I03-Macromolecular Crystallography
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David
Hargreaves
,
Rodrigo J.
Carbajo
,
Michael S.
Bodnarchuk
,
Kevin
Embrey
,
Philip B.
Rawlins
,
Martin
Packer
,
Sébastien L.
Degorce
,
Alexander W.
Hird
,
Jeffrey W.
Johannes
,
Elisabetta
Chiarparin
,
Markus
Schade
Abstract: The structure-based design of small-molecule inhibitors targeting protein–protein interactions (PPIs) remains a huge challenge as the drug must bind typically wide and shallow protein sites. A PPI target of high interest for hematological cancer therapy is myeloid cell leukemia 1 (Mcl-1), a prosurvival guardian protein from the Bcl-2 family. Despite being previously considered undruggable, seven small-molecule Mcl-1 inhibitors have recently entered clinical trials. Here, we report the crystal structure of the clinical-stage inhibitor AMG-176 bound to Mcl-1 and analyze its interaction along with clinical inhibitors AZD5991 and S64315. Our X-ray data reveal high plasticity of Mcl-1 and a remarkable ligand-induced pocket deepening. Nuclear Magnetic Resonance (NMR)-based free ligand conformer analysis demonstrates that such unprecedented induced fit is uniquely achieved by designing highly rigid inhibitors, preorganized in their bioactive conformation. By elucidating key chemistry design principles, this work provides a roadmap for targeting the largely untapped PPI class more successfully.
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May 2023
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I03-Macromolecular Crystallography
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Simon
Stael
,
Igor
Sabljic
,
Dominique
Audenaert
,
Thilde
Andersson
,
Liana
Tsiatsiani
,
Robert P.
Kumpf
,
Andreu
Vidal-Albalat
,
Cecilia
Lindgren
,
Dominique
Vercammen
,
Silke
Jacques
,
Long
Nguyen
,
Maria
Njo
,
Álvaro D.
Fernández-Fernández
,
Tine
Beunens
,
Evy
Timmerman
,
Kris
Gevaert
,
Marc
Van Montagu
,
Jerry
Stahlberg
,
Peter V.
Bozhkov
,
Anna
Linusson
,
Tom
Beeckman
,
Frank
Van Breusegem
Diamond Proposal Number(s):
[23773]
Abstract: Metacaspases are part of an evolutionarily broad family of multifunctional cysteine proteases, involved in disease and normal development. As the structure–function relationship of metacaspases remains poorly understood, we solved the X-ray crystal structure of an Arabidopsis thaliana type II metacaspase (AtMCA-IIf) belonging to a particular subgroup not requiring calcium ions for activation. To study metacaspase activity in plants, we developed an in vitro chemical screen to identify small molecule metacaspase inhibitors and found several hits with a minimal thioxodihydropyrimidine-dione structure, of which some are specific AtMCA-IIf inhibitors. We provide mechanistic insight into the basis of inhibition by the TDP-containing compounds through molecular docking onto the AtMCA-IIf crystal structure. Finally, a TDP-containing compound (TDP6) effectively hampered lateral root emergence in vivo, probably through inhibition of metacaspases specifically expressed in the endodermal cells overlying developing lateral root primordia. In the future, the small compound inhibitors and crystal structure of AtMCA-IIf can be used to study metacaspases in other species, such as important human pathogens, including those causing neglected diseases.
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May 2023
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23620]
Open Access
Abstract: The cell cycle checkpoint kinase Mec1ATR and its integral partner Ddc2ATRIP are vital for the DNA damage and replication stress response. Mec1–Ddc2 “senses” single-stranded DNA (ssDNA) by being recruited to the ssDNA binding Replication Protein A (RPA) via Ddc2. In this study, we show that a DNA damage–induced phosphorylation circuit modulates checkpoint recruitment and function. We demonstrate that Ddc2–RPA interactions modulate the association between RPA and ssDNA and that Rfa1-phosphorylation aids in the further recruitment of Mec1–Ddc2. We also uncover an underappreciated role for Ddc2 phosphorylation that enhances its recruitment to RPA-ssDNA that is important for the DNA damage checkpoint in yeast. The crystal structure of a phosphorylated Ddc2 peptide in complex with its RPA interaction domain provides molecular details of how checkpoint recruitment is enhanced, which involves Zn2+. Using electron microscopy and structural modeling approaches, we propose that Mec1–Ddc2 complexes can form higher order assemblies with RPA when Ddc2 is phosphorylated. Together, our results provide insight into Mec1 recruitment and suggest that formation of supramolecular complexes of RPA and Mec1–Ddc2, modulated by phosphorylation, would allow for rapid clustering of damage foci to promote checkpoint signaling.
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Apr 2023
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Krios I-Titan Krios I at Diamond
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David J. K.
Swainsbury
,
Frederick
Hawkings
,
Elizabeth C.
Martin
,
Sabina
Musial
,
Jack H.
Salisbury
,
Philip J.
Jackson
,
David A.
Farmer
,
Matthew P.
Johnson
,
C. Alistair
Siebert
,
Andrew
Hitchcock
,
C. Neil
Hunter
Diamond Proposal Number(s):
[29785]
Open Access
Abstract: Cytochrome bc1 complexes are ubiquinol:cytochrome c oxidoreductases, and as such, they are centrally important components of respiratory and photosynthetic electron transfer chains in many species of bacteria and in mitochondria. The minimal complex has three catalytic components, which are cytochrome b, cytochrome c1, and the Rieske iron–sulfur subunit, but the function of mitochondrial cytochrome bc1 complexes is modified by up to eight supernumerary subunits. The cytochrome bc1 complex from the purple phototrophic bacterium Rhodobacter sphaeroides has a single supernumerary subunit called subunit IV, which is absent from current structures of the complex. In this work we use the styrene–maleic acid copolymer to purify the R. sphaeroides cytochrome bc1 complex in native lipid nanodiscs, which retains the labile subunit IV, annular lipids, and natively bound quinones. The catalytic activity of the four-subunit cytochrome bc1 complex is threefold higher than that of the complex lacking subunit IV. To understand the role of subunit IV, we determined the structure of the four-subunit complex at 2.9 Å using single particle cryogenic electron microscopy. The structure shows the position of the transmembrane domain of subunit IV, which lies across the transmembrane helices of the Rieske and cytochrome c1 subunits. We observe a quinone at the Qo quinone-binding site and show that occupancy of this site is linked to conformational changes in the Rieske head domain during catalysis. Twelve lipids were structurally resolved, making contacts with the Rieske and cytochrome b subunits, with some spanning both of the two monomers that make up the dimeric complex.
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Mar 2023
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William
Mccorkindale
,
Kadi L.
Saar
,
Daren
Fearon
,
Melissa
Boby
,
Haim
Barr
,
Amir
Ben-Shmuel
,
Nir
London
,
Frank
Von Delft
,
John D.
Chodera
,
Alpha. A.
Lee
,
The
Covid Moonshot Consortium
Open Access
Abstract: A common challenge in drug design pertains to finding chemical modifications to a ligand that increases its affinity to the target protein. An underutilized advance is the increase in structural biology throughput, which has progressed from an artisanal endeavor to a monthly throughput of hundreds of different ligands against a protein in modern synchrotrons. However, the missing piece is a framework that turns high-throughput crystallography data into predictive models for ligand design. Here, we designed a simple machine learning approach that predicts protein–ligand affinity from experimental structures of diverse ligands against a single protein paired with biochemical measurements. Our key insight is using physics-based energy descriptors to represent protein–ligand complexes and a learning-to-rank approach that infers the relevant differences between binding modes. We ran a high-throughput crystallography campaign against the SARS-CoV-2 main protease (MPro), obtaining parallel measurements of over 200 protein–ligand complexes and their binding activities. This allows us to design one-step library syntheses which improved the potency of two distinct micromolar hits by over 10-fold, arriving at a noncovalent and nonpeptidomimetic inhibitor with 120 nM antiviral efficacy. Crucially, our approach successfully extends ligands to unexplored regions of the binding pocket, executing large and fruitful moves in chemical space with simple chemistry.
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Mar 2023
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I03-Macromolecular Crystallography
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Open Access
Abstract: Acinetobacter baumannii is a gram-negative bacterial pathogen that causes challenging nosocomial infections. β-lactam targeting of penicillin-binding protein (PBP)–mediated cell wall peptidoglycan (PG) formation is a well-established antimicrobial strategy. Exposure to carbapenems or zinc (Zn)-deprived growth conditions leads to a rod-to-sphere morphological transition in A. baumannii, an effect resembling that caused by deficiency in the RodA–PBP2 PG synthesis complex required for cell wall elongation. While it is recognized that carbapenems preferentially acylate PBP2 in A. baumannii and therefore block the transpeptidase function of the RodA–PBP2 system, the molecular details underpinning cell wall elongation inhibition upon Zn starvation remain undefined. Here, we report the X-ray crystal structure of A. baumannii PBP2, revealing an unexpected Zn coordination site in the transpeptidase domain required for protein stability. Mutations in the Zn-binding site of PBP2 cause a loss of bacterial rod shape and increase susceptibility to β-lactams, therefore providing a direct rationale for cell wall shape maintenance and Zn homeostasis in A. baumannii. Furthermore, the Zn-coordinating residues are conserved in various β- and γ-proteobacterial PBP2 orthologs, consistent with a widespread Zn-binding requirement for function that has been previously unknown. Due to the emergence of resistance to virtually all marketed antibiotic classes, alternative or complementary antimicrobial strategies need to be explored. These findings offer a perspective for dual inhibition of Zn-dependent PG synthases and metallo-β-lactamases by metal chelating agents, considered the most sought-after adjuvants to restore β-lactam potency against gram-negative bacteria.
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Feb 2023
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Stefan
Gahbauer
,
Galen J.
Correy
,
Marion
Schuller
,
Matteo P.
Ferla
,
Yagmur Umay
Doruk
,
Moira
Rachman
,
Taiasean
Wu
,
Morgan
Diolaiti
,
Siyi
Wang
,
R. Jeffrey
Neitz
,
Daren
Fearon
,
Dmytro S.
Radchenko
,
Yurii S.
Moroz
,
John J.
Irwin
,
Adam R.
Renslo
,
Jenny C.
Taylor
,
Jason E.
Gestwicki
,
Frank
Von Delft
,
Alan
Ashworth
,
Ivan
Ahel
,
Brian K.
Shoichet
,
James S.
Fraser
Open Access
Abstract: The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small-molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic, there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high-resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 153 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated conformational changes within the active site, and key inhibitor motifs that will template future drug development against Mac1.
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Jan 2023
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I23-Long wavelength MX
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Eugene
Kuatsjah
,
Michael
Zahn
,
Xiangyang
Chen
,
Ryo
Kato
,
Daniel J.
Hinchen
,
Mikhail O.
Konev
,
Rui
Katahira
,
Christian
Orr
,
Armin
Wagner
,
Yike
Zou
,
Stefan J.
Haugen
,
Kelsey J.
Ramirez
,
Joshua K.
Michener
,
Andrew R.
Pickford
,
Naofumi
Kamimura
,
Eiji
Masai
,
Kendall N.
Houk
,
John
Mcgeehan
,
Gregg T.
Beckham
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon–carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived β-1 dimer, was reported in Novosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C1 reaction product, and we further demonstrated that both enantiomers of erythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.
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Jan 2023
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[17221, 23620]
Open Access
Abstract: Type 4 filaments (T4F)—of which type 4 pili (T4P) are the archetype—are a superfamily of nanomachines nearly ubiquitous in prokaryotes. T4F are polymers of one major pilin, which also contain minor pilins whose roles are often poorly understood. Here, we complete the structure/function analysis of the full set of T4P pilins in the opportunistic bacterial pathogen Streptococcus sanguinis. We determined the structure of the minor pilin PilA, which is unexpectedly similar to one of the subunits of a tip-located complex of four minor pilins, widely conserved in T4F. We found that PilA interacts and dramatically stabilizes the minor pilin PilC. We determined the structure of PilC, showing that it is a modular pilin with a lectin module binding a subset of glycans prevalent in the human glycome, the host of S. sanguinis. Altogether, our findings support a model whereby the minor pilins in S. sanguinis T4P form a tip-located complex promoting adhesion to various host receptors. This has general implications for T4F.
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Jan 2023
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Abstract: A family of leucine-rich-repeat-containing G-protein-coupled receptors (LGRs) mediate diverse physiological responses when complexed with their cognate ligands. LGRs are present in all metazoan animals. In humans, the LGR ligands include glycoprotein hormones (GPHs) chorionic gonadotropin (hCG), luteinizing hormone, follicle-stimulating hormone (hFSH), and thyroid-stimulating hormone (hTSH). These hormones are αβ heterodimers of cystine-knot protein chains. LGRs and their ligand chains have coevolved. Ancestral hormone homologs, present in both bilaterian animals and chordates, are identified as α2β5. We have used single-wavelength anomalous diffraction and molecular replacement to determine structures of the α2β5 hormone from Caenorhabditis elegans (Ceα2β5). Ceα2β5 is unglycosylated, as are many other α2β5 hormones. Both Hsα2β5, the human homolog of Ceα2β5, and hTSH activate the same receptor (hTSHR). Despite having little sequence similarity to vertebrate GPHs, apart from the cysteine patterns from core disulfide bridges, Ceα2β5 is generally similar in structure to these counterparts; however, its α2 and β5 subunits are more symmetric as compared with α and β of hCG and hFSH. This quasisymmetry suggests a hypothetical homodimeric antecedent of the α2β5 and αβ heterodimers. Known structures together with AlphaFold models from the sequences for other LGR ligands provide representatives for the molecular evolution of LGR ligands from early metazoans through the present-day GPHs. The experimental Ceα2β5 structure validates its AlphaFold model, and thus also that for Hsα2β5; and interfacial characteristics in a model for the Hsα2β5:hTSHR complex are similar to those found in an experimental hTSH:hTSHR structure.
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Dec 2022
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