Balancing Specificity and Promiscuity in Enzyme Evolution: Multidimensional Activity Transitions in the Alkaline Phosphatase Superfamily

Bert van Loo, Christopher D. Bayer, Gerhard Fischer, Stefanie Jonas, Eugene Valkov, Mark F. Mohamed, Anastassia Vorobieva, Celine Dutruel, Marko Hyvönen and Florian Hollfelder

Journal of American Chemical Society 141:370-387 (2019)
DOI: 10.1021/jacs.8b10290
Pubmed: 30497259

PDB coordinates:
4UPH (3D view), 4UPI (3D view), 4UPL (3D view), 4UPK (3D view)


Highly proficient, promiscuous enzymes can be springboards for functional evolution, able to avoid loss of function during adaptation by their capacity to promote multiple reactions. We employ a systematic comparative study of structure, sequence, and substrate specificity to track the evolution of specificity and reactivity between promiscuous members of clades of the alkaline phosphatase (AP) superfamily. Construction of a phylogenetic tree of protein sequences maps out the likely transition zone between arylsulfatases (ASs) and phosphonate monoester hydrolases (PMHs). Kinetic analysis shows that all enzymes characterized have four chemically distinct phospho- and sulfoesterase activities, with rate accelerations ranging from 1011- to 1017-fold for their primary and 109- to 1012-fold for their promiscuous reactions, suggesting that catalytic promiscuity is widespread in the AP-superfamily. Continue reading →

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A general approach for the site-selective modification of native proteins, enabling the generation of stable and functional antibody–drug conjugates

Stephen J. Walsh, Soleilmane Omarjee, Warren R. J. D. Galloway, Terence T.-L. Kwan, Hannah F. Sore, Jeremy S. Parker, Marko Hyvönen, Jason S. Carroll and David R. Spring

Chem. Sci., 2019,10, 694-700

DOI: 10.1039/C8SC04645J
Pubmed: 0774870


<Antibody–drug conjugates (ADCs) are a class of targeted therapeutics that utilize the specificity of antibodies to selectively deliver highly potent cytotoxins to target cells. Although recent years have witnessed significant interest in ADCs, problems remain with the standard linkage chemistries used for cytotoxin-antibody bioconjugation. These typically (1) generate unstable constructs, which may lead to premature cytotoxin release, (2) often give a wide variance in drug–antibody ratios (DAR) and (3) have poor control of attachment location on the antibody, resulting in a variable pharmacokinetic profile. Herein, we report a novel divinylpyrimidine (DVP) linker platform for selective bioconjugation via covalent re-bridging of reduced disulfide bonds on native antibodies. Continue reading →

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Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset

Charlotte M. Miton, Stefanie Jonas, Gerhard Fischer, Fernanda Duarte, Mark F. Mohamed, Bert van Loo, Bálint Kintses, Shina C. L. Kamerlin, Nobuhiko Tokuriki, Marko Hyvönen, and Florian Hollfelder

Proceedings of the National Academy of Sciences of the USA (2017)

DOI: 10.1073/pnas.1607817115
Pubmed: 30012610

PDB coordinates:

4CYR (3D view), 4CXS (3D view), 4CXU (3D view), 4CYS (3D view)
5AJ9 (3D view), 4CXK (3D view)


The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono- and diester hydrolyses were only marginally affected (≤50-fold). Continue reading →

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Novel non-ATP competitive small molecules targeting the CK2 α/β interface

Paul Brear, Andrew North, Jessica Iegre, Kathy Hadje Georgiou, Alexandra Lubin, Laura Carro, William Green, Hannah F.Sore, Marko Hyvönen, David R.Spring

Bioorganic & Medicinal Chemistry 26:3016-3020 (2018)
DOI: 10.1016/j.bmc.2018.05.011
Pubmed: 29759799

PDB coordinates:


Increased CK2 levels are prevalent in many cancers. Combined with the critical role CK2 plays in many cell-signaling pathways, this makes it a prime target for down regulation to fight tumour growth. Herein, we report a fragment-based approach to inhibiting the interaction between CK2α and CK2β at the α-β interface of the holoenzyme. A fragment, CAM187, with an IC50 of 44 μM and a molecular weight of only 257 gmol-1 has been identified as the most promising compound. Importantly, the lead fragment only bound at the interface and was not observed in the ATP binding site of the protein when co-crystallised with CK2α. The fragment-like molecules discovered in this study represent unique scaffolds to CK2 inhibition and leave room for further optimisation.

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Second-generation CK2α inhibitors targeting the αD pocket

Jessica Iegre, Paul Brear, Claudia De Fusco, Masao Yoshida, Sophie L. Mitchell, Maxim Rossmann, Laura Carro Santos, Hannah F. Sore, Marko Hyvӧnen and David R. Spring

Chemical Science (2018) (9:3041-)
DOI: 10.1039/C7SC05122K
Pubmed: 29732088

PDB coordinates:

6EHU (3D view ), 5OTQ (3D view ), 5OTY (3D view ), 6EHK (3D view ), 5OTI (3D view ), 5OTL (3D view ), 5OTO (3D view ), 5OYF (3D view ), 5OSZ (3D view ), 5OT5 (3D view ), 5OTD (3D view ), 5OTH (3D view ), 5OT6 (3D view ), 5OUE (3D view ), 5OUM (3D view ), 5OUU (3D view ), 5OS8 (3D view ), 5OTR (3D view ), 6EII (3D view ), 5OQU (3D view ), 5ORK (3D view ), 5OSL (3D view ), 5OUL (3D view ), 5ORH (3D view ), 5ORJ (3D view ), 5OS7 (3D view )


CK2 is a critical cell cycle regulator that also promotes various anti-apoptotic mechanisms. Development of ATP-non-competitive inhibitors of CK2 is a very attractive strategy considering that the ATP binding site is highly conserved among other kinases. We have previously utilised a pocket outside the active site to develop a novel CK2 inhibitor, CAM4066. Whilst CAM4066 bound to this new pocket it was also interacting with the ATP site: herein, we describe an example of a CK2α inhibitor that binds completely outside the active site. Continue reading →

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Structural and Mechanistic Analysis of the Choline Sulfatase from Sinorhizobium melliloti: A Class I Sulfatase Specific for an Alkyl Sulfate Ester

Bert van Loo, Markus Schober, Eugene Valkov, Magdalena Heberlein, Erich Bornberg-Bauer, Kurt Faber, Marko Hyvönen, Florian Hollfelder

Journal of Molecular Biology, 430:1004-1023, 2018 
DOI: 10.1016/j.jmb.2018.02.010
Pubmed: 29458126
PDB coordinates:
6FNY (3D view )



Hydrolysis of organic sulfate esters proceeds by two distinct mechanisms, water attacking at either sulfur (S-O bond cleavage) or carbon (C-O bond cleavage). In primary and secondary alkyl sulfates attack at carbon is favored, whereas in aromatic sulfates and sulfated sugars attack at sulfur is preferred. This mechanistic distinction is mirrored in the classification of enzymes that catalyze sulfate ester hydrolysis: arylsulfatases catalyze S-O cleavage in sulfate sugars and arylsulfates and alkyl sulfatases break the C-O bond of alkyl sulfates. Continue reading →

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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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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:
PDB coordinates: 5NTU (3D view ), 5NXS (3D view )


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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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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