International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by E. Arnold, D. M. Himmel and M. G. Rossmann

International Tables for Crystallography (2012). Vol. F, ch. 4.3, pp. 132-133   | 1 | 2 |

Section 4.3.6. Noncovalent crystallization chaperones

Z. S. Derewendaa*

aDepartment of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908–0736, USA
Correspondence e-mail: zsd4n@virginia.edu

4.3.6. Noncovalent crystallization chaperones

| top | pdf |

Noncovalent crystallization chaperones, i.e. engineered binding proteins that produce noncovalent complexes with target macromolecules, constitute an exciting alternative to fusion carrier proteins. Complexes with such chaperones often exhibit enhanced solubility and/or crystallizability in com­parison to the isolated targets. The Fab and Fv fragments of antibodies are most commonly used for this purpose (Kovari et al., 1995[link]; Hunte & Michel, 2002[link]; Prongay et al., 1990[link]; Ostermeier et al., 1995[link]; Jiang et al., 2003[link]; Dutzler et al., 2003[link]; Lee et al., 2005[link]). In its canonical version, the technique requires animal immunization with subsequent purification of hybridoma-derived antibodies and their proteolytic digestion to obtain pure homogeneous Fab fragments (Karpusas et al., 2001[link]; Kovari et al., 1995[link]). Alternatively, the Fab fragment can be directly sequenced and a synthetic gene can be used for E. coli expression, although this is not trivial owing to the presence of disulfides and two separate polypeptide chains in an Fab molecule. To overcome this bottleneck, a more efficient method of recombinant production of antibody fragments using mammalian HEK 293T has recently been proposed (Nettleship et al., 2008[link]). Another possibility is the use of so-called nanobodies, i.e. single-chain fragments derived from camelid antibodies (Koide, Tereshko et al., 2007[link]; Lam et al., 2009[link]; Korotkov et al., 2009[link]). However, this strategy requires immunization of camels or llamas, which is not technically easy.

Regardless of the specific strategy, the use of hybridoma technology and animal immunization is always time-consuming and expensive. In principle, a more efficient approach is to carry out in vitro selection of Fab fragments using phage display (Lee et al., 2004[link]) or ribosome display (Lipovsek & Pluckthun, 2004[link]). However, since a typical antibody–antigen interface involves ∼30 amino acids, the total number of possible sequences of a given template Fab significantly exceeds the available combinatorial libraries. Consequently, traditional phage-display libraries greatly diminish diversity at the mutated sites, which explains why syn­thetic antibodies were initially weaker binders than natural ones (Hawkins et al., 1992[link]; Koide, 2009[link]). This problem was successfully overcome using a different type of phage-display library based on a `reduced genetic code' and comprised of only a few amino acids, e.g. four, which produces high-affinity binders based on a single Fab scaffold (Fellouse et al., 2004[link]; Lee et al., 2004[link]). In contrast to natural antibodies, such synthetic Fab fragments can be generated against unique conformations, complexes or weak antigens such as RNA. Among recent examples are the crystallization and structure determination of the closed form of the full-length KcsA potassium channel with its cognate synthetic Fab (Uysal et al., 2009[link]; Fig. 4.3.6.1[link]) and the crystallographic study of the ΔC209 P4-P6 domain of the Tetrahymena group I intron, a structured RNA molecule (Ye et al., 2008[link]).

[Figure 4.3.6.1]

Figure 4.3.6.1 | top | pdf |

Phage-display-generated Fab fragments as crystallization chaperones: the structure of the KcsA channel in the closed conformation in complex with a synthetic Fab (PDB code 3eff ; Uysal et al., 2009[link]). (a) A diagram showing how the Fab binds to the cytosolic portion in reference to the transmembrane domain. (b) The crystal structure of the complex showing how the synthetic Fab molecules mediate the major crystal contacts. (Figure courtesy of Dr Anthony Kossiakoff, University of Chicago.)

The in vitro display methods also allow the engineering of non-antibody scaffolds as alternative protein binders and crystallization chaperones (Koide, 2009[link]). For example, a fibronectin type III domain (FN3) scaffold was successfully used to generate binders with a reduced genetic code phage-display library (Koide, Gilbreth et al., 2007[link]; Gilbreth et al., 2008[link]). A similar approach was used for DARPins, i.e. designed ankyrin-repeat proteins (Sennhauser & Grütter, 2008[link]), based on ribosome-display selection (Lipovsek & Pluckthun, 2004[link]; Sennhauser & Grütter, 2008[link]). Several new protein structures have been solved as complexes with DARPin chaperones, including polo-like kinase 1 (Bandeiras et al., 2008[link]), the trimeric integral membrane multidrug transporter AcrB (Sennhauser et al., 2007[link]) and the receptor-binding protein (RBP, the BppL trimer) of the baseplate complex of the lactococcal phage TP901–1 (Veesler et al., 2009[link]).

References

Bandeiras, T. M. et al. (2008). Structure of wild-type Plk-1 kinase domain in complex with a selective DARPin. Acta Cryst. D64, 339–353.
Dutzler, R., Campbell, E. B. & MacKinnon, R. (2003). Gating the selectivity filter in ClC chloride channels. Science, 300, 108–112.
Fellouse, F. A., Wiesmann, C. & Sidhu, S. S. (2004). Synthetic antibodies from a four-amino-acid code: a dominant role for tyrosine in antigen recognition. Proc. Natl Acad. Sci. USA, 101, 12467–12472.
Gilbreth, R. N., Esaki, K., Koide, A., Sidhu, S. S. & Koide, S. (2008). A dominant conformational role for amino acid diversity in minimalist protein–protein interfaces. J. Mol. Biol. 381, 407–418.
Hawkins, R. E., Russell, S. J. & Winter, G. (1992). Selection of phage antibodies by binding-affinity – mimicking affinity maturation. J. Mol. Biol. 226, 889–896.
Hunte, C. & Michel, H. (2002). Crystallisation of membrane proteins mediated by antibody fragments. Curr. Opin. Struct. Biol. 12, 503–508.
Jiang, Y., Lee, A., Chen, J., Ruta, V., Cadene, M., Chait, B. T. & MacKinnon, R. (2003). X-ray structure of a voltage-dependent K+ channel. Nature (London), 423, 33–41.
Karpusas, M., Lucci, J., Ferrant, J., Benjamin, C., Taylor, F. R., Strauch, K., Garber, E. & Hsu, Y. M. (2001). Structure of CD40 ligand in complex with the Fab fragment of a neutralizing humanized antibody. Structure, 9, 321–329.
Koide, A., Gilbreth, R. N., Esaki, K., Tereshko, V. & Koide, S. (2007). High-affinity single-domain binding proteins with a binary-code interface. Proc. Natl Acad. Sci. USA, 104, 6632–6637.
Koide, A., Tereshko, V., Uysal, S., Margalef, K., Kossiakoff, A. A. & Koide, S. (2007). Exploring the capacity of minimalist protein interfaces: interface energetics and affinity maturation to picomolar K-D of a single-domain antibody with a flat paratope. J. Mol. Biol. 373, 941–953.
Koide, S. (2009). Engineering of recombinant crystallization chaperones. Curr. Opin. Struct. Biol. 19, 449–457.
Korotkov, K. V., Pardon, E., Steyaert, J. & Hol, W. G. (2009). Crystal structure of the N-terminal domain of the secretin GspD from ETEC determined with the assistance of a nanobody. Structure, 17, 255–265.
Kovari, L. C., Momany, C. & Rossmann, M. G. (1995). The use of anti­body fragments for crystallization and structure determinations. Structure, 3, 1291–1293.
Lam, A. Y., Pardon, E., Korotkov, K. V., Hol, W. G. & Steyaert, J. (2009). Nanobody-aided structure determination of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificus. J. Struct. Biol. 166, 8–15.
Lee, C. V., Liang, W. C., Dennis, M. S., Eigenbrot, C., Sidhu, S. S. & Fuh, G. (2004). High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold. J. Mol. Biol. 340, 1073–1093.
Lee, S. Y., Lee, A., Chen, J. & MacKinnon, R. (2005). Structure of the KvAP voltage-dependent K+ channel and its dependence on the lipid membrane. Proc. Natl Acad. Sci. USA, 102, 15441–15446.
Lipovsek, D. & Pluckthun, A. (2004). In-vitro protein evolution by ribosome display and mRNA display. J. Immunol. Methods, 290, 51–67.
Nettleship, J. E., Ren, J., Rahman, N., Berrow, N. S., Hatherley, D., Barclay, A. N. & Owens, R. J. (2008). A pipeline for the production of antibody fragments for structural studies using transient expression in HEK 293T cells. Protein Expr. Purif. 62, 83–89.
Ostermeier, C., Iwata, S., Ludwig, B. & Michel, H. (1995). F-V fragment mediated crystallization of the membrane-protein bacterial cytochrome-c-oxidase. Nat. Struct. Biol. 2, 842–846.
Prongay, A. J., Smith, T. J., Rossmann, M. G., Ehrlich, L. S., Carter, C. A. & McClure, J. (1990). Fusion proteins as tools for crystallization: the lactose permease from Escherichia coli. Proc. Natl Acad. Sci. USA, 87, 9980–9984.
Sennhauser, G., Amstutz, P., Briand, C., Storchenegger, O. & Grütter, M. G. (2007). Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors. PLoS Biol. 5, 106–113.
Sennhauser, G. & Grütter, M. G. (2008). Chaperone-assisted crystallography with DARPins. Structure, 16, 1443–1453.
Uysal, S., Vasquez, V., Tereshko, V., Esaki, K., Fellouse, F. A., Sidhu, S. S., Koide, S., Perozo, E. & Kossiakoff, A. (2009). Crystal structure of full-length KcsA in its closed conformation. Proc. Natl Acad. Sci. USA, 106, 6644–6649.
Veesler, D., Dreier, B., Blangy, S., Lichière, J., Tremblay, D., Moineau, S., Spinelli, S., Tegoni, M., Plückthun, A., Campanacci, V. & Cambillau, C. (2009). Crystal structure and function of a DARPin neutralizing inhibitor of lactococcal phage TP901–1: comparison of DARPin and camelid VHH binding mode. J. Biol. Chem. 284, 30718–30726.
Ye, J.-D., Tereshko, V., Frederiksen, J. K., Koide, A., Fellouse, F. A., Sidhu, S. S., Koide, S., Kossiakoff, A. A. & Piccirilli, J. A. (2008). Synthetic antibodies for specific recognition and crystallization of structured RNA. Proc. Natl Acad. Sci. USA, 105, 82–87.








































to end of page
to top of page