Publications by authors named "Guillermo Calero"

35 Publications

Detection of Microcrystals for CryoEM.

Methods Mol Biol 2021 ;2215:299-307

Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

Here, we present a strategy to identify microcrystals from initial protein crystallization screen experiments and to optimize diffraction quality of those crystals using negative stain transmission electron microscopy (TEM) as a guiding technique. The use of negative stain TEM allows visualization along the process and thus enables optimization of crystal diffraction by monitoring the lattice quality of crystallization conditions. Nanocrystals bearing perfect lattices are seeded and can be used for MicroED as well as growing larger crystals for X-ray and free electron laser (FEL) data collection.
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http://dx.doi.org/10.1007/978-1-0716-0966-8_14DOI Listing
March 2021

Transcription with a laser: Radiation-damage-free diffraction of RNA Polymerase II crystals.

Methods 2019 04 25;159-160:23-28. Epub 2019 Apr 25.

Department of Structural Biology, University of Pittsburgh School of Medicine, United States. Electronic address:

Well-diffracting crystals are essential to obtain relevant structural data that will lead to understanding of RNA Polymerase II (Pol II) transcriptional processes at a molecular level. Here we present a strategy to study Pol II crystals using negative stain transmission electron microscopy (TEM) and a methodology to optimize radiation damage free data collection using free electron laser (FEL) at the Linac Coherent Light Source (LCLS). The use of negative stain TEM allowed visualization and optimization of crystal diffraction by monitoring the lattice quality of crystallization conditions. Nano crystals bearing perfect lattices were seeded and used to grow larger crystals for FEL data collection. Moreover, the use of in house designed crystal loops together with ultra-violet (UV) microscopy for crystal detection facilitated data collection. Such strategy permitted collection of multiple crystals of radiation-free-damage data, resulting in the highest resolution of wild type (WT) Pol II crystals ever observed.
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http://dx.doi.org/10.1016/j.ymeth.2019.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7193734PMC
April 2019

The crystal structure of dGTPase reveals the molecular basis of dGTP selectivity.

Proc Natl Acad Sci U S A 2019 05 24;116(19):9333-9339. Epub 2019 Apr 24.

Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260;

Deoxynucleotide triphosphohydrolases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools. While the vast majority of these enzymes display broad activity toward canonical dNTPs, such as the dNTPase SAMHD1 that blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels, ()dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)- and inhibitor (GTP)-bound structures of hexameric dGTPase, including an X-ray free-electron laser structure of the free -dGTPase enzyme to 3.2 Å. To obtain this structure, we developed a method that applied UV-fluorescence microscopy, video analysis, and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomly located on fixed target holders, resulting in the highest indexing rates observed for a serial femtosecond crystallography experiment. Our structures show a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo- form. Moreover, despite no sequence homology, -dGTPase and SAMHD1 share similar active-site and HD motif architectures; however, -dGTPase residues at the end of the substrate-binding pocket mimic Watson-Crick interactions providing guanine base specificity, while a 7-Å cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotide-type discrimination. Furthermore, the structures shed light on the mechanism by which long distance binding (25 Å) of single-stranded DNA in an allosteric site primes the active site by conformationally "opening" a tyrosine gate allowing enhanced substrate binding.
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http://dx.doi.org/10.1073/pnas.1814999116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511015PMC
May 2019

MicroED Structure of Au(p-MBA) at Subatomic Resolution Reveals a Twinned FCC Cluster.

J Phys Chem Lett 2017 Nov 31;8(22):5523-5530. Epub 2017 Oct 31.

Department of Structural Biology, University of Pittsburgh , Pittsburgh, Pennsylvania 15260, United States.

Solving the atomic structure of metallic clusters is fundamental to understanding their optical, electronic, and chemical properties. Herein we present the structure of the largest aqueous gold cluster, Au(p-MBA) (p-MBA: para-mercaptobenzoic acid), solved by electron micro-diffraction (MicroED) to subatomic resolution (0.85 Å) and by X-ray diffraction at atomic resolution (1.3 Å). The 146 gold atoms may be decomposed into two constituent sets consisting of 119 core and 27 peripheral atoms. The core atoms are organized in a twinned FCC structure, whereas the surface gold atoms follow a C rotational symmetry about an axis bisecting the twinning plane. The protective layer of 57 p-MBAs fully encloses the cluster and comprises bridging, monomeric, and dimeric staple motifs. Au(p-MBA) is the largest cluster observed exhibiting a bulk-like FCC structure as well as the smallest gold particle exhibiting a stacking fault.
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http://dx.doi.org/10.1021/acs.jpclett.7b02621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5769702PMC
November 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

Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED.

Nat Methods 2017 Feb;14(4):399-402

Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, USA.

Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography.
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http://dx.doi.org/10.1038/nmeth.4178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5376236PMC
February 2017

The DDB1-DCAF1-Vpr-UNG2 crystal structure reveals how HIV-1 Vpr steers human UNG2 toward destruction.

Nat Struct Mol Biol 2016 Oct 29;23(10):933-940. Epub 2016 Aug 29.

Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

The HIV-1 accessory protein Vpr is required for efficient viral infection of macrophages and promotion of viral replication in T cells. Vpr's biological activities are closely linked to the interaction with human DCAF1, a cellular substrate receptor of the Cullin4-RING E3 ubiquitin ligase (CRL4) of the host ubiquitin-proteasome-mediated protein degradation pathway. The molecular details of how Vpr usurps the protein degradation pathway have not been delineated. Here we present the crystal structure of the DDB1-DCAF1-HIV-1-Vpr-uracil-DNA glycosylase (UNG2) complex. The structure reveals how Vpr engages with DCAF1, creating a binding interface for UNG2 recruitment in a manner distinct from the recruitment of SAMHD1 by Vpx proteins. Vpr and Vpx use similar N-terminal and helical regions to bind the substrate receptor, whereas different regions target the specific cellular substrates. Furthermore, Vpr uses molecular mimicry of DNA by a variable loop for specific recruitment of the UNG2 substrate.
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http://dx.doi.org/10.1038/nsmb.3284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5385928PMC
October 2016

Transmission electron microscopy for the evaluation and optimization of crystal growth.

Acta Crystallogr D Struct Biol 2016 05 26;72(Pt 5):603-15. Epub 2016 Apr 26.

Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.

The crystallization of protein samples remains the most significant challenge in structure determination by X-ray crystallography. Here, the effectiveness of transmission electron microscopy (TEM) analysis to aid in the crystallization of biological macromolecules is demonstrated. It was found that the presence of well ordered lattices with higher order Bragg spots, revealed by Fourier analysis of TEM images, is a good predictor of diffraction-quality crystals. Moreover, the use of TEM allowed (i) comparison of lattice quality among crystals from different conditions in crystallization screens; (ii) the detection of crystal pathologies that could contribute to poor X-ray diffraction, including crystal lattice defects, anisotropic diffraction and crystal contamination by heavy protein aggregates and nanocrystal nuclei; (iii) the qualitative estimation of crystal solvent content to explore the effect of lattice dehydration on diffraction and (iv) the selection of high-quality crystal fragments for microseeding experiments to generate reproducibly larger sized crystals. Applications to X-ray free-electron laser (XFEL) and micro-electron diffraction (microED) experiments are also discussed.
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http://dx.doi.org/10.1107/S2059798316001546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854312PMC
May 2016

The collection of MicroED data for macromolecular crystallography.

Nat Protoc 2016 May 14;11(5):895-904. Epub 2016 Apr 14.

Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.

The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is ∼10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals.
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http://dx.doi.org/10.1038/nprot.2016.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5357465PMC
May 2016

Assessment of microcrystal quality by transmission electron microscopy for efficient serial femtosecond crystallography.

Arch Biochem Biophys 2016 Jul 2;602:61-68. Epub 2016 Mar 2.

Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA.

Serial femtosecond crystallography (SFX) employing high-intensity X-ray free-electron laser (XFEL) sources has enabled structural studies on microcrystalline protein samples at non-cryogenic temperatures. However, the identification and optimization of conditions that produce well diffracting microcrystals remains an experimental challenge. Here, we report parallel SFX and transmission electron microscopy (TEM) experiments using fragmented microcrystals of wild type (WT) homoprotocatechuate 2,3-dioxygenase (HPCD) and an active site variant (H200Q). Despite identical crystallization conditions and morphology, as well as similar crystal size and density, the indexing efficiency of the diffraction data collected using the H200Q variant sample was over 7-fold higher compared to the diffraction results obtained using the WT sample. TEM analysis revealed an abundance of protein aggregates, crystal conglomerates and a smaller population of highly ordered lattices in the WT sample as compared to the H200Q variant sample. While not reported herein, the 1.75 Å resolution structure of the H200Q variant was determined from ∼16 min of beam time, demonstrating the utility of TEM analysis in evaluating sample monodispersity and lattice quality, parameters critical to the efficiency of SFX experiments.
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http://dx.doi.org/10.1016/j.abb.2016.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5215478PMC
July 2016

High-density grids for efficient data collection from multiple crystals.

Acta Crystallogr D Struct Biol 2016 Jan 1;72(Pt 1):2-11. Epub 2016 Jan 1.

Physical Bioscences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.

Higher throughput methods to mount and collect data from multiple small and radiation-sensitive crystals are important to support challenging structural investigations using microfocus synchrotron beamlines. Furthermore, efficient sample-delivery methods are essential to carry out productive femtosecond crystallography experiments at X-ray free-electron laser (XFEL) sources such as the Linac Coherent Light Source (LCLS). To address these needs, a high-density sample grid useful as a scaffold for both crystal growth and diffraction data collection has been developed and utilized for efficient goniometer-based sample delivery at synchrotron and XFEL sources. A single grid contains 75 mounting ports and fits inside an SSRL cassette or uni-puck storage container. The use of grids with an SSRL cassette expands the cassette capacity up to 7200 samples. Grids may also be covered with a polymer film or sleeve for efficient room-temperature data collection from multiple samples. New automated routines have been incorporated into the Blu-Ice/DCSS experimental control system to support grids, including semi-automated grid alignment, fully automated positioning of grid ports, rastering and automated data collection. Specialized tools have been developed to support crystallization experiments on grids, including a universal adaptor, which allows grids to be filled by commercial liquid-handling robots, as well as incubation chambers, which support vapor-diffusion and lipidic cubic phase crystallization experiments. Experiments in which crystals were loaded into grids or grown on grids using liquid-handling robots and incubation chambers are described. Crystals were screened at LCLS-XPP and SSRL BL12-2 at room temperature and cryogenic temperatures.
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http://dx.doi.org/10.1107/S2059798315020847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756618PMC
January 2016

Microfluidic sorting of protein nanocrystals by size for X-ray free-electron laser diffraction.

Struct Dyn 2015 Jul 19;2(4):041719. Epub 2015 Aug 19.

Department of Chemistry and Biochemistry, Arizona State University , Tempe, Arizona 85287, USA.

The advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still needed to improve data quality and analysis efficiency. One area is sample heterogeneity, where variations in crystal size (among other factors) lead to the requirement of large data sets (and thus 10-100 mg of protein) for determining accurate structure factors. To decrease sample dispersity, we developed a high-throughput microfluidic sorter operating on the principle of dielectrophoresis, whereby polydisperse particles can be transported into various fluid streams for size fractionation. Using this microsorter, we isolated several milliliters of photosystem I nanocrystal fractions ranging from 200 to 600 nm in size as characterized by dynamic light scattering, nanoparticle tracking, and electron microscopy. Sorted nanocrystals were delivered in a liquid jet via the gas dynamic virtual nozzle into the path of the XFEL at the Linac Coherent Light Source. We obtained diffraction to ∼4 Å resolution, indicating that the small crystals were not damaged by the sorting process. We also observed the shape transforms of photosystem I nanocrystals, demonstrating that our device can optimize data collection for the shape transform-based phasing method. Using simulations, we show that narrow crystal size distributions can significantly improve merged data quality in serial crystallography. From this proof-of-concept work, we expect that the automated size-sorting of protein crystals will become an important step for sample production by reducing the amount of protein needed for a high quality final structure and the development of novel phasing methods that exploit inter-Bragg reflection intensities or use variations in beam intensity for radiation damage-induced phasing. This method will also permit an analysis of the dependence of crystal quality on crystal size.
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http://dx.doi.org/10.1063/1.4928688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4711642PMC
July 2015

Crystal Structure of a Transcribing RNA Polymerase II Complex Reveals a Complete Transcription Bubble.

Mol Cell 2015 Jul;59(2):258-69

Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA. Electronic address:

Notwithstanding numerous published structures of RNA Polymerase II (Pol II), structural details of Pol II engaging a complete nucleic acid scaffold have been lacking. Here, we report the structures of TFIIF-stabilized transcribing Pol II complexes, revealing the upstream duplex and full transcription bubble. The upstream duplex lies over a wedge-shaped loop from Rpb2 that engages its minor groove, providing part of the structural framework for DNA tracking during elongation. At the upstream transcription bubble fork, rudder and fork loop 1 residues spatially coordinate strand annealing and the nascent RNA transcript. At the downstream fork, a network of Pol II interactions with the non-template strand forms a rigid domain with the trigger loop (TL), allowing visualization of its open state. Overall, our observations suggest that "open/closed" conformational transitions of the TL may be linked to interactions with the non-template strand, possibly in a synchronized ratcheting manner conducive to polymerase translocation.
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http://dx.doi.org/10.1016/j.molcel.2015.06.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4643057PMC
July 2015

WNT5A inhibits hepatocyte proliferation and concludes β-catenin signaling in liver regeneration.

Am J Pathol 2015 Aug 19;185(8):2194-205. Epub 2015 Jun 19.

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. Electronic address:

Activation of Wnt/β-catenin signaling during liver regeneration (LR) after partial hepatectomy (PH) is observed in several species. However, how this pathway is turned off when hepatocyte proliferation is no longer required is unknown. We assessed LR in liver-specific knockouts of Wntless (Wls-LKO), a protein required for Wnt secretion from a cell. When subjected to PH, Wls-LKO showed prolongation of hepatocyte proliferation for up to 4 days compared with littermate controls. This coincided with increased β-catenin-T-cell factor 4 interaction and cyclin-D1 expression. Wls-LKO showed decreased expression and secretion of inhibitory Wnt5a during LR. Wnt5a expression increased between 24 and 48 hours, and Frizzled-2 between 24 and 72 hours, after PH in normal mice. Treatment of primary mouse hepatocytes and liver tumor cells with Wnt5a led to a notable decrease in β-catenin-T-cell factor activity, cyclin-D1 expression, and cell proliferation. Intriguingly, Wnt5a-LKO did not display any prolongation of LR because of compensation by other cells. In addition, Wnt5a-LKO hepatocytes failed to respond to exogenous Wnt5a treatment in culture because of a compensatory decrease in Frizzled-2 expression. In conclusion, we demonstrate Wnt5a to be, by default, a negative regulator of β-catenin signaling and hepatocyte proliferation, both in vitro and in vivo. We also provide evidence that the Wnt5a/Frizzled-2 axis suppresses β-catenin signaling in hepatocytes in an autocrine manner, thereby contributing to timely conclusion of the LR process.
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http://dx.doi.org/10.1016/j.ajpath.2015.04.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4530131PMC
August 2015

Structural Basis of Clade-specific Engagement of SAMHD1 (Sterile α Motif and Histidine/Aspartate-containing Protein 1) Restriction Factors by Lentiviral Viral Protein X (Vpx) Virulence Factors.

J Biol Chem 2015 Jul 4;290(29):17935-17945. Epub 2015 Jun 4.

Department of Structural Biology and Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260. Electronic address:

Sterile α motif (SAM) and histidine/aspartate (HD)-containing protein 1 (SAMHD1) restricts human/simian immunodeficiency virus infection in certain cell types and is counteracted by the virulence factor Vpx. Current evidence indicates that Vpx recruits SAMHD1 to the Cullin4-Ring Finger E3 ubiquitin ligase (CRL4) by facilitating an interaction between SAMHD1 and the substrate receptor DDB1- and Cullin4-associated factor 1 (DCAF1), thereby targeting SAMHD1 for proteasome-dependent down-regulation. Host-pathogen co-evolution and positive selection at the interfaces of host-pathogen complexes are associated with sequence divergence and varying functional consequences. Two alternative interaction interfaces are used by SAMHD1 and Vpx: the SAMHD1 N-terminal tail and the adjacent SAM domain or the C-terminal tail proceeding the HD domain are targeted by different Vpx variants in a unique fashion. In contrast, the C-terminal WD40 domain of DCAF1 interfaces similarly with the two above complexes. Comprehensive biochemical and structural biology approaches permitted us to delineate details of clade-specific recognition of SAMHD1 by lentiviral Vpx proteins. We show that not only the SAM domain but also the N-terminal tail engages in the DCAF1-Vpx interaction. Furthermore, we show that changing the single Ser-52 in human SAMHD1 to Phe, the residue found in SAMHD1 of Red-capped monkey and Mandrill, allows it to be recognized by Vpx proteins of simian viruses infecting those primate species, which normally does not target wild type human SAMHD1 for degradation.
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http://dx.doi.org/10.1074/jbc.M115.665513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505041PMC
July 2015

Goniometer-based femtosecond crystallography with X-ray free electron lasers.

Proc Natl Acad Sci U S A 2014 Dec 31;111(48):17122-7. Epub 2014 Oct 31.

Stanford Synchrotron Radiation Lightsource.

The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.
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http://dx.doi.org/10.1073/pnas.1418733111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4260607PMC
December 2014

Novel binding motif and new flexibility revealed by structural analyses of a pyruvate dehydrogenase-dihydrolipoyl acetyltransferase subcomplex from the Escherichia coli pyruvate dehydrogenase multienzyme complex.

J Biol Chem 2014 Oct 10;289(43):30161-76. Epub 2014 Sep 10.

From the Departments of Pharmacology and Chemical Biology and the Veterans Affairs Medical Center, Pittsburgh, Pennsylvania 15240

The Escherichia coli pyruvate dehydrogenase multienzyme complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subcomplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subcomplex, both of which probably have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain subcomplex involving a homodimeric E1p, and the results may be applicable to a large range of complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subcomplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly.
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http://dx.doi.org/10.1074/jbc.M114.592915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4208021PMC
October 2014

Identifying, studying and making good use of macromolecular crystals.

Acta Crystallogr F Struct Biol Commun 2014 Aug 25;70(Pt 8):993-1008. Epub 2014 Jul 25.

Hauptman-Woodward Medical Research Institute, 700 Ellicott Street, Buffalo, NY 14203, USA.

Structural biology has contributed tremendous knowledge to the understanding of life on the molecular scale. The Protein Data Bank, a depository of this structural knowledge, currently contains over 100,000 protein structures, with the majority stemming from X-ray crystallography. As the name might suggest, crystallography requires crystals. As detectors become more sensitive and X-ray sources more intense, the notion of a crystal is gradually changing from one large enough to embellish expensive jewellery to objects that have external dimensions of the order of the wavelength of visible light. Identifying these crystals is a prerequisite to their study. This paper discusses developments in identifying these crystals during crystallization screening and distinguishing them from other potential outcomes. The practical aspects of ensuring that once a crystal is identified it can then be positioned in the X-ray beam for data collection are also addressed.
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http://dx.doi.org/10.1107/S2053230X14016574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4118793PMC
August 2014

Endosomal GPCR signaling turned off by negative feedback actions of PKA and v-ATPase.

Nat Chem Biol 2014 Sep 27;10(9):707-9. Epub 2014 Jul 27.

Laboratory for GPCR Biology, Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

The PTH receptor is to our knowledge one of the first G protein-coupled receptor (GPCR) found to sustain cAMP signaling after internalization of the ligand-receptor complex in endosomes. This unexpected model is adding a new dimension on how we think about GPCR signaling, but its mechanism is incompletely understood. We report here that endosomal acidification mediated by the PKA action on the v-ATPase provides a negative feedback mechanism by which endosomal receptor signaling is turned off.
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http://dx.doi.org/10.1038/nchembio.1589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4138287PMC
September 2014

Use of transmission electron microscopy to identify nanocrystals of challenging protein targets.

Proc Natl Acad Sci U S A 2014 Jun 28;111(23):8470-5. Epub 2014 May 28.

Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261;

The current practice for identifying crystal hits for X-ray crystallography relies on optical microscopy techniques that are limited to detecting crystals no smaller than 5 μm. Because of these limitations, nanometer-sized protein crystals cannot be distinguished from common amorphous precipitates, and therefore go unnoticed during screening. These crystals would be ideal candidates for further optimization or for femtosecond X-ray protein nanocrystallography. The latter technique offers the possibility to solve high-resolution structures using submicron crystals. Transmission electron microscopy (TEM) was used to visualize nanocrystals (NCs) found in crystallization drops that would classically not be considered as "hits." We found that protein NCs were readily detected in all samples tested, including multiprotein complexes and membrane proteins. NC quality was evaluated by TEM visualization of lattices, and diffraction quality was validated by experiments in an X-ray free electron laser.
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http://dx.doi.org/10.1073/pnas.1400240111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060711PMC
June 2014

Structure and function of the catalytic domain of the dihydrolipoyl acetyltransferase component in Escherichia coli pyruvate dehydrogenase complex.

J Biol Chem 2014 May 17;289(22):15215-30. Epub 2014 Apr 17.

From the Department of Chemistry, Rutgers University, Newark, New Jersey 07102,

The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s(-1), comparable with the rate of NADH production in the PDHc reaction. Hence, neither reductive acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4'-aminopyrimidine tautomer of bound thiamin diphosphate (AP).
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http://dx.doi.org/10.1074/jbc.M113.544080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140881PMC
May 2014

Formation and fate of a complete 31-protein RNA polymerase II transcription preinitiation complex.

J Biol Chem 2013 Mar 9;288(9):6325-32. Epub 2013 Jan 9.

Department of Structural Biology, Stanford University, Stanford, California 94305, USA.

Whereas individual RNA polymerase II (pol II)-general transcription factor (GTF) complexes are unstable, an assembly of pol II with six GTFs and promoter DNA could be isolated in abundant homogeneous form. The resulting complete pol II transcription preinitiation complex (PIC) contained equimolar amounts of all 31 protein components. An intermediate in assembly, consisting of four GTFs and promoter DNA, could be isolated and supplemented with the remaining components for formation of the PIC. Nuclease digestion and psoralen cross-linking mapped the PIC between positions -70 and -9, centered on the TATA box. Addition of ATP to the PIC resulted in quantitative conversion to an open complex, which retained all 31 proteins, contrary to expectation from previous studies. Addition of the remaining NTPs resulted in run-off transcription, with an efficiency that was promoter-dependent and was as great as 17.5% with the promoters tested.
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http://dx.doi.org/10.1074/jbc.M112.433623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3585067PMC
March 2013

Noncanonical GPCR signaling arising from a PTH receptor-arrestin-Gβγ complex.

Proc Natl Acad Sci U S A 2013 Jan 7;110(4):1530-5. Epub 2013 Jan 7.

Laboratory for G Protein-Coupled Receptor Biology, Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA.

G protein-coupled receptors (GPCRs) participate in ubiquitous transmembrane signal transduction processes by activating heterotrimeric G proteins. In the current "canonical" model of GPCR signaling, arrestins terminate receptor signaling by impairing receptor-G-protein coupling and promoting receptor internalization. However, parathyroid hormone receptor type 1 (PTHR), an essential GPCR involved in bone and mineral metabolism, does not follow this conventional desensitization paradigm. β-Arrestins prolong G protein (G(S))-mediated cAMP generation triggered by PTH, a process that correlates with the persistence of arrestin-PTHR complexes on endosomes and which is thought to be associated with prolonged physiological calcemic and phosphate responses. This presents an inescapable paradox for the current model of arrestin-mediated receptor-G-protein decoupling. Here we show that PTHR forms a ternary complex that includes arrestin and the Gβγ dimer in response to PTH stimulation, which in turn causes an accelerated rate of G(S) activation and increases the steady-state levels of activated G(S), leading to prolonged generation of cAMP. This work provides the mechanistic basis for an alternative model of GPCR signaling in which arrestins contribute to sustaining the effect of an agonist hormone on the receptor.
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http://dx.doi.org/10.1073/pnas.1205756110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3557057PMC
January 2013

A general path for large-scale solubilization of cellular proteins: from membrane receptors to multiprotein complexes.

Protein Expr Purif 2013 Feb 5;87(2):111-9. Epub 2012 Nov 5.

Department of Structural Biology, University of Pittsburgh School of Medicine, USA.

Expression of recombinant proteins in bacterial or eukaryotic systems often results in aggregation rendering them unavailable for biochemical or structural studies. Protein aggregation is a costly problem for biomedical research. It forces research laboratories and the biomedical industry to search for alternative, more soluble, non-human proteins and limits the number of potential "druggable" targets. In this study we present a highly reproducible protocol that introduces the systematic use of an extensive number of detergents to solubilize aggregated proteins expressed in bacterial and eukaryotic systems. We validate the usefulness of this protocol by solubilizing traditionally difficult human protein targets to milligram quantities and confirm their biological activity. We use this method to solubilize monomeric or multimeric components of multi-protein complexes and demonstrate its efficacy to reconstitute large cellular machines. This protocol works equally well on cytosolic, nuclear and membrane proteins and can be easily adapted to a high throughput format.
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http://dx.doi.org/10.1016/j.pep.2012.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4265565PMC
February 2013

Interaction of the mediator head module with RNA polymerase II.

Structure 2012 May;20(5):899-910

Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Mediator, a large (21 polypeptides, MW ∼1 MDa) complex conserved throughout eukaryotes, plays an essential role in control of gene expression by conveying regulatory signals that influence the activity of the preinitiation complex. However, the precise mode of interaction between Mediator and RNA polymerase II (RNAPII), and the mechanism of regulation by Mediator remain elusive. We used cryo-electron microscopy and reconstituted in vitro transcription assays to characterize a transcriptionally-active complex including the Mediator Head module and components of a minimum preinitiation complex (RNAPII, TFIIF, TFIIB, TBP, and promoter DNA). Our results reveal how the Head interacts with RNAPII, affecting its conformation and function.
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http://dx.doi.org/10.1016/j.str.2012.02.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383055PMC
May 2012

Structural and binding studies of the C-terminal domains of yeast TFIIF subunits Tfg1 and Tfg2.

Proteins 2012 Feb 17;80(2):519-29. Epub 2011 Nov 17.

Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260.

The general transcription factor TFIIF plays essential roles at several steps during eukaryotic transcription. While several studies have offered insights into the structure/function relationship in human TFIIF, much less is known about the yeast system. Here, we describe the first NMR structural and binding studies of the C-terminal domains (CTDs) of Tfg1 and Tfg2 subunits of Saccharomyces cerevisiae TFIIF. We used the program CS-ROSETTA to determine the three-dimensional folds of these domains in solution, and performed binding studies with DNA and protein targets. CS-ROSETTA models indicate that the Tfg1 and Tfg2 C-terminal domains have winged-helix architectures, similar to the human homologs. We showed that both Tfg1 and Tfg2 CTDs interact with double-stranded DNA oligonucleotides, and mapped the DNA binding interfaces using solution NMR. Tfg1-CTD, but not Tfg2-CTD, also binds to yeast FCP1, an RNA polymerase II-specific phosphatase, and we delineated the interaction surface with the CTD of FCP1. Our results provide insights into the structural basis of yeast TFIIF function and the differential roles of Tfg1 and Tfg2 subunits during transcription.
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http://dx.doi.org/10.1002/prot.23217DOI Listing
February 2012

Architecture of the Mediator head module.

Nature 2011 Jul 3;475(7355):240-3. Epub 2011 Jul 3.

Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, Indiana 46202, USA.

Mediator is a key regulator of eukaryotic transcription, connecting activators and repressors bound to regulatory DNA elements with RNA polymerase II (Pol II). In the yeast Saccharomyces cerevisiae, Mediator comprises 25 subunits with a total mass of more than one megadalton (refs 5, 6) and is organized into three modules, called head, middle/arm and tail. Our understanding of Mediator assembly and its role in regulating transcription has been impeded so far by limited structural information. Here we report the crystal structure of the essential Mediator head module (seven subunits, with a mass of 223 kilodaltons) at a resolution of 4.3 ångströms. Our structure reveals three distinct domains, with the integrity of the complex centred on a bundle of ten helices from five different head subunits. An intricate pattern of interactions within this helical bundle ensures the stable assembly of the head subunits and provides the binding sites for general transcription factors and Pol II. Our structural and functional data suggest that the head module juxtaposes transcription factor IIH and the carboxy-terminal domain of the largest subunit of Pol II, thereby facilitating phosphorylation of the carboxy-terminal domain of Pol II. Our results reveal architectural principles underlying the role of Mediator in the regulation of gene expression.
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http://dx.doi.org/10.1038/nature10162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4109712PMC
July 2011

Structure of an RNA polymerase II-TFIIB complex and the transcription initiation mechanism.

Science 2010 Jan 12;327(5962):206-9. Epub 2009 Nov 12.

Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.

Previous x-ray crystal structures have given insight into the mechanism of transcription and the role of general transcription factors in the initiation of the process. A structure of an RNA polymerase II-general transcription factor TFIIB complex at 4.5 angstrom resolution revealed the amino-terminal region of TFIIB, including a loop termed the "B finger," reaching into the active center of the polymerase where it may interact with both DNA and RNA, but this structure showed little of the carboxyl-terminal region. A new crystal structure of the same complex at 3.8 angstrom resolution obtained under different solution conditions is complementary with the previous one, revealing the carboxyl-terminal region of TFIIB, located above the polymerase active center cleft, but showing none of the B finger. In the new structure, the linker between the amino- and carboxyl-terminal regions can also be seen, snaking down from above the cleft toward the active center. The two structures, taken together with others previously obtained, dispel long-standing mysteries of the transcription initiation process.
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http://dx.doi.org/10.1126/science.1182015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2813267PMC
January 2010

Schizosacharomyces pombe RNA polymerase II at 3.6-A resolution.

Proc Natl Acad Sci U S A 2009 Jun 20;106(23):9185-90. Epub 2009 May 20.

Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.

The second structure of a eukaryotic RNA polymerase II so far determined, that of the enzyme from the fission yeast Schizosaccharomyces pombe, is reported here. Comparison with the previous structure of the enzyme from the budding yeast Saccharomyces cerevisiae reveals differences in regions implicated in start site selection and transcription factor interaction. These aspects of the transcription mechanism differ between S. pombe and S. cerevisiae, but are conserved between S. pombe and humans. Amino acid changes apparently responsible for the structural differences are also conserved between S. pombe and humans, suggesting that the S. pombe structure may be a good surrogate for that of the human enzyme.
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http://dx.doi.org/10.1073/pnas.0903361106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2684843PMC
June 2009

A unified view of ligand-protected gold clusters as superatom complexes.

Proc Natl Acad Sci U S A 2008 Jul 1;105(27):9157-62. Epub 2008 Jul 1.

Departments of Physics and Chemistry, Nanoscience Center, University of Jyväskylä, FI-40014 Jyväskylä, Finland.

Synthesis, characterization, and functionalization of self-assembled, ligand-stabilized gold nanoparticles are long-standing issues in the chemistry of nanomaterials. Factors driving the thermodynamic stability of well documented discrete sizes are largely unknown. Herein, we provide a unified view of principles that underlie the stability of particles protected by thiolate (SR) or phosphine and halide (PR(3), X) ligands. The picture has emerged from analysis of large-scale density functional theory calculations of structurally characterized compounds, namely Au(102)(SR)(44), Au(39)(PR(3))(14)X(6)(-), Au(11)(PR(3))(7)X(3), and Au(13)(PR(3))(10)X(2)(3+), where X is either a halogen or a thiolate. Attributable to a compact, symmetric core and complete steric protection, each compound has a filled spherical electronic shell and a major energy gap to unoccupied states. Consequently, the exceptional stability is best described by a "noble-gas superatom" analogy. The explanatory power of this concept is shown by its application to many monomeric and oligomeric compounds of precisely known composition and structure, and its predictive power is indicated through suggestions offered for a series of anomalously stable cluster compositions which are still awaiting a precise structure determination.
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http://dx.doi.org/10.1073/pnas.0801001105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2442568PMC
July 2008