Dataset Information

Resurfaced shape complementary proteins that selectively bind the oncoprotein gankyrin.

ABSTRACT: Increased cellular levels of protein-protein interactions involving the ankyrin repeat oncoprotein gankyrin are directly linked to aberrant cellular events and numerous cancers. Inhibition of these protein-protein interactions is thus an attractive therapeutic strategy. However, the relatively featureless topology of gankyrin's putative binding face and large surface areas involved in gankyrin-dependent protein-protein interactions present a dramatic challenge to small molecule discovery. The size, high folding energies, and well-defined surfaces present in many proteins overcome some of the challenges faced by small molecule discovery. We used split-superpositive Green Fluorescent Protein (split-spGFP) reassembly to screen a 5×10(9) library of resurfaced proteins that are shape complementary to the putative binding face of gankyrin and identified mutants that potently and selectively bind this oncoprotein in vitro and in living cells. Collectively, our findings represent the first synthetic proteins that bind gankyrin and may represent a general strategy for developing protein basic research tools and drug leads that bind disease-relevant ankyrin repeats.

SUBMITTER: Chapman AM

PROVIDER: S-EPMC4201333 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Resurfaced shape complementary proteins that selectively bind the oncoprotein gankyrin.

Chapman Alex M AM McNaughton Brian R BR

ACS chemical biology 20140821 10

Increased cellular levels of protein-protein interactions involving the ankyrin repeat oncoprotein gankyrin are directly linked to aberrant cellular events and numerous cancers. Inhibition of these protein-protein interactions is thus an attractive therapeutic strategy. However, the relatively featureless topology of gankyrin's putative binding face and large surface areas involved in gankyrin-dependent protein-protein interactions present a dramatic challenge to small molecule discovery. The si ...[more]

PMID: 25137482

Similar Datasets

Project description:Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments and that lipids can bind to specific sites, for example, in potassium channels. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be important not only for defining the selectivity of membrane proteins towards lipids, but also for understanding the role of lipids in modulating protein function or drug binding.

Dataset Information

Resurfaced shape complementary proteins that selectively bind the oncoprotein gankyrin.

Publications

Resurfaced shape complementary proteins that selectively bind the oncoprotein gankyrin.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets