Marko

The thrombospondin module 1 domain of the matricellular protein CCN3 shows an atypical disulfide pattern and incomplete CWR layers

Emma-Ruoqi Xu, Aleix Lafita, Alex Bateman, Marko Hyvönen

Acta Crystallographica Section D Structural Biology D76:124-134 (2020)
DOI: 10.1107/S2059798319016747
Pubmed: 32038043

PDB coordinates:

6RK1 (3D view)

Abstract

The members of the CCN (Cyr61/CTGF/Nov) family are a group of matricellular regulatory proteins that are essential to a wide range of functional pathways in cell signalling. Through interacting with extracellular matrix components and growth factors via one of their four domains, the CCN proteins are involved in critical biological processes such as angiogenesis, cell proliferation, bone development, fibrogenesis and tumorigenesis. Here, the crystal structure of the thrombospondin module 1 (TSP1) domain of CCN3 (previously known as Nov) is presented, which shares a similar three-stranded fold with the thrombo­spondin type 1 repeats of thrombospondin-1 and spondin-1, but with variations in the disulfide connectivity. Continue reading →

Posted by Marko in Publications, 0 comments

A cryptic hydrophobic pocket in the polo-box domain of the polo-like kinase PLK1 regulates substrate recognition and mitotic chromosome segregation

Pooja Sharma, Robert Mahen, Maxim Rossmann, Jamie E. Stokes, Bryn Hardwick, David J. Huggins, Amy Emery, Dominique L. Kunciw, Marko Hyvönen, David R. Spring, Grahame J. McKenzie & Ashok R. Venkitaraman

Scientific Reports, 9:15930 (2019)
DOI: s41598-019-50702-2
Pubmed: tbd
PDB coordinates: 5NMM (3D view), 5NEI (3D view)

Abstract

The human polo-like kinase PLK1 coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins. How PLK1 activity is directed to specific substrates via phosphopeptide recognition by its carboxyl-terminal polo-box domain (PBD) is poorly understood. Here, we combine molecular, structural and chemical biology to identify a determinant for PLK1 substrate recognition that is essential for proper chromosome segregation. We show that mutations ablating an evolutionarily conserved, Tyr-lined pocket in human PLK1 PBD trigger cellular anomalies in mitotic progression and timing. Continue reading →

Posted by Marko in Publications, 0 comments

Organoid culture media formulated with growth factors of defined cellular activity

Manuela Urbischek, Helena Rannikmae, Thomas Foets, Katharina Ravn, Marko Hyvönen & Marc de la Roche

Scientific Reports 6193 (2019)
DOI: 0.1038/s41598-019-42604-0
Pubmed: 30996238

Abstract


The media formulations necessary for deriving and sustaining organoids from epithelial tissues such as prostate, colon, gastric, liver, pancreas, and others have been established. Critical components of organoid media are a set of growth factors that include R-spondins and BMP signalling antagonists such as Noggin or Gremlin 1. Currently, the practical limitations for formulating organoid media of reproducible potency and larger-scale media production that have hampered further technological applications of organoid technology include: the cost of growth factors such as R-spondins and Gremlin 1/Noggin and their production as defined specific activities free of contaminants that may affect organoid growth. Continue reading →

Posted by Marko in Publications, 0 comments

Efficient development of stable and highly functionalised peptides targeting the CK2α/CK2β protein–protein interaction

Jessica Iegre, Paul Brear, David J. Baker, Yaw Sing Tan, Eleanor L. Atkinson, Hannah F. Sore, Daniel H. O’ Donovan, Chandra S. Verma, Marko Hyvönen and David R. Spring

Chem. Sci., advance publication (2019)

DOI: 10.1039/C9SC00798A
Pubmed: TBC

PDB coordinates:

6Q38 (3D view)
6Q4Q (3D view)

Abstract

The discovery of new Protein–Protein Interaction (PPI) modulators is currently limited by the difficulties associated with the design and synthesis of selective small molecule inhibitors. Peptides are a potential solution for disrupting PPIs; however, they typically suffer from poor stability in vivo and limited tissue penetration hampering their wide spread use as new chemical biology tools and potential therapeutics. In this work, a combination of CuAAC chemistry, molecular modelling, X-ray crystallography, and biological validation allowed us to develop highly functionalised peptide PPI inhibitors of the protein CK2. Continue reading →

Posted by Marko in Publications, 0 comments

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)

Abstract

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 →

Posted by Marko in Publications, 0 comments

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

Abstract

<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 →

Posted by Marko in Publications, 0 comments

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)

Abstract

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 →

Posted by Marko in Publications, 0 comments

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:

Abstract

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.

Posted by Marko in Publications, 0 comments

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 )

Abstract

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 →

Posted by Marko in Publications, 0 comments

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 )

 

Abstract

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 →

Posted by Marko in Publications, 0 comments