I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Petra
Lukacik
,
C. David
Owen
,
Gemma
Harris
,
Jani Reddy
Bolla
,
Sarah
Picaud
,
Irfan
Alibay
,
Joanne E.
Nettleship
,
Louise E.
Bird
,
Raymond
Owens
,
Philip C.
Biggin
,
Panagis
Filippakopoulos
,
Carol V.
Robinson
,
Martin A.
Walsh
Diamond Proposal Number(s):
[4990, 5073, 4988]
Open Access
Abstract: Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in respiratory disease and otitis media. Important for NTHi survival, colonization and persistence in vivo is the Sap (sensitivity to antimicrobial peptides) ABC transporter system. Current models propose a direct role for Sap in heme and antimicrobial peptide (AMP) transport. Here, the crystal structure of SapA, the periplasmic component of Sap, in a closed, ligand bound conformation, is presented. Phylogenetic and cavity volume analysis predicts that the small, hydrophobic SapA central ligand binding cavity is most likely occupied by a hydrophobic di- or tri- peptide. The cavity is of insufficient volume to accommodate heme or folded AMPs. Crystal structures of SapA have identified surface interactions with heme and dsRNA. Heme binds SapA weakly (Kd 282 μM) through a surface exposed histidine, while the dsRNA is coordinated via residues which constitute part of a conserved motif (estimated Kd 4.4 μM). The RNA affinity falls within the range observed for characterized RNA/protein complexes. Overall, we describe in molecular-detail the interactions of SapA with heme and dsRNA and propose a role for SapA in the transport of di- or tri-peptides.
|
Oct 2021
|
|
I24-Microfocus Macromolecular Crystallography
|
Angeliki
Ditsiou
,
Chiara
Cilibrasi
,
Nikiana
Simigdala
,
Athanasios
Papakyriakou
,
Leanne
Milton-Harris
,
Viviana
Vella
,
Joanne E.
Nettleship
,
Jae Ho
Lo
,
Shivani
Soni
,
Goar
Smbatyan
,
Panagiota
Ntavelou
,
Teresa
Gagliano
,
Maria Chiara
Iachini
,
Sahir
Khurshid
,
Thomas
Simon
,
Lihong
Zhou
,
Storm
Hassell-Hart
,
Philip
Carter
,
Laurence H.
Pearl
,
Robin L.
Owen
,
Raymond J.
Owens
,
S. Mark
Roe
,
Naomi E.
Chayen
,
Heinz-Josef
Lenz
,
John
Spencer
,
Chrisostomos
Prodromou
,
Apostolos
Klinakis
,
Justin
Stebbing
,
Georgios
Giamas
Diamond Proposal Number(s):
[14493]
Open Access
Abstract: Elucidating signaling driven by lemur tyrosine kinase 3 (LMTK3) could help drug development. Here, we solve the crystal structure of LMTK3 kinase domain to 2.1Å resolution, determine its consensus motif and phosphoproteome, unveiling in vitro and in vivo LMTK3 substrates. Via high-throughput homogeneous time-resolved fluorescence screen coupled with biochemical, cellular, and biophysical assays, we identify a potent LMTK3 small-molecule inhibitor (C28). Functional and mechanistic studies reveal LMTK3 is a heat shock protein 90 (HSP90) client protein, requiring HSP90 for folding and stability, while C28 promotes proteasome-mediated degradation of LMTK3. Pharmacologic inhibition of LMTK3 decreases proliferation of cancer cell lines in the NCI-60 panel, with a concomitant increase in apoptosis in breast cancer cells, recapitulating effects of LMTK3 gene silencing. Furthermore, LMTK3 inhibition reduces growth of xenograft and transgenic breast cancer mouse models without displaying systemic toxicity at effective doses. Our data reinforce LMTK3 as a druggable target for cancer therapy.
|
Nov 2020
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
|
Jingshan
Ren
,
Joanne E.
Nettleship
,
Gemma
Harris
,
William
Mwangi
,
Nahid
Rhaman
,
Clare
Grant
,
Abhay
Kotecha
,
Elizabeth
Fry
,
Bryan
Charleston
,
David I.
Stuart
,
John
Hammond
,
Raymond J.
Owens
Diamond Proposal Number(s):
[10627, 14744]
Open Access
Abstract: Cattle antibodies have unusually long CDR3 loops in their heavy chains (HCs), and limited light chain (LC) diversity, raising the question of whether these mask the effect of LC variation on antigen recognition. We have investigated the role of the LC in the structure and activity of two neutralizing cattle antibodies (B4 and B13) that bind the F protein of bovine respiratory syncytial virus (bRSV). Recombinant Fab fragments of B4 and B13 bound bRSV infected cells and showed similar affinities for purified bRSV F protein. Exchanging the LCs between the Fab fragments produced hybrid Fabs: B13* (B13 HC/B4 LC) and B4* (B4 HC/B13 LC). The affinity of B13* to the F protein was found to be two-fold lower than B13 whilst the binding affinity of B4* was reduced at least a hundred-fold compared to B4 such that it no longer bound to bRSV infected cells. Comparison of the structures of B4 and B13 with their LC exchanged counterparts B4* and B13* showed that paratope of the HC variable domain (VH) of B4 was disrupted on pairing with the B13 LC, consistent with the loss of binding activity. By contrast, B13 H3 adopts a similar conformation when paired with either B13 or B4 LCs. These observations confirm the expected key role of the extended H3 loop in antigen-binding by cattle antibodies but also show that the quaternary LC/HC subunit interaction can be crucial for its presentation and thus the LC variable domain (VL) is also important for antigen recognition.
|
Aug 2019
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Juliana Roverta
Torini
,
Adriano
De Freitas Fernandes
,
Vitor Hugo
Balasco Serrao
,
Larissa
Romanello
,
Louise
Bird
,
Joanne E.
Nettleship
,
Raymond J.
Owens
,
Jose
Brandao-Neto
,
Ana Eliza
Zeraik
,
Ricardo
Demarco
,
Humberto
D'Muniz Pereira
Diamond Proposal Number(s):
[5073, 14493]
Abstract: Nucleoside diphosphate kinases (NDPKs) are crucial to keep the high triphosphate nucleotide levels in the biological process. The enzymatic mechanism has been extensively described; however, the structural characteristics and kinetic parameters have never been fully determined. In Schistosoma mansoni, NDPK (SmNDPK) is directly involved in the pyrimidine and purine salvage pathways, being essential for nucleotide metabolism. The SmNDPK enzymatic activity is the highest of the known purine metabolisms when compared to the mammalian NDPKs, suggesting the importance of this enzyme in the worm metabolism. Here, we report the recombinant expression of SmNDPK that resulted in 1.7 and 1.9 Å apo-form structure in different space-groups, as well as the 2.1 Å SmNDPK.ADP complex. The binding and kinetic assays reveal the ATP-dependence for enzyme activation. Moreover, in situ hybridization showed that SmNDPK transcripts are found in reproductive organs and in the esophagus gland of adult worms, which can be intrinsically related with the oviposition and digestive processes. These results will help us fully understand the crucial participation of this enzyme in Schistosoma mansoni and its importance for the pathology of the disease.
|
Jul 2019
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
|
Larissa
Romanello
,
Ana Eliza
Zeraik
,
Adriano
De Freitas Fernandes
,
Juliana Roberta
Torini
,
Louise E.
Bird
,
Joanne E.
Nettleship
,
Heather
Rada
,
Yamini
Reddivari
,
Ray J.
Owens
,
Vitor Hugo Balasco
Serrão
,
Ricardo
Demarco
,
Jose
Brandao-Neto
,
Humberto
D'Muniz Pereira
Diamond Proposal Number(s):
[5073]
Abstract: Schistosoma mansoni, the parasite responsible for schistosomiasis, lacks the “de novo” purine biosynthetic pathway and depends entirely on the purine salvage pathway for the supply of purines. Numerous reports of praziquantel resistance have been described, as well as stimulated efforts to develop new drugs against schistosomiasis. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme of the purine salvage pathway. Here, we describe a crystallographic structure of the S. mansoni HPGRT-1 (SmHGPRT), complexed with IMP at a resolution of 2.8Ǻ. Four substitutions were identified in the region of the active site between SmHGPRT-1 and human HGPRT. We also present data from RNA-Seq and WISH, suggesting that some isoforms of HGPRT might be involved in the process related to sexual maturation and reproduction in worms; furthermore, its enzymatic assays show that the isoform SmHGPRT-3 does not present the same catalytic efficiency as other isoforms. Finally, although other studies have previously suggested this enzyme as a potential antischistosomal chemotherapy target, the kinetics parameters reveal the impossibility to use SmHGPRT as an efficient chemotherapeutic target.
|
Feb 2019
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Juliana Roberta
Torini
,
Larissa
Romanello
,
Fernanda Aparecida Heleno
Batista
,
Vitor Hugo Balasco
Serrao
,
Muhamamd
Faheem
,
Ana Eliza
Zeraik
,
Louise
Bird
,
Joanne E.
Nettleship
,
Yamini
Reddivari
,
Ray
Owens
,
Ricardo
Demarco
,
Júlio César
Borges
,
Jose
Brandao-Neto
,
Humberto
D'Muniz Pereira
Open Access
Abstract: Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, SmPNP2. Thermofluorescence screening of different ligands suggested cytidine and cytosine are potential ligands. The binding of cytosine and cytidine were confirmed by isothermal titration calorimetry, with a KD of 27 μM for cytosine, and a KM of 76.3 μM for cytidine. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystal structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capacity of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site thus permitting an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site enhances our understanding of the correlation between structure and nucleotide selectivity, thus suggesting new ways to analyse PNP activity.
|
Sep 2018
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[14744]
Open Access
Abstract: The genomes of the malaria-causing Plasmodium parasites encode a protein fused of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) domains that catalyze sequential reactions in the folate biosynthetic pathway. Whereas higher organisms derive folate from their diet and lack the enzymes for its synthesis, most eubacteria and a number of lower eukaryotes including malaria parasites synthesize tetrahydrofolate via DHPS. Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) HPPK-DHPSs are currently targets of drugs like sulfadoxine (SDX). The SDX effectiveness as an antimalarial drug is increasingly diminished by rise and spread of drug resistance mutations. Here, we present the crystal structure of PvHPPK-DHPS in complex with four substrates/analogs, revealing the bifunctional PvHPPK-DHPS architecture in an unprecedented state of enzymatic activation. SDX’s effect on HPPK-DHPS is due to 4-amino benzoic acid (pABA) mimicry, and the PvHPPK-DHPS structure sheds light on the SDX-binding cavity as well as on mutations that effect SDX potency. We mapped five dominant drug resistance mutations in PvHPPK-DHPS: S382A, A383G, K512E/D, A553G, and V585A, most of which occur individually or in clusters proximal to the pABA-binding site. We found that these resistance mutations subtly alter the intricate enzyme/pABA/SDX interactions such that DHPS affinity for pABA is diminished only moderately, but its affinity for SDX is changed substantially. In conclusion, the PvHPPK-DHPS structure rationalizes and unravels the structural bases for SDX resistance mutations and highlights architectural features in HPPK-DHPSs from malaria parasites that can form the basis for developing next-generation anti-folate agents to combat malaria parasites.
|
Aug 2018
|
|
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[12346]
Open Access
Abstract: Lens epithelium-derived growth factor (LEDGF)/p75 is the dominant binding partner of HIV-1 integrase in human cells. The crystal structure of the HIV integrase-binding domain (IBD) of LEDGF has been determined in the absence of ligand. IBD was overexpressed in Escherichia coli, purified and crystallized by sitting-drop vapour diffusion. X-ray diffraction data were collected at Diamond Light Source to a resolution of 2.05 Å. The crystals belonged to space group P21, with eight polypeptide chains in the asymmetric unit arranged as an unusual octamer composed of four domain-swapped IBD dimers. IBD exists as a mixture of monomers and dimers in concentrated solutions, but the dimers are unlikely to be biologically relevant.
|
Mar 2018
|
|
I24-Microfocus Macromolecular Crystallography
|
James I.
Robinson
,
Euan W.
Baxter
,
Robin L.
Owen
,
Maren
Thomsen
,
Darren C.
Tomlinson
,
Mark P.
Waterhouse
,
Stephanie J.
Win
,
Joanne E.
Nettleship
,
Christian
Tiede
,
Richard J.
Foster
,
Raymond J.
Owens
,
Colin W. G.
Fishwick
,
Sarah A.
Harris
,
Adrian
Goldman
,
Michael J.
Mcpherson
,
Ann W.
Morgan
Diamond Proposal Number(s):
[5969]
Abstract: Protein–protein interactions are essential for the control of cellular functions and are critical for regulation of the immune system. One example is the binding of Fc regions of IgG to the Fc gamma receptors (FcγRs). High sequence identity (98%) between the genes encoding FcγRIIIa (expressed on macrophages and natural killer cells) and FcγRIIIb (expressed on neutrophils) has prevented the development of monospecific agents against these therapeutic targets. We now report the identification of FcγRIIIa-specific artificial binding proteins called “Affimer” that block IgG binding and abrogate FcγRIIIa-mediated downstream effector functions in macrophages, namely TNF release and phagocytosis. Cocrystal structures and molecular dynamics simulations have revealed the structural basis of this specificity for two Affimer proteins: One binds directly to the Fc binding site, whereas the other acts allosterically.
|
Jan 2018
|
|
I02-Macromolecular Crystallography
|
Larissa
Romanello
,
Vitor Hugo Balasco
Serrao
,
Juliana Roberta Torini
De Souza
,
Louise E.
Bird
,
Joanne E.
Nettleship
,
Heather
Rada
,
Yamini
Reddivari
,
Ray J.
Owens
,
Ricardo
De Marco
,
Jose
Brandao-Neto
,
Humberto
D' Muniz Pereira
Diamond Proposal Number(s):
[5073, 5894]
Abstract: Schistosoma mansoni is the parasite responsible for schistosomiasis, a disease that affects about 218 million people worldwide. Currently, both direct treatment and disease control initiatives rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, have stimulated efforts to develop new drugs for the treatment of schistosomiasis. Schistosomes do not have the de novo purine biosynthetic pathway, and instead depend entirely on the purine salvage pathway to supply its need for purines. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenylosuccinate lyase (SmADSL) is an enzyme in this pathway, which cleaves adenylosuccinate (ADS) into adenosine 5'-monophosphate (AMP) and fumarate. SmADSL kinetic characterization was performed by isothermal titration calorimetry (ITC) using both ADS and SAICAR as substrates. Structures of SmADSL in Apo form and in complex with AMP were elucidated by x-ray crystallography revealing a highly conserved tetrameric structure required for their function since the active sites is formed from residues of three different subunits. The active sites are also highly conserved between species and it is difficult to identify a potent species-specific inhibitor for the development of new therapeutic agents. In contrast, several mutagenesis studies have demonstrated the importance of dimeric interface residues in the stability of the quaternary structure of the enzyme. The lower conservation of these residues between SmADSL and human ADSL could be used to lead the development of anti-schistosomiasis drugs based on disruption of subunit interfaces. These structures and kinetics data add another layer of information to Schistosoma mansoni purine salvage pathway.
|
Mar 2017
|
|
