Crystallization chaperones

We generate nanobodies that facilitate the crystallization of challenging targets and identifies nanobodies that stabilize unique conformations to study the structure, function and dynamics of these proteins.

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Structural enzymology

We are interested in understanding how enzymes function at the molecular and atomic level. We combine structural biology with protein engineering and enzyme kinetics to study how structure determines function. 

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Protein-protein interactions

Protein-protein complexes play an important role in biology and disease. In order to characterise these "transient" protein-protein interactions, we are developping Nanobody-based methods to stabilize PPIs.
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Crystallization chaperones



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




Case studies

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. 



Protein protein complexes: In collaboration with the Kobilka lab, we identified nanobodies that stabilize the ß2AR-Gs complex. One of these nanobodies (red) that inhibits the GTP driven dissociation of  ß2AR-Gs was used to obtain the high-resolution crystal structure of this complex, providing the first view of transmembrane signaling by a GPCR  (Rasmussen et al. 2011a, PDB entry 3SN6).
GPCRs: We generatednanobodies (purple) which selectively recognize an active state of the human β2 adrenergic receptor (gray). Such Nanobodies that faithfully mimic the effects of G protein binding were used to obtain diffraction quality crystals and to solve the first structure of an active agonist-bound state of the human β2 adrenergic receptor (Rasmussen et al, 2011b, PDB entry 3P0G).
ABC transporters: In collaboration with the Chang lab, we solved a crystal structure of the P-glycoprotein, a mediator of efflux-based multidrug resistance in many cancers. The multidrug transporter has a nanobody bound to the C-terminal side of the first nucleotide-binding domain. This nanobody strongly inhibits the ATP hydrolysis activity of mouse P-gp by hindering the formation of a dimeric complex between the ATP-binding domains, which is essential for nucleotide hydrolysis. (Ward et al., 2013, PDB entry 4KSD).
Intrinsically disordered proteins: We solved the structure of the intrinsically flexible addiction antidote MazE (cyan), using a nM affinity nanobody as crystallization a crystallization chaperone. 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. The nanobody contributes to generate a crystal latice that leaves enough space for the disordered part of MazE. (Loris et al, 2003, PDB entry 1MVF).



  Proteins from larger molecular complexes: The interface areas between Xaperones and their antigens are ranging from 600 to 900 Å2, very similar to the contact area of the interfaces of protein-protein interactions. It follows that Xaperones are suitable to stabilize the protomers of larger protein assemblies in one-to-one heterodimers.
We proved this principle by solving the structures of EpsI (mangenta) and EpsJ (cyan). Using a Xaperone (green) as a crystallization aid, the EpsI:EpsJ pseudopilin heterodimer (two components of the bacterial type 2 secretion system) was crystallized in 15 days compared with 11 months and 17 variants required for crystallization without Xaperone.  (Lam et al., 2008, PDB entry 3CFI).


Amyloidogenic proteins: The identification and characterization of oligomers preceding the formation of fibrils is of particular interest because of an increasing awareness that these species are likely to play a critical role in the pathogenesis of protein deposition diseases. We selected Nanobodies that block the fibrillogenesis of a proteolytic amyloidogenic ΔN6 variant of β2m. We found that one of the fibrillogenesis inhibitors (gray) traps a domain swapped dimer of ΔN6β2m in the crystal (green). The crystal structure of this dimer has several properties that have been attributed to prefibrillar intermediates of β2m fibrillogenesis (Domanska et al. 2011, PDB entry 2X89).
Flexible multidomain proteins: In collaboration with Wim Hol, we solved the crystal structure of the periplasmic N-terminal domain of GspD (green) from the bacterial type 2 secretion system secretin in complex with a nanobody (yellow). 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, PDB entry 3EZJ).