Structure of a GRK5-Calmodulin Complex Reveals Molecular Mechanism of GRK Activation and Substrate Targeting.

The phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) facilitates arrestin binding and receptor desensitization. Although this process can be regulated by Ca2+-binding proteins such as calmodulin (CaM) and recoverin, the molecular mechanisms are poorly understood. Here, we report structural, computational, and biochemical analysis of a CaM complex with GRK5, revealing how CaM shapes GRK5 response to calcium. The CaM N and C domains bind independently to two helical regions at the GRK5 N and C termini to inhibit GPCR phosphorylation, though only the C domain interaction disrupts GRK5 membrane association, thereby facilitating cytoplasmic translocation. The CaM N domain strongly activates GRK5 via ordering of the amphipathic αN-helix of GRK5 and allosteric disruption of kinase-RH domain interaction for phosphorylation of cytoplasmic GRK5 substrates. These results provide a framework for understanding how two functional effects, GRK5 activation and localization, can cooperate under control of CaM for selective substrate targeting by GRK5.

Efficient refolding of a hydrophobic protein with multiple S-S bonds by on-resin immobilized metal affinity chromatography.

The efficient refolding of recombinant proteins produced in the form of inclusion bodies (IBs) in Escherichia coli still is a complicated experimental problem especially for large hydrophobic highly disulfide-bonded proteins. The aim of this work was to develop highly efficient and simple refolding procedure for such a protein. The recombinant C-terminal fragment of human alpha-fetoprotein (rAFP-Cterm), which has molecular weight of 26 kDa and possesses 6 S-S bonds, was expressed in the form of IBs in E. coli. The C-terminal 7× His tag was introduced to facilitate protein purification and refolding. The refolding procedure of the immobilized protein by immobilized metal chelating chromatography (IMAC) was developed. Such hydrophobic highly disulfide-bonded proteins tend to irreversibly bind to traditionally used agarose-based matrices upon attempted refolding of the immobilized protein. Indeed, the yield of rAFP-Cterm upon its refolding by IMAC on agarose-based matrix was negligible with bulk of the protein irreversibly stacked to the resin. The key has occurred to be using IMAC based on silica matrix. This increased on-resin refolding yield of the target protein from almost 0 to 60% with purity 98%. Compared to dilution refolding of the same protein, the productivity of the developed procedure was two orders higher. There was no need for further purification or concentration of the renatured protein. The usage of silica-based matrix for the refolding of immobilized proteins by IMAC can improve and facilitate the experimental work for difficult-to-refold proteins.

High-efficient expression, refolding and purification of functional recombinant C-terminal fragment of human alpha-fetoprotein.

Human alpha-fetoprotein (hAFP) is an oncofetal protein which is a common cancer marker. Conjugates of native hAFP with different cytostatic agents inhibit growth of cancer cells in vivo and in vitro. The hAFP interacts with its receptor (AFPR) on the surface of cancer cells via its C-terminal domain. The aim of this work was to develop a highly efficient expression system in Escherichia coli and efficient refolding procedure for the recombinant C-terminal fragment of hAFP (rAFP-Cterm) and to characterize its functional properties. C-terminal fragment of hAFP (rAFP-Cterm) comprising amino acids from 404 to 609 was expressed in E. coli BL21 (DE3) strain with high yield. High efficient purification and refolding procedures were developed giving yield of refolded protein about 80% with purity about 95%. The refolded rAFP-Cterm bound specifically with cancer cells carrying AFPR and was accumulated by them with the same efficiency as native hAFP. This rAFP-Cterm can be used as a vehicle for the targeted delivery of drugs to cancer cells.