Multidomain proteins
We solved the crystal structure of the periplasmic N-terminal domain of GspD (blue) from the bacterial type 2 secretion system secretin in complex with a Xaperone (red). The prime function of the Xaperone in promoting crystal growth is probably formation of a compact 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 KV, Pardon E, Steyaert J & Hol WG (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. 
Intrinsically disordered proteins
Our lab solved the structure of the intrinsically flexible addiction antidote MazE (blue), using a nM affinity Xaperone (red) as a crystallization chaperon. The Xaperone generates a crystal lattice with a cavity that accomodates the intrinsically unfolded part of MazE. Remarkably, more than half of MazE is disordered in the crystal, shedding light on the difficulty of crystallizing MazE in isolation. In complex with the antibody fragment, the total amount of structured polypeptide (126 amino acids of Xaperone 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 R, Marianovsky I, Lah J, Laeremans T, Engelberg-Kulka H, Glaser G, Muyldermans S & Wyns L (2003) Crystal structure of the intrinsically flexible addiction antidote MazE. J Biol Chem 278, 28252-28257.
Parts of multiprotein assemblies
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 (red) and EpsJ (blue). 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 AY, Pardon E, Korotkov KV, Hol WG & Steyaert J (2009) Nanobody- aided structure determination of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificus. J Struct Biol 166, 8-15.
In trypanosomes, many key RNA editing steps occur in 20S editosomes, which have a core of 12 proteins. Among these, the ‘‘interaction protein” KREPA6 (blue) performs a central role in maintaining the integrity of the editosome core. The use of Xaperones accelerated crystal growth of KREPA6 from Trypanosoma brucei dramatically. All three structures obtained are heterotetramers with a KREPA6 dimer in the center, and one Xaperone (red) bound to each KREPA6 subunit.
Wu M, Park Y-J, Pardon E, Turley S, Hayhurst A, Deng J, Steyaert J & Hol WGJ (2010) Structures of a key interaction protein from the Trypanosoma brucei editosome in complex with single domain antibodies. J. Struct. Biol., in press.
Integral membrane proteins
The complex behavior of GPCRs in response to natural or synthetic ligands and proteins can be attributed to the receptor’s structural plasticity manifested in multiple functionally distinct conformational states. We generated Xaperones (blue) which selectively recognize an active state of the human β2 adrenergic receptor (green). Such Xaperones 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, SGF, Choi, H.-J., Fung, J.-J., Pardon, E, Casarosa P, Seok Chae P, DeVree BT, Rosenbaum DM, Thian FS, Kobilka TS, Schnapp A, Konetzki I, Sunahara RK, Gellman SH, Pautsch A, Steyaert J, Weis WI, Kobilka BK (2010). Structure of a nanobody-stabilized active state of the β2 adrenoceptor, Nature, in press.
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 Xaperones that block the fibrillogenesis of a proteolytic amyloidogenic ΔN6 variant of β2m (blue). We found that one of the fibrillogenesis inhibitors (red) traps a domain swapped dimer of ΔN6β2m in the crystal. The crystal structure of this dimer has several properties that have been attributed to prefibrillar intermediates of β2m fibrillogenesis.
Domanska K, Vanderhaegen S, Srinivasan V, Pardon E & Dupeux F (2011) Atomic structure of a nanobody-trapped domain-swapped dimer of an amyloidogenic β2-microglobulin variant. PNAS. Published online before print.
