Anti-CD20: tales of identical twins?

atomic resolution structural information is X-ray crystallography, a technique accounting for 88% of structures deposited in the Protein Data Bank. Unfortunately, this technique requires milligram amounts of purified protein , which has to subsequently be induced to form crystals. Both protein production and crystallization can be difficult tasks particularly for membrane-bound, multidomain and multisubunit macromolecular complexes. GPIb-IX-V demonstrates all these difficulty criteria. In such cases structural biologists use the divide-and-conquer approach, dividing the proteins into smaller, more manageable fragments, for example, deleting the membrane-spanning regions. These approaches previously led to structure determination of the extracellular domain of GPIb␣ and its complex with VWF-A1 domain. 4,5 A strategy that can be used in even more stubborn cases is to crystallize the protein of interest in the presence of large fusion-tag, as recently exemplified for ␤-adrenergic and Toll-like receptors. 6 This approach has been taken a step further by McEwan et al, who grafted 3 segments of the GPIX subunit onto the homologous GPIb␤ subunit (see figure). 7 McEwan et al now describe the structures of the extracellular domains of both GPIb␤ and this GPIb␤/IX chimera. 1 The observation that GPIX cannot be expressed on cell surface by itself, but only in the presence of GPIb␤, provides an assay for the effects of mutations on the interaction between these proteins through the analysis of surface expression in transiently transfected cells. One of the key advances provided by McEwan and colleagues is the description of the quaternary association of GPIb-IX sub-complex (the GPV subunit is not essential for most functions of the receptor). Clearly, one needs to be cautious when interpreting the interfaces observed in the GPIb␤/IX chimera crystals as reflective of the functional interaction between GPIb␤ and GPIX in the intact receptor complex. However, this conclusion is the simplest explanation of the available data and is supported by several pieces of evidence: (1) it was demonstrated, based on surface expression analysis and coimmunoprecipitation experiments, that the GPIX loops grafted in the GPIb␤/IX chimera are sufficient to mediate association with GPIb␤ 7 ; (2) the crystal structure of the GPIb␤/IX chimera reveals interactions of GPIX loops with GPIb␤ sequences that that are consistent with association in the native complex; (3) analogous interactions are observed in at least 2 crystal forms of the GPIb␤/IX chimeric protein; and (4) mutagenesis of residues in the observed interface results in a loss of the ability of GPIb␤ …