Publications by authors named "Karl Harlos"

93 Publications

Inhibition of Plasmodium falciparum Lysyl-tRNA Synthetase via a Piperidine-Ring Scaffold Inspired Cladosporin Analogues.

Chembiochem 2021 Jul 28;22(14):2468-2477. Epub 2021 May 28.

Molecular Medicine - Structural Parasitology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi, 110067, India.

Plasmodium falciparum lysyl-tRNA synthetase (PfKRS) represents a promising therapeutic anti-malarial target. Cladosporin 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 reveal compounds that are moderate in potency. Co-crystals of Cla-B and Cla-C with PfKRS reveal key atomic configurations that allow drug binding to and inhibition of the enzyme. Collectively these piperidine ring scaffold inhibitors lay a framework for further structural editing and functional modifications of the cladosporin scaffold to obtain a potent lead.
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http://dx.doi.org/10.1002/cbic.202100212DOI Listing
July 2021

Structural basis of malaria parasite phenylalanine tRNA-synthetase inhibition by bicyclic azetidines.

Nat Commun 2021 01 12;12(1):343. Epub 2021 Jan 12.

Molecular Medicine, Structural Parasitology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India.

The inhibition of Plasmodium cytosolic phenylalanine tRNA-synthetase (cFRS) by a novel series of bicyclic azetidines has shown the potential to prevent malaria transmission, provide prophylaxis, and offer single-dose cure in animal models of malaria. To date, however, the molecular basis of Plasmodium cFRS inhibition by bicyclic azetidines has remained unknown. Here, we present structural and biochemical evidence that bicyclic azetidines are competitive inhibitors of L-Phe, one of three substrates required for the cFRS-catalyzed aminoacylation reaction that underpins protein synthesis in the parasite. Critically, our co-crystal structure of a PvcFRS-BRD1389 complex shows that the bicyclic azetidine ligand binds to two distinct sub-sites within the PvcFRS catalytic site. The ligand occupies the L-Phe site along with an auxiliary cavity and traverses past the ATP binding site. Given that BRD1389 recognition residues are conserved amongst apicomplexan FRSs, this work lays a structural framework for the development of drugs against both Plasmodium and related apicomplexans.
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http://dx.doi.org/10.1038/s41467-020-20478-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803973PMC
January 2021

Structure of the Human Cation-Independent Mannose 6-Phosphate/IGF2 Receptor Domains 7-11 Uncovers the Mannose 6-Phosphate Binding Site of Domain 9.

Structure 2020 12 1;28(12):1300-1312.e5. Epub 2020 Sep 1.

School of Chemistry, Cantock's Close, University of Bristol, Bristol BS8 1TS, UK; BrisSynBio, Life Sciences Building, Tyndall Avenue, Bristol BS8 1TQ, UK. Electronic address:

The cation-independent mannose 6-phosphate (M6P)/Insulin-like growth factor-2 receptor (CI-MPR/IGF2R) is an ∼300 kDa transmembrane protein responsible for trafficking M6P-tagged lysosomal hydrolases and internalizing IGF2. The extracellular region of the CI-MPR has 15 homologous domains, including M6P-binding domains (D) 3, 5, 9, and 15 and IGF2-binding domain 11. We have focused on solving the first structures of human D7-10 within two multi-domain constructs, D9-10 and D7-11, and provide the first high-resolution description of the high-affinity M6P-binding D9. Moreover, D9 stabilizes a well-defined hub formed by D7-11 whereby two penta-domains intertwine to form a dimeric helical-type coil via an N-glycan bridge on D9. Remarkably the D7-11 structure matches an IGF2-bound state of the receptor, suggesting this may be an intrinsically stable conformation at neutral pH. Interdomain clusters of histidine and proline residues may impart receptor rigidity and play a role in structural transitions at low pH.
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http://dx.doi.org/10.1016/j.str.2020.08.002DOI Listing
December 2020

Natural Killer Cell Activation Receptor NKp30 Oligomerization Depends on Its -Glycosylation.

Cancers (Basel) 2020 Jul 21;12(7). Epub 2020 Jul 21.

Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, 12840 Prague, Czech Republic.

NKp30 is one of the main human natural killer (NK) cell activating receptors used in directed immunotherapy. The oligomerization of the NKp30 ligand binding domain depends on the length of the C-terminal stalk region, but our structural knowledge of NKp30 oligomerization and its role in signal transduction remains limited. Moreover, ligand binding of NKp30 is affected by the presence and type of -glycosylation. In this study, we assessed whether NKp30 oligomerization depends on its -glycosylation. Our results show that NKp30 forms oligomers when expressed in HEK293S GnTI cell lines with simple -glycans. However, NKp30 was detected only as monomers after enzymatic deglycosylation. Furthermore, we characterized the interaction between NKp30 and its best-studied cognate ligand, B7-H6, with respect to glycosylation and oligomerization, and we solved the crystal structure of this complex with glycosylated NKp30, revealing a new glycosylation-induced mode of NKp30 dimerization. Overall, this study provides new insights into the structural basis of NKp30 oligomerization and explains how the stalk region and glycosylation of NKp30 affect its ligand affinity. This furthers our understanding of the molecular mechanisms involved in NK cell activation, which is crucial for the successful design of novel NK cell-based targeted immunotherapeutics.
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http://dx.doi.org/10.3390/cancers12071998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409301PMC
July 2020

Structural basis of semaphorin-plexin cis interaction.

EMBO J 2020 07 5;39(13):e102926. Epub 2020 Jun 5.

Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.

Semaphorin ligands interact with plexin receptors to contribute to functions in the development of myriad tissues including neurite guidance and synaptic organisation within the nervous system. Cell-attached semaphorins interact in trans with plexins on opposing cells, but also in cis on the same cell. The interplay between trans and cis interactions is crucial for the regulated development of complex neural circuitry, but the underlying molecular mechanisms are uncharacterised. We have discovered a distinct mode of interaction through which the Drosophila semaphorin Sema1b and mouse Sema6A mediate binding in cis to their cognate plexin receptors. Our high-resolution structural, biophysical and in vitro analyses demonstrate that monomeric semaphorins can mediate a distinctive plexin binding mode. These findings suggest the interplay between monomeric vs dimeric states has a hereto unappreciated role in semaphorin biology, providing a mechanism by which Sema6s may balance cis and trans functionalities.
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http://dx.doi.org/10.15252/embj.2019102926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327498PMC
July 2020

Crystal structures of the two domains that constitute the Plasmodium vivax p43 protein.

Acta Crystallogr D Struct Biol 2020 Feb 30;76(Pt 2):135-146. Epub 2020 Jan 30.

Molecular Medicine - Structural Parasitology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Road, New Delhi 110 067, India.

Scaffold modules known as aminoacyl-tRNA synthetase (aaRS)-interacting multifunctional proteins (AIMPs), such as AIMP1/p43, AIMP2/p38 and AIMP3/p18, are important in driving the assembly of multi-aaRS (MARS) complexes in eukaryotes. Often, AIMPs contain an N-terminal glutathione S-transferase (GST)-like domain and a C-terminal OB-fold tRNA-binding domain. Recently, the apicomplexan-specific Plasmodium falciparum p43 protein (Pfp43) has been annotated as an AIMP and its tRNA binding, tRNA import and membrane association have been characterized. The crystal structures of both the N- and C-terminal domains of the Plasmodium vivax p43 protein (Pvp43), which is an ortholog of Pfp43, have been resolved. Analyses reveal the overall oligomeric structure of Pvp43 and highlight several notable features that show Pvp43 to be a soluble, cytosolic protein. The dimeric assembly of the N-terminal GST-like domain of Pvp43 differs significantly from canonical GST dimers, and it is tied to the C-terminal tRNA-binding domain via a linker region. This work therefore establishes a framework for dissecting the additional roles of p43 orthologs in eukaryotic multi-protein MARS complexes.
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http://dx.doi.org/10.1107/S2059798319016413DOI Listing
February 2020

Disruption of the dimerization interface of the sensing domain in the dimeric heme-based oxygen sensor GcHK abolishes bacterial signal transduction.

J Biol Chem 2020 02 30;295(6):1587-1597. Epub 2019 Dec 30.

Department of Biochemistry, Faculty of Science, Charles University, Prague 2, 128 43 Czech Republic. Electronic address:

The heme-based oxygen sensor protein GcHK is a globin-coupled histidine kinase in the soil bacterium sp. Fw109-5. Its C-terminal functional domain exhibits autophosphorylation activity induced by oxygen binding to the heme-Fe(II) complex located in the oxygen-sensing N-terminal globin domain. A detailed understanding of the signal transduction mechanisms in heme-containing sensor proteins remains elusive. Here, we investigated the role of the globin domain's dimerization interface in signal transduction in GcHK. We present a crystal structure of a monomeric imidazole-bound GcHK globin domain at 1.8 Å resolution, revealing that the helices of the WT globin dimer are under tension and suggesting that Tyr-15 plays a role in both this tension and the globin domain's dimerization. Biophysical experiments revealed that whereas the isolated WT globin domain is dimeric in solution, the Y15A and Y15G variants in which Tyr-15 is replaced with Ala or Gly, respectively, are monomeric. Additionally, we found that although the dimerization of the full-length protein is preserved via the kinase domain dimerization interface in all variants, full-length GcHK variants bearing the Y15A or Y15G substitutions lack enzymatic activity. The combined structural and biophysical results presented here indicate that Tyr-15 plays a key role in the dimerization of the globin domain of GcHK and that globin domain dimerization is essential for internal signal transduction and autophosphorylation in this protein. These findings provide critical insights into the signal transduction mechanism of the histidine kinase GcHK from .
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http://dx.doi.org/10.1074/jbc.RA119.011574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7008379PMC
February 2020

A key region of molecular specificity orchestrates unique ephrin-B1 utilization by Cedar virus.

Life Sci Alliance 2020 01 20;3(1). Epub 2019 Dec 20.

Icahn School of Medicine at Mount Sinai, New York, NY, USA

The emergent zoonotic henipaviruses, Hendra, and Nipah are responsible for frequent and fatal disease outbreaks in domestic animals and humans. Specificity of henipavirus attachment glycoproteins (G) for highly species-conserved ephrin ligands underpins their broad host range and is associated with systemic and neurological disease pathologies. Here, we demonstrate that Cedar virus (CedV)-a related henipavirus that is ostensibly nonpathogenic-possesses an idiosyncratic entry receptor repertoire that includes the common henipaviral receptor, ephrin-B2, but, distinct from pathogenic henipaviruses, does not include ephrin-B3. Uniquely among known henipaviruses, CedV can use ephrin-B1 for cellular entry. Structural analyses of CedV-G reveal a key region of molecular specificity that directs ephrin-B1 utilization, while preserving a universal mode of ephrin-B2 recognition. The structural and functional insights presented uncover diversity within the known henipavirus receptor repertoire and suggest that only modest structural changes may be required to modulate receptor specificities within this group of lethal human pathogens.
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http://dx.doi.org/10.26508/lsa.201900578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925387PMC
January 2020

Diversity of oligomerization in Drosophila semaphorins suggests a mechanism of functional fine-tuning.

Nat Commun 2019 08 15;10(1):3691. Epub 2019 Aug 15.

Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK.

Semaphorin ligands and their plexin receptors are one of the major cell guidance factors that trigger localised changes in the cytoskeleton. Binding of semaphorin homodimer to plexin brings two plexins in close proximity which is a prerequisite for plexin signalling. This model appears to be too simplistic to explain the complexity and functional versatility of these molecules. Here, we determine crystal structures for all members of Drosophila class 1 and 2 semaphorins. Unlike previously reported semaphorin structures, Sema1a, Sema2a and Sema2b show stabilisation of sema domain dimer formation via a disulfide bond. Unexpectedly, our structural and biophysical data show Sema1b is a monomer suggesting that semaphorin function may not be restricted to dimers. We demonstrate that semaphorins can form heterodimers with members of the same semaphorin class. This heterodimerization provides a potential mechanism for cross-talk between different plexins and co-receptors to allow fine-tuning of cell signalling.
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http://dx.doi.org/10.1038/s41467-019-11683-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6695400PMC
August 2019

The structure of a prokaryotic viral envelope protein expands the landscape of membrane fusion proteins.

Nat Commun 2019 02 19;10(1):846. Epub 2019 Feb 19.

Molecular and Integrative Biosciences Research Program, University of Helsinki, Viikinkaari 1, 00014, Helsinki, Finland.

Lipid membrane fusion is an essential function in many biological processes. Detailed mechanisms of membrane fusion and the protein structures involved have been mainly studied in eukaryotic systems, whereas very little is known about membrane fusion in prokaryotes. Haloarchaeal pleomorphic viruses (HRPVs) have a membrane envelope decorated with spikes that are presumed to be responsible for host attachment and membrane fusion. Here we determine atomic structures of the ectodomains of the 57-kDa spike protein VP5 from two related HRPVs revealing a previously unreported V-shaped fold. By Volta phase plate cryo-electron tomography we show that VP5 is monomeric on the viral surface, and we establish the orientation of the molecules with respect to the viral membrane. We also show that the viral membrane fuses with the host cytoplasmic membrane in a process mediated by VP5. This sheds light on protein structures involved in prokaryotic membrane fusion.
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http://dx.doi.org/10.1038/s41467-019-08728-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381117PMC
February 2019

Insights from the crystal structure of the chicken CREB3 bZIP suggest that members of the CREB3 subfamily transcription factors may be activated in response to oxidative stress.

Protein Sci 2019 04 6;28(4):779-787. Epub 2019 Feb 6.

Division of Structural Biology, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, United Kingdom.

cAMP response element binding Protein 3 (CREB3) is an endoplasmic reticulum (ER) membrane-bound transcription factor, which belongs to the basic leucine zipper (bZIP) superfamily of eukaryotic transcription factors. CREB3 plays a role in the ER-stress induced unfolded protein response (UPR) and is a multifunctional cellular factor implicated in a number of biological processes including cell proliferation and migration, tumor suppression, and immune-related gene expression. To gain structural insights into the transcription factor, we determined the crystal structure of the conserved bZIP domain of chicken CREB3 (chCREB3) to a resolution of 3.95 Å. The X-ray structure provides evidence that chCREB3 can form a stable homodimer. The chCREB3 bZIP has a structured, pre-formed DNA binding region, even in the absence of DNA, a feature that could potentially enhance both the DNA binding specificity and affinity of chCREB3. Significantly, the homodimeric bZIP possesses an intermolecular disulfide bond that connects equivalent cysteine residues of the parallel helices in the leucine zipper region. This disulfide bond in the hydrophobic core of the bZIP may increase the stability of the homodimer under oxidizing conditions. Moreover, sequence alignment of bZIP sequences from chicken, human, and mouse reveals that only members of the CREB3 subfamily contain this cysteine residue, indicating that it could act as a redox-sensor. Taken together, these results suggest that the activity of these transcription factors may be redox-regulated and they may be activated in response to oxidative stress.
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http://dx.doi.org/10.1002/pro.3573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6423718PMC
April 2019

A Protective Monoclonal Antibody Targets a Site of Vulnerability on the Surface of Rift Valley Fever Virus.

Cell Rep 2018 12;25(13):3750-3758.e4

Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK; Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address:

The Gn subcomponent of the Gn-Gc assembly that envelopes the human and animal pathogen, Rift Valley fever virus (RVFV), is a primary target of the neutralizing antibody response. To better understand the molecular basis for immune recognition, we raised a class of neutralizing monoclonal antibodies (nAbs) against RVFV Gn, which exhibited protective efficacy in a mouse infection model. Structural characterization revealed that these nAbs were directed to the membrane-distal domain of RVFV Gn and likely prevented virus entry into a host cell by blocking fusogenic rearrangements of the Gn-Gc lattice. Genome sequence analysis confirmed that this region of the RVFV Gn-Gc assembly was under selective pressure and constituted a site of vulnerability on the virion surface. These data provide a blueprint for the rational design of immunotherapeutics and vaccines capable of preventing RVFV infection and a model for understanding Ab-mediated neutralization of bunyaviruses more generally.
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http://dx.doi.org/10.1016/j.celrep.2018.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315105PMC
December 2018

Author Correction: Pathogen-derived HLA-E bound epitopes reveal broad primary anchor pocket tolerability and conformationally malleable peptide binding.

Nat Commun 2018 11 13;9(1):4833. Epub 2018 Nov 13.

Nuffield Department of Medicine Research Building, Roosevelt Drive, Nuffield Department of Medicine, University of Oxford, Oxford, OX3 7FZ, UK.

The original version of this Article contained an error in the spelling of the author Jonah B Sacha, which was incorrectly given as Jonah Sacha. These errors have now been corrected in both the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41467-018-07304-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232182PMC
November 2018

Structure of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase-dihydropteroate synthase from sheds light on drug resistance.

J Biol Chem 2018 09 13;293(39):14962-14972. Epub 2018 Aug 13.

From the Molecular Medicine-Structural Parasitology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India.

The genomes of the malaria-causing 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. () and () HPPK-DHPSs are currently targets of drugs like sulfadoxine (SDX). The SDX effectiveness as an antimalarial drug is increasingly diminished by the rise and spread of drug-resistant mutations. Here, we present the crystal structure of HPPK-DHPS in complex with four substrates/analogs, revealing the bifunctional HPPK-DHPS architecture in an unprecedented state of enzymatic activation. SDX's effect on HPPK-DHPS is due to 4-amino benzoic acid (ABA) mimicry, and the HPPK-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 HPPK-DHPS: S382A, A383G, K512E/D, A553G, and V585A, most of which occur individually or in clusters proximal to the ABA-binding site. We found that these resistance mutations subtly alter the intricate enzyme/ABA/SDX interactions such that DHPS affinity for ABA is diminished only moderately, but its affinity for SDX is changed substantially. In conclusion, the HPPK-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.
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http://dx.doi.org/10.1074/jbc.RA118.004558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166723PMC
September 2018

Pathogen-derived HLA-E bound epitopes reveal broad primary anchor pocket tolerability and conformationally malleable peptide binding.

Nat Commun 2018 08 7;9(1):3137. Epub 2018 Aug 7.

Nuffield Department of Medicine Research Building, Roosevelt Drive, Nuffield Department of Medicine, University of Oxford, Oxford, OX3 7FZ, UK.

Through major histocompatibility complex class Ia leader sequence-derived (VL9) peptide binding and CD94/NKG2 receptor engagement, human leucocyte antigen E (HLA-E) reports cellular health to NK cells. Previous studies demonstrated a strong bias for VL9 binding by HLA-E, a preference subsequently supported by structural analyses. However, Mycobacteria tuberculosis (Mtb) infection and Rhesus cytomegalovirus-vectored SIV vaccinations revealed contexts where HLA-E and the rhesus homologue, Mamu-E, presented diverse pathogen-derived peptides to CD8 T cells, respectively. Here we present crystal structures of HLA-E in complex with HIV and Mtb-derived peptides. We show that despite the presence of preferred primary anchor residues, HLA-E-bound peptides can adopt alternative conformations within the peptide binding groove. Furthermore, combined structural and mutagenesis analyses illustrate a greater tolerance for hydrophobic and polar residues in the primary pockets than previously appreciated. Finally, biochemical studies reveal HLA-E peptide binding and exchange characteristics with potential relevance to its alternative antigen presenting function in vivo.
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http://dx.doi.org/10.1038/s41467-018-05459-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6081459PMC
August 2018

Structure of the Wnt signaling enhancer LYPD6 and its interactions with the Wnt coreceptor LRP6.

FEBS Lett 2018 09 24;592(18):3152-3162. Epub 2018 Aug 24.

Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, UK.

Ly6/urokinase-type plasminogen activator receptor (uPAR) (LU) domain containing 6 (LYPD6) is a Wnt signaling enhancer that promotes phosphorylation of the Wnt coreceptor low density lipoprotein receptor-related protein 6 (LRP6). It also binds the nicotinic acetylcholine receptor (nAChR). We report here the 1.25 Å resolution structure of the LYPD6 extracellular LU domain and map its interaction with LRP6 by mutagenesis and surface plasmon resonance. The LYPD6 structure reveals a 'trifingered protein domain' fold with the middle fingertip bearing an 'NxI' motif, a tripeptide motif associated with LRP5/6 binding by Wnt inhibitors. Of the Ly6 protein family members, only LYPD6 has an NxI motif. Since mutations in the LYPD6 NxI motif abolish or severely reduce interaction with LRP6, our results indicate its key role in the interaction of LYPD6 with LRP6.
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http://dx.doi.org/10.1002/1873-3468.13212DOI Listing
September 2018

Specific Stereoisomeric Conformations Determine the Drug Potency of Cladosporin Scaffold against Malarial Parasite.

J Med Chem 2018 07 4;61(13):5664-5678. Epub 2018 Jun 4.

Organic Chemistry Division , CSIR-National Chemical Laboratory , Dr. Homi Bhabha Road , Pune 411008 , India.

The dependence of drug potency on diastereomeric configurations is a key facet. Using a novel general divergent synthetic route for a three-chiral center antimalarial natural product cladosporin, we built its complete library of stereoisomers (cladologs) and assessed their inhibitory potential using parasite-, enzyme-, and structure-based assays. We show that potency is manifest via tetrahyropyran ring conformations that are housed in the ribose binding pocket of parasite lysyl tRNA synthetase (KRS). Strikingly, drug potency between top and worst enantiomers varied 500-fold, and structures of KRS-cladolog complexes reveal that alterations at C3 and C10 are detrimental to drug potency whereas changes at C3 are sensed by rotameric flipping of glutamate 332. Given that scores of antimalarial and anti-infective drugs contain chiral centers, this work provides a new foundation for focusing on inhibitor stereochemistry as a facet of antimicrobial drug development.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00565DOI Listing
July 2018

Structures of monomeric and oligomeric forms of the perforin-like protein 1.

Sci Adv 2018 03 21;4(3):eaaq0762. Epub 2018 Mar 21.

Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.

and are the parasitic agents of toxoplasmosis and malaria, respectively, and use perforin-like proteins (PLPs) to invade host organisms and complete their life cycles. The PLP1 (PLP1) is required for efficient exit from parasitophorous vacuoles in which proliferation occurs. We report structures of the membrane attack complex/perforin (MACPF) and Apicomplexan PLP C-terminal β-pleated sheet (APCβ) domains of PLP1. The MACPF domain forms hexameric assemblies, with ring and helix geometries, and the APCβ domain has a novel β-prism fold joined to the MACPF domain by a short linker. Molecular dynamics simulations suggest that the helical MACPF oligomer preserves a biologically important interface, whereas the APCβ domain binds preferentially through a hydrophobic loop to membrane phosphatidylethanolamine, enhanced by the additional presence of inositol phosphate lipids. This mode of membrane binding is supported by site-directed mutagenesis data from a liposome-based assay. Together, these structural and biophysical findings provide insights into the molecular mechanism of membrane targeting by PLP1.
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http://dx.doi.org/10.1126/sciadv.aaq0762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943054PMC
March 2018

Shielding and activation of a viral membrane fusion protein.

Nat Commun 2018 01 24;9(1):349. Epub 2018 Jan 24.

Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK.

Entry of enveloped viruses relies on insertion of hydrophobic residues of the viral fusion protein into the host cell membrane. However, the intermediate conformations during fusion remain unknown. Here, we address the fusion mechanism of Rift Valley fever virus. We determine the crystal structure of the Gn glycoprotein and fit it with the Gc fusion protein into cryo-electron microscopy reconstructions of the virion. Our analysis reveals how the Gn shields the hydrophobic fusion loops of the Gc, preventing premature fusion. Electron cryotomography of virions interacting with membranes under acidic conditions reveals how the fusogenic Gc is activated upon removal of the Gn shield. Repositioning of the Gn allows extension of Gc and insertion of fusion loops in the outer leaflet of the target membrane. These data show early structural transitions that enveloped viruses undergo during host cell entry and indicate that analogous shielding mechanisms are utilized across diverse virus families.
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http://dx.doi.org/10.1038/s41467-017-02789-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5783950PMC
January 2018

Targeting Prolyl-tRNA Synthetase to Accelerate Drug Discovery against Malaria, Leishmaniasis, Toxoplasmosis, Cryptosporidiosis, and Coccidiosis.

Structure 2017 10 31;25(10):1495-1505.e6. Epub 2017 Aug 31.

Molecular Medicine - Structural Parasitology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, India. Electronic address:

Developing anti-parasitic lead compounds that act on key vulnerabilities are necessary for new anti-infectives. Malaria, leishmaniasis, toxoplasmosis, cryptosporidiosis and coccidiosis together kill >500,000 humans annually. Their causative parasites Plasmodium, Leishmania, Toxoplasma, Cryptosporidium and Eimeria display high conservation in many housekeeping genes, suggesting that these parasites can be attacked by targeting invariant essential proteins. Here, we describe selective and potent inhibition of prolyl-tRNA synthetases (PRSs) from the above parasites using a series of quinazolinone-scaffold compounds. Our PRS-drug co-crystal structures reveal remarkable active site plasticity that accommodates diversely substituted compounds, an enzymatic feature that can be leveraged for refining drug-like properties of quinazolinones on a per parasite basis. A compound we termed In-5 exhibited a unique double conformation, enhanced drug-like properties, and cleared malaria in mice. It thus represents a new lead for optimization. Collectively, our data offer insights into the structure-guided optimization of quinazolinone-based compounds for drug development against multiple human eukaryotic pathogens.
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http://dx.doi.org/10.1016/j.str.2017.07.015DOI Listing
October 2017

Structural Transitions of the Conserved and Metastable Hantaviral Glycoprotein Envelope.

J Virol 2017 11 13;91(21). Epub 2017 Oct 13.

Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom

Hantaviruses are zoonotic pathogens that cause severe hemorrhagic fever and pulmonary syndrome. The outer membrane of the hantavirus envelope displays a lattice of two glycoproteins, Gn and Gc, which orchestrate host cell recognition and entry. Here, we describe the crystal structure of the Gn glycoprotein ectodomain from the Asiatic Hantaan virus (HTNV), the most prevalent pathogenic hantavirus. Structural overlay analysis reveals that the HTNV Gn fold is highly similar to the Gn of Puumala virus (PUUV), a genetically and geographically distinct and less pathogenic hantavirus found predominantly in northeastern Europe, confirming that the hantaviral Gn fold is architecturally conserved across hantavirus clades. Interestingly, HTNV Gn crystallized at acidic pH, in a compact tetrameric configuration distinct from the organization at neutral pH. Analysis of the Gn, both in solution and in the context of the virion, confirms the pH-sensitive oligomeric nature of the glycoprotein, indicating that the hantaviral Gn undergoes structural transitions during host cell entry. These data allow us to present a structural model for how acidification during endocytic uptake of the virus triggers the dissociation of the metastable Gn-Gc lattice to enable insertion of the Gc-resident hydrophobic fusion loops into the host cell membrane. Together, these data reveal the dynamic plasticity of the structurally conserved hantaviral surface. Although outbreaks of Korean hemorrhagic fever were first recognized during the Korean War (1950 to 1953), it was not until 1978 that they were found to be caused by Hantaan virus (HTNV), the most prevalent pathogenic hantavirus. Here, we describe the crystal structure of HTNV envelope glycoprotein Gn, an integral component of the Gn-Gc glycoprotein spike complex responsible for host cell entry. HTNV Gn is structurally conserved with the Gn of a genetically and geographically distal hantavirus, Puumala virus, indicating that the observed α/β fold is well preserved across the family. The combination of our crystal structure with solution state analysis of recombinant protein and electron cryo-microscopy of acidified hantavirus allows us to propose a model for endosome-induced reorganization of the hantaviral glycoprotein lattice. This provides a molecular-level rationale for the exposure of the hydrophobic fusion loops on the Gc, a process required for fusion of viral and cellular membranes.
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http://dx.doi.org/10.1128/JVI.00378-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640846PMC
November 2017

Convergent immunological solutions to Argentine hemorrhagic fever virus neutralization.

Proc Natl Acad Sci U S A 2017 07 19;114(27):7031-7036. Epub 2017 Jun 19.

Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom;

Transmission of hemorrhagic fever New World arenaviruses from their rodent reservoirs to human populations poses substantial public health and economic dangers. These zoonotic events are enabled by the specific interaction between the New World arenaviral attachment glycoprotein, GP1, and cell surface human transferrin receptor (hTfR1). Here, we present the structural basis for how a mouse-derived neutralizing antibody (nAb), OD01, disrupts this interaction by targeting the receptor-binding surface of the GP1 glycoprotein from Junín virus (JUNV), a hemorrhagic fever arenavirus endemic in central Argentina. Comparison of our structure with that of a previously reported nAb complex (JUNV GP1-GD01) reveals largely overlapping epitopes but highly distinct antibody-binding modes. Despite differences in GP1 recognition, we find that both antibodies present a key tyrosine residue, albeit on different chains, that inserts into a central pocket on JUNV GP1 and effectively mimics the contacts made by the host TfR1. These data provide a molecular-level description of how antibodies derived from different germline origins arrive at equivalent immunological solutions to virus neutralization.
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http://dx.doi.org/10.1073/pnas.1702127114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5502616PMC
July 2017

High-speed fixed-target serial virus crystallography.

Nat Methods 2017 Aug 19;14(8):805-810. Epub 2017 Jun 19.

Photon Science, Deutsches Elektronen-Synchrotron DESY, Hamburg, Germany.

We report a method for serial X-ray crystallography at X-ray free-electron lasers (XFELs), which allows for full use of the current 120-Hz repetition rate of the Linear Coherent Light Source (LCLS). Using a micropatterned silicon chip in combination with the high-speed Roadrunner goniometer for sample delivery, we were able to determine the crystal structures of the picornavirus bovine enterovirus 2 (BEV2) and the cytoplasmic polyhedrosis virus type 18 polyhedrin, with total data collection times of less than 14 and 10 min, respectively. Our method requires only micrograms of sample and should therefore broaden the applicability of serial femtosecond crystallography to challenging projects for which only limited sample amounts are available. By synchronizing the sample exchange to the XFEL repetition rate, our method allows for most efficient use of the limited beam time available at XFELs and should enable a substantial increase in sample throughput at these facilities.
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http://dx.doi.org/10.1038/nmeth.4335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5588887PMC
August 2017

Ligand-Induced Conformational Change of Insulin-Regulated Aminopeptidase: Insights on Catalytic Mechanism and Active Site Plasticity.

J Med Chem 2017 04 3;60(7):2963-2972. Epub 2017 Apr 3.

National Center for Scientific Research Demokritos, Agia Paraskevi , Athens 15310, Greece.

Insulin-regulated aminopeptidase (IRAP) is an enzyme with several important biological functions that is known to process a large variety of different peptidic substrates, although the mechanism behind this wide specificity is not clearly understood. We describe a crystal structure of IRAP in complex with a recently developed bioactive and selective inhibitor at 2.53 Å resolution. In the presence of this inhibitor, the enzyme adopts a novel conformation in which domains II and IV are juxtaposed, forming a hollow structure that excludes external solvent access to the catalytic center. A loop adjacent to the enzyme's GAMEN motif undergoes structural reconfiguration, allowing the accommodation of bulky inhibitor side chains. Atomic interactions between the inhibitor and IRAP that are unique to this conformation can explain the strong selectivity compared to homologous aminopeptidases ERAP1 and ERAP2. This conformation provides insight on IRAP's catalytic cycle and reveals significant active-site plasticity that may underlie its substrate permissiveness.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01890DOI Listing
April 2017

A Two-Tailed Phosphopeptide Crystallizes to Form a Lamellar Structure.

Angew Chem Int Ed Engl 2017 03 13;56(12):3252-3255. Epub 2017 Feb 13.

Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, 76100, Israel.

The crystal structure of a designed phospholipid-inspired amphiphilic phosphopeptide at 0.8 Å resolution is presented. The phosphorylated β-hairpin peptide crystallizes to form a lamellar structure that is stabilized by intra- and intermolecular hydrogen bonding, including an extended β-sheet structure, as well as aromatic interactions. This first reported crystal structure of a two-tailed peptidic bilayer reveals similarities in thickness to a typical phospholipid bilayer. However, water molecules interact with the phosphopeptide in the hydrophilic region of the lattice. Additionally, solid-state NMR was used to demonstrate correlation between the crystal structure and supramolecular nanostructures. The phosphopeptide was shown to self-assemble into semi-elliptical nanosheets, and solid-state NMR provides insight into the self-assembly mechanisms. This work brings a new dimension to the structural study of biomimetic amphiphilic peptides with determination of molecular organization at the atomic level.
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http://dx.doi.org/10.1002/anie.201609877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412914PMC
March 2017

Toremifene interacts with and destabilizes the Ebola virus glycoprotein.

Nature 2016 07 29;535(7610):169-172. Epub 2016 Jun 29.

Division of Structural Biology, University of Oxford, The Henry Wellcome Building for Genomic Medicine, Headington, Oxford, OX3 7BN, UK.

Ebola viruses (EBOVs) are responsible for repeated outbreaks of fatal infections, including the recent deadly epidemic in West Africa. There are currently no approved therapeutic drugs or vaccines for the disease. EBOV has a membrane envelope decorated by trimers of a glycoprotein (GP, cleaved by furin to form GP1 and GP2 subunits), which is solely responsible for host cell attachment, endosomal entry and membrane fusion. GP is thus a primary target for the development of antiviral drugs. Here we report the first, to our knowledge, unliganded structure of EBOV GP, and high-resolution complexes of GP with the anticancer drug toremifene and the painkiller ibuprofen. The high-resolution apo structure gives a more complete and accurate picture of the molecule, and allows conformational changes introduced by antibody and receptor binding to be deciphered. Unexpectedly, both toremifene and ibuprofen bind in a cavity between the attachment (GP1) and fusion (GP2) subunits at the entrance to a large tunnel that links with equivalent tunnels from the other monomers of the trimer at the three-fold axis. Protein–drug interactions with both GP1 and GP2 are predominately hydrophobic. Residues lining the binding site are highly conserved among filoviruses except Marburg virus (MARV), suggesting that MARV may not bind these drugs. Thermal shift assays show up to a 14 °C decrease in the protein melting temperature after toremifene binding, while ibuprofen has only a marginal effect and is a less potent inhibitor. These results suggest that inhibitor binding destabilizes GP and triggers premature release of GP2, thereby preventing fusion between the viral and endosome membranes. Thus, these complex structures reveal the mechanism of inhibition and may guide the development of more powerful anti-EBOV drugs.
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http://dx.doi.org/10.1038/nature18615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4947387PMC
July 2016

Structure of a phleboviral envelope glycoprotein reveals a consolidated model of membrane fusion.

Proc Natl Acad Sci U S A 2016 06 20;113(26):7154-9. Epub 2016 Jun 20.

Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom;

An emergent viral pathogen termed severe fever with thrombocytopenia syndrome virus (SFTSV) is responsible for thousands of clinical cases and associated fatalities in China, Japan, and South Korea. Akin to other phleboviruses, SFTSV relies on a viral glycoprotein, Gc, to catalyze the merger of endosomal host and viral membranes during cell entry. Here, we describe the postfusion structure of SFTSV Gc, revealing that the molecular transformations the phleboviral Gc undergoes upon host cell entry are conserved with otherwise unrelated alpha- and flaviviruses. By comparison of SFTSV Gc with that of the prefusion structure of the related Rift Valley fever virus, we show that these changes involve refolding of the protein into a trimeric state. Reverse genetics and rescue of site-directed histidine mutants enabled localization of histidines likely to be important for triggering this pH-dependent process. These data provide structural and functional evidence that the mechanism of phlebovirus-host cell fusion is conserved among genetically and patho-physiologically distinct viral pathogens.
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http://dx.doi.org/10.1073/pnas.1603827113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4932967PMC
June 2016

Structure of astrotactin-2: a conserved vertebrate-specific and perforin-like membrane protein involved in neuronal development.

Open Biol 2016 05 4;6(5). Epub 2016 May 4.

Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK

The vertebrate-specific proteins astrotactin-1 and 2 (ASTN-1 and ASTN-2) are integral membrane perforin-like proteins known to play critical roles in neurodevelopment, while ASTN-2 has been linked to the planar cell polarity pathway in hair cells. Genetic variations associated with them are linked to a variety of neurodevelopmental disorders and other neurological pathologies, including an advanced onset of Alzheimer's disease. Here we present the structure of the majority endosomal region of ASTN-2, showing it to consist of a unique combination of polypeptide folds: a perforin-like domain, a minimal epidermal growth factor-like module, a unique form of fibronectin type III domain and an annexin-like domain. The perforin-like domain differs from that of other members of the membrane attack complex-perforin (MACPF) protein family in ways that suggest ASTN-2 does not form pores. Structural and biophysical data show that ASTN-2 (but not ASTN-1) binds inositol triphosphates, suggesting a mechanism for membrane recognition or secondary messenger regulation of its activity. The annexin-like domain is closest in fold to repeat three of human annexin V and similarly binds calcium, and yet shares no sequence homology with it. Overall, our structure provides the first atomic-resolution description of a MACPF protein involved in development, while highlighting distinctive features of ASTN-2 responsible for its activity.
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http://dx.doi.org/10.1098/rsob.160053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4892435PMC
May 2016

The crystal structure of human dopamine β-hydroxylase at 2.9 Å resolution.

Sci Adv 2016 Apr 8;2(4):e1500980. Epub 2016 Apr 8.

Department of Chemistry, Kemitorvet 207, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.

The norepinephrine pathway is believed to modulate behavioral and physiological processes, such as mood, overall arousal, and attention. Furthermore, abnormalities in the pathway have been linked to numerous diseases, for example hypertension, depression, anxiety, Parkinson's disease, schizophrenia, Alzheimer's disease, attention deficit hyperactivity disorder, and cocaine dependence. We report the crystal structure of human dopamine β-hydroxylase, which is the enzyme converting dopamine to norepinephrine. The structure of the DOMON (dopamine β-monooxygenase N-terminal) domain, also found in >1600 other proteins, reveals a possible metal-binding site and a ligand-binding pocket. The catalytic core structure shows two different conformations: an open active site, as also seen in another member of this enzyme family [the peptidylglycine α-hydroxylating (and α-amidating) monooxygenase], and a closed active site structure, in which the two copper-binding sites are only 4 to 5 Å apart, in what might be a coupled binuclear copper site. The dimerization domain adopts a conformation that bears no resemblance to any other known protein structure. The structure provides new molecular insights into the numerous devastating disorders of both physiological and neurological origins associated with the dopamine system.
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http://dx.doi.org/10.1126/sciadv.1500980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846438PMC
April 2016
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