Publications by authors named "Alexander B Taylor"

69 Publications

Nucleic acid binding by SAMHD1 contributes to the antiretroviral activity and is enhanced by the GpsN modification.

Nat Commun 2021 02 2;12(1):731. Epub 2021 Feb 2.

Department of Biochemistry and Structural Biology, UT Health San Antonio, San Antonio, TX, USA.

SAMHD1 impedes infection of myeloid cells and resting T lymphocytes by retroviruses, and the enzymatic activity of the protein-dephosphorylation of deoxynucleotide triphosphates (dNTPs)-implicates enzymatic dNTP depletion in innate antiviral immunity. Here we show that the allosteric binding sites of the enzyme are plastic and can accommodate oligonucleotides in place of the allosteric activators, GTP and dNTP. SAMHD1 displays a preference for oligonucleotides containing phosphorothioate bonds in the Rp configuration located 3' to G nucleotides (GpsN), the modification pattern that occurs in a mechanism of antiviral defense in prokaryotes. In the presence of GTP and dNTPs, binding of GpsN-containing oligonucleotides promotes formation of a distinct tetramer with mixed occupancy of the allosteric sites. Mutations that impair formation of the mixed-occupancy complex abolish the antiretroviral activity of SAMHD1, but not its ability to deplete dNTPs. The findings link nucleic acid binding to the antiretroviral activity of SAMHD1, shed light on the immunomodulatory effects of synthetic phosphorothioated oligonucleotides and raise questions about the role of nucleic acid phosphorothioation in human innate immunity.
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http://dx.doi.org/10.1038/s41467-021-21023-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854603PMC
February 2021

An iterative process produces oxamniquine derivatives that kill the major species of schistosomes infecting humans.

PLoS Negl Trop Dis 2020 08 18;14(8):e0008517. Epub 2020 Aug 18.

Departments of Biochemistry and Structural Biology, the University of Texas Health Science Center, San Antonio, Texas, United States of America.

Currently there is only one method of treatment for human schistosomiasis, the drug praziquantel. Strong selective pressure has caused a serious concern for a rise in resistance to praziquantel leading to the necessity for additional pharmaceuticals, with a distinctly different mechanism of action, to be used in combination therapy with praziquantel. Previous treatment of Schistosoma mansoni included the use of oxamniquine (OXA), a prodrug that is enzymatically activated in S. mansoni but is ineffective against S. haematobium and S. japonicum. The oxamniquine activating enzyme was identified as a S. mansoni sulfotransferase (SmSULT-OR). Structural data have allowed for directed drug development in reengineering oxamniquine to be effective against S. haematobium and S. japonicum. Guided by data from X-ray crystallographic studies and Schistosoma worm killing assays on oxamniquine, our structure-based drug design approach produced a robust SAR program that tested over 300 derivatives and identified several new lead compounds with effective worm killing in vitro. Previous studies resulted in the discovery of compound CIDD-0066790, which demonstrated broad-species activity in killing of schistosome species. As these compounds are racemic mixtures, we tested and demonstrate that the R enantiomer CIDD-007229 kills S. mansoni, S. haematobium and S. japonicum better than the parent drug (CIDD-0066790). The search for derivatives that kill better than CIDD-0066790 has resulted in a derivative (CIDD- 149830) that kills 100% of S. mansoni, S. haematobium and S. japonicum adult worms within 7 days. We hypothesize that the difference in activation and thus killing by the derivatives is due to the ability of the derivative to fit in the binding pocket of each sulfotransferase (SmSULT-OR, ShSULT-OR, SjSULT-OR) and to be efficiently sulfated. The purpose of this research is to develop a second drug to be used in conjunction with praziquantel to treat the major human species of Schistosoma. Collectively, our findings show that CIDD-00149830 and CIDD-0072229 are promising novel drugs for the treatment of human schistosomiasis and strongly support further development and in vivo testing.
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http://dx.doi.org/10.1371/journal.pntd.0008517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7454593PMC
August 2020

Why does oxamniquine kill Schistosoma mansoni and not S. haematobium and S. japonicum?

Int J Parasitol Drugs Drug Resist 2020 08 10;13:8-15. Epub 2020 Apr 10.

Department of Biochemistry and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, 78229, USA; Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, 78229, USA. Electronic address:

Human schistosomiasis is a disease which globally affects over 229 million people. Three major species affecting humans are Schistosoma mansoni, S. haematobium and S. japonicum. Previous treatment of S. mansoni includes the use of oxamniquine (OXA), a prodrug that is enzymatically activated in S. mansoni but is ineffective against S. haematobium and S. japonicum. The OXA activating enzyme was identified and crystallized, as being a S. mansoni sulfotransferase (SmSULT). S. haematobium and S. japonicum possess homologs of SmSULT (ShSULT and SjSULT) begging the question; why does oxamniquine fail to kill S. haematobium and S. japonicum adult worms? Investigation of the molecular structures of the sulfotransferases indicates that structural differences, specifically in OXA contact residues, do not abrogate OXA binding in the active sites as previously hypothesized. Data presented argue that the ability of SULTs to sulfate and thus activate OXA and its derivatives is linked to the ability of OXA to fit in the binding pocket to allow the transfer of a sulfur group.
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http://dx.doi.org/10.1016/j.ijpddr.2020.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7167500PMC
August 2020

Structure of a Zinc Porphyrin-Substituted Bacterioferritin and Photophysical Properties of Iron Reduction.

Biochemistry 2020 04 16;59(16):1618-1629. Epub 2020 Apr 16.

Department of Chemistry, University of Texas at San Antonio, San Antonio, Texas 78249, United States.

The iron storage protein bacterioferritin (Bfr) binds up to 12 hemes at specific sites in its protein shell. The heme can be substituted with the photosensitizer Zn(II)-protoporphyrin IX (ZnPP), and photosensitized reductive iron release from the ferric oxyhydroxide {[FeO(OH)]} core inside the ZnPP-Bfr protein shell was demonstrated [Cioloboc, D., et al. (2018) , 178-187]. This report describes the X-ray crystal structure of ZnPP-Bfr and the effects of loaded iron on the photophysical properties of the ZnPP. The crystal structure of ZnPP-Bfr shows a unique six-coordinate zinc in the ZnPP with two axial methionine sulfur ligands. Steady state and transient ultraviolet-visible absorption and luminescence spectroscopies show that irradiation with light overlapping the Soret absorption causes oxidation of ZnPP to the cation radical ZnPP only when the ZnPP-Bfr is loaded with [FeO(OH)]. Femtosecond transient absorption spectroscopy shows that this photooxidation occurs from the singlet excited state (ZnPP*) on the picosecond time scale and is consistent with two oxidizing populations of Fe, which do not appear to involve the ferroxidase center iron. We propose that [FeO(OH)] clusters at or near the inner surface of the protein shell are responsible for ZnPP photooxidation. Hopping of the photoinjected electrons through the [FeO(OH)] would effectively cause migration of Fe through the inner cavity to pores where it exits the protein. Reductive iron mobilization is presumed to be a physiological function of Bfrs. The phototriggered Fe reduction could be used to identify the sites of iron mobilization within the Bfr protein shell.
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http://dx.doi.org/10.1021/acs.biochem.9b01103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927158PMC
April 2020

Molecular basis for hycanthone drug action in schistosome parasites.

Mol Biochem Parasitol 2020 03 3;236:111257. Epub 2020 Feb 3.

Department of Biochemistry & Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, United States; X-Ray Crystallography Core Laboratory, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, United States. Electronic address:

Hycanthone (HYC) is a retired drug formerly used to treat schistosomiasis caused by infection from Schistosoma mansoni and S. haematobium. Resistance to HYC was first observed in S. mansoni laboratory strains and in patients in the 1970s and the use of this drug was subsequently discontinued with the substitution of praziquantel (PZQ) as the single antischistosomal drug in the worldwide formulary. In endemic regions, multiple organizations have partnered with the World Health Organization to deliver PZQ for morbidity control and prevention. While the monotherapy reduces the disease burden, additional drugs are needed to use in combination with PZQ to stay ahead of potential drug resistance. HYC will not be reintroduced into the schistosomiasis drug formulary as a combination drug because it was shown to have adverse properties including mutagenic, teratogenic and carcinogenic activities. Oxamniquine (OXA) was used to treat S. mansoni infection in Brazil during the brief period of HYC use, until the 1990s. Its antischistosomal efficacy has been shown to work through the same mechanism as HYC and it does not possess the undesirable properties linked to HYC. OXA demonstrates cross-resistance in Schistosoma strains with HYC resistance and both are prodrugs requiring metabolic activation in the worm to toxic sulfated forms. The target activating enzyme has been identified as a sulfotransferase enzyme and is currently used as the basis for a structure-guided drug design program. Here, we characterize the sulfotransferases from S. mansoni and S. haematobium in complexes with HYC to compare and contrast with OXA-bound sulfotransferase crystal structures. Although HYC is discontinued for antischistosomal treatment, it can serve as a resource for design of derivative compounds without contraindication.
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http://dx.doi.org/10.1016/j.molbiopara.2020.111257DOI Listing
March 2020

Oxamniquine resistance alleles are widespread in Old World Schistosoma mansoni and predate drug deployment.

PLoS Pathog 2019 10 25;15(10):e1007881. Epub 2019 Oct 25.

Texas Biomedical Research Institute, San Antonio, Texas, United States of America.

Do mutations required for adaptation occur de novo, or are they segregating within populations as standing genetic variation? This question is key to understanding adaptive change in nature, and has important practical consequences for the evolution of drug resistance. We provide evidence that alleles conferring resistance to oxamniquine (OXA), an antischistosomal drug, are widespread in natural parasite populations under minimal drug pressure and predate OXA deployment. OXA has been used since the 1970s to treat Schistosoma mansoni infections in the New World where S. mansoni established during the slave trade. Recessive loss-of-function mutations within a parasite sulfotransferase (SmSULT-OR) underlie resistance, and several verified resistance mutations, including a deletion (p.E142del), have been identified in the New World. Here we investigate sequence variation in SmSULT-OR in S. mansoni from the Old World, where OXA has seen minimal usage. We sequenced exomes of 204 S. mansoni parasites from West Africa, East Africa and the Middle East, and scored variants in SmSULT-OR and flanking regions. We identified 39 non-synonymous SNPs, 4 deletions, 1 duplication and 1 premature stop codon in the SmSULT-OR coding sequence, including one confirmed resistance deletion (p.E142del). We expressed recombinant proteins and used an in vitro OXA activation assay to functionally validate the OXA-resistance phenotype for four predicted OXA-resistance mutations. Three aspects of the data are of particular interest: (i) segregating OXA-resistance alleles are widespread in Old World populations (4.29-14.91% frequency), despite minimal OXA usage, (ii) two OXA-resistance mutations (p.W120R, p.N171IfsX28) are particularly common (>5%) in East African and Middle-Eastern populations, (iii) the p.E142del allele has identical flanking SNPs in both West Africa and Puerto Rico, suggesting that parasites bearing this allele colonized the New World during the slave trade and therefore predate OXA deployment. We conclude that standing variation for OXA resistance is widespread in S. mansoni.
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http://dx.doi.org/10.1371/journal.ppat.1007881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834289PMC
October 2019

Structural Adaptation in Its Orphan Domain Engenders Betaglycan with an Alternate Mode of Growth Factor Binding Relative to Endoglin.

Structure 2019 09 18;27(9):1427-1442.e4. Epub 2019 Jul 18.

Department of Structural Biology, University of Pittsburgh School of Medicine, Biomedical Science Tower 3, Room 2051, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA. Electronic address:

Betaglycan (BG) and endoglin (ENG), homologous co-receptors of the TGF-β family, potentiate the signaling activity of TGF-β2 and inhibin A, and BMP-9 and BMP-10, respectively. BG exists as monomer and forms 1:1 growth factor (GF) complexes, while ENG exists as a dimer and forms 2:1 GF complexes. Herein, the structure of the BG orphan domain (BG) reveals an insertion that blocks the region that the endoglin orphan domain (ENG) uses to bind BMP-9, preventing it from binding in the same manner. Using binding studies with domain-deleted forms of TGF-β and BG, as well as small-angle X-ray scattering data, BG is shown to bind its cognate GF in an entirely different manner compared with ENG. The alternative interfaces likely engender BG and ENG with the ability to selectively bind and target their cognate GFs in a unique temporal-spatial manner, without interfering with one another or other TGF-β family GFs.
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http://dx.doi.org/10.1016/j.str.2019.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726503PMC
September 2019

Disulfide bond of Mycoplasma pneumoniae community-acquired respiratory distress syndrome toxin is essential to maintain the ADP-ribosylating and vacuolating activities.

Cell Microbiol 2019 08 9;21(8):e13032. Epub 2019 May 9.

Department of Microbiology, Immunology and Molecular Genetics, The University of Texas Health Science Center at San Antonio, San Antonio, Texas.

Mycoplasma pneumoniae is the leading cause of bacterial community-acquired pneumonia among hospitalised children in United States and worldwide. Community-acquired respiratory distress syndrome (CARDS) toxin is a key virulence determinant of M. pneumoniae. The N-terminus of CARDS toxin exhibits ADP-ribosyltransferase (ADPRT) activity, and the C-terminus possesses binding and vacuolating activities. Thiol-trapping experiments of wild-type (WT) and cysteine-to-serine-mutated CARDS toxins with alkylating agents identified disulfide bond formation at the amino terminal cysteine residues C230 and C247. Compared with WT and other mutant toxins, C247S was unstable and unusable for comparative studies. Although there were no significant variations in binding, entry, and retrograde trafficking patterns of WT and mutated toxins, C230S did not elicit vacuole formation in intoxicated cells. In addition, the ADPRT domain of C230S was more sensitive to all tested proteases when compared with WT toxin. Despite its in vitro ADPRT activity, the reduction of C230S CARDS toxin-mediated ADPRT activity-associated IL-1β production in U937 cells and the recovery of vacuolating activity in the protease-released carboxy region of C230S indicated that the disulfide bond was essential not only to maintain the conformational stability of CARDS toxin but also to properly execute its cytopathic effects.
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http://dx.doi.org/10.1111/cmi.13032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612593PMC
August 2019

A mutually induced conformational fit underlies Ca-directed interactions between calmodulin and the proximal C terminus of KCNQ4 K channels.

J Biol Chem 2019 04 26;294(15):6094-6112. Epub 2019 Feb 26.

From the Departments of Cell and Integrative Physiology, University of Texas Health San Antonio, San Antonio, Texas 78229. Electronic address:

Calmodulin (CaM) conveys intracellular Ca signals to KCNQ (Kv7, "M-type") K channels and many other ion channels. Whether this "calmodulation" involves a dramatic structural rearrangement or only slight perturbations of the CaM/KCNQ complex is as yet unclear. A consensus structural model of conformational shifts occurring between low nanomolar and physiologically high intracellular [Ca] is still under debate. Here, we used various techniques of biophysical chemical analyses to investigate the interactions between CaM and synthetic peptides corresponding to the A and B domains of the KCNQ4 subtype. We found that in the absence of CaM, the peptides are disordered, whereas Ca/CaM imposed helical structure on both KCNQ A and B domains. Isothermal titration calorimetry revealed that Ca/CaM has higher affinity for the B domain than for the A domain of KCNQ2-4 and much higher affinity for the B domain when prebound with the A domain. X-ray crystallography confirmed that these discrete peptides spontaneously form a complex with Ca/CaM, similar to previous reports of CaM binding KCNQ-AB domains that are linked together. Microscale thermophoresis and heteronuclear single-quantum coherence NMR spectroscopy indicated the C-lobe of Ca-free CaM to interact with the KCNQ4 B domain ( ∼10-20 μm), with increasing Ca molar ratios shifting the CaM-B domain interactions via only the CaM C-lobe to also include the N-lobe. Our findings suggest that in response to increased Ca, CaM undergoes lobe switching that imposes a dramatic mutually induced conformational fit to both the proximal C terminus of KCNQ4 channels and CaM, likely underlying Ca-dependent regulation of KCNQ gating.
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http://dx.doi.org/10.1074/jbc.RA118.006857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463706PMC
April 2019

Design, Synthesis, and Characterization of Novel Small Molecules as Broad Range Antischistosomal Agents.

ACS Med Chem Lett 2018 Oct 14;9(10):967-973. Epub 2018 Sep 14.

Center for Innovative Drug Discovery, University of Texas at San Antonio, Department of Chemistry, One UTSA Circle, San Antonio, Texas 78249, United States.

Schistosomiasis is a major human parasitic disease afflicting more than 250 million people, historically treated with chemotherapies praziquantel or oxamniquine. Since oxamniquine is species-specific, killing but not other schistosome species ( or ) and evidence for drug resistant strains is growing, research efforts have focused on identifying novel approaches. Guided by data from X-ray crystallographic studies and worm killing assays on oxamniquine, our structure-based drug design approach produced a robust structure-activity relationship (SAR) program that identified several new lead compounds with effective worm killing. These studies culminated in the discovery of compound , which demonstrated broad-species activity in killing (75%), (40%), and (83%).
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http://dx.doi.org/10.1021/acsmedchemlett.8b00257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6187409PMC
October 2018

High affinity interactions of Pb with synaptotagmin I.

Metallomics 2018 09;10(9):1211-1222

Department of Biochemistry and Biophysics, Texas A&M University, 300 Olsen Boulevard, College Station, TX 77843, USA.

Lead (Pb) is a potent neurotoxin that disrupts synaptic neurotransmission. We report that Synaptotagmin I (SytI), a key regulator of Ca2+-evoked neurotransmitter release, has two high-affinity Pb2+ binding sites that belong to its cytosolic C2A and C2B domains. The crystal structures of Pb2+-complexed C2 domains revealed that protein-bound Pb2+ ions have holodirected coordination geometries and all-oxygen coordination spheres. The on-rate constants of Pb2+ binding to the C2 domains of SytI are comparable to those of Ca2+ and are diffusion-limited. In contrast, the off-rate constants are at least two orders of magnitude smaller, indicating that Pb2+ can serve as both a thermodynamic and kinetic trap for the C2 domains. We demonstrate, using NMR spectroscopy, that population of these sites by Pb2+ ions inhibits further Ca2+ binding despite the existing coordination vacancies. Our work offers a unique insight into the bioinorganic chemistry of Pb(ii) and suggests a mechanism by which low concentrations of Pb2+ ions can interfere with the Ca2+-dependent function of SytI in the cell.
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http://dx.doi.org/10.1039/c8mt00135aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6146066PMC
September 2018

Community-Acquired Respiratory Distress Syndrome Toxin Uses a Novel KELED Sequence for Retrograde Transport and Subsequent Cytotoxicity.

mBio 2018 01 23;9(1). Epub 2018 Jan 23.

Department of Microbiology, Immunology and Molecular Genetics, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA

is an atypical bacterium that causes respiratory illnesses in humans, including pharyngitis, tracheobronchitis, and community-acquired pneumonia (CAP). It has also been directly linked to reactive airway disease, asthma, and extrapulmonary pathologies. During its colonization, expresses a unique ADP-ribosylating and vacuolating cytotoxin designated ommunity-cquired espiratory istress yndrome (CARDS) toxin. CARDS toxin persists and localizes in the airway in CAP patients, asthmatics, and trauma patients with ventilator-associated pneumonia. Although CARDS toxin binds to specific cellular receptors, is internalized, and induces hyperinflammation, histopathology, mucus hyperplasia, and other airway injury, the intracellular trafficking of CARDS toxin remains unclear. Here, we show that CARDS toxin translocates through early and late endosomes and the Golgi complex and concentrates at the perinuclear region to reach the endoplasmic reticulum (ER). Using ER-targeted SNAP-tag, we confirmed the association of CARDS toxin with the ER and determined that CARDS toxin follows the retrograde pathway. In addition, we identified a novel CARDS toxin amino acid fingerprint, KELED, that is required for toxin transport to the ER and subsequent toxin-mediated cytotoxicity., a leading cause of bacterial community-acquired pneumonia (CAP) among children and adults in the United States, synthesizes a 591-amino-acid ADP-ribosylating and vacuolating protein, designated ommunity-cquired espiratory istress yndrome (CARDS) toxin. CARDS toxin alone is sufficient to induce and mimic major inflammatory and histopathological phenotypes associated with infection in rodents and primates. In order to elicit its ADP-ribosylating and vacuolating activities, CARDS toxin must bind to host cell receptors, be internalized via clathrin-mediated pathways, and subsequently be transported to specific intracellular organelles. Here, we demonstrate how CARDS toxin utilizes its unique KELED sequence to exploit the retrograde pathway machinery to reach the endoplasmic reticulum (ER) and fulfill its cytopathic potential. The knowledge generated from these studies may provide important clues to understand the mode of action of CARDS toxin and develop interventions that reduce or eliminate -associated airway and extrapulmonary pathologies.
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http://dx.doi.org/10.1128/mBio.01663-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5784248PMC
January 2018

Structure-Based Design and Synthesis of Potent and Selective Matrix Metalloproteinase 13 Inhibitors.

J Med Chem 2017 07 27;60(13):5816-5825. Epub 2017 Jun 27.

Department of Chemistry, Scripps Florida , 130 Scripps Way, Jupiter, Florida 33458, United States.

We describe the use of comparative structural analysis and structure-guided molecular design to develop potent and selective inhibitors (10d and (S)-17b) of matrix metalloproteinase 13 (MMP-13). We applied a three-step process, starting with a comparative analysis of the X-ray crystallographic structure of compound 5 in complex with MMP-13 with published structures of known MMP-13·inhibitor complexes followed by molecular design and synthesis of potent but nonselective zinc-chelating MMP inhibitors (e.g., 10a and 10b). After demonstrating that the pharmacophores of the chelating inhibitors (S)-10a, (R)-10a, and 10b were binding within the MMP-13 active site, the Zn chelating unit was replaced with nonchelating polar residues that bridged over the Zn binding site and reached into a solvent accessible area. After two rounds of structural optimization, these design approaches led to small molecule MMP-13 inhibitors 10d and (S)-17b, which bind within the substrate-binding site of MMP-13 and surround the catalytically active Zn ion without chelating to the metal. These compounds exhibit at least 500-fold selectivity versus other MMPs.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420151PMC
July 2017

Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions.

Biochemistry 2017 06 15;56(25):3283-3295. Epub 2017 Jun 15.

Department of Biochemistry and Biophysics, Texas A&M University , 300 Olsen Boulevard, College Station, Texas 77843, United States.

C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca-dependent manner. In these cases, membrane association is triggered by Ca binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd, in lieu of Ca to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding. Using X-ray crystallography, NMR, Förster resonance energy transfer, and vesicle cosedimentation assays, we demonstrate that, although Cd binds to the loop region of C2A/B domains of synaptotagmin 1 with high affinity, long-range Coulombic interactions are too weak to support membrane binding of individual domains. We attribute this behavior to two factors: the stoichiometry of Cd binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd to directly coordinate the lipids. In contrast, electron paramagnetic resonance experiments revealed that Cd does support membrane binding of the C2 domains in full-length synaptotagmin 1, where the high local lipid concentrations that result from membrane tethering can partially compensate for lack of a full complement of divalent metal ions and specific lipid coordination in Cd-complexed C2A/B domains. Our data suggest that long-range Coulombic interactions alone can drive the initial association of C2A/B with anionic membranes and that Ca further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca ion binding to the C2 domain loop regions.
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http://dx.doi.org/10.1021/acs.biochem.7b00188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5600830PMC
June 2017

Structural and enzymatic insights into species-specific resistance to schistosome parasite drug therapy.

J Biol Chem 2017 07 23;292(27):11154-11164. Epub 2017 May 23.

From the Departments of Biochemistry and Structural Biology and

The antischistosomal prodrug oxamniquine is activated by a sulfotransferase (SULT) in the parasitic flatworm Of the three main human schistosome species, only is sensitive to oxamniquine therapy despite the presence of SULT orthologs in and The reason for this species-specific drug action has remained a mystery for decades. Here we present the crystal structures of and SULTs, including SULT in complex with oxamniquine. We also examined the activity of the three enzymes ; surprisingly, all three are active toward oxamniquine, yet we observed differences in catalytic efficiency that implicate kinetics as the determinant for species-specific toxicity. These results provide guidance for designing oxamniquine derivatives to treat infection caused by all species of schistosome to combat emerging resistance to current therapy.
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http://dx.doi.org/10.1074/jbc.M116.766527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500785PMC
July 2017

Copper-zinc superoxide dismutase is activated through a sulfenic acid intermediate at a copper ion entry site.

J Biol Chem 2017 07 22;292(29):12025-12040. Epub 2017 May 22.

Department of Biological Sciences, University of Texas at Dallas, Richardson, Texas 75080. Electronic address:

Metallochaperones are a diverse family of trafficking molecules that provide metal ions to protein targets for use as cofactors. The copper chaperone for superoxide dismutase (Ccs1) activates immature copper-zinc superoxide dismutase (Sod1) by delivering copper and facilitating the oxidation of the Sod1 intramolecular disulfide bond. Here, we present structural, spectroscopic, and cell-based data supporting a novel copper-induced mechanism for Sod1 activation. Ccs1 binding exposes an electropositive cavity and proposed "entry site" for copper ion delivery on immature Sod1. Copper-mediated sulfenylation leads to a sulfenic acid intermediate that eventually resolves to form the Sod1 disulfide bond with concomitant release of copper into the Sod1 active site. Sod1 is the predominant disulfide bond-requiring enzyme in the cytoplasm, and this copper-induced mechanism of disulfide bond formation obviates the need for a thiol/disulfide oxidoreductase in that compartment.
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http://dx.doi.org/10.1074/jbc.M117.775981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519355PMC
July 2017

An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling.

J Biol Chem 2017 04 22;292(17):7173-7188. Epub 2017 Feb 22.

From the Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260,

The transforming growth factor β isoforms, TGF-β1, -β2, and -β3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-β pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-βs in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-β monomer, lacking the heel helix, a structural motif essential for binding the TGF-β type I receptor (TβRI) but dispensable for binding the other receptor required for TGF-β signaling, the TGF-β type II receptor (TβRII), as an alternative therapeutic modality for blocking TGF-β signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-β monomers and bound TβRII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-β signaling with a of 20-70 nm Investigation of the mechanism showed that the high affinity of the engineered monomer for TβRII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit TβRI, enabled it to bind endogenous TβRII but prevented it from binding and recruiting TβRI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-β signaling and may inform similar modifications of other TGF-β family members.
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http://dx.doi.org/10.1074/jbc.M116.768754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409485PMC
April 2017

Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1.

Proc Natl Acad Sci U S A 2016 12 6;113(51):14727-14732. Epub 2016 Dec 6.

Department of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229;

Intron lariats are circular, branched RNAs (bRNAs) produced during pre-mRNA splicing. Their unusual chemical and topological properties arise from branch-point nucleotides harboring vicinal 2',5'- and 3',5'-phosphodiester linkages. The 2',5'-bonds must be hydrolyzed by the RNA debranching enzyme Dbr1 before spliced introns can be degraded or processed into small nucleolar RNA and microRNA derived from intronic RNA. Here, we measure the activity of Dbr1 from Entamoeba histolytica by using a synthetic, dark-quenched bRNA substrate that fluoresces upon hydrolysis. Purified enzyme contains nearly stoichiometric equivalents of Fe and Zn per polypeptide and demonstrates turnover rates of ∼3 s Similar rates are observed when apo-Dbr1 is reconstituted with Fe(II)+Zn(II) under aerobic conditions. Under anaerobic conditions, a rate of ∼4.0 s is observed when apoenzyme is reconstituted with Fe(II). In contrast, apo-Dbr1 reconstituted with Mn(II) or Fe(II) under aerobic conditions is inactive. Diffraction data from crystals of purified enzyme using X-rays tuned to the Fe absorption edge show Fe partitions primarily to the β-pocket and Zn to the α-pocket. Structures of the catalytic mutant H91A in complex with 7-mer and 16-mer synthetic bRNAs reveal bona fide RNA branchpoints in the Dbr1 active site. A bridging hydroxide is in optimal position for nucleophilic attack of the scissile phosphate. The results clarify uncertainties regarding structure/function relationships in Dbr1 enzymes, and the fluorogenic probe permits high-throughput screening for inhibitors that may hold promise as treatments for retroviral infections and neurodegenerative disease.
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http://dx.doi.org/10.1073/pnas.1612729114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5187747PMC
December 2016

KDM2B Recruitment of the Polycomb Group Complex, PRC1.1, Requires Cooperation between PCGF1 and BCORL1.

Structure 2016 Oct 25;24(10):1795-1801. Epub 2016 Aug 25.

Department of Biochemistry, Midwestern University, 19555 North 59(th) Avenue, Glendale, AZ 85308, USA. Electronic address:

KDM2B recruits H2A-ubiquitinating activity of a non-canonical Polycomb Repression Complex 1 (PRC1.1) to CpG islands, facilitating gene repression. We investigated the molecular basis of recruitment using in vitro assembly assays to identify minimal components, subcomplexes, and domains required for recruitment. A minimal four-component PRC1.1 complex can be assembled by combining two separately isolated subcomplexes: the DNA-binding KDM2B/SKP1 heterodimer and the heterodimer of BCORL1 and PCGF1, a core component of PRC1.1. The crystal structure of the KDM2B/SKP1/BCORL1/PCGF1 complex illustrates the crucial role played by the PCGF1/BCORL1 heterodimer. The BCORL1 PUFD domain positions residues preceding the RAWUL domain of PCGF1 to create an extended interface for interaction with KDM2B, which is unique to the PCGF1-containing PRC1.1 complex. The structure also suggests how KDM2B might simultaneously function in PRC1.1 and an SCF ubiquitin ligase complex and the possible molecular consequences of BCOR PUFD internal tandem duplications found in pediatric kidney and brain tumors.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5088048PMC
http://dx.doi.org/10.1016/j.str.2016.07.011DOI Listing
October 2016

The Phylogeny and Active Site Design of Eukaryotic Copper-only Superoxide Dismutases.

J Biol Chem 2016 Sep 17;291(40):20911-20923. Epub 2016 Aug 17.

From the Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205,

In eukaryotes the bimetallic Cu/Zn superoxide dismutase (SOD) enzymes play important roles in the biology of reactive oxygen species by disproportionating superoxide anion. Recently, we reported that the fungal pathogen Candida albicans expresses a novel copper-only SOD, known as SOD5, that lacks the zinc cofactor and electrostatic loop (ESL) domain of Cu/Zn-SODs for substrate guidance. Despite these abnormalities, C. albicans SOD5 can disproportionate superoxide at rates limited only by diffusion. Here we demonstrate that this curious copper-only SOD occurs throughout the fungal kingdom as well as in phylogenetically distant oomycetes or "pseudofungi" species. It is the only form of extracellular SOD in fungi and oomycetes, in stark contrast to the extracellular Cu/Zn-SODs of plants and animals. Through structural biology and biochemical approaches we demonstrate that these copper-only SODs have evolved with a specialized active site consisting of two highly conserved residues equivalent to SOD5 Glu-110 and Asp-113. The equivalent positions are zinc binding ligands in Cu/Zn-SODs and have evolved in copper-only SODs to control catalysis and copper binding in lieu of zinc and the ESL. Similar to the zinc ion in Cu/Zn-SODs, SOD5 Glu-110 helps orient a key copper-coordinating histidine and extends the pH range of enzyme catalysis. SOD5 Asp-113 connects to the active site in a manner similar to that of the ESL in Cu/Zn-SODs and assists in copper cofactor binding. Copper-only SODs are virulence factors for certain fungal pathogens; thus this unique active site may be a target for future anti-fungal strategies.
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http://dx.doi.org/10.1074/jbc.M116.748251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5076504PMC
September 2016

Dopamine Receptor Gene DRD4 7-Repeat Allele X Maternal Sensitivity Interaction on Child Externalizing Behavior Problems: Independent Replication of Effects at 18 Months.

PLoS One 2016 5;11(8):e0160473. Epub 2016 Aug 5.

Psychiatry, University of Michigan, Ann Arbor, Michigan, United States of America.

The DRD4 VNTR has been associated with child behavior problems in interaction with maternal insensitivity in European and American cohorts of preschoolers, with the 7-repeat (7R) allele associated with greater problems. We sought to replicate and expand these findings by examining effects on reports of child behavior problems at 18 months. A 63 family sample with data for observed maternal sensitivity ratings, DRD4 VNTR genotype, and maternal report of child behavior problems at 18-months was used in this preliminary analysis. Maternal sensitivity was measured at 6-months of age using laboratory observational measures (free-play and a teaching task). Maternal report of toddler behavior was obtained at 18-months via the standard Child Behavior Checklist, and infant genotype on the DRD4 VNTR was obtained using PCR. Infants carrying the DRD4 7R allele showed greater effects of maternal insensitivity than non-carriers for behavioral problems at 18-months. We replicated previous findings of association of infant DRD4 x maternal sensitivity interactions with child Externalizing problems in the European-ancestry sample (N = 42) in a median split of maternal sensitivity (p = .00011, eta2 = .329) and in regression analyses controlling for maternal age, maternal depression, and child gender in European ancestry (B = -3.4, SE 1.33, p = .01) and the total sample (B = -2.2, SE 1.02, p = .02). Exploratory analyses also found evidence of DRD4 x maternal sensitivity interaction with the CBCL ADHD scale. These findings replicate in an independent cohort DRD4 x maternal insensitivity interaction effect on child externalizing behavior problems at 18 months, further supporting the role of the DRD4 genotype in differential sensitivity to parenting.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160473PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4975398PMC
August 2017

Domain Movements upon Activation of Phenylalanine Hydroxylase Characterized by Crystallography and Chromatography-Coupled Small-Angle X-ray Scattering.

J Am Chem Soc 2016 05 12;138(20):6506-16. Epub 2016 May 12.

Department of Chemistry, Princeton University , Princeton, New Jersey 08544, United States.

Mammalian phenylalanine hydroxylase (PheH) is an allosteric enzyme that catalyzes the first step in the catabolism of the amino acid phenylalanine. Following allosteric activation by high phenylalanine levels, the enzyme catalyzes the pterin-dependent conversion of phenylalanine to tyrosine. Inability to control elevated phenylalanine levels in the blood leads to increased risk of mental disabilities commonly associated with the inherited metabolic disorder, phenylketonuria. Although extensively studied, structural changes associated with allosteric activation in mammalian PheH have been elusive. Here, we examine the complex allosteric mechanisms of rat PheH using X-ray crystallography, isothermal titration calorimetry (ITC), and small-angle X-ray scattering (SAXS). We describe crystal structures of the preactivated state of the PheH tetramer depicting the regulatory domains docked against the catalytic domains and preventing substrate binding. Using SAXS, we further describe the domain movements involved in allosteric activation of PheH in solution and present the first demonstration of chromatography-coupled SAXS with Evolving Factor Analysis (EFA), a powerful method for separating scattering components in a model-independent way. Together, these results support a model for allostery in PheH in which phenylalanine stabilizes the dimerization of the regulatory domains and exposes the active site for substrate binding and other structural changes needed for activity.
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http://dx.doi.org/10.1021/jacs.6b01563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4896396PMC
May 2016

Structural and Functional Characterization of the Enantiomers of the Antischistosomal Drug Oxamniquine.

PLoS Negl Trop Dis 2015 20;9(10):e0004132. Epub 2015 Oct 20.

Departments of Biochemistry, the University of Texas Health Science Center, San Antonio, Texas, United States of America; Department of Pathology, the University of Texas Health Science Center, San Antonio, Texas, United States of America.

Background: For over two decades, a racemic mixture of oxamniquine (OXA) was administered to patients infected by Schistosoma mansoni, but whether one or both enantiomers exert antischistosomal activity was unknown. Recently, a ~30 kDa S. mansoni sulfotransferase (SmSULT) was identified as the target of OXA action.

Methodology/principal Findings: Here, we separate the OXA enantiomers using chromatographic methods and assign their optical activities as dextrorotary [(+)-OXA] or levorotary [(-)-OXA]. Crystal structures of the parasite enzyme in complex with optically pure (+)-OXA and (-)-OXA) reveal their absolute configurations as S- and R-, respectively. When tested in vitro, S-OXA demonstrated the bulk of schistosomicidal activity, while R-OXA had antischistosomal effects when present at relatively high concentrations. Crystal structures R-OXA•SmSULT and S-OXA•SmSULT complexes reveal similarities in the modes of OXA binding, but only the S-OXA enantiomer is observed in the structure of the enzyme exposed to racemic OXA.

Conclusions/significance: Together the data suggest the higher schistosomicidal activity of S-OXA is correlated with its ability to outcompete R-OXA binding the sulfotransferase active site. These findings have important implications for the design, syntheses, and dosing of new OXA-based antischistosomal compounds.
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http://dx.doi.org/10.1371/journal.pntd.0004132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4618941PMC
March 2016

RING Dimerization Links Higher-Order Assembly of TRIM5α to Synthesis of K63-Linked Polyubiquitin.

Cell Rep 2015 Aug 23;12(5):788-97. Epub 2015 Jul 23.

Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229, USA. Electronic address:

Members of the tripartite motif (TRIM) protein family of RING E3 ubiquitin (Ub) ligases promote innate immune responses by catalyzing synthesis of polyubiquitin chains linked through lysine 63 (K63). Here, we investigate the mechanism by which the TRIM5α retroviral restriction factor activates Ubc13, the K63-linkage-specific E2. Structural, biochemical, and functional characterization of the TRIM5α:Ubc13-Ub interactions reveals that activation of the Ubc13-Ub conjugate requires dimerization of the TRIM5α RING domain. Our data explain how higher-order oligomerization of TRIM5α, which is promoted by the interaction with the retroviral capsid, enhances the E3 Ub ligase activity of TRIM5α and contributes to its antiretroviral function. This E3 mechanism, in which RING dimerization is transient and depends on the interaction of the TRIM protein with the ligand, is likely to be conserved in many members of the TRIM family and may have evolved to facilitate recognition of repetitive epitope patterns associated with infection.
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http://dx.doi.org/10.1016/j.celrep.2015.06.072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4526822PMC
August 2015

Structure of CARDS toxin, a unique ADP-ribosylating and vacuolating cytotoxin from Mycoplasma pneumoniae.

Proc Natl Acad Sci U S A 2015 Apr 6;112(16):5165-70. Epub 2015 Apr 6.

Department of Biochemistry, X-Ray Crystallography Core Laboratory, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229; South Texas Veterans Health Care System, US Department of Veterans Affairs, San Antonio, TX 78229

Mycoplasma pneumoniae (Mp) infections cause tracheobronchitis and "walking" pneumonia, and are linked to asthma and other reactive airway diseases. As part of the infectious process, the bacterium expresses a 591-aa virulence factor with both mono-ADP ribosyltransferase (mART) and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). CARDS TX binds to human surfactant protein A and annexin A2 on airway epithelial cells and is internalized, leading to a range of pathogenetic events. Here we present the structure of CARDS TX, a triangular molecule in which N-terminal mART and C-terminal tandem β-trefoil domains associate to form an overall architecture distinct from other well-recognized ADP-ribosylating bacterial toxins. We demonstrate that CARDS TX binds phosphatidylcholine and sphingomyelin specifically over other membrane lipids, and that cell surface binding and internalization activities are housed within the C-terminal β-trefoil domain. The results enhance our understanding of Mp pathogenicity and suggest a novel avenue for the development of therapies to treat Mp-associated asthma and other acute and chronic airway diseases.
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http://dx.doi.org/10.1073/pnas.1420308112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4413325PMC
April 2015

Structural basis of HIV-1 capsid recognition by PF74 and CPSF6.

Proc Natl Acad Sci U S A 2014 Dec 17;111(52):18625-30. Epub 2014 Dec 17.

Department of Molecular Physiology and Biological Physics, Center for Membrane Biology, Cardiovascular Research Center, and Division of Cardiovascular Medicine, Department of Medicine, University of Virginia School of Medicine, Charlottesville, VA 22908

Upon infection of susceptible cells by HIV-1, the conical capsid formed by ∼250 hexamers and 12 pentamers of the CA protein is delivered to the cytoplasm. The capsid shields the RNA genome and proteins required for reverse transcription. In addition, the surface of the capsid mediates numerous host-virus interactions, which either promote infection or enable viral restriction by innate immune responses. In the intact capsid, there is an intermolecular interface between the N-terminal domain (NTD) of one subunit and the C-terminal domain (CTD) of the adjacent subunit within the same hexameric ring. The NTD-CTD interface is critical for capsid assembly, both as an architectural element of the CA hexamer and pentamer and as a mechanistic element for generating lattice curvature. Here we report biochemical experiments showing that PF-3450074 (PF74), a drug that inhibits HIV-1 infection, as well as host proteins cleavage and polyadenylation specific factor 6 (CPSF6) and nucleoporin 153 kDa (NUP153), bind to the CA hexamer with at least 10-fold higher affinities compared with nonassembled CA or isolated CA domains. The crystal structure of PF74 in complex with the CA hexamer reveals that PF74 binds in a preformed pocket encompassing the NTD-CTD interface, suggesting that the principal inhibitory target of PF74 is the assembled capsid. Likewise, CPSF6 binds in the same pocket. Given that the NTD-CTD interface is a specific molecular signature of assembled hexamers in the capsid, binding of NUP153 at this site suggests that key features of capsid architecture remain intact upon delivery of the preintegration complex to the nucleus.
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http://dx.doi.org/10.1073/pnas.1419945112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4284599PMC
December 2014

Insights into the role of the unusual disulfide bond in copper-zinc superoxide dismutase.

J Biol Chem 2015 Jan 28;290(4):2405-18. Epub 2014 Nov 28.

From the Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, the Department of Bioinspired Science, Ewha Womans University, Seoul 120-750, Korea

The functional and structural significance of the intrasubunit disulfide bond in copper-zinc superoxide dismutase (SOD1) was studied by characterizing mutant forms of human SOD1 (hSOD) and yeast SOD1 lacking the disulfide bond. We determined x-ray crystal structures of metal-bound and metal-deficient hC57S SOD1. C57S hSOD1 isolated from yeast contained four zinc ions per protein dimer and was structurally very similar to wild type. The addition of copper to this four-zinc protein gave properly reconstituted 2Cu,2Zn C57S hSOD, and its spectroscopic properties indicated that the coordination geometry of the copper was remarkably similar to that of holo wild type hSOD1. In contrast, the addition of copper and zinc ions to apo C57S human SOD1 failed to give proper reconstitution. Using pulse radiolysis, we determined SOD activities of yeast and human SOD1s lacking disulfide bonds and found that they were enzymatically active at ∼10% of the wild type rate. These results are contrary to earlier reports that the intrasubunit disulfide bonds in SOD1 are essential for SOD activity. Kinetic studies revealed further that the yeast mutant SOD1 had less ionic attraction for superoxide, possibly explaining the lower rates. Saccharomyces cerevisiae cells lacking the sod1 gene do not grow aerobically in the absence of lysine, but expression of C57S SOD1 increased growth to 30-50% of the growth of cells expressing wild type SOD1, supporting that C57S SOD1 retained a significant amount of activity.
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http://dx.doi.org/10.1074/jbc.M114.588798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303690PMC
January 2015

Characterization of selective exosite-binding inhibitors of matrix metalloproteinase 13 that prevent articular cartilage degradation in vitro.

J Med Chem 2014 Nov 11;57(22):9598-611. Epub 2014 Nov 11.

Lead Identification Division, Translational Research Institute, ‡Department of Molecular Therapeutics, and §Department of Chemistry, Scripps Florida, The Scripps Research Institute , Jupiter, Florida 33458, United States.

Matrix metalloproteinase 13 (MMP-13) has been shown to be the main collagenase responsible for degradation of articular cartilage during osteoarthritis and therefore represents a target for drug development. As a result of high-throughput screening and structure-activity relationship studies, we identified a novel, highly selective class of MMP-13 inhibitors (compounds 1 (Q), 2 (Q1), and 3 (Q2)). Mechanistic characterization revealed a noncompetitive nature of these inhibitors with binding constants in the low micromolar range. Crystallographic analyses revealed two binding modes for compound 2 in the MMP-13 S1' subsite and in an S1/S2* subsite. Type II collagen- and cartilage-protective effects exhibited by compounds 1, 2, and 3 suggested that these compounds might be efficacious in future in vivo studies. Finally, these compounds were also highly selective when tested against a panel of 30 proteases, which, in combination with a good CYP inhibition profile, suggested low off-target toxicity and drug-drug interactions in humans.
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http://dx.doi.org/10.1021/jm501284eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4255739PMC
November 2014

Structural basis of lariat RNA recognition by the intron debranching enzyme Dbr1.

Nucleic Acids Res 2014 14;42(16):10845-55. Epub 2014 Aug 14.

Department of Biochemistry, The University of Texas Health Science Center, San Antonio, TX 78229, USA X-ray Crystallography Core Laboratory, The University of Texas Health Science Center, San Antonio, TX 78229, USA Geriatric Research, Education, and Clinical Center, Department of Veterans Affairs, South Texas Veterans Health Care System, San Antonio, TX 78229, USA

The enzymatic processing of cellular RNA molecules requires selective recognition of unique chemical and topological features. The unusual 2',5'-phosphodiester linkages in RNA lariats produced by the spliceosome must be hydrolyzed by the intron debranching enzyme (Dbr1) before they can be metabolized or processed into essential cellular factors, such as snoRNA and miRNA. Dbr1 is also involved in the propagation of retrotransposons and retroviruses, although the precise role played by the enzyme in these processes is poorly understood. Here, we report the first structures of Dbr1 alone and in complex with several synthetic RNA compounds that mimic the branchpoint in lariat RNA. The structures, together with functional data on Dbr1 variants, reveal the molecular basis for 2',5'-phosphodiester recognition and explain why the enzyme lacks activity toward 3',5'-phosphodiester linkages. The findings illuminate structure/function relationships in a unique enzyme that is central to eukaryotic RNA metabolism and set the stage for the rational design of inhibitors that may represent novel therapeutic agents to treat retroviral infections and neurodegenerative disease.
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http://dx.doi.org/10.1093/nar/gku725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176325PMC
January 2015

Multiple polymer architectures of human polyhomeotic homolog 3 sterile alpha motif.

Proteins 2014 Oct 5;82(10):2823-30. Epub 2014 Aug 5.

Department of Biochemistry, The University of Texas Health Science Center San Antonio, MSC 7760, 7703 Floyd Curl Dr., San Antonio, Texas, 78229.

The self-association of sterile alpha motifs (SAMs) into a helical polymer architecture is a critical functional component of many different and diverse array of proteins. For the Drosophila Polycomb group (PcG) protein Polyhomeotic (Ph), its SAM polymerization serves as the structural foundation to cluster multiple PcG complexes, helping to maintain a silenced chromatin state. Ph SAM shares 64% sequence identity with its human ortholog, PHC3 SAM, and both SAMs polymerize. However, in the context of their larger protein regions, PHC3 SAM forms longer polymers compared with Ph SAM. Motivated to establish the precise structural basis for the differences, if any, between Ph and PHC3 SAM, we determined the crystal structure of the PHC3 SAM polymer. PHC3 SAM uses the same SAM-SAM interaction as the Ph SAM sixfold repeat polymer. Yet, PHC3 SAM polymerizes using just five SAMs per turn of the helical polymer rather than the typical six per turn observed for all SAM polymers reported to date. Structural analysis suggested that malleability of the PHC3 SAM would allow formation of not just the fivefold repeat structure but also possibly others. Indeed, a second PHC3 SAM polymer in a different crystal form forms a sixfold repeat polymer. These results suggest that the polymers formed by PHC3 SAM, and likely others, are dynamic. The functional consequence of the variable PHC3 SAM polymers may be to create different chromatin architectures.
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http://dx.doi.org/10.1002/prot.24645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4198450PMC
October 2014