scholarly journals Orchestrating serine resolvases

2010 ◽  
Vol 38 (2) ◽  
pp. 384-387 ◽  
Author(s):  
Phoebe A. Rice ◽  
Kent. W. Mouw ◽  
Sherwin P. Montaño ◽  
Martin R. Boocock ◽  
Sally-J. Rowland ◽  
...  
Keyword(s):  
Dna Bending ◽  
Strand Exchange ◽  
Wild Type ◽  
Remarkable Feature ◽  
Synaptic Complex ◽  
X Ray ◽  
X Ray Crystallography ◽  
Crossover Site ◽  
Net Release ◽  
Reaction Direction

A remarkable feature of the serine resolvases is their regulation: the wild-type enzymes will catalyse intra- but not inter-molecular recombination, can sense the relative orientation of their sites and can exchange strands directionally, despite the fact that there is no net release of chemical bond energy. The key to this regulation is that they are only active within a large intertwined complex called the ‘synaptosome’. Because substrate topology greatly facilitates (or, in other cases, inhibits) formation of the synaptosome, it acts as a ‘topological filter’. Within the defined topology of the synaptosome, strand exchange releases supercoiling tension, providing an energy source to bias the reaction direction. The regulatory portion of this complex contains additional copies of the recombinase and sometimes other DNA-bending proteins. We are using a combination of X-ray crystallography, biochemistry and genetics to model the full synaptic complex and to understand how the regulatory portion activates the crossover-site-bound recombinases.

scholarly journals Identification and Mutagenesis of the Adeno-Associated Virus 5 Sialic Acid Binding Region

2014 ◽  
Vol 89 (3) ◽  
pp. 1660-1672 ◽  
Author(s):  
Sandra Afione ◽  
Michael A. DiMattia ◽  
Sujata Halder ◽  
Giovanni Di Pasquale ◽  
Mavis Agbandje-McKenna ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Wild Type Virus ◽  
Fold Axis ◽  
Cell Tropism ◽  
Type Virus ◽  
Wild Type ◽  
X Ray ◽  
X Ray Crystallography ◽  
Sialic Acid Binding

ABSTRACTAs a genus, the dependoviruses use a diverse group of cell surface carbohydrates for attachment and entry. Despite the fact that a majority of adeno-associated viruses (AAVs) utilize sialic acid (SIA) for binding and transduction, this virus-carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, two SIA binding regions were mapped for AAV5. The first site mapped to the depression in the center of the 3-fold axis of symmetry, while the second site was located under the βHI loop close to the 5-fold axis. Mutagenesis of amino acids 569 and 585 or 587 within the 3-fold depression resulted in elimination or alteration in SIA-dependent transduction, respectively. This change in SIA binding was confirmed using glycan microarrays. Mutagenesis of the second site identified a role in transduction that was SIA independent. Further studies of the mutants at the 3-fold site demonstrated a change in transduction activity and cell tropismin vivoas well as resistance to neutralization by a polyclonal antibody raised against the wild-type virus.IMPORTANCEDespite the fact that a majority of AAVs utilize sialic acid for binding and transduction, this virus-carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, the sialic acid binding regions of AAV5 were identified and studied using a variety of approaches. Mutagenesis of this region resulted in elimination or alteration in sialic acid-dependent transduction in cell lines. This change in sialic acid glycan binding was confirmed using glycan arrays. Further study also demonstrated a change in transduction and activity and cell tropismin vivoas well as resistance to neutralization by antibodies raised against the wild-type virus.

scholarly journals Roles of individual EF-hands in the activation of m-calpain by calcium

2000 ◽  
Vol 348 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Previn DUTT ◽  
J. C. Simon ARTHUR ◽  
Pawel GROCHULSKI ◽  
Miroslaw CYGLER ◽  
John S. ELCE
Keyword(s):  
Driving Force ◽  
Specific Activity ◽  
Small Subunit ◽  
Wild Type ◽  
X Ray ◽  
X Ray Crystallography ◽  
Kd Values ◽  
Ef Hand ◽  
Ef Hands ◽  
Made In

m-Calpain is a heterodimeric, cytosolic, thiol protease, which is activated by Ca2+-binding to EF-hands in the C-terminal domains of both subunits. There are four potential Ca2+-binding EF-hands in each subunit, but their relative affinities for Ca2+ are not known. In the present study mutations were made in both subunits to reduce the Ca2+-binding affinity at one or more EF-hands in one or both subunits. X-ray crystallography of some of the mutated small subunits showed that Ca2+ did not bind to the mutated EF-hands, but that its binding at other sites was not affected. The structures of the mutant small subunits in the presence of Ca2+ were otherwise identical to that of the Ca2+-bound wild-type small subunit. In the whole enzyme the wild-type macroscopic Ca2+ requirement (Kd) was approx. 350 μM. The mutations did not affect the maximum specific activity of the enzyme, but caused increases in Kd, which were characteristic of each site. All the EF-hands could be mutated in various combinations without loss of activity, but preservation of at least one wild-type EF-hand 3 sequence was required to maintain Kd values lower than 1 mM. The results suggest that all the EF-hands can contribute co-operatively to calpain activation, but that EF-hand 3, in both subunits, has the highest intrinsic affinity for Ca2+ and provides the major driving force for conformational change.

scholarly journals The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography

2004 ◽  
Vol 385 (1) ◽  
pp. 75-83 ◽  
Author(s):  
Barbara PETSCHACHER ◽  
Stefan LEITGEB ◽  
Kathryn L. KAVANAGH ◽  
David K. WILSON ◽  
Bernd NIDETZKY
Keyword(s):  
Double Mutant ◽  
Profile Analysis ◽  
Xylose Reductase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binary Complex ◽  
Wild Type ◽  
Coenzyme Specificity ◽  
X Ray ◽  
X Ray Crystallography

CtXR (xylose reductase from the yeast Candida tenuis; AKR2B5) can utilize NADPH or NADH as co-substrate for the reduction of D-xylose into xylitol, NADPH being preferred approx. 33-fold. X-ray structures of CtXR bound to NADP+ and NAD+ have revealed two different protein conformations capable of accommodating the presence or absence of the coenzyme 2′-phosphate group. Here we have used site-directed mutagenesis to replace interactions specific to the enzyme–NADP+ complex with the aim of engineering the co-substrate-dependent conformational switch towards improved NADH selectivity. Purified single-site mutants K274R (Lys274→Arg), K274M, K274G, S275A, N276D, R280H and the double mutant K274R–N276D were characterized by steady-state kinetic analysis of enzymic D-xylose reductions with NADH and NADPH at 25 °C (pH 7.0). The results reveal between 2- and 193-fold increases in NADH versus NADPH selectivity in the mutants, compared with the wild-type, with only modest alterations of the original NADH-linked xylose specificity and catalytic-centre activity. Catalytic reaction profile analysis demonstrated that all mutations produced parallel effects of similar magnitude on ground-state binding of coenzyme and transition state stabilization. The crystal structure of the double mutant showing the best improvement of coenzyme selectivity versus wild-type and exhibiting a 5-fold preference for NADH over NADPH was determined in a binary complex with NAD+ at 2.2 Å resolution.

scholarly journals Chemical and structural investigation of the paroxetine-human serotonin transporter complex

2020 ◽  
Author(s):  
Jonathan A. Coleman ◽  
Vikas Navratna ◽  
Daniele Antermite ◽  
Dongxue Yang ◽  
James A. Bull ◽  
...  
Keyword(s):  
Serotonin Transporter ◽  
Structural Investigation ◽  
Anomalous Scattering ◽  
Wild Type ◽  
Inhibitor Binding ◽  
X Ray ◽  
X Ray Crystallography ◽  
Open Conformation ◽  
Bona Fide ◽  
Serotonin Transport

ABSTRACTAntidepressants target the serotonin transporter (SERT) by inhibiting serotonin reuptake. Structural and biochemical studies aiming to understand the binding of small-molecules to conformationally dynamic transporters like SERT often require thermostabilizing mutations and antibodies to stabilize a specific conformation. Such modifications to SERT have led to questions about the relationships of these structures to the bona fide conformation and inhibitor binding poses of the wild-type transporter. To address these concerns, we characterized wild-type SERT with truncated N- and C-termini and thermostabilized variants of SERT bound with paroxetine using x-ray crystallography, single particle cryo-EM and biochemical techniques. Moreover, using a C–H functionalization approach to synthesize enantiopure analogues, we replaced the halide of the fluorophenyl group in paroxetine with either bromine or iodine. We then exploited the anomalous scattering of Br and I to define the pose of the respective paroxetine analog. These structures provide mutually consistent insights into how paroxetine and its analogs bind to the central substrate-binding site of SERT, stabilize the outward-open conformation, and inhibit serotonin transport.

Addition von Schwefeldioxid an Ruthenium-Thiolat-Komplexe, Struktur eines bemerkenswert stabilen Thiolat-SO2-Addukts [1]/ Addition of Sulfur Dioxide to Ruthenium Thiolate Complexes, Structure of a Remarkably Stable Thiolate-SO2-Addukt [1]

1992 ◽  
Vol 47 (5) ◽  
pp. 732-740 ◽  
Author(s):  
Wolfdieter A. Schenk ◽  
Eberhard Dombrowski ◽  
Irene Reuther ◽  
Thomas Stur
Keyword(s):  
Sulfur Dioxide ◽  
Monoclinic Space Group ◽  
Single Bond ◽  
Remarkable Feature ◽  
Space Group ◽  
X Ray ◽  
X Ray Crystallography ◽  
Thiolate Ligand ◽  
Sulfur Monoxide ◽  
Thiolate Complexes

Ruthenium thiolates [cpRu(PPh3),(SMe)] and [cp*Ru(PPh2Me)2(SR)] (R = Me, Ph) react with sulfur dioxide in two different ways, substitution of phosphine for SO2, and addition of SO2 to the thiolate ligand. The structure of [cpRu(CO)(PPh3)(SMe · SO2)] was determined by X-ray crystallography: Crystals are monoclinic, space group P21/n, a = 10.26(9), b = 16.00(6), c = 14.73(6) Å, β = 90.141(5)°, Ζ = 4. The most remarkable feature is a very long (247,8 pm) S—S single bond. [cp*Ru(PPh2Me)(SO2)(SPh)] and the corresponding sulfur monoxide complex [cp*Ru(PPh2Me)(SO)(SPh)] undergo [3+2] cycloaddition with dimethylacetylene-dicarboxylate.

scholarly journals STAG1 vulnerabilities for exploiting cohesin synthetic lethality in STAG2-deficient cancers

2020 ◽  
Vol 3 (7) ◽  
pp. e202000725
Author(s):  
Petra van der Lelij ◽  
Joseph A Newman ◽  
Simone Lieb ◽  
Julian Jude ◽  
Vittorio Katis ◽  
...  
Keyword(s):  
Synthetic Lethality ◽  
Wild Type ◽  
Synthetic Lethal ◽  
X Ray ◽  
X Ray Crystallography ◽  
Chemical Genetic ◽  
Biochemical Assays ◽  
Genome Wide ◽  
Chromatid Cohesion ◽  
Cohesin Subunit

The cohesin subunit STAG2 has emerged as a recurrently inactivated tumor suppressor in human cancers. Using candidate approaches, recent studies have revealed a synthetic lethal interaction between STAG2 and its paralog STAG1. To systematically probe genetic vulnerabilities in the absence of STAG2, we have performed genome-wide CRISPR screens in isogenic cell lines and identified STAG1 as the most prominent and selective dependency of STAG2-deficient cells. Using an inducible degron system, we show that chemical genetic degradation of STAG1 protein results in the loss of sister chromatid cohesion and rapid cell death in STAG2-deficient cells, while sparing STAG2–wild-type cells. Biochemical assays and X-ray crystallography identify STAG1 regions that interact with the RAD21 subunit of the cohesin complex. STAG1 mutations that abrogate this interaction selectively compromise the viability of STAG2-deficient cells. Our work highlights the degradation of STAG1 and inhibition of its interaction with RAD21 as promising therapeutic strategies. These findings lay the groundwork for the development of STAG1-directed small molecules to exploit synthetic lethality in STAG2-mutated tumors.

scholarly journals Description of Transport Tunnel in Haloalkane Dehalogenase Variant LinB D147C+L177C from Sphingobium japonicum

Catalysts ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 5
Author(s):  
Iuliia Iermak ◽  
Oksana Degtjarik ◽  
Petra Havlickova ◽  
Michal Kuty ◽  
Radka Chaloupkova ◽  
...  
Keyword(s):  
Active Sites ◽  
Data Bank ◽  
Structural Basis ◽  
Protein Fold ◽  
Wild Type ◽  
Haloalkane Dehalogenase ◽  
Wild Type Enzyme ◽  
X Ray ◽  
X Ray Crystallography ◽  
Activity Of Enzymes

The activity of enzymes with active sites buried inside their protein core highly depends on the efficient transport of substrates and products between the active site and the bulk solvent. The engineering of access tunnels in order to increase or decrease catalytic activity and specificity in a rational way is a challenging task. Here, we describe a combined experimental and computational approach to characterize the structural basis of altered activity in the haloalkane dehalogenase LinB D147C+L177C variant. While the overall protein fold is similar to the wild type enzyme and the other LinB variants, the access tunnels have been altered by introduced cysteines that were expected to form a disulfide bond. Surprisingly, the mutations have allowed several conformations of the amino acid chain in their vicinity, interfering with the structural analysis of the mutant by X-ray crystallography. The duration required for the growing of protein crystals changed from days to 1.5 years by introducing the substitutions. The haloalkane dehalogenase LinB D147C+L177C variant crystal structure was solved to 1.15 Å resolution, characterized and deposited to Protein Data Bank under PDB ID 6s06.

scholarly journals Chemical and structural investigation of the paroxetine-human serotonin transporter complex

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jonathan A Coleman ◽  
Vikas Navratna ◽  
Daniele Antermite ◽  
Dongxue Yang ◽  
James A Bull ◽  
...  
Keyword(s):  
Small Molecules ◽  
Serotonin Transporter ◽  
Structural Investigation ◽  
Anomalous Scattering ◽  
Wild Type ◽  
Inhibitor Binding ◽  
X Ray ◽  
X Ray Crystallography ◽  
Open Conformation ◽  
Serotonin Transport

Antidepressants target the serotonin transporter (SERT) by inhibiting serotonin reuptake. Structural and biochemical studies aiming to understand binding of small-molecules to conformationally dynamic transporters like SERT often require thermostabilizing mutations and antibodies to stabilize a specific conformation, leading to questions about relationships of these structures to the bonafide conformation and inhibitor binding poses of wild-type transporter. To address these concerns, we determined the structures of ∆N72/∆C13 and ts2-inactive SERT bound to paroxetine analogues using single-particle cryo-EM and x-ray crystallography, respectively. We synthesized enantiopure analogues of paroxetine containing either bromine or iodine instead of fluorine. We exploited the anomalous scattering of bromine and iodine to define the pose of these inhibitors and investigated inhibitor binding to Asn177 mutants of ts2-active SERT. These studies provide mutually consistent insights into how paroxetine and its analogues bind to the central substrate-binding site of SERT, stabilize the outward-open conformation, and inhibit serotonin transport.

Difference Fourier Analysis of Glucose Embedded and Frozen Hydrated Purple Membrane

1982 ◽  
Vol 40 ◽  
pp. 74-75
Author(s):  
Jules S. Jaffe ◽  
Robert M. Glaeser
Keyword(s):  
Purple Membrane ◽  
Data Set ◽  
High Resolution Data ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fourier Techniques ◽  
Versus Protein ◽  
The Difference ◽  
Difference Fourier ◽  
Ideal Method

Although difference Fourier techniques are standard in X-ray crystallography it has only been very recently that electron crystallographers have been able to take advantage of this method. We have combined a high resolution data set for frozen glucose embedded Purple Membrane (PM) with a data set collected from PM prepared in the frozen hydrated state in order to visualize any differences in structure due to the different methods of preparation. The increased contrast between protein-ice versus protein-glucose may prove to be an advantage of the frozen hydrated technique for visualizing those parts of bacteriorhodopsin that are embedded in glucose. In addition, surface groups of the protein may be disordered in glucose and ordered in the frozen state. The sensitivity of the difference Fourier technique to small changes in structure provides an ideal method for testing this hypothesis.

3-D reconstruction of molecular shape from microcrystal thin sections

1983 ◽  
Vol 41 ◽  
pp. 748-749
Author(s):  
S. Cusack ◽  
J.-C. Jésior
Keyword(s):  
Three Dimensional ◽  
Molecular Shape ◽  
Biological Molecules ◽  
Fourier Space ◽  
Single Particles ◽  
Thin Sections ◽  
Standard Methods ◽  
X Ray ◽  
X Ray Crystallography ◽  
Dimensional Reconstruction

Three-dimensional reconstruction techniques using electron microscopy have been principally developed for application to 2-D arrays (i.e. monolayers) of biological molecules and symmetrical single particles (e.g. helical viruses). However many biological molecules that crystallise form multilayered microcrystals which are unsuitable for study by either the standard methods of 3-D reconstruction or, because of their size, by X-ray crystallography. The grid sectioning technique enables a number of different projections of such microcrystals to be obtained in well defined directions (e.g. parallel to crystal axes) and poses the problem of how best these projections can be used to reconstruct the packing and shape of the molecules forming the microcrystal.Given sufficient projections there may be enough information to do a crystallographic reconstruction in Fourier space. We however have considered the situation where only a limited number of projections are available, as for example in the case of catalase platelets where three orthogonal and two diagonal projections have been obtained (Fig. 1).

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