Locking the Elbow: Improved Antibody Fab Fragments as Chaperones for Structure Determination

Bailey, L.J.; Sheehy, Kimberly M.; Dominik, Pawel K.; Liang, Wenyuan; Rui, Huan; Clark, Michael; Jaskółowski, Mateusz; Kim, Yejoon; Deneka, Dawid; Tang, Wei-Jen; Kossiakoff, Anthony A.

doi:10.1016/j.jmb.2017.12.012

Cited by 58 publications

(61 citation statements)

References 41 publications

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“…S2A and B of [20]) ( Supplementary Figure 2). Nonetheless, the CR3022 elbow angles, which are distant from the antibody-antigen interface, differ in the two structures, as we mutated the elbow region (as described in [25]) of CR3022 to promote crystallization with SARS-CoV RBD. The conserved binding mode of CR3022 to SARS-CoV-2 RBD and SARS-CoV RBD indicates that the difference in binding affinity of CR3022 between SARS-CoV-2 RBD and SARS-CoV RBD is only due to a very subtle structural difference.…”

Section: Crystal Structure Reveals the Impact Of P384a In Cr3022 Bindingmentioning

confidence: 99%

See 1 more Smart Citation

A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

Yuan

Bangaru

et al. 2020

Preprint

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Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that can cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential for cross-neutralizing epitopes on SARS-like viruses.

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Section: Crystal Structure Reveals the Impact Of P384a In Cr3022 Bindingmentioning

confidence: 99%

“…The Fab was purified with a CaptureSelect™ CH1-XL Pre-packed Column (Thermo Fisher Scientific) followed by size exclusion chromatography. For crystallization, a VSRRLP variant of the elbow region was used to reduce the conformational flexibility between the constant and variable domains [25].…”

Section: Expression and Purification Of Cr3022 Fabmentioning

confidence: 99%

A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

Yuan

Bangaru

et al. 2020

Preprint

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“…Six substitutions and one deletion (from 112 SSASTKG 118 to 112 VSRRLP 117 ) were introduced into the elbow region of Fab395 and Fab366 heavy chains, and different mutations (from 112 SSASTKG to 112 FNQIKG 117 ) were introduced to the elbow region of Fab364 heavy chain to stabilize the Fab and facilitate crystallization as previously described (44). Additionally, Fab239-NPNA2, Fab364-NPNA2 and Fab250-NPNA3 co-complexes were mixed with Streptococcal immunoglobulin G-binding protein G (domain III) in the Fab to protein G ratio of 1:1.…”

Section: X-ray Crystallography and Structural Analysismentioning

confidence: 99%

Structural and biophysical correlation of anti-NANP antibodies with in vivo protection against P. falciparum

Pholcharee

Oyen

Flores-García

et al. 2020

Preprint

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The most advanced P. falciparum circumsporozoite protein (PfCSP)-based malaria vaccine, RTS,S/AS01 (RTS,S), confers partial protection but with antibody titers that wane relatively rapidly, highlighting the need to elicit more potent and durable antibody responses. Here, we elucidate crystal structures, binding affinities and kinetics, and in vivo protection of eight anti-NANP antibodies (Abs) derived from an RTS,S phase 2a trial and encoded by three different heavy-chain germline genes. The structures reinforce the importance of homotypic Fab-Fab interactions in protective Abs and the overwhelmingly dominant preference for a germline-encoded aromatic residue for recognition of the NANP motif. A number of biophysical properties were analyzed and antibody affinity correlated best with protection in an in vivo mouse model, with the more potent antibodies also recognizing epitopes with repeating secondary structural motifs of type I β- and Asn pseudo 310 turns. Such insights can be incorporated into design of more effective immunogens as well as antibodies for passive immunization.

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“…High variability in the orientation of light and heavy chains of the netakimab Fab fragment in the asymmetric unit may indicate their high mobility in vivo. Comparative analysis of structures of Fab antibody fragments from the PDB reveals high interdomain mobility in molecules of various Fab fragments alone [38] and complexed [39]. In our case, there is a high range of Fab conformations between the constant and variable domains in a single crystal structure.…”

Section: Region Namementioning

confidence: 58%

Fab Fragment of VHH-Based Antibody Netakimab: Crystal Structure and Modeling Interaction with Cytokine IL-17A

et al. 2019

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Interleukin 17A (IL-17A) is a proinflammatory cytokine produced by Th17 cells. Antibody BCD-085 (netakimab) against human IL-17A is one of the new inhibitors of this cytokine. In netakimab, the VH domain is replaced by the VHH domain of Lama glama possessing a long complementarity determining region (CDR-H3) in its heavy chain. Here we demonstrate the high affinity of IL-17A to the Fab fragment of netakimab and to its integral part, the VHH domain. We have determined the crystal structure of the Fab fragment of netakimab at 1.9 Å resolution. High variability in the orientation of light and heavy chains of the Fab fragment of netakimab was shown, which is determined by the peculiarity of the structural organization of the CDR-H3. As the high conformational plasticity of the molecule hampers modeling the Fab fragment of netakimab complexed to IL-17A, we have carried out modeling the complex between the antigen and the integral part of the Fab fragment, the VHH domain. We explain the high netakimab Fab fragment affinity for IL-17A by a large number of protein–protein contacts due to additional interactions between CDR-H3 and the cytokine dimer.

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Locking the Elbow: Improved Antibody Fab Fragments as Chaperones for Structure Determination

Cited by 58 publications

References 41 publications

A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

Structural and biophysical correlation of anti-NANP antibodies with in vivo protection against P. falciparum

Fab Fragment of VHH-Based Antibody Netakimab: Crystal Structure and Modeling Interaction with Cytokine IL-17A

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