Guillermo Calero

University of Pittsburgh, Pittsburgh, Pennsylvania, United States

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Publications (33)303.18 Total impact

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    ABSTRACT: 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.
    Full-text · Article · Jan 2016
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    ABSTRACT: 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 10–100mg 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 A ̊ 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.
    Full-text · Article · Aug 2015 · Structural Dynamics
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    ABSTRACT: 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. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Jul 2015 · Molecular cell
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    ABSTRACT: 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. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
    No preview · Article · Jun 2015 · American Journal Of Pathology
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    ABSTRACT: Sterile Alpha Motif (SAM) and Histidine/Aspartate (HD) containing protein 1 (SAMHD1) restricts HIV/SIV 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 coevolution 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: SamHD1's 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. Further, we show that changing the single Ser52 in human SAMHD1 to Phe, the residue found in SAMHD1 of Red-capped monkey (RCM) and Mandril (MND), 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. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    No preview · Article · Jun 2015 · Journal of Biological Chemistry
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    ABSTRACT: 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.
    Full-text · Article · Oct 2014 · Proceedings of the National Academy of Sciences
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    ABSTRACT: 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 ofβ2-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
    Full-text · Article · Oct 2014 · Proceedings of the National Academy of Sciences
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    ABSTRACT: 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 sub-complex between E1p and an E2p didomain containing a hybrid lipoyl domain (LDh) along with the peripheral subunit binding domain (PSBD) 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 sub-complex; both of which likely have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain sub-complex 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 sub-complex, 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.
    No preview · Article · Sep 2014 · Journal of Biological Chemistry
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    ABSTRACT: 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.
    Full-text · Article · Sep 2014 · Nature Chemical Biology
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    Guillermo Calero · Aina E Cohen · Joseph R Luft · Janet Newman · Edward H Snell
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    ABSTRACT: 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.
    Full-text · Article · Jul 2014 · Acta Crystallographica Section F: Structural Biology Communications
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    ABSTRACT: Recent advancements at the Linac Coherent Light Source X-ray free-electron laser (XFEL) enabling successful serial femtosecond diffraction experiments using nanometre-sized crystals (NCs) have opened up the possibility of X-ray structure determination of proteins that produce only submicrometre crystals such as many membrane proteins. Careful crystal pre-characterization including compatibility testing of the sample delivery method is essential to ensure efficient use of the limited beamtime available at XFEL sources. This work demonstrates the utility of transmission electron microscopy for detecting and evaluating NCs within the carrier solutions of liquid injectors. The diffraction quality of these crystals may be assessed by examining the crystal lattice and by calculating the fast Fourier transform of the image. Injector reservoir solutions, as well as solutions collected post-injection, were evaluated for three types of protein NCs (i) the membrane protein PTHR1, (ii) the multi-protein complex Pol II-GFP and (iii) the soluble protein lysozyme. Our results indicate that the concentration and diffraction quality of NCs, particularly those with high solvent content and sensitivity to mechanical manipulation may be affected by the delivery process.
    Full-text · Article · Jul 2014 · Philosophical Transactions of The Royal Society B Biological Sciences
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    ABSTRACT: 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.
    Full-text · Article · Jun 2014 · Proceedings of the National Academy of Sciences
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    ABSTRACT: The E. 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 (LD), a peripheral subunit binding domain (PSBD) and catalytic domain (E2pCD). Herein are reported: (1) The X-ray structure of E2pCD revealed both intra- and inter-trimer interactions, similar to those reported for other E2pCDs. (2) Reconstitution of recombinant LD, E2pCD with E1p, E3p to PDHc could maintain at least 6.4% activity (NADH production) confirming 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 coenzymeA catalyzed by E2pCD was observed with a rate constant of 199 s-1, comparable to 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 H/D exchange mass spectrometry. Inclusion of each additional domain of E2p strengthened the interaction with E1p, and was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifest by the appearance of a circular dichroism band characteristic of the canonical 4-aminopyrimidine tautomer of bound thiamin diphosphate.
    Full-text · Article · Apr 2014 · Journal of Biological Chemistry
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    ABSTRACT: 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.
    Full-text · Article · Jan 2013 · Journal of Biological Chemistry
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    ABSTRACT: 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.
    Full-text · Article · Jan 2013 · Proceedings of the National Academy of Sciences
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    ABSTRACT: 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.
    No preview · Article · Nov 2012 · Protein Expression and Purification
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    ABSTRACT: 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.
    Preview · Article · May 2012 · Structure
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    ABSTRACT: 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. Proteins 2011. © 2011 Wiley Periodicals, Inc.
    Full-text · Article · Jan 2012 · Proteins Structure Function and Bioinformatics
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    Full-text · Article · Jan 2012 · Biophysical Journal
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    Preview · Article · Jan 2012 · Biophysical Journal

Publication Stats

2k Citations
303.18 Total Impact Points

Institutions

  • 2012-2015
    • University of Pittsburgh
      • Department of Structural Biology
      Pittsburgh, Pennsylvania, United States
  • 2008-2013
    • Stanford University
      • • Department of Structural Biology
      • • Department of Medicine
      Palo Alto, California, United States
  • 2007-2008
    • Stanford Medicine
      • Department of Structural Biology
      Stanford, California, United States
  • 2003-2005
    • Cornell University
      • Department of Chemistry and Chemical Biology
      Итак, New York, United States