A general protocol for the generation of Nanobodies for structural biology

Pardon, Els; Laeremans, Toon; Triest, Sarah; Rasmussen, Søren G. F.; Wohlkonig, A.; Ruf, A.; Muyldermans, Serge; Hól, Wim G. J.; Kobilka, Brian K.; Steyaert, Jan

doi:10.1038/nprot.2014.039

Cited by 591 publications

(603 citation statements)

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“…Several authors have suggested that V H Hs, at least, are even less likely than conventional antibodies to bind linear peptides with high affinity. 102,103 Although this is a plausible hypothesis based on the typical structures of sdAb paratopes (see below), it has not yet been substantiated by any data. Moreover, the relatively large number of studies reporting sdAb reactivity by western blotting suggests that sdAbs directed against continuous epitopes are probably not vanishingly rare.…”

Section: Single-domain Antibodies Direct Against Linear Protein Epitopesmentioning

confidence: 93%

Antigen recognition by single-domain antibodies: structural latitudes and constraints

Henry

MacKenzie

2018

mAbs

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Single-domain antibodies (sdAbs), the autonomous variable domains of heavy chain-only antibodies produced naturally by camelid ungulates and cartilaginous fishes, have evolved to bind antigen using only three complementarity-determining region (CDR) loops rather than the six present in conventional VH:VL antibodies. It has been suggested, based on limited evidence, that sdAbs may adopt paratope structures that predispose them to preferential recognition of recessed protein epitopes, but poor or non-recognition of protuberant epitopes and small molecules. Here, we comprehensively surveyed the evidence in support of this hypothesis. We found some support for a global structural difference in the paratope shapes of sdAbs compared with those of conventional antibodies: sdAb paratopes have smaller molecular surface areas and diameters, more commonly have non-canonical CDR1 and CDR2 structures, and have elongated CDR3 length distributions, but have similar amino acid compositions and are no more extended (interatomic distance measured from CDR base to tip) than conventional antibody paratopes. Comparison of X-ray crystal structures of sdAbs and conventional antibodies in complex with cognate antigens showed that sdAbs and conventional antibodies bury similar solvent-exposed surface areas on proteins and form similar types of non-covalent interactions, although these are more concentrated in the compact sdAb paratope. Thus, sdAbs likely have privileged access to distinct antigenic regions on proteins, but only owing to their small molecular size and not to general differences in molecular recognition mechanism. The evidence surrounding the purported inability of sdAbs to bind small molecules was less clear. The available data provide a structural framework for understanding the evolutionary emergence and function of autonomous heavy chain-only antibodies.

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Section: Single-domain Antibodies Direct Against Linear Protein Epitopesmentioning

confidence: 93%

Antigen recognition by single-domain antibodies: structural latitudes and constraints

Henry

MacKenzie

2018

mAbs

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“…These states may represent natural functional intermediates in overall transport cycle, as the Nbs do not interfere with sugar binding and therefore with protonation, because effective sugar binding requires the protonated form of LacY (39). Moreover, in vivo-matured Nbs do not apparently induce nonnative conformations of antigens (28). Thus, Nbs developed against the outward-open LacY mutant may be useful for crystallization of WT LacY in different conformations without the use of mutagenesis.…”

Section: Discussionmentioning

confidence: 99%

“…Nbs were prepared against the G46W/G262W LacY mutant using a previously published protocol (28). In brief, one llama (Lama glama) received six weekly injections of 100 μg of purified G46W/G262W LacY reconstituted into PLs with lipid to protein ratio 5 (0.4 mg/mL LacY and 2 mg/mL phospholipids).…”

Section: Methodsmentioning

confidence: 99%

Outward-facing conformers of LacY stabilized by nanobodies

Смирнова

Kasho

Jiang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The lactose permease of Escherichia coli (LacY), a highly dynamic polytopic membrane protein, catalyzes stoichiometric galactoside/ H + symport by an alternating access mechanism and exhibits multiple conformations, the distribution of which is altered by sugar binding. We have developed single-domain camelid nanobodies (Nbs) against a LacY mutant in an outward (periplasmic)-open conformation to stabilize this state of the WT protein. Twelve purified Nbs inhibit lactose transport in right-side-out membrane vesicles, indicating that the Nbs recognize epitopes on the periplasmic side of LacY. Stopped-flow kinetics of sugar binding by WT LacY in detergent micelles or reconstituted into proteoliposomes reveals dramatic increases in galactoside-binding rates induced by interaction with the Nbs. Thus, WT LacY in complex with the great majority of the Nbs exhibits varied increases in access of sugar to the binding site with an increase in association rate constants (k on ) of up to ∼50-fold (reaching 10 7 M −1 ·s −1 ). In contrast, with the double-Trp mutant, which is already open on the periplasmic side, the Nbs have little effect. The findings are clearly consistent with stabilization of WT conformers with an open periplasmic cavity. Remarkably, some Nbs drastically decrease the rate of dissociation of bound sugar leading to increased affinity (greater than 200-fold for lactose).membrane transport proteins | fluorescence | major facilitator superfamily T ypical of many transport proteins, from organisms as widely separated evolutionarily as Archaea and Homo sapiens, the lactose permease of Escherichia coli (LacY), a paradigm for the Major Facilitator Superfamily (1), catalyzes the coupled, stoichiometric translocation of a galactopyranoside and an H + (galactoside/H + symport) across the cytoplasmic membrane (reviewed in refs. 2 and 3). Although it is now generally accepted that membrane transport proteins operate by an alternating access mechanism, this has been documented almost exclusively for LacY (reviewed in refs. 4 and 5). By this means, galactoside-and H + -binding sites become alternatively accessible to either side of the membrane as the result of reciprocal opening/closing of cavities on the periplasmic and cytoplasmic sides of the molecule. LacY is highly dynamic, and alternates between different conformations (6, 7).Until recently, six X-ray structures of LacY have exhibited the same inward-facing conformation with an aqueous cavity open to the cytoplasmic side, a tightly sealed periplasmic side, and sugarand H + -binding sites in the middle of the molecule (8-11). Numerous studies confirm that this conformation prevails in the absence of sugar (12-16). Recently, however, the X-ray structure of double-Trp mutant G46W/G262W with bound sugar reveals a conformation with a narrowly open periplasmic pathway and a tightly sealed cytoplasmic side (PDB ID code 4OAA) (17), thereby providing structural evidence that an intermediate occluded conformation occurs between the outward-and inwardfacing conformations in ...

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“…Such V HH are often called "Xaperones TM " when used for crystallization and have been applied to conformationally stabilize unstructured protein domains, transmembrane proteins, and protein complexes for crystallization (21). C-DCX-specific Xaperones were generated by a protocol similar to that described previously (22). Two llamas were immunized four times with T-DCX.…”

Section: Methodsmentioning

confidence: 99%