Heparin-aggregated RANTES can be crystallised

Crystals of RANTES (regulated on activation normal T-cell expressed) have been grown in the presence of heparin-derived sulphated oligosaccharides which cause RANTES to aggregate severely. The crystals have a tendency to be polycrystalline but diffract to 3.8 - 1.8 Å resolution. Oligosaccharides length and stoichiometry influence aggregation, nucleation and crystal growth and quality. Surprising, rather than inhibiting crystallisation, aggregation appears to stimulate nucleation. We have co-crystallised RANTES in the presence of oligosaccharides ranging in size from 1 to 12 sugar moieties. The best crystals, both according to size and diffraction quality have been obtained with a six moiety sugar. Crystals grow in the same space group with similar cell parameters as previously reported. We find no homogeneous binding of the sulphated sugars to RANTES even after eliminating sulphate from the crystallisation conditions to avoid competition with sulphates from the sulphated sugars. There is no electron density at the sulphate positions characterised in the original structure and the residues involved in sulphate binding adopt a different side chain orientation. Either heterogeneous binding of the sulphated sugars or an active ratchet-like mechanism by which the sulphated sugars are eliminated from the crystal lattice as the crystals grow may be responsible for the absence of sugars in the structure. The fact that aggregated proteins can be crystallised is important, since it is generally accepted that proteins that are polydisperse by light dynamic scattering are poor candidates for crystallisation trials.

Scaffolds for protein crystallisation

In the absence of a method to ensure that crystals can be obtained for any given protein, the possibility of developing scaffolds for protein crystallisation becomes attractive. Among several approaches that could yield scaffolds, two are particularly promising: the first is based on immunoglobulin Fab fragments and immunoglobulin binding proteins while the second is based on fusion proteins. In the Fab based scaffold, the protein of interest is the antigen recognised by the antibody. In the second case, it is a protein fused to one of the scaffold components. The operational difference between the two methods is the existence of a flexible covalent tether compared to a highly specific interaction. The relative merits and disadvantages of each approach are discussed here. We also describe a lattice obtained through a combinatorial approach which appears to have the required properties to be considered a scaffold. The system makes use of an Fab derived from a rheumatoid factor and an Fc-fusion protein. The Fc-fusion system is ideal for enhanced expression of glycoproteins in mammalian cells and provides a useful tag for their purification. The molecular replacement shows a mode of binding for this rheumatoid factor that is not competitive with bacterial Fc-binding proteins. Hence it may be possible to generalise the method to include bacterial expression of fusion proteins with either protein A or protein G as the fusion partner.

Disulphide dimerised peptide creates a crystal contact in an anti-peptide antibody

Although many Fab derived from human and murine antibodies have been crystallised, several such fragments, including anti-peptide Fab 4x11, remain problematic for crystallisation. Diffracting crystals have been obtained for this Fab only in complex with a cysteine containing peptide CGGIRGERA. The IRGERA portion contained in the C-terminus of histone H3 is recognised by the monoclonal antibody and the CGG portion is an essential spacer. Crystallisation experiments and seeding studies show that disulphide bond formation is essential for obtaining crystals. Data has been collected to 2.7 Å resolution, and the structure solved by molecular replacement. The asymmetric unit contains two antibodies whose binding sites are face-to-face in an non-crystallographic approximate two-fold rotational axis. We find two Fab in the asymmetric unit with the two antigen combining sites facing each other. This result was unexpected since it has been common practice to avoid the cysteine-containing peptides because of heterogeneity, since the peptide solution is likely to contain both dimeric and monomeric peptides. This result suggests that the cysteine containing peptides used in immunisation for coupling them to a carrier protein could also be used to screen for crystals. Structural data obtained for the same peptides as those used in the immunisation is valuable to evaluate to what extent the linker to the carrier protein may have contributed to the shaping of the antibody binding site. The introduction of exposed cysteines on the surface of proteins by site directed mutagenesis could help resolve difficult crystallisation cases. Should these not form a disulphide bridge in the crystal but nonetheless crystallise, the free cysteine could be used to make a heavy atom derivative for isomorphous replacement.

Protein L mutants for the crystallization of antibody fragments

In many cases, antibody and their complexes can be crystallized and their structure determined without major difficulties. The remaining problematic cases may be approached through techniques such as of combinatorial complex crystallization which uses immunoglobulin binding proteins (IBP). The range of lattices that can be made using this method can be expanded by engineering mutants of IBP domains. We have designed Peptostreptococcus magnus protein L (PpL) mutants with altered immunoglobulin light chain binding characteristics. While the wild type PpL has two binding sites, some of the mutants contact the light chain via only one site. Other mutants have combinations of weakened first and second binding sites that modify their crystallization properties and their packing mode. In this study, we have selected PpL mutants with different behavior and that are most useful for crystallization and we present the various packing modes obtained so far.