Molecular characterization of latent GDF8 reveals mechanisms of activation

Ryan G. Walker, Jason C. McCoy, Magdalena Czepnik, Melanie J. Mills, Adam Haggd, Kelly L. Walton, Thomas R. Cotton, Marko Hyvönen, Richard T. Lee, Paul Gregorevic, Craig A. Harrison, and Thomas B. Thompson



Growth/differentiation factor 8 (GDF8), or myostatin, negatively regulates muscle mass. GDF8 is held in a latent state through interactions with its N-terminal prodomain, much like TGF-β. Using a combination of small-angle X-ray scattering and mutagenesis, we characterized the interactions of GDF8 with its prodomain. Our results show that the prodomain:GDF8 complex can exist in a fully latent state and an activated or “triggered” state where the prodomain remains in complex with the mature domain. Continue reading →

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Paul & co’s work on CK2α inhibition highlighted

Paul & co’s work on CK2α inhibition highlighted

Dan Erlanson has highlighted our project on Ck2α inhibitor development in his Practical Fragments blog  “Fragment Linking to selective Ck2 inhibitor“.

This a project we have done in close collaboration with David Spring’s group at the Department of Chemistry. The story started from a serendipitous observation that a fragment expected to bind on the top of the N-lobe of CK2α, on the interaction site with scaffolding protein Ck2β bound in a number of different sites on the kinase, including in a new pocket close to the ATP binding site of the kinase. This previously unidentified site was observed only thanks to a new crystal form Paul had obtained. In this crystal form, the so-called αD helix was mobile enough to be displaced by a fragment that, soaked at high concentration,  found a new home in the very hydrophobic pocket that the displacement of the helix revealed. After some optimisation of the fragment and a crystallographic screen to identify ATP-site binding “war heads”, Claudia and others in the Spring lab were able to create the linked molecule CAM4066.

The main story was published in Chemical Science and the detailed description of the design process came out this year in Bioorganic and Medicinal Chemistry.

If interested more in this story, do check in Youtube some of the movies Paul has made from this project:

Development of CAM4066

Optimisation of the fragment in the aD pocket

Growth of the linker from the aD site to the active site

And it should not go forgotten that all the work has been guided continuous crystallographic assessement of the process, with ~30 unique crystal structures in the two papers: 5CVH (3D view), 5CVG (3D view), 5CVF (3D view), 5CU3 (3D view), 5CU4 (3D view), 5CU6 (3D view), 5CSH (3D view), 5CSP (3D view), 5CSV (3D view), 5CSH (3D view), 5CS6 (3D view), 5CLP (3D view), 5MMF (3D view ), 5MMR (3D view ), 5MO5 (3D view ), 5MO6 (3D view ), 5MO7 (3D view ), 5MO8 (3D view ), 5MOD (3D view ), 5MOE (3D view ), 5MOH (3D view ), 5MOT (3D view ), 5MOV (3D view ), 5MOW (3D view ), 5CU0 (3D view ), 5CU2 (3D view ), 5CT0 (3D view ), 5CTP (3D view ), 5CX9 (3D view ).

And there is more to come! Keep your eyes open.

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Computationally-guided optimization of small-molecule inhibitors of the Aurora A kinase–TPX2 protein–protein interaction

Daniel J. Cole, Matej Janecek, Jamie E. Stokes, Maxim Rossmann, John C. Faver, Grahame J. McKenzie, Ashok R. Venkitaraman, Marko Hyvönen, David R. Spring, David J. Huggins and William L. Jorgensen

Chemical Communications 53, 9372-9375 (2017)
DOI 10.1039/C7CC05379G
Pubmed: 28787041

PDB coordinates: 5OBR (3D view)


Free energy perturbation theory, in combination with enhanced sampling of protein–ligand binding modes, is evaluated in the context of fragment-based drug design, and used to design two new small-molecule inhibitors of the Aurora A kinase–TPX2 protein–protein interaction.

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A fragment-based approach leading to the discovery of a novel binding site and the selective CK2 inhibitor CAM4066.

Claudia De Fusco, Paul Brear, Jessie Iegre, Kathy H. Georgiou, Hannah F. Sore, Marko Hyvönen, David R. Spring

Bioorganic and Medicinal Chemistry, 25:3471-3482 (2017)
DOI: 10.1016/j.bmc.2017.04.037
Pubmed: 28495381

PDB coordinates: 5MMF (3D view ), 5MMR (3D view ), 5MO5 (3D view ), 5MO6 (3D view ), 5MO7 (3D view ), 5MO8 (3D view ), 5MOD (3D view ), 5MOE (3D view ), 5MOH (3D view ), 5MOT (3D view ), 5MOV (3D view ), 5MOW (3D view ), 5CU0 (3D view ), 5CU2 (3D view ), 5CT0 (3D view ), 5CTP (3D view ), 5CX9 (3D view )


Recently we reported the discovery of a potent and selective CK2α inhibitor CAM4066. This compound inhibits CK2 activity by exploiting a pocket located outside the ATP binding site (αD pocket). Here we describe in detail the journey that led to the discovery of CAM4066 using the challenging fragment linking strategy. Specifically, we aimed to develop inhibitors by linking a high-affinity fragment anchored in the αD site to a weakly binding warhead fragment occupying the ATP site. Moreover, we describe the remarkable impact that molecular modelling had on the development of this novel chemical tool. The work described herein shows potential for the development of a novel class of CK2 inhibitors.

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Structural analyses of von Willebrand factor C domains of collagen 2A and CCN3 reveal an alternative mode of binding to bone morphogenetic protein-2

Emma-Ruoqi Xu, Emily E. Blythe, Gerhard Fischer and  Marko Hyvönen

Journal of Biological Chemistry, 292:12516-12527, 2017
Pubmed ID: 28584056
PDB coordinates: 5NIR (3D view), 5NB8 (3D view)


Bone morphogenetic proteins (BMPs) are secreted growth factors that promote differentiation processes in embryogenesis and tissue development. Regulation of BMP signalling involves binding to a variety of extracellular proteins, among which are many von Willebrand factor C (vWC) domain-containing proteins. While the crystal structure of the complex of crossveinless-2 (CV-2) vWC1 and BMP-2 previously revealed one mode of the vWC:BMP binding mechanism, other vWC domains may bind to BMP differently.  Continue reading →

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Development of a multipurpose scaffold for the display of peptide loops

Maxim Rossmann, Sandra J. Greive, Tommaso Moschetti, Michael Dinan, Marko Hyvönen

Protein Engineering, Design and Selection 30: 419–430 (2017)
DOI: 10.1093/protein/gzx017
Pubmed: 228444399


Protein-protein interactions (PPIs) determine a wide range of biological processes and analysis of these dynamic networks is increasingly becoming a mandatory tool for studying protein function. Using the globular ATPase domain of recombinase RadA as a scaffold, we have developed a peptide display system (RAD display), which allows for the presentation of target peptides, protein domains or full-length proteins and their rapid recombinant production in bacteria. Continue reading →

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Specific inhibition of CK2a from an anchor outside the active site

Paul Brear, Claudia De Fusco, Kathy Hadje Georgiou, Nicola J. Francis-Newton, Christopher J. Stubbs,  Hannah F. Sore, Ashok R. Venkitaraman, Chris Abell, David R. Spring and Marko Hyvönen

Chemical Science, 7(11):6839-6845, 2016
Pubmed: 28451126
PDB coordinates:
5CVH (3D view), 5CVG (3D view), 5CVF (3D view), 5CU3 (3D view), 5CU4 (3D view), 5CU6 (3D view), 5CSH (3D view), 5CSP (3D view), 5CSV (3D view), 5CSH (3D view), 5CS6 (3D view), 5CLP (3D view)


The development of selective inhibitors of protein kinases is challenging because of the significant conservation of the ATP binding site. Here, we describe a new mechanism by which the protein kinase CK2α can be selectively inhibited using features outside the ATP site. We have identified a new binding site for small molecules on CK2α adjacent to the ATP site and behind the αD loop, termed the αD pocket. An elaborated fragment anchored in this site has been linked with a low affinity fragment binding in the ATP site, creating a novel and selective inhibitor (CAM4066) that binds CK2α with a Kd of 320 nM and shows significantly improved selectivity compared to other CK2α inhibitors. CAM4066 shows target engagement in several cell lines and similar potency to clinical trial candidate CX4945. Our data demonstrate that targeting a poorly conserved, cryptic pocket allows inhibition of CK2α via a novel mechanism, enabling the development of a new generation of selective CK2α inhibitors.

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Allosteric modulation of AURKA kinase activity by a small-molecule inhibitor of its protein-protein interaction with TPX2

Matej Janeček, Maxim Rossmann, Pooja Sharma, Amy Emery, David J. Huggins, Simon R. Stockwell, Jamie E. Stokes, Yaw S. Tan, Estrella Guarino Almeida, Bryn Hardwick, Ana J. Narvaez, Marko Hyvönen, David R. Spring, Grahame J. McKenzie & Ashok R. Venkitaraman

Scientific Reports 6, Article number: 28528 (2016)
DOI: 10.1038/srep28528
Pubmed: 27339427

PDB coordinates: 5DNR (3D view ), 5DT3 (3D view ), 5DT0 (3D view ), 5RDR (3D view ), 5DR9 (3D view ), 5DR2 (3D view ), 5DR6 (3D view ), 5DOS (3D view ), 5DPV (3D view ), 5DT4 (3D view ), 5DN3 (3D view )


The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the ‘Y pocket’) that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases.

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Structure of Gremlin-1 and analysis of its interaction with BMP-2

Miglė Kišonaitė, Xuelu Wang and Marko Hyvönen

Biochemical Journal 473, 1593-1604 (2016)
DOI: 10.1042/BCJ20160254
Pubmed: 27373274
PDB coordinates: 5AEJ (3D view)


Bone morphogenetic protein 2 (BMP-2) is a member of the transforming growth factor-β (TGF-β) signalling family and has a very broad biological role in development. Its signalling is regulated by many effectors: transmembrane proteins, membrane-attached proteins and soluble secreted antagonists such as Gremlin-1. Very little is known about the molecular mechanism by which Gremlin-1 and other DAN (differential screening-selected gene aberrative in neuroblastoma) family proteins inhibit BMP signalling. We analysed the interaction of Gremlin-1 with BMP-2 using a range of biophysical techniques, and used mutagenesis to map the binding site on BMP-2. We have also determined the crystal structure of Gremlin-1, revealing a similar conserved dimeric structure to that seen in other DAN family inhibitors. Measurements using biolayer interferometry (BLI) indicate that Gremlin-1 and BMP-2 can form larger complexes, beyond the expected 1:1 stoichiometry of dimers, forming oligomers that assemble in alternating fashion. These results suggest that inhibition of BMP-2 by Gremlin-1 occurs by a mechanism that is distinct from other known inhibitors such as Noggin and Chordin and we propose a novel model of BMP-2–Gremlin-1 interaction yet not seen among any BMP antagonists, and cannot rule out that several different oligomeric states could be found, depending on the concentration of the two proteins.

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ATP half‐sites in RadA and RAD51 recombinases bind nucleotides

May E. Marsh, Duncan E. Scott, Matthias T. Ehebauer, Chris Abell, Tom L. Blundell and Marko Hyvönen

FEBS OpenBio 6:372–385 (2016)
DOI: 10.1002/2211-5463.12052
Pubmed: 27419043

PDB coordinates: 4UQO (3D view),4D6P (3D view),4B2P (3D view),4A6X (3D view),4A6P (3D view)


Homologous recombination is essential for repair of DNA double-strand breaks. Central to this process is a family of recombinases, including archeal RadA and human RAD51, which form nucleoprotein filaments on damaged single-stranded DNA ends and facilitate their ATP-dependent repair. ATP binding and hydrolysis are dependent on the formation of a nucleoprotein filament comprising RadA/RAD51 and single-stranded DNA, with ATP bound between adjacent protomers. We demonstrate that truncated, monomeric Pyrococcus furiosus RadA and monomerised human RAD51 retain the ability to bind ATP and other nucleotides with high affinity. We present crystal structures of both apo and nucleotide-bound forms of monomeric RadA. These structures reveal that while phosphate groups are tightly bound, RadA presents a shallow, poorly defined binding surface for the nitrogenous bases of nucleotides. We suggest that RadA monomers would be constitutively bound to nucleotides in the cell and that the bound nucleotide might play a structural role in filament assembly.

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