You are hereNanobody-aided crystallography
Chaperon (social): an adult who supervises one or more unmarried men or women during social occasions
Chaperon (headgear): a form of hood or versatile hat worn in Western Europe in the Middle Ages
Chaperon (protein): a protein that assists the non-covalent folding/unfolding in molecular biology
Chaperon (crystallography): a protein that assists another protein to crystallize
The occurrence of bona fide antibodies devoid of light chains in Camelidae was one of the major discoveries within our department. These so-called heavy-chain antibodies bind antigens solely with one single variable domain, referred to as VHH domain or Nanobody (Nb). Methods were developed to clone the VHH repertoire of an immunised dromedary (or llama) in phage display vectors, and to select the antigen-specific VHHs from these 'immune' VHH libraries. Recombinant Nanobodies are small (15kDa) and strictly monomeric. They bind the target with nM affinity, are stable and easy to manipulate. Moreover, the Nanobodies often bind to epitopes that are less immunogenic for conventional antibodies, such as the active sites of enzymes. Due to their small size, they also target areas that are not accessible to standard antibodies. Another advantage is that they generally bind conformational epitopes and that they are well expressed in bacterial expression systems so that they are cheaper and easier to produce in all kind of formats than standard monoclonal antibodies.
There are a number of strong arguments in favor of specific nanobodies as crystallization chaperones for many challenging targets:
- Specific nanobodies may increase the solubility of insoluble targets
- Specific nanobodies may increase the stability of unstable (solubilized membrane) proteins
- Specific nanobodies can stabilize individual components or parts of multiprotein complexes
- Specific nanobodies can be used to produce affinity columns aimed to purify difficult proteins to homogeneity.
- Nanobodies bound to these membrane proteins may increase the polar surface thus enabling better growing crystals
- Nanobodies bound to these membrane proteins may allow the resolution of their structure by molecular replacement
In the last years, we proved the principle that Nanobodies can be used as crystallization chaperones in the crystallization and structure determination of challenging target proteins that would prove unsolvable using more conventional strategies including intrinsically disordered proteins, proteins from larger molecular complexes, aggregating proteins, oligomerizing proteins and membrane proteins.
| Flexible multidomain proteins: We solved the crystal structure of the periplasmic N-terminal domain ofGspD(peri-GspD) from the bacterial type 2 secretion
system secretin in complex with a nanobody (Nb7). The prime function of Nb7 in promoting crystal growth is probably formation of the heterotetramer. Peri-GspD in the tetramer is more rigid than peri-GspD by itself, given the potentially flexible linker between the N1 and N2 subdomains (Korotkov et al., 2009).
|Intrinsically disordered proteins: In 2003, we solved the structure of the intrinsically flexible addiction antidote MazE (green), using a nM affinity nanobody as crystallization target. In complex with the antibody fragment (blue), the total amount of structured polypeptide (126 amino acids of antibody and 44 amino acids of MazE) rises to 73% compared with 45% of free MazE, thus providing a much better starting point for crystallization (Loris et al, 2003).|
|Proteins from larger molecular complexes: The interface areas between Nanobodies and their antigens are ranging from 600 to 900 Å2, very similar to the contact area of the interfaces of non-obligate and obligate protein-protein interactions. It thus appears that Nanobodies are suitable to stabilize the protomers of larger protein assemblies in one-to-one heterodimers. Recently, we proved this principle by solving the structures EpsI and EpsJ, three proteins of secretion system II (Lam et al., 2008)|
The examples described above convincingly show that nanobody-assisted crystallography helps reduce the crystallization bottleneck in structural biology. Building on our growing expertise and our IP position1 in this field, we are open for collaborations with academic and industrial partners. For any inquiries, please contact Jan Steyaert.
Loris, R., Marianovsky, I., Lah, J., Laeremans, T., Engelberg-Kulka, H., Glaser, G., Muyldermans, S., and Wyns, L. (2003) J.Biol.Chem 278, 28252-28257
Korotkov, K. V., Pardon, E., Steyaert, J., and Hol, W. G. (2009) Structure 17, 255-265
Lam, A. Y., Pardon, E., Korotkov, K. V., Hol, W. G., and Steyaert, J. (2009) J.Struct.Biol. 166, 8-15
Dumoulin, M., Last, A. M., Desmyter, A., Decanniere, K., Canet, D., Larsson, G., Spencer, A., Archer, D. B., Sasse, J., Muyldermans, S., Wyns, L., Redfield, C., Matagne, A., Robinson, C. V., and Dobson, C. M. (2003) Nature 424, 783-788
1VUB and VIB have a strong and broad IP position with granted US and European patents that cover the basic structure, composition, preparation and uses of both heavy chain antibodies and Nanobodies (the “Hamers patents”).