I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[22808]
Open Access
Abstract: Antibody therapeutics are one of the most important classes of drugs. Antibody structures have become an integral part of predicting the behavior of potential therapeutics, either directly or as the basis of modeling. Structures of Fab:antigen complexes have even greater value. While the crystallization and structure determination of Fabs is easy relative to many other protein classes, especially membrane proteins, broad screening and optimization of crystalline hits is still necessary. Through a comprehensive review of rabbit Fab crystal contacts and their incompatibility with human Fabs, we identified a small secondary structural element from the rabbit light chain constant domain potentially responsible for hindering the crystallization of human Fabs. Upon replacing the human kappa constant domain FG loop (HQGLSSP) with the two residue shorter rabbit loop (QGTTS), we dramatically improved the crystallization of human Fabs and Fab:antigen complexes. Our design, which we call “Crystal Kappa”, enables rapid crystallization of human fabs and fab complexes in a broad range of conditions, with less material in smaller screens or from dilute solutions.
|
Sep 2020
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[12788]
Open Access
Abstract: We have used a combination of computational and structure-based redesign of the low molecular weight protein tyrosine phosphatase, LMW-PTP, to create new activity towards phosphoinositide substrates for which the wild-type enzyme had little or no activity. The redesigned enzymes retain catalytic activity despite residue alterations in the active site, and kinetic experiments confirmed specificity for up to four phosphoinositide substrates. Changes in the shape and overall volume of the active site where critical to facilitate access of the new substrates for catalysis. The kinetics data suggest that both the position and the combination of amino acid mutations are important for specificity towards the phosphoinositide substrates. The introduction of basic residues proved essential to establish new interactions with the multiple phosphate groups in the inositol head, thus promoting catalytically productive complexes. The crystallographic structures of the top-ranking designs confirmed the computational predictions and showed that residue substitutions do not alter the overall folding of the phosphatase or the conformation of the active site P-loop. The engineered LMW-PTP mutants with new activities can be useful reagents in investigating cell signalling pathways and offer the potential for therapeutic applications.
|
Jun 2020
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[19951]
Open Access
Abstract: Lactate oxidases belong to a group of FMN-dependent enzymes and they catalyze a conversion of lactate to pyruvate with a release of hydrogen peroxide. Hydrogen peroxide is also utilized as a read out in biosensors to quantitate lactate levels in biological samples. Aerococcus viridans lactate oxidase is the best characterized lactate oxidase and our knowledge of lactate oxidases relies largely to studies conducted with that particular enzyme. Pediococcus acidilactici lactate oxidase is also commercially available for e.g. lactate measurements, but this enzyme has not been characterized in detail before. Here we report structural characterization of the recombinant enzyme and its co-factor dependent oligomerization. The crystal structures revealed two distinct conformations in the loop closing the active site, consistent with previous biochemical studies implicating the role of loop in catalysis. Despite the structural conservation of active site residues, we were not able to detect either oxidase or monooxygenase activity when L-lactate was used as a substrate. Pediococcus acidilactici lactate oxidase is therefore an example of a misannotation of an FMN-dependent enzyme, which catalyzes likely a so far unknown oxidation reaction.
|
Feb 2020
|
|
I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[7641]
Open Access
Abstract: Plant NLRs are modular immune receptors that trigger rapid cell death in response to
attempted infection by pathogens. A highly conserved nucleotide-binding domain shared
with APAF-1, various R-proteins and CED-4 (NB-ARC domain) is proposed to act as a
molecular switch, cycling between ADP (repressed) and ATP (active) bound forms. Studies
of plant NLR NB-ARC domains have revealed functional similarities to mammalian homologues,
and provided insight into potential mechanisms of regulation. However, further
advances have been limited by difficulties in obtaining sufficient yields of protein suitable for
structural and biochemical techniques. From protein expression screens in Escherichia coli
and Sf9 insect cells, we defined suitable conditions to produce the NB-ARC domain from the
tomato NLR NRC1. Biophysical analyses of this domain showed it is a folded, soluble protein.
Structural studies revealed the NRC1 NB-ARC domain had co-purified with ADP, and
confirmed predicted structural similarities between plant NLR NB-ARC domains and their
mammalian homologues.
|
Aug 2019
|
|
I02-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[14757]
Open Access
Abstract: This study aimed to analyse microstructure data on the density and orientation of collagen fibrils in whole eye globes and to propose an effective method for the preparation of data for use in numerical simulations of the eye’s biomechanical performance. Wide-angle X-ray scattering was applied to seven healthy ex-vivo human eyes. Each eye was dissected into an anterior and a posterior cup, and radial incisions were used to flatten the tissue before microstructure characterisation. A method was developed to use the microstructure data obtained for the dissected tissue to build realistic 3D maps of fibril density and orientation covering the whole eye globe. At the central cornea, 61.5±2.3% of fibrils were aligned within 45° sectors surrounding the two orthogonal directions. In contrast, more than one-third of the total fibril content was concentrated along the circumferential direction at the limbus (37.0±2.4%) and around the optic nerve head (34.8±2.1%). The insertion locations of the four recti muscles exhibited a preference in the meridional direction near the equator (38.6±3.9%). There was also a significant difference in fibril density between the limbus and other regions (ratio = 1.91±0.45, p <0.01 at the central cornea and ratio = 0.80±0.21, p <0.01 at the posterior pole). Characterisation of collagen fibril density and orientation across the whole ocular surface has been possible but required the use of a technique that involved tissue dissection and hence caused tissue damage. The method presented in this paper aimed to minimise the effect of dissection on the quality of obtained data and was successful in identifying fibril distribution trends that were compatible with earlier studies, which concentrated on localised areas of the ocular globe.
|
Apr 2019
|
|
I02-Macromolecular Crystallography
|
Nadine
Daou
,
Yuanguo
Wang
,
Vladimir M.
Levdikov
,
Madhumitha
Nandakumar
,
Jonathan
Livny
,
Laurent
Bouillaut
,
Elena
Blagova
,
Keshan
Zhang
,
Boris R.
Belitsky
,
Kyu
Rhee
,
Anthony J.
Wilkinson
,
Xingmin
Sun
,
Abraham L.
Sonenshein
Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Toxin synthesis and endospore formation are two of the most critical factors that determine the outcome of infection by Clostridioides difficile. The two major toxins, TcdA and TcdB, are the principal factors causing damage to the host. Spores are the infectious form of C. difficile, permit survival of the bacterium during antibiotic treatment and are the predominant cell form that leads to recurrent infection. Toxin production and sporulation have their own specific mechanisms of regulation, but they share negative regulation by the global regulatory protein CodY. Determining the extent of such regulation and its detailed mechanism is important for understanding the linkage between two apparently independent biological phenomena and raises the possibility of creating new ways of limiting infection. The work described here shows that a codY null mutant of a hypervirulent (ribotype 027) strain is even more virulent than its parent in a mouse model of infection and that the mutant expresses most sporulation genes prematurely during exponential growth phase. Moreover, examining the expression patterns of mutants producing CodY proteins with different levels of residual activity revealed that expression of the toxin genes is dependent on total CodY inactivation, whereas most sporulation genes are turned on when CodY activity is only partially diminished. These results suggest that, in wild-type cells undergoing nutrient limitation, sporulation genes can be turned on before the toxin genes.
|
Jan 2019
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[14794]
Open Access
Abstract: Most glycosyltransferases, including B4GalT1 (EC 2.4.1.38), are known to assemble into enzyme homomers and functionally relevant heteromers in vivo. However, it remains unclear why and how these enzymes interact at the molecular/atomic level. Here, we solved the crystal structure of the wild-type human B4GalT1 homodimer. We also show that B4GalT1 exists in a dynamic equilibrium between monomer and dimer, since a purified monomer reappears as a mixture of both and as we obtained crystal forms of the monomer and dimer assemblies in the same crystallization conditions. These two crystal forms revealed the unliganded B4GalT1 in both the open and the closed conformation of the Trp loop and the lid regions, responsible for donor and acceptor substrate binding, respectively. The present structures also show the lid region in full in an open conformation, as well as a new conformation for the GlcNAc acceptor loop (residues 272–288). The physiological relevance of the homodimer in the crystal was validated by targeted mutagenesis studies coupled with FRET assays. These showed that changing key catalytic amino acids impaired homomer formation in vivo. The wild-type human B4GalT1 structure also explains why the variant proteins used for crystallization in earlier studies failed to reveal the homodimers described in this study.
|
Oct 2018
|
|
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
|
Mohanram
Sivaraja
,
Nicola
Pozzi
,
Matthew
Rienzo
,
Kenneth
Lin
,
Timothy P.
Shiau
,
Daniel M.
Clemens
,
Lev
Igoudin
,
Piotr
Zalicki
,
Stephanie S.
Chang
,
M. Angels
Estiarte
,
Kevin M.
Short
,
David C.
Williams
,
Anirban
Datta
,
Enrico
Di Cera
,
David B.
Kita
Open Access
Abstract: Introduction. In recent years, the traditional treatments for thrombotic diseases, heparin and warfarin, are increasingly being replaced by novel oral anticoagulants offering convenient dosing regimens, more predictable anticoagulant responses, and less frequent monitoring. However, these drugs can be contraindicated for some patients and, in particular, their bleeding liability remains high. Methods. We have developed a new class of direct thrombin inhibitors (VE-DTIs) and have utilized kinetics, biochemical, and X-ray structural studies to characterize the mechanism of action and in vitro pharmacology of an exemplary compound from this class, Compound 1. Results. We demonstrate that Compound 1, an exemplary VE-DTI, acts through reversible covalent inhibition. Compound 1 inhibits thrombin by transiently acylating the active site S195 with high potency and significant selectivity over other trypsin-like serine proteases. The compound inhibits the binding of a peptide substrate with both clot-bound and free thrombin with nanomolar potency. Compound 1 is a low micromolar inhibitor of thrombin activity against endogenous substrates such as fibrinogen and a nanomolar inhibitor of the activation of protein C and thrombin-activatable fibrinolysis inhibitor. In the thrombin generation assay, Compound 1 inhibits thrombin generation with low micromolar potency but does not increase the lag time for thrombin formation. In addition, Compound 1 showed weak inhibition of clotting in PT and aPTT assays consistent with its distinctive profile in the thrombin generation assay. Conclusion. Compound 1, while maintaining strong potency comparable to the current DTIs, has a distinct mechanism of action which produces a differentiating pharmacological profile. Acting through reversible covalent inhibition, these direct thrombin inhibitors could lead to new anticoagulants with better combined efficacy and bleeding profiles.
|
Aug 2018
|
|
B21-High Throughput SAXS
|
Diamond Proposal Number(s):
[14794]
Open Access
Abstract: NHLRC2 (NHL repeat-containing protein 2) is an essential protein. Mutations of NHLRC2, including Asp148Tyr, have been recently associated with a novel FINCA disease (fibrosis, neurodegeneration, cerebral angiomatosis), which is fatal in early childhood. To gain insight into the mechanisms of action of this essential protein, we determined the crystal structure of the Trx-like and NHL repeat β-propeller domains of human NHLRC2 to a resolution of 2.7 Å. The structure reveals two domains adjacent to each other that form a cleft containing a conserved CCINC motif. A SAXS structure of full-length NHLRC2 reveals that the non-conserved C-terminal domain does not pack against the N-terminal domains. Analysis of the surface properties of the protein identifies an extended negative electrostatic potential in the surface of the cleft formed by the two domains, which likely forms a binding site for a ligand or interaction partner(s). Bioinformatics analysis discovers homologs across a range of eukaryotic and prokaryotic species and conserved residues map mostly to the adjacent surfaces of the Trx-like and β-propeller domains that form the cleft, suggesting both that this forms the potential functional site of NHLRC2 and that the function is conserved across species. Asp148 is located in the Trx-like domain and is not conserved across species. The Asp148Tyr mutation destabilizes the structure of the protein by 2°C. The NHLRC2 structure, the first of any of its homologs, provides an important step towards more focused structure-function studies of this essential protein.
|
Aug 2018
|
|