I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Mathieu
Ferrari
,
Matteo
Righi
,
Vania
Baldan
,
Patrycja
Wawrzyniecka
,
Anna
Bulek
,
Alexander
Kinna
,
Biao
Ma
,
Reyisa
Bughda
,
Zulaikha
Akbar
,
Saket
Srivastava
,
Isaac
Gannon
,
Mathew
Robson
,
James
Sillibourne
,
Ram
Jha
,
Mohamed
El-Kholy
,
Oliver Muhammad
Amin
,
Evangelia
Kokalaki
,
Mohammed Amin
Banani
,
Rehan
Hussain
,
William
Day
,
Wen Chean
Lim
,
Priyanka
Ghongane
,
Jade R.
Hopkins
,
Dennis
Jungherz
,
Marco
Herling
,
Martin
Welin
,
Sachin
Surade
,
Michael
Dyson
,
John
Mccafferty
,
Derek
Logan
,
Shaun
Cordoba
,
Simon
Thomas
,
Andrew
Sewell
,
Paul
Maciocia
,
Shimobi
Onuoha
,
Martin
Pule
Open Access
Abstract: Peripheral T cell lymphomas are typically aggressive with a poor prognosis. Unlike other hematologic malignancies, the lack of target antigens to discriminate healthy from malignant cells limits the efficacy of immunotherapeutic approaches. The T cell receptor expresses one of two highly homologous chains [T cell receptor β-chain constant (TRBC) domains 1 and 2] in a mutually exclusive manner, making it a promising target. Here we demonstrate specificity redirection by rational design using structure-guided computational biology to generate a TRBC2-specific antibody (KFN), complementing the antibody previously described by our laboratory with unique TRBC1 specificity (Jovi-1) in targeting broader spectrum of T cell malignancies clonally expressing either of the two chains. This permits generation of paired reagents (chimeric antigen receptor-T cells) specific for TRBC1 and TRBC2, with preclinical evidence to support their efficacy in T cell malignancies.
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Feb 2024
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I03-Macromolecular Crystallography
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Josefin
Ahlqvist
,
Javier A.
Linares-Pastén
,
Andrius
Jasilionis
,
Martin
Welin
,
Maria
Håkansson
,
L. Anders
Svensson
,
Lei
Wang
,
Hildegard
Watzlawick
,
Arnþór
Ævarsson
,
Ólafur H.
Friðjónsson
,
Guðmundur Ó.
Hreggviðsson
,
Bernd
Ketelsen Striberny
,
Eirin
Glomsaker
,
Olav
Lanes
,
Salam
Al-Karadaghi
,
Eva
Nordberg Karlsson
Diamond Proposal Number(s):
[20028, 23282]
Open Access
Abstract: This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3′–5′ exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5′–3′ exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SβαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SβαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3′–5′ exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SβαR motif, was first determined to 2.19 Å resolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Å resolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.
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Nov 2022
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I04-Macromolecular Crystallography
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Katarzyna
Składanowska
,
Yehudi
Bloch
,
Jamie
Strand
,
Kerry F.
White
,
Jing
Hua
,
Daniel
Aldridge
,
Martin
Welin
,
Derek T.
Logan
,
Arne
Soete
,
Romain
Merceron
,
Casey
Murphy
,
Mathias
Provost
,
J. Fernando
Bazan
,
Christopher A.
Hunter
,
Jonathan A.
Hill
,
Savvas N.
Savvidis
Diamond Proposal Number(s):
[23282]
Open Access
Abstract: Interleukin-27 (IL-27) uniquely assembles p28 and EBI3 subunits to a heterodimeric cytokine that signals via IL-27Rα and gp130. To provide the structural framework for receptor activation by IL-27 and its emerging therapeutic targeting, we report here crystal structures of mouse IL-27 in complex with IL-27Rα and of human IL-27 in complex with SRF388, a monoclonal antibody undergoing clinical trials with oncology indications. One face of the helical p28 subunit interacts with EBI3, while the opposite face nestles into the interdomain elbow of IL-27Rα to juxtapose IL-27Rα to EBI3. This orients IL-27Rα for paired signaling with gp130, which only uses its immunoglobulin domain to bind to IL-27. Such a signaling complex is distinct from those mediated by IL-12 and IL-23. The SRF388 binding epitope on IL-27 overlaps with the IL-27Rα interaction site explaining its potent antagonistic properties. Collectively, our findings will facilitate the mechanistic interrogation, engineering, and therapeutic targeting of IL-27.
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Oct 2022
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I04-Macromolecular Crystallography
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Martina
Durcik
,
Ákos
Nyerges
,
Žiga
Skok
,
Darja Gramec
Skledar
,
Jurij
Trontelj
,
Nace
Zidar
,
Janez
Ilaš
,
Anamarija
Zega
,
Cristina D.
Cruz
,
Päivi
Tammela
,
Martin
Welin
,
Yengo R.
Kimbung
,
Dorota
Focht
,
Ondřej
Benek
,
Tamás
Révész
,
Gábor
Draskovits
,
Petra Éva
Szili
,
Lejla
Daruka
,
Csaba
Pál
,
Danijel
Kikelj
,
Lucija Peterlin
Mašič
,
Tihomir
Tomašič
Diamond Proposal Number(s):
[20028]
Abstract: The rise in multidrug-resistant bacteria defines the need for identification of new antibacterial agents that are less prone to resistance acquisition. Compounds that simultaneously inhibit multiple bacterial targets are more likely to suppress the evolution of target-based resistance than monotargeting compounds. The structurally similar ATP binding sites of DNA gyrase and topoisomerase Ⅳ offer an opportunity to accomplish this goal. Here we present the design and structure-activity relationship analysis of balanced, low nanomolar inhibitors of bacterial DNA gyrase and topoisomerase IV that show potent antibacterial activities against the ESKAPE pathogens. For inhibitor 31c, a crystal structure in complex with Staphylococcus aureus DNA gyrase B was obtained that confirms the mode of action of these compounds. The best inhibitor, 31h, does not show any in vitro cytotoxicity and has excellent potency against Gram-positive (MICs: range, 0.0078–0.0625 μg/mL) and Gram-negative pathogens (MICs: range, 1–2 μg/mL). Furthermore, 31h inhibits GyrB mutants that can develop resistance to other drugs. Based on these data, we expect that structural derivatives of 31h will represent a step toward clinically efficacious multitargeting antimicrobials that are not impacted by existing antimicrobial resistance.
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Mar 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Stefanie
Freitag-Pohl
,
Andrius
Jasilionis
,
Maria
Håkansson
,
L. Anders
Svensson
,
Rebeka
Kovačič
,
Martin
Welin
,
Hildegard
Watzlawick
,
Lei
Wang
,
Josef
Altenbuchner
,
Magdalena
Płotka
,
Anna Karina
Kaczorowska
,
Tadeusz
Kaczorowski
,
Eva
Nordberg Karlsson
,
Salam
Al-Karadaghi
,
Björn
Walse
,
Arnthór
Aevarsson
,
Ehmke
Pohl
Open Access
Abstract: As part of the Virus-X Consortium that aims to identify and characterize novel proteins and enzymes from bacteriophages and archaeal viruses, the genes of the putative lytic proteins XepA from Bacillus subtilis prophage PBSX and YomS from prophage SPβ were cloned and the proteins were subsequently produced and functionally characterized. In order to elucidate the role and the molecular mechanism of XepA and YomS, the crystal structures of these proteins were solved at resolutions of 1.9 and 1.3 Å, respectively. XepA consists of two antiparallel β-sandwich domains connected by a 30-amino-acid linker region. A pentamer of this protein adopts a unique dumbbell-shaped architecture consisting of two discs and a central tunnel. YomS (12.9 kDa per monomer), which is less than half the size of XepA (30.3 kDa), shows homology to the C-terminal part of XepA and exhibits a similar pentameric disc arrangement. Each β-sandwich entity resembles the fold of typical cytoplasmic membrane-binding C2 domains. Only XepA exhibits distinct cytotoxic activity in vivo, suggesting that the N-terminal pentameric domain is essential for this biological activity. The biological and structural data presented here suggest that XepA disrupts the proton motive force of the cytoplasmatic membrane, thus supporting cell lysis.
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Nov 2019
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Jessica B.
Casaletto
,
Melissa L.
Geddie
,
Adnan O.
Abu-Yousif
,
Kristina
Masson
,
Aaron
Fulgham
,
Antoine
Boudot
,
Tim
Maiwald
,
Jeffrey D.
Kearns
,
Neeraj
Kohli
,
Stephen
Su
,
Maja
Razlog
,
Andreas
Raue
,
Ashish
Kalra
,
Maria
Håkansson
,
Derek T.
Logan
,
Martin
Welin
,
Shrikanta
Chattopadhyay
,
Brian D.
Harms
,
Ulrik B.
Nielsen
,
Birgit
Schoeberl
,
Alexey A.
Lugovskoy
,
Gavin
Macbeath
Diamond Proposal Number(s):
[12427]
Abstract: Activation of the Met receptor tyrosine kinase, either by its ligand, hepatocyte growth factor (HGF), or via ligand-independent mechanisms, such as MET amplification or receptor overexpression, has been implicated in driving tumor proliferation, metastasis, and resistance to therapy. Clinical development of Met-targeted antibodies has been challenging, however, as bivalent antibodies exhibit agonistic properties, whereas monovalent antibodies lack potency and the capacity to down-regulate Met. Through computational modeling, we found that the potency of a monovalent antibody targeting Met could be dramatically improved by introducing a second binding site that recognizes an unrelated, highly expressed antigen on the tumor cell surface. Guided by this prediction, we engineered MM-131, a bispecific antibody that is monovalent for both Met and epithelial cell adhesion molecule (EpCAM). MM-131 is a purely antagonistic antibody that blocks ligand-dependent and ligand-independent Met signaling by inhibiting HGF binding to Met and inducing receptor down-regulation. Together, these mechanisms lead to inhibition of proliferation in Met-driven cancer cells, inhibition of HGF-mediated cancer cell migration, and inhibition of tumor growth in HGF-dependent and -independent mouse xenograft models. Consistent with its design, MM-131 is more potent in EpCAM-high cells than in EpCAM-low cells, and its potency decreases when EpCAM levels are reduced by RNAi. Evaluation of Met, EpCAM, and HGF levels in human tumor samples reveals that EpCAM is expressed at high levels in a wide range of Met-positive tumor types, suggesting a broad opportunity for clinical development of MM-131.
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Mar 2019
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I03-Macromolecular Crystallography
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Abstract: Automated molecular de novo design led to thediscovery of an innovative inhibitor of death-associated proteinkinase 3 (DAPK3). An unprecedented crystal structure of theinactive DAPK3 homodimer shows the fragment-like hitbound to the ATP pocket. Target prediction software basedon machine learning models correctly identified additionalmacromolecular targets of the computationally designed com-pound and the structurally related marketed drug azosemide.The study validates computational de novo design as a primemethod for generating chemical probes and starting points for drug discovery.
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Oct 2015
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[480, 6603]
Open Access
Abstract: Human NUDT16 is a member of the NUDIX hydrolase superfamily. After having been initially described as an mRNA decapping enzyme, recent studies conferred it a role as an “housecleaning” enzyme specialized in the removal of hazardous (deoxy)inosine diphosphate from the nucleotide pool. Here we present the crystal structure of human NUDT16 both in its apo-form and in complex with its product inosine monophosphate (IMP). NUDT16 appears as a dimer whose formation generates a positively charged trench to accommodate substrate-binding. Complementation of the structural data with detailed enzymatic and biophysical studies revealed the determinants of substrate recognition and particularly the importance of the substituents in position 2 and 6 on the purine ring. The affinity for the IMP product, harboring a carbonyl in position 6 on the base, compared to purine monophosphates lacking a H-bond acceptor in this position, implies a catalytic cycle whose rate is primarily regulated by the product-release step. Finally, we have also characterized a phenomenon of inhibition by the product of the reaction, IMP, which might exclude non-deleterious nucleotides from NUDT16-mediated hydrolysis regardless of their cellular concentration. Taken together, this study details structural and regulatory mechanisms explaining how substrates are selected for hydrolysis by human NUDT16.
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Jun 2015
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[480, 6603]
Abstract: SHIP2, OCRL, and INPP5B belong to inositol polyphosphate 5-phophatase subfamilies involved in insulin regulation and Lowes syndrome. The structural basis for membrane recognition, substrate specificity, and regulation of inositol polyphosphate 5-phophatases is still poorly understood. We determined the crystal structures of human SHIP2, OCRL, and INPP5B, the latter in complex with phosphoinositide substrate analogs, which revealed a membrane interaction patch likely to assist in sequestering substrates from the lipid bilayer. Residues recognizing the 1-phosphate of the substrates are highly conserved among human family members, suggesting similar substrate binding modes. However, 3- and 4-phosphate recognition varies and determines individual substrate specificity profiles. The high conservation of the environment of the scissile 5-phosphate suggests a common reaction geometry for all members of the human 5-phosphatase family.
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May 2014
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