01GL The of glycan Asn460 with VRC01GL and the absence of a resolved proximal in the crystal structure of 426c core†-VRC01GL also a likely preference for the Asn460 of the 426c gp120 for binding (Figure Table 2 data and 426c or We observed that of the Asn463 NLGS or also did not in a reduction in VRC01GL binding relative to the 426c S278A/T462A/T465A core (Figure Supplementary file 1). This was as V5 glycosylation of Env trimers containing all variable loops have been reported to VRC01GL recognition of the CD4BS (Huang et al., 2016; Li et al., 2011; McGuire et al., 2013; Zhou et al., 2010). We observed density for the proximal linked to Asn463 in one of the two of the asymmetric of the 426c core†-VRC01GL crystal structure and the presence of this using LC-MS/MS (Figure VRC01GL-based IP experiments by LC-MS/MS these structural observations by an Asn463 glycosylation which was between and fractions (Figure This our BLI data the Asn463 glycan does not hinder VRC01GL binding in the context of the 426c core and that this site is VRC01GL Fab bound to the Asn276 426c core construct A of VRC01-class is the shortening of the CDRL1 loop and/or the of of which have been to of the Asn276 glycan near the CD4BS (Jardine et al., et al., 2016; Wu et al., 2015; Zhou et al., 2010). Although VRC01MAT was shown to bind to the trimeric Env CD4BS in the presence of glycan Asn276, its removal significantly increased binding affinity and neutralization potency (Jardine et al., 2013; McGuire et al., 2016; McGuire et al., 2013; Medina-Ramírez et al., 2017a; Stamatatos et al., 2017). Removal of the Asn276 NLGS from trimeric SOSIP constructs by either or mutations significantly enhanced the antigenicity of the VRC01 epitope (McGuire et al., 2016; McGuire et al., 2013). However, when such glycan-depleted trimeric Env constructs were used as the antibodies they to overcome the glycan present at position Asn276 of wild-type (Briney et al., 2016; Dosenovic et al., 2015). Removal of glycan Asn276 through VRC01GL indicating this amino acid residue was for binding (McGuire et al., 2016). These observations suggest that initial engagement of VRC01-class precursors in individuals with an at position and may also be at in the presence of a glycan at this NLGS (Scharf et al., 2016). the glycan of the 426c the deletion of variable loops 1, 2, and in our 426c core and the minimal impact V5 loop NLGSs had on VRC01GL we tested the 426c core construct could interact with VRC01GL in the presence of the Asn276 NLGS (Figure 1—figure supplement 1). BLI analysis revealed VRC01GL bound to the and 426c with equilibrium dissociation constants of and (Figure The crystal structure of the disulfide engineered 426c