I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Julian M.
Ludäscher
,
Emma
Scaletti Hutchinson
,
Guillem
Vila-Julià
,
Ann-Sofie
Jemth
,
Saher
Shahid
,
Elisee
Wiita
,
Israel
Cabeza De Vaca
,
Szymon
Pach
,
Lukas
Gajdos
,
Swati
Aggarwal
,
Ellen
Walse
,
Oliver
Mortusewicz
,
Thomas
Helleday
,
Jens
Carlsson
,
Pal
Stenmark
Diamond Proposal Number(s):
[29948]
Open Access
Abstract: Human single-strand-selective monofunctional uracil DNA glycosylase 1 (hSMUG1) removes uracil, 5-hydroxymethyluracil (5hmU) and 5-fluorouracil (5FU) from DNA, thereby initiating the base excision repair (BER) process. hSMUG1 is important for maintaining genomic integrity and plays a significant role in cancer biology. Here, we present the structures of hSMUG1, including complexes with products (uracil and 5FU) and an enzyme-product complex of hSMUG1 with double-stranded DNA (dsDNA). Analysis of our hSMUG1-dsDNA complex reveals how uracil is flipped out of the dsDNA for excision and identifies key residues that we confirm to be critical for both DNA binding and enzymatic activity. Furthermore, our hSMUG1 substrate complexes, molecular dynamics simulations and neutron diffraction data suggest a mechanism by which the substrate uracil rotates following base excision. The structural and functional information presented here will be highly useful for the future development of inhibitors and/or activators targeting hSMUG1.
|
Jun 2026
|
|
I03-Macromolecular Crystallography
|
Emily C.
Hank
,
Nicholas D.
D'Arcy-Evans
,
Emma R.
Scaletti
,
Carlos
Benítez-Buelga
,
Olov
Wallner
,
Florian
Ortis
,
Kaixin
Zhou
,
Liuzhen
Meng
,
Alicia
Del Prado
,
Patricia
Calvo
,
Ingrid
Almlöf
,
Elisée
Wiita
,
Karen
Nierlin
,
Sara
Kosenina
,
Andreas
Krämer
,
Alice
Eddershaw
,
Mario
Kehler
,
Maeve
Long
,
Ann-Sofie
Jemth
,
Holly
Dawson
,
Josephine
Stewart
,
Adam
Dickey
,
Mikhael E.
Astorga
,
Marek
Varga
,
Evert J.
Homan
,
Martin
Scobie
,
Stefan
Knapp
,
Leandro
Sastre
,
Pal
Stenmark
,
Miguel
De Vega
,
Thomas
Helleday
,
Maurice
Michel
Diamond Proposal Number(s):
[15806, 21625]
Open Access
Abstract: Bifunctional DNA glycosylases employ an active site lysine or the N-terminus to form a Schiff base with an abasic (AP) site base excision repair intermediate. For 8-oxoguanine DNA glycosylase 1 (OGG1), cleaving this reversible structure is the rate-determining step in the initiation of 8-oxoguanine (8-oxoG) repair in DNA. Evolution has led OGG1 to use a product-assisted catalysis approach, where the excised 8-oxoG acts as a Brønsted base for cleavage of a Schiff base intermediate. However, the physicochemical properties of 8-oxoG significantly limit the inherent enzymatic turnover leading to a weak, cellularly absent, AP lyase activity. We hypothesized that chemical synthesis of purine analogues enables access to complex structures that are suitable as product-like catalysts. Herein, the nucleobase landscape is profiled for its potential to increase OGG1 Schiff base cleavage. 8-Substituted 6-thioguanines emerge as potent and selective scaffolds enabling OGG1 to cleave AP sites opposite any canonical nucleobase by β-elimination. This effectively broadens the enzymatic substrate scope of OGG1, shaping a complete, artificial AP-lyase function. In addition, a second class of compounds, 6-substituted pyrazolo-[3,4-d]-pyrimidines, stimulate OGG1 function at high pH, while thioguanines govern enzymatic control at acidic pH. This enables up to 20-fold increased enzyme turnover and a de novo OGG1 β-elimination in conditions commonly not tolerated. The tool compounds employed here are non-toxic in cells and stimulate the repair of AP sites through a natural, APE1 dependent pathway, as opposed to previously reported β,δ-lyase stimulator TH10785.
|
Oct 2025
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[21625]
Open Access
Abstract: Isoprene pyrophosphates play a crucial role in the synthesis of a diverse array of essential nonsterol and sterol biomolecules and serve as substrates for posttranslational isoprenylation of proteins, enabling specific anchoring to cellular membranes. Hydrolysis of isoprene pyrophosphates would be a means to modulate their levels, downstream products, and protein isoprenylation. While NUDIX hydrolases from plants have been described to catalyze the hydrolysis of isoprene pyrophosphates, homologous enzymes with this function in animals have not yet been reported. In this study, we screened an extensive panel of human NUDIX hydrolases for activity in hydrolyzing isoprene pyrophosphates. We found that human nucleotide triphosphate diphosphatase NUDT15 and 8-oxo-dGDP phosphatase NUDT18 efficiently catalyze the hydrolysis of several physiologically relevant isoprene pyrophosphates. Notably, we demonstrate that geranyl pyrophosphate is an excellent substrate for NUDT18, with a catalytic efficiency of 2.1 × 105 m−1·s−1, thus making it the best substrate identified for NUDT18 to date. Similarly, geranyl pyrophosphate proved to be the best isoprene pyrophosphate substrate for NUDT15, with a catalytic efficiency of 4.0 × 104 M−1·s−1. LC–MS analysis of NUDT15 and NUDT18 catalyzed isoprene pyrophosphate hydrolysis revealed the generation of the corresponding monophosphates and inorganic phosphate. Furthermore, we solved the crystal structure of NUDT15 in complex with the hydrolysis product geranyl phosphate at a resolution of 1.70 Å. This structure revealed that the active site nicely accommodates the hydrophobic isoprenoid moiety and helped identify key binding residues. Our findings imply that isoprene pyrophosphates are endogenous substrates of NUDT15 and NUDT18, suggesting they are involved in animal isoprene pyrophosphate metabolism.
|
Jun 2024
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Maurice
Michel
,
Carlos
Benítez-Buelga
,
Patricia A.
Calvo
,
Bishoy M. F.
Hanna
,
Oliver
Mortusewicz
,
Geoffrey
Masuyer
,
Jonathan
Davies
,
Olov
Wallner
,
Sanjiv
Kumar
,
Julian J.
Albers
,
Sergio
Castañeda-Zegarra
,
Ann-Sofie
Jemth
,
Torkild
Visnes
,
Ana
Sastre-Perona
,
Akhilesh N.
Danda
,
Evert J.
Homan
,
Karthick
Marimuthu
,
Zhao
Zhenjun
,
Celestine N.
Chi
,
Antonio
Sarno
,
Elisée
Wiita
,
Catharina
Von Nicolai
,
Anna J.
Komor
,
Varshni
Rajagopal
,
Sarah
Müller
,
Emily C.
Hank
,
Marek
Varga
,
Emma R.
Scaletti
,
Monica
Pandey
,
Stella
Karsten
,
Hanne
Haslene-Hox
,
Simon
Loevenich
,
Petra
Marttila
,
Azita
Rasti
,
Kirill
Mamonov
,
Florian
Ortis
,
Fritz
Schömberg
,
Olga
Loseva
,
Josephine
Stewart
,
Nicholas
D’arcy-Evans
,
Tobias
Koolmeister
,
Martin
Henriksson
,
Dana
Michel
,
Ana
De Ory
,
Lucia
Acero
,
Oriol
Calvete
,
Martin
Scobie
,
Christian
Hertweck
,
Ivan
Vilotijevic
,
Christina
Kalderén
,
Ana
Osorio
,
Rosario
Perona
,
Alexandra
Stolz
,
Pal
Stenmark
,
Ulrika
Warpman Berglund
,
Miguel
De Vega
,
Thomas
Helleday
Diamond Proposal Number(s):
[15806, 21625]
Abstract: Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed β,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.
|
Jun 2022
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Nadilly
Bonagas
,
Nina M. S.
Gustafsson
,
Martin
Henriksson
,
Petra
Marttila
,
Robert
Gustafsson
,
Elisée
Wiita
,
Sanjay
Borhade
,
Alanna C.
Green
,
Karl S. A.
Vallin
,
Antonio
Sarno
,
Richard
Svensson
,
Camilla
Göktürk
,
Therese
Pham
,
Ann-Sofie
Jemth
,
Olga
Loseva
,
Victoria
Cookson
,
Nicole
Kiweler
,
Lars
Sandberg
,
Azita
Rasti
,
Judith E.
Unterlass
,
Martin
Haraldsson
,
Yasmin
Andersson
,
Emma R.
Scaletti
,
Christoffer
Bengtsson
,
Cynthia B. J.
Paulin
,
Kumar
Sanjiv
,
Eldar
Abdurakhmanov
,
Linda
Pudelko
,
Ben
Kunz
,
Matthieu
Desroses
,
Petar
Iliev
,
Katarina
Färnegårdh
,
Andreas
Krämer
,
Neeraj
Garg
,
Maurice
Michel
,
Sara
Häggblad
,
Malin
Jarvius
,
Christina
Kalderén
,
Amanda Bögedahl
Jensen
,
Ingrid
Almlöf
,
Stella
Karsten
,
Si Min
Zhang
,
Maria
Häggblad
,
Anders
Eriksson
,
Jianping
Liu
,
Björn
Glinghammar
,
Natalia
Nekhotiaeva
,
Fredrik
Klingegård
,
Tobias
Koolmeister
,
Ulf
Martens
,
Sabin
Llona-Minguez
,
Ruth
Moulson
,
Helena
Nordström
,
Vendela
Parrow
,
Leif
Dahllund
,
Birger
Sjöberg
,
Irene L.
Vargas
,
Duy Duc
Vo
,
Johan
Wannberg
,
Stefan
Knapp
,
Hans E.
Krokan
,
Per I.
Arvidsson
,
Martin
Scobie
,
Johannes
Meiser
,
Pal
Stenmark
,
Ulrika Warpman
Berglund
,
Evert J.
Homan
,
Thomas
Helleday
Open Access
Abstract: The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors.
|
Feb 2022
|
|
I24-Microfocus Macromolecular Crystallography
|
Daniel
Rehling
,
Si Min
Zhang
,
Ann-Sofie
Jemth
,
Tobias
Koolmeister
,
Adam
Throup
,
Olov
Wallner
,
Emma
Scaletti
,
Takaya
Moriyama
,
Rina
Nishii
,
Jonathan
Davies
,
Matthieu
Desroses
,
Sean G.
Rudd
,
Martin
Scobie
,
Evert
Homan
,
Ulrika Warpman
Berglund
,
Jun J.
Yang
,
Thomas
Helleday
,
Pal
Stenmark
Diamond Proposal Number(s):
[21625]
Open Access
Abstract: The enzyme NUDT15 efficiently hydrolyses the active metabolites of thiopurine drugs, which are routinely used for treating cancer and inflammatory diseases. Loss-of-function variants in NUDT15 are strongly associated with thiopurine intolerance, such as leukopenia, and pre-emptive NUDT15 genotyping has been clinically implemented to personalize thiopurine dosing. However, understanding the molecular consequences of these variants has been difficult, as no structural information was available for NUDT15 proteins encoded by clinically actionable pharmacogenetic variants due to their inherent instability. Recently, the small molecule NUDT15 inhibitor TH1760 has been shown to sensitize cells to thiopurines, through enhanced accumulation of 6-thio-guanine in DNA. Building upon this, we herein report the development of the potent and specific NUDT15 inhibitor, TH7755. TH7755 demonstrates a greatly improved cellular target engagement and 6-thioguanine potentiation compared to TH1760, while showing no cytotoxicity on its own. This potent inhibitor also stabilized NUDT15, enabling analysis by X-ray crystallography. We have determined high-resolution structures of the clinically relevant NUDT15 variants Arg139Cys, Arg139His, Val18Ile and V18_V19insGlyVal. These structures provide clear insights into the structural basis for the thiopurine intolerance phenotype observed in patients carrying these pharmacogenetic variants. These findings will aid in predicting the effects of new NUDT15 sequence variations yet to be discovered in the clinic.
|
Mar 2021
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[11171, 15868]
Open Access
Abstract: Phosphoglucomutase 5 (PGM5) in humans is known as a structural muscle protein without enzymatic activity, but detailed understanding of its function is lacking. PGM5 belongs to the alpha-D-phosphohexomutase family and is closely related to the enzymatically active metabolic enzyme PGM1. In the Atlantic herring, Clupea harengus, PGM5 is one of the genes strongly associated with ecological adaptation to the brackish Baltic Sea. We here present the first crystal structures of PGM5, from the Atlantic and Baltic herring, differing by a single substitution Ala330Val. The structure of PGM5 is overall highly similar to structures of PGM1. The structure of the Baltic herring PGM5 in complex with the substrate glucose-1-phosphate shows conserved substrate binding and active site compared to human PGM1, but both PGM5 variants lack phosphoglucomutase activity under the tested conditions. Structure comparison and sequence analysis of PGM5 and PGM1 from fish and mammals suggest that the lacking enzymatic activity of PGM5 is related to differences in active-site loops that are important for flipping of the reaction intermediate. The Ala330Val substitution does not alter structure or biophysical properties of PGM5 but, due to its surface-exposed location, could affect interactions with protein-binding partners.
|
Dec 2020
|
|
I04-Macromolecular Crystallography
|
Torkild
Visnes
,
Carlos
Benítez-Buelga
,
Armando
Cázares-Körner
,
Kumar
Sanjiv
,
Bishoy M. F.
Hanna
,
Oliver
Mortusewicz
,
Varshni
Rajagopal
,
Julian J.
Albers
,
Daniel W
Hagey
,
Tove
Bekkhus
,
Saeed
Eshtad
,
Juan Miguel
Baquero
,
Geoffrey
Masuyer
,
Olov
Wallner
,
Sarah
Müller
,
Therese
Pham
,
Camilla
Göktürk
,
Azita
Rasti
,
Sharda
Suman
,
Raúl
Torres-Ruiz
,
Antonio
Sarno
,
Elisée
Wiita
,
Evert J.
Homan
,
Stella
Karsten
,
Karthick
Marimuthu
,
Maurice
Michel
,
Tobias
Koolmeister
,
Martin
Scobie
,
Olga
Loseva
,
Ingrid
Almlöf
,
Judith Edda
Unterlass
,
Aleksandra
Pettke
,
Johan
Boström
,
Monica
Pandey
,
Helge
Gad
,
Patrick
Herr
,
Ann-Sofie
Jemth
,
Samir
El andaloussi
,
Christina
Kalderén
,
Sandra
Rodriguez-Perales
,
Javier
Benítez
,
Hans E
Krokan
,
Mikael
Altun
,
Pal
Stenmark
,
Ulrika Warpman
Berglund
,
Thomas
Helleday
Diamond Proposal Number(s):
[15806]
Open Access
Abstract: Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.
|
Nov 2020
|
|
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[15806]
Open Access
Abstract: The bifunctional human enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) catalyzes two essential steps in the de novo purine biosynthesis pathway. PAICS is overexpressed in many cancers and could be a promising target for the development of cancer therapeutics. Here, using gene knockdowns and clonogenic survival and cell viability assays, we demonstrate that PAICS is required for growth and survival of prostate cancer cells. PAICS catalyzes the carboxylation of aminoimidazole ribonucleotide (AIR) and the subsequent conversion of carboxyaminoimidazole ribonucleotide (CAIR) and L-aspartate to N-succinylcarboxamide-5-aminoimidazole ribonucleotide (SAICAR). Of note, we present the first structures of human octameric PAICS in complexes with native ligands. In particular, we report the structure of PAICS with CAIR bound in the active sites of both domains and SAICAR bound in one of the SAICAR synthetase domains. Moreover, we report the PAICS structure with SAICAR and an ATP analog occupying the SAICAR synthetase active site. These structures provide insight into substrate and product binding and the architecture of the active sites, disclosing important structural information for rational design of PAICS inhibitors as potential anticancer drugs.
|
Jun 2020
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[15806]
Open Access
Abstract: MutT homologue 1 (MTH1) removes oxidized nucleotides from the nucleotide pool and thereby prevents their incorporation into the genome and thereby reduces genotoxicity. We previously reported that MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis suggesting that MTH1 may also sanitize the nucleotide pool from other methylated nucleotides. We here show that MTH1 efficiently catalyzes the hydrolysis of N6-methyl-dATP to N6-methyl-dAMP and further report that N6-methylation of dATP drastically increases the MTH1 activity. We also observed MTH1 activity with N6-methyl-ATP, albeit at a lower level. We show that N6-methyl-dATP is incorporated into DNA in vivo, as indicated by increased N6-methyl-dA DNA levels in embryos developed from MTH1 knock-out zebrafish eggs microinjected with N6-methyl-dATP compared with noninjected embryos. N6-methyl-dATP activity is present in MTH1 homologues from distantly related vertebrates, suggesting evolutionary conservation and indicating that this activity is important. Of note, N6-methyl-dATP activity is unique to MTH1 among related NUDIX hydrolases. Moreover, we present the structure of N6-methyl-dAMP-bound human MTH1, revealing that the N6-methyl group is accommodated within a hydrophobic active-site sub-pocket explaining why N6-methyl-dATP is a good MTH1 substrate. N6-methylation of DNA and RNA has been reported to have epigenetic roles and to affect mRNA metabolism. We propose that MTH1 acts in concert with adenosine deaminase-like protein isoform 1 (ADAL1) to prevent incorporation of N6-methyl-(d)ATP into DNA and RNA. This would hinder potential dysregulation of epigenetic control and RNA metabolism via conversion of N6-methyl-(d)ATP to N6-methyl-(d)AMP, followed by ADAL1 catalyzed deamination producing (d)IMP that can enter the nucleotide salvage pathway.
|
Mar 2020
|
|
