B21-High Throughput SAXS
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Open Access
Abstract: Human immunoglobulin IgG3 possesses a uniquely long hinge region that separates its Fab antigen-binding and Fc receptor-binding regions. Owing to this hinge length, the molecular structure of full-length IgG3 remains elusive, and the role of the two conserved glycosylation sites in the Fc region is unknown. To address these issues, we subjected glycosylated and deglycosylated human myeloma IgG3 to multidisciplinary solution structure studies. Using analytical ultracentrifugation, the elongated structure of IgG3 was determined from the reduced sedimentation coefficients s020,w of 5.82-6.29 S for both glycosylated and deglycosylated IgG3. X-ray and neutron scattering showed that the Guinier RG values were 6.95 nm for glycosylated IgG3 and were unchanged after deglycosylation, again indicating an elongated structure. The distance distribution function P(r) of both forms of IgG3 showed a maximum length of 25-28 nm and three distinct maxima. The molecular structure of IgG3 was determined using atomistic modelling based on molecular dynamics simulations of the IgG3 hinge and Monte Carlo simulations to identify physically-realistic arrangements of the Fab and Fc regions. This resulted in libraries containing 135,135 and 73,905 glycosylated and deglycosylated IgG3 structures respectively. Comparisons with the X-ray and neutron scattering curves gave 100 best-fit models for each of the two forms of IgG3 that accounted for the experimental scattering curves. These models revealed the first molecular structures for full-length IgG3. The structures exhibited relatively-restricted Fab and Fc conformations joined by an extended semi-rigid hinge, which explains the potent effector functions of IgG3 relative to the other subclasses IgG1, IgG2 and IgG4.
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Jul 2021
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[20113]
Open Access
Abstract: N-glycosylation is one of the most abundant post-translational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of glycosylation. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology, but also for the development of new drugs for a wide range of human diseases. Chemoenzymatic synthesis using engineered endo-β-N-acetylglucosaminidases (ENGases) has been used extensively to modulate the chemistry of N-glycosylated proteins. However, defining the molecular mechanisms by which ENGases specifically recognize and process N-glycans remains a major challenge. Here we present the X-ray crystal structure of the ENGase EndoBT-3987 from Bacteroides thetaiotaomicron in complex with a hybrid type (Hy-type) glycan product. In combination with alanine scanning mutagenesis, molecular docking calculations and enzymatic activity measurements conducted on a chemically engineered monoclonal antibody substrate unveil two mechanisms for Hy-type recognition and processing by paradigmatic ENGases. Altogether, the experimental data provide pivotal insight into the molecular mechanism of substrate recognition and specificity for GH18 ENGases and further advance our understanding of chemoenzymatic synthesis and remodeling of homogeneous N-glycan glycoproteins.
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Jul 2021
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[22113]
Open Access
Abstract: Streptococcus pyogenes, or Group A Streptococcus, is a Gram-positive bacterium that can be both a human commensal and pathogen. Central to this dichotomy are temperate bacteriophages that incorporate into the bacterial genome as a prophage. These genetic elements encode both the phage proteins and the toxins harmful to the human host. One such conserved phage protein, paratox (Prx), is always found encoded adjacent to the toxin genes and this linkage is preserved during all stages of the phage life cycle. Within Streptococcus pyogenes, Prx functions to inhibit the quorum-sensing receptor-signal pair ComRS, the master regulator of natural competence or the ability to uptake endogenous DNA. However, the mechanism by which Prx directly binds and inhibits the receptor ComR is unknown. To understand how Prx inhibits ComR at the molecular level we pursued an X-ray crystal structure of Prx bound to ComR. The structural data supported by solution X-ray scattering data demonstrate that Prx induces a conformational change in ComR to directly access its DNA binding domain. Furthermore, electromobility shift assays and competition binding assays reveal that Prx effectively uncouples the inter-domain conformational change required for activation of ComR via the signaling molecule XIP. Although to our knowledge the molecular mechanism of quorum-sensing inhibition by Prx is unique, it is analogous to the mechanism employed by the phage protein Aqs1 in Pseudomonas aeruginosa. Together, this demonstrates an example of convergent evolution between Gram-positive and Gram-negative phages to inhibit quorum-sensing, and highlights the versatility of small phage proteins.
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Jul 2021
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[21741]
Open Access
Abstract: Pyridoxal 5’-phosphate (PLP), the catalytically active form of vitamin B6, plays a pivotal role in metabolism as an enzyme cofactor. PLP is a very reactive molecule and it can be very toxic unless its intracellular concentration is finely regulated. In Escherichia coli, PLP formation is catalyzed by pyridoxine 5’-phosphate oxidase (PNPO), a homodimeric FMN-dependent enzyme that is responsible for the last step of PLP biosynthesis and is also involved in the PLP salvage pathway. We have recently observed that E. coli PNPO undergoes an allosteric feedback inhibition by PLP, caused by a strong allosteric coupling between PLP binding at the allosteric site and substrate binding at the active site. Here we report the crystallographic identification of the PLP allosteric site, located at the interface between the enzyme subunits and mainly circumscribed by three arginine residues (Arg23, Arg24 and Arg215) that form an “arginine-cage” and efficiently trap PLP. The crystal structure of the PNPO-PLP complex, characterized by a marked structural asymmetry, presents only one PLP molecule bound at the allosteric site of one monomer, and sheds light on the allosteric inhibition mechanism that makes the enzyme-substrate-PLP ternary complex catalytically incompetent. Site directed mutagenesis studies focused on the “arginine cage” validate the identity of the allosteric site and provide an effective means to modulate the allosteric properties of the enzyme, from the loosening of the allosteric coupling (in the R23L/R24L and R23L/R215L variants) to the complete loss of allosteric properties (in the R23L/R24L/R215L variant).
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May 2021
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[22113]
Open Access
Abstract: Approximately 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing cirrhosis and hepatocellular carcinoma. The HBV genome persists as covalently closed circular DNA (cccDNA), which serves as the template for all HBV mRNA transcripts. Current nucleos(t)ide analogs used to treat HBV do not directly target the HBV cccDNA genome, and thus cannot eradicate HBV infection. Here, we report the discovery of a unique G-quadruplex structure in the pre-core promoter region of the HBV genome that is conserved amongst nearly all genotypes. This region is central to critical steps in the viral life-cycle, including the generation of pre-genomic RNA, synthesis of core and polymerase proteins, and genome encapsidation; thus, an increased understanding of the HBV pre-core region may lead to the identification of novel anti-HBV cccDNA targets. We utilized biophysical methods (circular dichroism, and small-angle X-ray scattering) to characterize the HBV G-quadruplex and the effect of three distinct G to A mutants. We also used microscale thermophoresis to quantify the binding affinity of G-quadruplex and its mutants with a known quadruplex-binding protein (DHX36). To investigate the physiological relevance of HBV G-quadruplex, we employed assays using DHX36 to pull-down cccDNA and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids by monitoring the levels of genomic DNA, pre-genomic RNA, and antigens. Further evaluation of this critical host-protein interaction site in the HBV cccDNA genome may facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.
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Mar 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Claire C.
Munier
,
Leonardo
De Maria
,
Karl
Edman
,
Anders
Gunnarsson
,
Marianna
Longo
,
Carol
Mackintosh
,
Saleha
Patel
,
Arjan
Snijder
,
Lisa
Wissler
,
Luc
Brunsveld
,
Christian
Ottmann
,
Matthew W. D.
Perry
Diamond Proposal Number(s):
[20016]
Open Access
Abstract: Glucocorticoid receptor (GR) is a ligand-dependent transcription factor that plays a central role in inflammation. GR activity is also modulated via protein–protein interactions, including binding of 14-3-3 proteins induced by GR phosphorylation. However, the specific phosphorylation sites on GR that trigger these interactions and their functional consequences are less clear. Hence, we sought to examine this system in more detail. We used phosphorylated GR peptides, biophysical studies and X-ray crystallography to identify key residues within the ligand binding domain of GR, T524 and S617, whose phosphorylation results in binding of the representative 14-3-3 protein 14-3-3ζ. A kinase screen identified MINK1 as responsible for phosphorylating T524 and ROCK1 for phosphorylating S617; cell-based approaches confirmed the importance of both GR phosphosites and MINK1 but not ROCK1 alone in inducing GR–14-3-3 binding. Together our results provide molecular-level insight into 14-3-3-mediated regulation of GR and highlight both MINK1 and the GR–14-3-3 axis as potential targets for future therapeutic intervention.
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Mar 2021
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Open Access
Abstract: The human dedicator of cytokinesis (DOCK) family consists of 11 structurally conserved proteins that serve as atypical RHO guanine nucleotide exchange factors (RHO GEFs). These regulatory proteins act as mediators in numerous cellular cascades that promote cytoskeletal remodelling, playing roles in various crucial processes such as differentiation, migration, polarisation and axon growth in neurons. At the molecular level, DOCK DHR2 domains facilitate nucleotide dissociation from small GTPases, a process which is otherwise too slow for rapid spatiotemporal control of cellular signalling. Here, we provide an overview of the biological and structural characteristics for the various DOCK proteins and describe how they differ from other RHO GEFs and between DOCK sub-families. The expression of the family varies depending on cell or tissue type, and they are consequently implicated in a broad range of disease phenotypes, particularly in the brain. A growing body of available structural information reveals the mechanism by which the catalytic DHR2 domain elicits nucleotide dissociation and also indicates strategies for the discovery and design of high-affinity small molecule inhibitors. Such compounds could serve as chemical probes to interrogate the cellular function and provide starting points for drug discovery of this important class of enzymes.
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Mar 2021
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I24-Microfocus Macromolecular Crystallography
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Daniel
Rehling
,
Si Min
Zhang
,
Ann-Sofie
Jemth
,
Tobias
Koolmeister
,
Adam
Throup
,
Olov
Wallner
,
Emma
Scaletti
,
Takaya
Moriyama
,
Rina
Nishii
,
Jonathan
Davies
,
Matthieu
Desroses
,
Sean G.
Rudd
,
Martin
Scobie
,
Evert
Homan
,
Ulrika Warpman
Berglund
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Jun J.
Yang
,
Thomas
Helleday
,
Pal
Stenmark
Diamond Proposal Number(s):
[21625]
Open Access
Abstract: The enzyme NUDT15 efficiently hydrolyses the active metabolites of thiopurine drugs, which are routinely used for treating cancer and inflammatory diseases. Loss-of-function variants in NUDT15 are strongly associated with thiopurine intolerance, such as leukopenia, and pre-emptive NUDT15 genotyping has been clinically implemented to personalize thiopurine dosing. However, understanding the molecular consequences of these variants has been difficult, as no structural information was available for NUDT15 proteins encoded by clinically actionable pharmacogenetic variants due to their inherent instability. Recently, the small molecule NUDT15 inhibitor TH1760 has been shown to sensitize cells to thiopurines, through enhanced accumulation of 6-thio-guanine in DNA. Building upon this, we herein report the development of the potent and specific NUDT15 inhibitor, TH7755. TH7755 demonstrates a greatly improved cellular target engagement and 6-thioguanine potentiation compared to TH1760, while showing no cytotoxicity on its own. This potent inhibitor also stabilized NUDT15, enabling analysis by X-ray crystallography. We have determined high-resolution structures of the clinically relevant NUDT15 variants Arg139Cys, Arg139His, Val18Ile and V18_V19insGlyVal. These structures provide clear insights into the structural basis for the thiopurine intolerance phenotype observed in patients carrying these pharmacogenetic variants. These findings will aid in predicting the effects of new NUDT15 sequence variations yet to be discovered in the clinic.
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Mar 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23620]
Open Access
Abstract: UTP-glucose-1-phosphate uridylyltransferases (UGPases) are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In Bacillus subtilis 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The B. subtilis UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and gtaB mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UGPases, are present in B. subtilis. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated in vitro using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a B. subtilis gtaB mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase in vivo. When wild-type and mutant B. subtilis strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing yngB and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with glucose residues during oxygen limited growth condition.
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Feb 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Open Access
Abstract: Microbial plant pathogens secrete effector proteins which manipulate the host to promote infection. Effectors can be recognised by plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors, initiating an immune response. The AVR-Pik effector from the rice blast fungus Magnaporthe oryzae is recognised by a pair of rice NLR receptors, Pik-1 and Pik-2. Pik-1 contains a non-canonical integrated heavy metal-associated (HMA) domain, which directly binds AVR-Pik to activate plant defences. The host targets of AVR-Pik are also HMA domain-containing proteins, namely heavy metal-associated isoprenylated plant proteins (HIPPs) and heavy metal-associated plant proteins (HPPs). Here, we demonstrate that one of these targets interacts with a wider set of AVR-Pik variants compared to the Pik-1 HMA domains. We define the biochemical and structural basis of the interaction between AVR-Pik and OsHIPP19, and compare the interaction to that formed with the HMA domain of Pik-1. Using analytical gel filtration and surface plasmon resonance, we show that multiple AVR-Pik variants, including the stealthy variants AVR-PikC and AVR-PikF which do not interact with any characterised Pik-1 alleles, bind to OsHIPP19 with nanomolar affinity. The crystal structure of OsHIPP19 in complex with AVR-PikF reveals differences at the interface that underpin high-affinity binding of OsHIPP19-HMA to a wider set of AVR-Pik variants than achieved by the integrated HMA domain of Pik-1. Our results provide a foundation for engineering the HMA domain of Pik-1 to extend binding to currently unrecognised AVR-Pik variants and expand disease resistance in rice to divergent pathogen strains.
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Feb 2021
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