I24-Microfocus Macromolecular Crystallography
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Shuo
Chen
,
Jiale
Wu
,
Shan
Zhong
,
Yuntong
Li
,
Ping
Zhang
,
Jingyi
Ma
,
Jingshan
Ren
,
Yun
Tan
,
Yunhao
Wang
,
Kin Fai
Au
,
Christian
Siebold
,
Gareth L.
Bond
,
Zhu
Chen
,
Min
Lu
,
E. Yvonne
Jones
,
Xin
Lu
Open Access
Abstract: The most frequently mutated protein in human cancer is p53, a transcription factor (TF) that regulates myriad genes instrumental in diverse cellular outcomes including growth arrest and cell death. Cell context-dependent p53 modulation is critical for this life-or-death balance, yet remains incompletely understood. Here we identify sequence signatures enriched in genomic p53-binding sites modulated by the transcription cofactor iASPP. Moreover, our p53–iASPP crystal structure reveals that iASPP displaces the p53 L1 loop—which mediates sequence-specific interactions with the signature-corresponding base—without perturbing other DNA-recognizing modules of the p53 DNA-binding domain. A TF commonly uses multiple structural modules to recognize its cognate DNA, and thus this mechanism of a cofactor fine-tuning TF–DNA interactions through targeting a particular module is likely widespread. Previously, all tumor suppressors and oncoproteins that associate with the p53 DNA-binding domain—except the oncogenic E6 from human papillomaviruses (HPVs)—structurally cluster at the DNA-binding site of p53, complicating drug design. By contrast, iASPP inhibits p53 through a distinct surface overlapping the E6 footprint, opening prospects for p53-targeting precision medicine to improve cancer therapy.
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Aug 2019
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15916]
Open Access
Abstract: Retroviruses can integrate their DNA into the host-cell genome. Inherited retroviral DNA and other transposable elements account for more than half of the human genome. Transposable elements must be tightly regulated to restrict their proliferation and prevent toxic gene expression. KAP1/TRIM28 is an essential regulator of transposable element transcription. We determined the crystal structure of the KAP1 TRIM. The structure identifies a protein–protein interaction site required for recruitment of KAP1 to transposable elements. An epigenetic gene silencing assay confirms the importance of this site for KAP1-dependent silencing. We also show that KAP1 self-assembles in solution, but this self-assembly is not required for silencing. Our work provides insights into KAP1-dependent silencing and tools for expanding our mechanistic understanding of this process.
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Jul 2019
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I02-Macromolecular Crystallography
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Open Access
Abstract: In the disease familial amyloidosis, Finnish type (FAF), also known as AGel amyloidosis (AGel), the mechanism by which point mutations in the calcium-regulated actin-severing protein gelsolin lead to furin cleavage is not understood in the intact protein. Here, we provide a structural and biochemical characterization of the FAF variants. X-ray crystallography structures of the FAF mutant gelsolins demonstrate that the mutations do not significantly disrupt the calcium-free conformations of gelsolin. Small-angle X-ray–scattering (SAXS) studies indicate that the FAF calcium-binding site mutants are slower to activate, whereas G167R is as efficient as the wild type. Actin-regulating studies of the gelsolins at the furin cleavage pH (6.5) show that the mutant gelsolins are functional, suggesting that they also adopt relatively normal active conformations. Deletion of gelsolin domains leads to sensitization to furin cleavage, and nanobody-binding protects against furin cleavage. These data indicate instability in the second domain of gelsolin (G2), since loss or gain of G2-stabilizing interactions impacts the efficiency of cleavage by furin. To demonstrate this principle, we engineered non-FAF mutations in G3 that disrupt the G2-G3 interface in the calcium-activated structure. These mutants led to increased furin cleavage. We carried out molecular dynamics (MD) simulations on the FAF and non-FAF mutant G2-G3 fragments of gelsolin. All mutants showed an increase in the distance between the center of masses of the 2 domains (G2 and G3). Since G3 covers the furin cleavage site on G2 in calcium-activated gelsolin, this suggests that destabilization of this interface is a critical step in cleavage.
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Jun 2019
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I04-Macromolecular Crystallography
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Melodie M.
Machovina
,
Sam J. B.
Mallinson
,
Brandon C.
Knott
,
Alexander W.
Meyers
,
Marc
Garcia-borràs
,
Lintao
Bu
,
Japheth E.
Gado
,
April
Oliver
,
Graham P.
Schmidt
,
Daniel J.
Hinchen
,
Michael F.
Crowley
,
Christopher W.
Johnson
,
Ellen L.
Neidle
,
Christina M.
Payne
,
Kendall N.
Houk
,
Gregg T.
Beckham
,
John E.
Mcgeehan
,
Jennifer L.
Dubois
Diamond Proposal Number(s):
[17212]
Open Access
Abstract: Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds.
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Jun 2019
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7141]
Open Access
Abstract: We have solved the X-ray crystal structure of the RNA chaperone protein Hfq from the alpha-proteobacterium Caulobacter crescentus to 2.15-Å resolution, resolving the conserved core of the protein and the entire C-terminal domain (CTD). The structure reveals that the CTD of neighboring hexamers pack in crystal contacts, and that the acidic residues at the C-terminal tip of the protein interact with positive residues on the rim of Hfq, as has been recently proposed for a mechanism of modulating RNA binding. De novo computational models predict a similar docking of the acidic tip residues against the core of Hfq. We also show that C. crescentus Hfq has sRNA binding and RNA annealing activities and is capable of facilitating the annealing of certain Escherichia coli sRNA:mRNA pairs in vivo. Finally, we describe how the Hfq CTD and its acidic tip residues provide a mechanism to modulate annealing activity and substrate specificity in various bacteria.
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May 2019
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I24-Microfocus Macromolecular Crystallography
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Beatriz
Baragaña
,
Barbara
Forte
,
Ryan
Choi
,
Stephen
Nakazawa Hewitt
,
Juan A.
Bueren-calabuig
,
Joao Pedro
Pisco
,
Caroline
Peet
,
David M.
Dranow
,
David A.
Robinson
,
Chimed
Jansen
,
Neil R.
Norcross
,
Sumiti
Vinayak
,
Mark
Anderson
,
Carrie F.
Brooks
,
Caitlin A.
Cooper
,
Sebastian
Damerow
,
Michael
Delves
,
Karen
Dowers
,
James
Duffy
,
Thomas E.
Edwards
,
Irene
Hallyburton
,
Benjamin G.
Horst
,
Matthew A.
Hulverson
,
Liam
Ferguson
,
María Belén
Jiménez-díaz
,
Rajiv S.
Jumani
,
Donald D.
Lorimer
,
Melissa S.
Love
,
Steven
Maher
,
Holly
Matthews
,
Case W.
Mcnamara
,
Peter
Miller
,
Sandra
O’neill
,
Kayode K.
Ojo
,
Maria
Osuna-cabello
,
Erika
Pinto
,
John
Post
,
Jennifer
Riley
,
Matthias
Rottmann
,
Laura M.
Sanz
,
Paul
Scullion
,
Arvind
Sharma
,
Sharon M.
Shepherd
,
Yoko
Shishikura
,
Frederick R. C.
Simeons
,
Erin E.
Stebbins
,
Laste
Stojanovski
,
Ursula
Straschil
,
Fabio K.
Tamaki
,
Jevgenia
Tamjar
,
Leah S.
Torrie
,
Amélie
Vantaux
,
Benoît
Witkowski
,
Sergio
Wittlin
,
Manickam
Yogavel
,
Fabio
Zuccotto
,
Iñigo
Angulo-barturen
,
Robert
Sinden
,
Jake
Baum
,
Francisco-javier
Gamo
,
Pascal
Mäser
,
Dennis E.
Kyle
,
Elizabeth A.
Winzeler
,
Peter J.
Myler
,
Paul G.
Wyatt
,
David
Floyd
,
David
Matthews
,
Amit
Sharma
,
Boris
Striepen
,
Christopher D.
Huston
,
David W.
Gray
,
Alan H.
Fairlamb
,
Andrei V.
Pisliakov
,
Chris
Walpole
,
Kevin D.
Read
,
Wesley C.
Van Voorhis
,
Ian H.
Gilbert
Diamond Proposal Number(s):
[10071]
Open Access
Abstract: Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.
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Apr 2019
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I02-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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I04-1-Macromolecular Crystallography (fixed wavelength)
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Oliver W.
Bayfield
,
Evgeny
Klimuk
,
Dennis C.
Winkler
,
Emma L.
Hesketh
,
Maria
Chechik
,
Naiqian
Cheng
,
Eric C.
Dykeman
,
Leonid
Minakhin
,
Neil A.
Ranson
,
Konstantin
Severinov
,
Alasdair C.
Steven
,
Alfred A.
Antson
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Understanding molecular events during virus assembly and genome packaging is important for understanding viral life cycles, and the functioning of other protein–nucleic acid machines. The model system developed for the thermophilic bacteriophage P23-45 offers advantages over other systems. Cryo-EM reconstructions reveal modifications to a canonical capsid protein fold, resulting in capsids that are abnormally large for this virus class. Structural information on the portal protein, through which the genome is packaged, demonstrates that the capsid influences the portal’s conformation. This has implications for understanding how processes inside and outside the capsid can be coordinated.
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Feb 2019
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12346]
Abstract: The RAS gene family is frequently mutated in human cancers, and the quest for compounds that bind to mutant RAS remains a major goal, as it also does for inhibitors of protein–protein interactions. We have refined crystallization conditions for KRAS169Q61H-yielding crystals suitable for soaking with compounds and exploited this to assess new RAS-binding compounds selected by screening a protein–protein interaction-focused compound library using surface plasmon resonance. Two compounds, referred to as PPIN-1 and PPIN-2, with related structures from 30 initial RAS binders showed binding to a pocket where compounds had been previously developed, including RAS effector protein–protein interaction inhibitors selected using an intracellular antibody fragment (called Abd compounds). Unlike the Abd series of RAS binders, PPIN-1 and PPIN-2 compounds were not competed by the inhibitory anti-RAS intracellular antibody fragment and did not show any RAS-effector inhibition properties. By fusing the common, anchoring part from the two new compounds with the inhibitory substituents of the Abd series, we have created a set of compounds that inhibit RAS-effector interactions with increased potency. These fused compounds add to the growing catalog of RAS protein–protein inhibitors and show that building a chemical series by crossing over two chemical series is a strategy to create RAS-binding small molecules.
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Jan 2019
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[8547]
Abstract: The crystal structure has been determined of the F1-catalytic domain of the ATP synthase from Mycobacterium smegmatis which hydrolyzes adenosine triphosphate (ATP) very poorly. The structure of the α3β3-component of the catalytic domain is similar to those in active F1-ATPases in Escherichia coli and Geobacillus stearothermophilus. However, its ε-subunit differs from those in these two active bacterial F1-ATPases as an ATP molecule is not bound to the two α-helices forming its C-terminal domain, probably because they are shorter than those in active enzymes and they lack an amino acid that contributes to the ATP binding site in active enzymes. In E. coli and G. stearothermophilus, the α-helices adopt an “up” state where the α-helices enter the α3β3-domain and prevent the rotor from turning. The mycobacterial F1-ATPase is most similar to the F1-ATPase from Caldalkalibacillus thermarum, which also hydrolyzes ATP poorly. The βE-subunits in both enzymes are in the usual “open” conformation, but appear to be occupied uniquely by the combination of an ADP molecule with no magnesium ion, plus phosphate. This occupation is consistent with the finding that their rotors have been arrested at the same point in their rotary catalytic cycles. These bound hydrolytic products are probably the basis of inhibition of ATP hydrolysis. It can be envisaged that specific as yet unidentified small molecules might bind to the F1-domain in M. tuberculosis, prevent ATP synthesis and inhibit the growth of the pathogen.
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Jan 2019
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