Lateral recognition of a dye hapten by a llama VHH domain

Spinelli, S.; Tegoni, Mariella; Frenken, Leon; Vliet, Cees van; Cambillau, Christian

doi:10.1006/jmbi.2001.4856

Cited by 82 publications

(71 citation statements)

References 27 publications

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“…Spinelli reported the structure of VHH-A52 with RR1. 25 Where VHH-R2 forms a cavity with its three CDRs and binds RR6 in a central combining site, VHH-A52 binds RR1 on a lateral combining site with CDR2, CDR3, and a framework residue. Thus, although the mechanism for binding their antigen is different between VHH-A52 and VHH-R2, both VHHs are able to refold upon addition of their antigen at 80°C.…”

Section: Resultsmentioning

confidence: 99%

Induced refolding of a temperature denatured llama heavy‐chain antibody fragment by its antigen

et al. 2005

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In a previous study we have shown that llama VHH antibody fragments are able to bind their antigen after a heat shock of 90°C, in contrast to the murine monoclonal antibodies. However, the molecular mechanism by which antibody:antigen interaction occurs under these extreme conditions remains unclear. To examine in more detail the structural and thermodynamic aspects of the binding mechanism, an extensive CD, ITC, and NMR study was initiated. In this study the interaction between the llama VHH -R2 fragment and its antigen, the dye Reactive Red-6 (RR6) has been explored. The data show clearly that most of the VHH-R2 population at 80°C is in an unfolded conformation. In contrast, CD spectra representing the complex between VHH-R2 and the dye remained the same up to 80°C. Interestingly, addition of the dye to the denatured VHH-R2 at 80°C yielded the spectrum of the native complex. These results suggest an induced refolding of denatured VHH-R2 by its antigen under these extreme conditions. This induced refolding showed some similarities with the well established "induced fit" mechanism of antibodyantigen interactions at ambient temperature. However, the main difference with the "induced fit" mechanism is that at the start of the addition of the antigen most of the VHH molecules are in an unfolded conformation. The refolding capability under these extreme conditions and the stable complex formation make VHHs useful in a wide variety of applications. Proteins 2005;59:555-564.

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Section: Resultsmentioning

confidence: 99%

Induced refolding of a temperature denatured llama heavy‐chain antibody fragment by its antigen

et al. 2005

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“…However, the extent of these interactions is much smaller than for the present VHHs, the amount of buried surfaces covered by framework residues being comprised of between 1 and 9% of the total interaction (29). Such interactions have also been observed to be important in VHH/hapten interactions (13). The main interactions, however, were provided by the three CDRs, CDR3 being the main contributor in the complexes of AMB7 and AMD10 VHHs and the least for AMD9 VHH.…”

Section: Structure Of the Complexesmentioning

confidence: 94%

Three Camelid VHH Domains in Complex with Porcine Pancreatic α-Amylase

Desmyter¹,

Spinelli²,

Payan³

et al. 2002

Journal of Biological Chemistry

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148

129

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Camelids produce functional antibodies devoid of light chains and CH1 domains. The antigen-binding fragment of such heavy chain antibodies is therefore comprised in one single domain, the camelid heavy chain antibody VH (VHH). Here we report on the structures of three dromedary VHH domains in complex with porcine pancreatic ␣-amylase. Two VHHs bound outside the catalytic site and did not inhibit or inhibited only partially the amylase activity. The third one, AMD9, interacted with the active site crevice and was a strong amylase inhibitor (K i ‫؍‬ 10 nM). In contrast with complexes of other proteinaceous amylase inhibitors, amylase kept its native structure. The water-accessible surface areas of VHHs covered by amylase ranged between 850 and 1150 Å 2 , values similar to or even larger than those observed in the complexes between proteins and classical antibodies. These values could certainly be reached because a surprisingly high extent of framework residues are involved in the interactions of VHHs with amylase. The framework residues that participate in the antigen recognition represented 25-40% of the buried surface. The inhibitory interaction of AMD9 involved mainly its complementarity-determining region (CDR) 2 loop, whereas the CDR3 loop was small and certainly did not protrude as it does in cAb-Lys3, a VHH-inhibiting lysozyme. AMD9 inhibited amylase, although it was outside the direct reach of the catalytic residues; therefore it is to be expected that inhibiting VHHs might also be elicited against proteases. These results illustrate the versatility and efficiency of VHH domains as protein binders and enzyme inhibitors and are arguments in favor of their use as drugs against diabetes.The fundamental molecular recognition molecules of the humoral immune response are remarkably homogeneous throughout the vertebrate phylum. All immunoglobulins are multimers of heterodimeric chains where each heavy (H) 1 chain of four or five domains is linked by disulfide bridges to a light (L) chain of two domains (1). The antigen-binding part of the immunoglobulins is formed invariably by the N-terminal domains of both the H and L chains. These domains display a large sequence variation concentrated in three regions per domain, the CDRs.However, important deviations of this conserved immunoglobulin organization have been observed. In some immunoglobulin isotypes of camelids from the old world (camels and dromedaries) or from the new world (llamas and vicunas) the L chain is missing (2). Furthermore their H chain is devoid of the CH1 domain (3, 4) due to an unconventional splicing event during the mRNA maturation. The antigen-binding fragment of the heavy chain antibodies is therefore comprised in one single domain, the unique N-terminal variable domain referred to as VHH that replaces a four-domain Fab fragment in the immunoglobulin structure (5). This VHH domain is obtained after a DNA recombination between dedicated VHH germline gene segments with D and J minigenes. The dromedary VHH germline genes are quite diverse. They...

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“…19,21 This protruding surface increases the actual interaction surface of the paratopes, extremely facilitating insertion of nanobodies in cavities on the surface of the antigens or ligand-binding sites of receptors. 22 In limited cases, a flat paratope surface 23 and occasionally a cavity for the antihapten binders 24,25 were also observed owing to the long loops in most nanobody structures folding over the FR2 region in VHH. 1 These different shapes of the paratope surfaces demonstrated the extreme flexibility and great diversity of both the sdAbs.…”

Section: Structure Of Nanobodiesmentioning

confidence: 99%

Nanobody-derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications

Wang

Fan

Shao

et al. 2016

IJN

122

107

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Owing to peculiar properties of nanobody, including nanoscale size, robust structure, stable and soluble behaviors in aqueous solution, reversible refolding, high affinity and specificity for only one cognate target, superior cryptic cleft accessibility, and deep tissue penetration, as well as a sustainable source, it has been an ideal research tool for the development of sophisticated nanobiotechnologies. Currently, the nanobody has been evolved into versatile research and application tool kits for diverse biomedical and biotechnology applications. Various nanobody-derived formats, including the nanobody itself, the radionuclide or fluorescent-labeled nanobodies, nanobody homo- or heteromultimers, nanobody-coated nanoparticles, and nanobody-displayed bacteriophages, have been successfully demonstrated as powerful nanobiotechnological tool kits for basic biomedical research, targeting drug delivery and therapy, disease diagnosis, bioimaging, and agricultural and plant protection. These applications indicate a special advantage of these nanobody-derived technologies, already surpassing the “me-too” products of other equivalent binders, such as the full-length antibodies, single-chain variable fragments, antigen-binding fragments, targeting peptides, and DNA-based aptamers. In this review, we summarize the current state of the art in nanobody research, focusing on the nanobody structural features, nanobody production approach, nanobody-derived nanobiotechnology tool kits, and the potentially diverse applications in biomedicine and biotechnology. The future trends, challenges, and limitations of the nanobody-derived nanobiotechnology tool kits are also discussed.

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Lateral recognition of a dye hapten by a llama VHH domain

Cited by 82 publications

References 27 publications

Induced refolding of a temperature denatured llama heavy‐chain antibody fragment by its antigen

Induced refolding of a temperature denatured llama heavy‐chain antibody fragment by its antigen

Three Camelid VHH Domains in Complex with Porcine Pancreatic α-Amylase

Nanobody-derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications

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