[Show abstract][Hide abstract] ABSTRACT: LRP5 and LRP6 are proteins predicted to contain four six-bladed β-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 β-propeller domain of
nidogen. However, the NXI motif is not involved in the interaction of sclerostin with LRP4 (another β-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.
No preview · Article · Jun 2012 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Structural biology studies typically require large quantities of pure, soluble protein. Currently the most widely-used method for obtaining such protein involves the use of bioinformatics and experimental methods to design constructs of the target, which are cloned and expressed. Recently an alternative approach has emerged, which involves random fragmentation of the gene of interest and screening for well-expressing fragments. Here we describe the application of one such fragmentation method, combinatorial domain hunting (CDH), to a target which historically was difficult to express, human MEK-1. We show how CDH was used to identify a fragment which covers the kinase domain of MEK-1 and which expresses and crystallizes significantly better than designed expression constructs, and we report the crystal structure of this fragment which explains some of its superior properties. Gene fragmentation methods, such as CDH, thus hold great promise for tackling difficult-to-express target proteins.
No preview · Article · Feb 2012 · Journal of Structural Biology