Theo

Structure of the human pro-myostatin precursor and determinants of growth factor latency

Thomas Cotton, Gerhard Fischer, Xuelu Wang, Jason McCoy, Magdalena Czepnik, Thomas B. Thompson, Marko Hyvönen

The EMBO Journal (2018) 37: 367-383
DOI: 10.15252/embj.201797883
Pre-publication in BiorXiv, DOI: doi.org/10.1101/153403
PDB coordinates: 5NTU (3D view ), 5NXS (3D view )


Abstract

Myostatin, a key regulator of muscle mass in vertebrates, is biosynthesised as a latent precursor in muscle and is activated by sequential proteolysis of the pro-domain. To investigate the molecular mechanism by which pro-myostatin remains latent, we have solved the structure of unprocessed pro-myostatin and analysed the properties of the protein in its different forms. Continue reading →

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Structure and activation of pro-activin A

Xuelu Wang, Gerhard Fischer and Marko Hyvönen

Nature Communications 7:1205, 2016
DOI: 10.1038/ncomms12052
Pubmed: 27373274

PDB coordinates: 5HLY (3D view), 5HLZ (3D view)

Abstract

Activins are growth factors with multiple roles in the development and homeostasis. Like all TGF-β family of growth factors, activins are synthesized as large precursors from which mature dimeric growth factors are released proteolytically. Here we have studied the activation of activin A and determined crystal structures of the unprocessed precursor and of the cleaved pro-mature complex. Replacing the natural furin cleavage site with a HRV 3C protease site, we show how the protein gains its bioactivity after proteolysis and is as active as the isolated mature domain. The complex remains associated in conditions used for biochemical analysis with a dissociation constant of 5 nM, but the pro-domain can be actively displaced from the complex by follistatin. Our high-resolution structures of pro-activin A share features seen in the pro-TGF-β1 and pro-BMP-9 structures, but reveal a new oligomeric arrangement, with a domain-swapped, cross-armed conformation for the protomers in the dimeric protein.

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Engineering Archeal Surrogate Systems for the Development of Protein-Protein Interaction Inhibitors against Human RAD51

Tommaso Moschetti, Timothy Sharpe, Gerhard Fischer, May E. Marsh, Hong Kin Ng, Matthew Morgan, Duncan E. Scott, Tom L. Blundell, Ashok R. Venkitaraman, John Skidmore, Chris Abell, and Marko Hyvönen

Journal of Molecular Biology 428(23): 4589–4607, 2016

DOI: 10.1016/j.jmb.2016.10.009

PDB coordinates: 5FOS, 5LB2, 5LBI, 5L8V, 5LB4, 5KDD, 5J4L, 5JEE, 5JED, 5JEC, 5JFG, 5J4H, 5J4K

 

Protein-protein interactions (PPIs) are increasingly important targets for drug discovery. Efficient fragment-based drug discovery approaches to tackle PPIs are often stymied by difficulties in the production of stable, unliganded target proteins. Here, we report an approach that exploits protein engineering to “humanise” thermophilic archeal surrogate proteins as targets for small-molecule inhibitor discovery and to exemplify this approach in the development of inhibitors against the PPI between the recombinase RAD51 and tumour suppressor BRCA2. As human RAD51 has proved impossible to produce in a form that is compatible with the requirements of fragment-based drug discovery, we have developed a surrogate protein system using RadA from Pyrococcus furiosus. Using a monomerised RadA as our starting point, we have adopted two parallel and mutually instructive approaches to mimic the human enzyme: firstly by mutating RadA to increase sequence identity with RAD51 in the BRC repeat binding sites, and secondly by generating a chimeric archaeal human protein. Both approaches generate proteins that interact with a fourth BRC repeat with affinity and stoichiometry comparable to human RAD51. Stepwise humanisation has also allowed us to elucidate the determinants of RAD51 binding to BRC repeats and the contributions of key interacting residues to this interaction. These surrogate proteins have enabled the development of biochemical and biophysical assays in our ongoing fragment-based small-molecule inhibitor programme and they have allowed us to determine hundreds of liganded structures in support of our structure-guided design process, demonstrating the feasibility and advantages of using archeal surrogates to overcome difficulties in handling human proteins.

Pubmed | ScienceDirect

J Mol Biol. 2016 Nov 20;428(23):4589-4607. doi: 10.1016/j.jmb.2016.10.009.

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