The rational design of affinity-attenuated OmCI for the purification of complement C5.

Complement component C5 is the target of the mAb eculizumab and is the focus of a sustained drug discovery effort to prevent complement-induced inflammation in a range of autoimmune diseases. The immune evasion protein OmCI binds to and potently inactivates C5; this tight-binding interaction can be exploited to affinity-purify C5 protein from serum, offering a vastly simplified protocol compared with existing methods. However, breaking the high-affinity interaction requires conditions that risk denaturing or activating C5. We performed structure-guided in silico mutagenesis to identify prospective OmCI residues that contribute significantly to the binding affinity. We tested our predictions in vitro, using site-directed mutagenesis, and characterized mutants using a range of biophysical techniques, as well as functional assays. Our biophysical analyses suggest that the C5-OmCI interaction is complex with potential for multiple binding modes. We present single mutations that lower the affinity of OmCI for C5 and combinations of mutations that significantly decrease or entirely abrogate formation of the complex. The affinity-attenuated forms of OmCI are suitable for affinity purification and allow elution under mild conditions that are nondenaturing or activating to C5. We present the rational design, biophysical characterization, and experimental validation of affinity-reduced forms of OmCI as tool reagents to enable the affinity purification of C5.

Characterization of the interaction of sclerostin with the low density lipoprotein receptor-related protein (LRP) family of Wnt co-receptors.

LRP5 and LRP6 are proteins predicted to contain four six-bladed ß-propeller domains and both bind the bone-specific Wnt signaling antagonist sclerostin. Here, we report the crystal structure of the amino-terminal region of LRP6 and using NMR show that the ability of sclerostin to bind to this molecule is mediated by the central core of sclerostin and does not involve the amino- and carboxyl-terminal flexible arm regions. We show that this structured core region interacts with LRP5 and LRP6 via an NXI motif (found in the sequence PNAIG) within a flexible loop region (loop 2) within the central core region. This sequence is related closely to a previously identified motif in laminin that mediates its interaction with the ß-propeller domain of nidogen. However, the NXI motif is not involved in the interaction of sclerostin with LRP4 (another ß-propeller containing protein in the LRP family). A peptide derived from the loop 2 region of sclerostin blocked the interaction of sclerostin with LRP5/6 and also inhibited Wnt1 but not Wnt3A or Wnt9B signaling. This suggests that these Wnts interact with LRP6 in different ways.