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Biochemistry of ovine bone and morphogenetic proteins and receptors

The transforming growth factor (TGF)-β superfamily mediates a wide range of differentiation and developmental processes across many genera. GDF9 and BMP15 are expressed exclusively in the mammalian ovary and are the only TGF-β ligands that lack the conserved cysteine residue used for dimerisation. As a platform for studying the interactions between GDF9 and BMP15 and their receptors, BMPRII and BMPRIb, a variety of strategies were attempted to produce soluble and active proteins from recombinant systems. Both ligands and receptors showed a tendency to form insoluble aggregates when expressed in prokaryotic systems; however after extensive screening, quantities of biologically active GDF9 were produced using in vitro refolding. When expressed alone, either containing a histidine tag or as an untagged protein, the BMPRII ectodomain was deposited as insoluble inclusion bodies. This protein, subjected to in vitro refolding procedures, exhibited multiple species following anion exchange chromatography and size exclusion chromatography, as visualised on native PAGE. Separation of these species could be achieved using a MonoP matrix. One of these separated fractions, representing about 5% of the starting material, was amenable to crystallisation, and furthermore exhibited activity in a rat granulosa cell thymidine incorporation assay. Two different crystals forms of the extracellular domain of BMPRII were grown from the same protein batch under similar crystallisation conditions. Notably, the tetragonal form that grew more slowly possessed several disordered finger regions, while electron density for the entire molecule was clear in the orthorhombic form. The hydrophobic core of the ligand binding surface of BMPRII , as seen in both structures, resembles that of ActRII bound to BMP2. The A-loop of BMPRII, which is involved in ligand binding, lies in two different conformations in the two structures of BMPRII, mediated by a rearrangement in disulfide Cys94-Cys117. It is proposed here that the tetragonal form represents the ligand-bound receptor structure. Although the majority of the hydrophobic binding surface is shared with ActRII(b), it is likely that His87 and Tyr40 are unique residues that confer specificity in BMPRII ligand binding.

Identiferoai:union.ndltd.org:ADTP/217578
Date January 2006
CreatorsMace, Peter, n/a
PublisherUniversity of Otago. Department of Biochemistry
Source SetsAustraliasian Digital Theses Program
LanguageEnglish
Detected LanguageEnglish
Rightshttp://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Peter Mace

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