I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13440]
Open Access
Abstract: The activity-regulated cytoskeleton-associated protein (Arc) is important for synaptic plasticity and the normal function of the brain. Arc interacts with neuronal postsynaptic proteins, but the mechanistic details of its function have not been fully established. The C-terminal domain of Arc consists of tandem domains, termed the N- and C-lobe. The N-lobe harbours a peptide binding site, able to bind multiple targets. By measuring the affinity of human Arc towards various peptides from stargazin and guanylate kinase-associated protein (GKAP), we have refined its specificity determinants. We found two sites in the GKAP repeat region that bind to Arc and confirmed these interactions by X-ray crystallography. Phosphorylation of the stargazin peptide did not affect binding affinity but caused changes in thermodynamic parameters. Comparison of the crystal structures of three high-resolution human Arc-peptide complexes identifies three conserved C–H…π interactions at the binding cavity, explaining the sequence specificity of short linear motif binding by Arc. We further characterise central residues of the Arc lobe fold, show the effects of peptide binding on protein dynamics, and identify acyl carrier proteins as structures similar to the Arc lobes. We hypothesise that Arc may affect protein-protein interactions and phase separation at the postsynaptic density, affecting protein turnover and re-modelling of the synapse. The present data on Arc structure and ligand binding will help in further deciphering these processes.
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Jul 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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John
Liddle
,
Andrew C.
Pearce
,
Christopher
Arico-Muendel
,
Svetlana
Belyanskaya
,
Andrew
Brewster
,
Murray
Brown
,
Chun-Wa
Chung
,
Alexis
Denis
,
Nerina
Dodic
,
Anthony
Dossang
,
Peter
Eddershaw
,
Diana
Klimaszewska
,
Imran
Haq
,
Duncan S.
Holmes
,
Alistair
Jagger
,
Toral
Jakhria
,
Emilie
Jigorel
,
Ken
Lind
,
Jeff
Messer
,
Margaret
Neu
,
Allison
Olszewski
,
Riccardo
Ronzoni
,
James
Rowedder
,
Martin
Rüdiger
,
Steve
Skinner
,
Kathrine J.
Smith
,
Lionel
Trottet
,
Iain
Uings
,
Zhengrong
Zhu
,
James A.
Irving
,
David A.
Lomas
Diamond Proposal Number(s):
[23853, 17201]
Abstract: α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α1-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors.
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Jun 2021
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Martina
Wirth
,
Stephane
Mouilleron
,
Wenxin
Zhang
,
Eva
Sjøttem
,
Yakubu
Princely Abudu
,
Ashish
Jain
,
Hallvard
Lauritz Olsvik
,
Jack-Ansgar
Bruun
,
Minoo
Razi
,
Harold B. J.
Jefferies
,
Rebecca
Lee
,
Dhira
Joshi
,
Nicola
O'Reilly
,
Terje
Johansen
,
Sharon A.
Tooze
Diamond Proposal Number(s):
[9826]
Open Access
Abstract: Autophagy is a highly conserved degradative pathway, essential for cellular homeostasis and implicated in diseases including cancer and neurodegeneration. Autophagy-related 8 (ATG8) proteins play a central role in autophagosome formation and selective delivery of cytoplasmic cargo to lysosomes by recruiting autophagy adaptors and receptors. The LC3-interacting region (LIR) docking site (LDS) of ATG8 proteins binds to LIR motifs present in autophagy adaptors and receptors. LIR-ATG8 interactions can be highly selective for specific mammalian ATG8 family members (LC3A-C, GABARAP, and GABARAPL1-2) and how this specificity is generated and regulated is incompletely understood.
We have identified a LIR motif in the Golgi protein SCOC (short coiled-coil protein) exhibiting strong binding to GABARAP, GABARAPL1, LC3A and LC3C. The residues within and surrounding the core LIR motif of the SCOC LIR domain were phosphorylated by autophagy-related kinases (ULK1-3, TBK1) increasing specifically LC3 family binding. More distant flanking residues also contributed to ATG8 binding. Loss of these residues was compensated by phosphorylation of serine residues immediately adjacent to the core LIR motif, indicating that the interactions of the flanking LIR regions with the LDS are important and highly dynamic.
Our comprehensive structural, biophysical and biochemical analyses support and provide novel mechanistic insights into how phosphorylation of LIR domain residues regulates the affinity and binding specificity of ATG8 proteins towards autophagy adaptors and receptors.
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Jun 2021
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I03-Macromolecular Crystallography
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Igor M.
Ferreira
,
José Edwin N.
Quesñay
,
Alliny C. S.
Bastos
,
Camila T.
Rodrigues
,
Melanie
Vollmar
,
Tobias
Krojer
,
Claire
Strain-Damerell
,
Nicola A.
Burgess-Brown
,
Frank
Von Delft
,
Wyatt W.
Yue
,
Sandra M G.
Dias
,
Andre L. B.
Ambrosio
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: Cancer cells exhibit an altered metabolic phenotype, consuming higher levels of the amino acid glutamine. This metabolic reprogramming depends on increased mitochondrial glutaminase activity to convert glutamine to glutamate, an essential precursor for bioenergetic and biosynthetic processes in cells. Mammals encode the kidney-type (GLS) and liver-type (GLS2) glutaminase isozymes. GLS is overexpressed in cancer and associated with enhanced malignancy. On the other hand, GLS2 is either a tumor suppressor or an oncogene, depending on the tumor type. The GLS structure and activation mechanism are well known, while the structural determinants for GLS2 activation remain elusive. Here, we describe the structure of the human glutaminase domain of GLS2, followed by the functional characterization of the residues critical for its activity. Increasing concentrations of GLS2 lead to tetramer stabilization, a process enhanced by phosphate. In GLS2, the so-called “lid loop” is in a rigid open conformation, which may be related to its higher affinity for phosphate and lower affinity for glutamine; hence, it has lower glutaminase activity than GLS. The lower affinity of GLS2 for glutamine is also related to its less electropositive catalytic site than GLS, as indicated by a Thr225Lys substitution within the catalytic site decreasing the GLS2 glutamine concentration corresponding to half-maximal velocity (K0.5). Finally, we show that the Lys253Ala substitution (corresponding to the Lys320Ala in the GLS “activation” loop, formerly known as the “gating” loop) renders a highly active protein in stable tetrameric form. We conclude that the “activation” loop, a known target for GLS inhibition, may also be a drug target for GLS2.
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Jun 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[24717]
Open Access
Abstract: This structural and biophysical study exploited a method of perdeuterating hen egg-white lysozyme based on the expression of insoluble protein in Escherichia coli followed by in-column chemical refolding. This allowed detailed comparisons with perdeuterated lysozyme produced in the yeast Pichia pastoris, as well as with unlabelled lysozyme. Both perdeuterated variants exhibit reduced thermal stability and enzymatic activity in comparison with hydrogenated lysozyme. The thermal stability of refolded perdeuterated lysozyme is 4.9°C lower than that of the perdeuterated variant expressed and secreted in yeast and 6.8°C lower than that of the hydrogenated Gallus gallus protein. However, both perdeuterated variants exhibit a comparable activity. Atomic resolution X-ray crystallographic analyses show that the differences in thermal stability and enzymatic function are correlated with refolding and deuteration effects. The hydrogen/deuterium isotope effect causes a decrease in the stability and activity of the perdeuterated analogues; this is believed to occur through a combination of changes to hydrophobicity and protein dynamics. The lower level of thermal stability of the refolded perdeuterated lysozyme is caused by the unrestrained Asn103 peptide-plane flip during the unfolded state, leading to a significant increase in disorder of the Lys97–Gly104 region following subsequent refolding. An ancillary outcome of this study has been the development of an efficient and financially viable protocol that allows stable and active perdeuterated lysozyme to be more easily available for scientific applications.
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May 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946]
Abstract: Plasmodium falciparum lysyl tRNA synthetase ( Pf KRS) represent a promising therapeutic anti‐malarial target. Cladosporin, a tool compound was identified as a selective and potent PfKRS inhibitor but lacks metabolic stability. Here, we report chemical synthesis, biological evaluation and structural characterization of analogues where the tetrahydropyran (THP) frame of cladosporin is replaced with the piperidine ring bearing functional group variations. Thermal binding, enzymatic, kinetic and parasitic assays complemented with x‐ray crystallography reveals compounds to be of moderate potency. Co‐crystals of Cla‐B and Cla‐C with PfKRS reveals key atomic configurations that allow drug binding to, and inhibition of the enzyme. Collectively, these piperidine ring scaffold inhibitors lays a framework for further structural editing and functional modifications of cladosporin scaffold to obtain a potent lead.
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May 2021
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I03-Macromolecular Crystallography
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Open Access
Abstract: SWI/SNF (BAF) chromatin remodelling complexes are key regulators of gene expression programs, and attractive drug targets for cancer therapies. Here we show that the N-terminus of the BAF155/SMARCC1 subunit contains a putative DNA-binding MarR-like domain, a chromodomain and a BRCT domain that are interconnected to each other to form a distinct module. In this structure the chromodomain makes interdomain interactions and has lost its canonical function to bind to methylated lysines. The structure provides new insights into the missense mutations that target this module in cancer. This study also reveals two adjacent, highly-conserved pockets in a cleft between the domains that form a potential binding site, which can be targeted with small molecules, offering a new strategy to target SWI/SNF complexes.
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May 2021
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I03-Macromolecular Crystallography
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Abstract: Synthetic peptides are attractive candidates to manipulate protein-protein interactions inside the cell as they mimic natural interactions to compete for binding. However, protein-peptide interactions are often dynamic and weak. A challenge is to design peptides that make improved interactions with the target. Here, we devise a fragment-linking strategy—“mash-up” design—to deliver a high-affinity ligand, KinTag, for the kinesin-1 motor. Using structural insights from natural micromolar-affinity cargo-adaptor ligands, we have identified and combined key binding features in a single, high-affinity ligand. An X-ray crystal structure demonstrates interactions as designed and reveals only a modest increase in interface area. Moreover, when genetically encoded, KinTag promotes transport of lysosomes with higher efficiency than natural sequences, revealing a direct link between motor-adaptor binding affinity and organelle transport. Together, these data demonstrate a fragment-linking strategy for peptide design and its application in a synthetic motor ligand to direct cellular cargo transport.
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Apr 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[12342, 23269]
Open Access
Abstract: The rational design of linear peptides that assemble controllably and predictably in water is challenging. Short sequences must encode unique target structures and avoid alternative states. However, the non-covalent forces that stabilize and discriminate between states are weak. Nonetheless, for α-helical coiled-coil assemblies considerable progress has been made in rational de novo design. In these, sequence repeats of nominally hydrophobic (h) and polar (p) residues, hpphppp, direct the assembly of amphipathic helices into dimeric to tetrameric bundles. Expanding this pattern to hpphhph can produce larger α-helical barrels. Here, we show that pentameric to nonameric barrels are accessed by varying the residue at one of the h sites. In peptides with four L/I–K–E–I–A–x–Z repeats, decreasing the size of Z from threonine to serine to alanine to glycine gives progressively larger oligomers. X-ray crystal structures of the resulting α-helical barrels rationalize this: side chains at Z point directly into the helical interfaces, and smaller residues allow closer helix contacts and larger assemblies.
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Apr 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Abstract: Aspartate/asparagine‐β‐hydroxylase (AspH) is a human 2‐oxoglutarate (2OG) and Fe(II) oxygenase that catalyzes C3 hydroxylations of aspartate/asparagine residues of epidermal growth factor‐like domains (EGFDs). Unusually, AspH employs two histidine residues to chelate Fe(II) rather than the typical triad of two histidine and one glutamate/aspartate residue. We report kinetic, inhibition, and crystallographic studies concerning human AspH variants in which either of its Fe(II) binding histidine residues are substituted for alanine. Both the H725A and, in particular, the H679A AspH variant retain substantial catalytic activity. Crystal structures clearly reveal metal‐ligation by only a single protein histidine ligand. The results have implications for the functional assignment of 2OG oxygenases and for the design of non‐protein biomimetic catalysts.
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Apr 2021
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