Publications by authors named "David G Myszka"

77 Publications

Development of a Model Protein Interaction Pair as a Benchmarking Tool for the Quantitative Analysis of 2-Site Protein-Protein Interactions.

J Biomol Tech 2015 Dec 29;26(4):125-41. Epub 2015 Oct 29.

1 Bristol-Myers Squibb, Princeton, New Jersey 08540, USA; 2 Tokyo Institute of Technology, Yokohama 226-8503, Japan; 3 Google[x], Google Life Sciences, Mountain View, California 94043, USA; 4 SystaMedic, Incorporated, Groton, Connecticut 06340, USA; 5 Biosensor Tools LLC, Salt Lake City, Utah 84103, USA; 6 National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA; 7 Polaris Pharmaceuticals, Incorporated, San Diego, California 92121, USA; and 8 Institute for Bioscience and Biotechnology Research, Fischell Department of Bioengineering, University of Maryland, Rockville, Maryland 20850, USA.

A significant challenge in the molecular interaction field is to accurately determine the stoichiometry and stepwise binding affinity constants for macromolecules having >1 binding site. The mission of the Molecular Interactions Research Group (MIRG) of the Association of Biomolecular Resource Facilities (ABRF) is to show how biophysical technologies are used to quantitatively characterize molecular interactions, and to educate the ABRF members and scientific community on the utility and limitations of core technologies [such as biosensor, microcalorimetry, or analytic ultracentrifugation (AUC)]. In the present work, the MIRG has developed a robust model protein interaction pair consisting of a bivalent variant of the Bacillus amyloliquefaciens extracellular RNase barnase and a variant of its natural monovalent intracellular inhibitor protein barstar. It is demonstrated that this system can serve as a benchmarking tool for the quantitative analysis of 2-site protein-protein interactions. The protein interaction pair enables determination of precise binding constants for the barstar protein binding to 2 distinct sites on the bivalent barnase binding partner (termed binase), where the 2 binding sites were engineered to possess affinities that differed by 2 orders of magnitude. Multiple MIRG laboratories characterized the interaction using isothermal titration calorimetry (ITC), AUC, and surface plasmon resonance (SPR) methods to evaluate the feasibility of the system as a benchmarking model. Although general agreement was seen for the binding constants measured using solution-based ITC and AUC approaches, weaker affinity was seen for surface-based method SPR, with protein immobilization likely affecting affinity. An analysis of the results from multiple MIRG laboratories suggests that the bivalent barnase-barstar system is a suitable model for benchmarking new approaches for the quantitative characterization of complex biomolecular interactions.
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http://dx.doi.org/10.7171/jbt.15-2604-001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4627511PMC
December 2015

Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus.

Acta Crystallogr D Biol Crystallogr 2015 May 25;71(Pt 5):1159-75. Epub 2015 Apr 25.

Department of Biochemistry and Molecular Genetics, Northwestern University, 303 East Chicago Avenue, Chicago, IL 60611, USA.

When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL (SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NAD(+)) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD(+), NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme.
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http://dx.doi.org/10.1107/S1399004715004228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4427200PMC
May 2015

A bacteriophage capsid protein provides a general amyloid interaction motif (GAIM) that binds and remodels misfolded protein assemblies.

J Mol Biol 2014 Jun 22;426(13):2500-19. Epub 2014 Apr 22.

Neurophage Pharmaceuticals, 222 Third Street, Suite 3120, Cambridge, MA 02142, USA. Electronic address:

Misfolded protein aggregates, characterized by a canonical amyloid fold, play a central role in the pathobiology of neurodegenerative diseases. Agents that bind and sequester neurotoxic intermediates of amyloid assembly, inhibit the assembly or promote the destabilization of such protein aggregates are in clinical testing. Here, we show that the gene 3 protein (g3p) of filamentous bacteriophage mediates potent generic binding to the amyloid fold. We have characterized the amyloid binding and conformational remodeling activities using an array of techniques, including X-ray fiber diffraction and NMR. The mechanism for g3p binding with amyloid appears to reflect its physiological role during infection of Escherichia coli, which is dependent on temperature-sensitive interdomain unfolding and cis-trans prolyl isomerization of g3p. In addition, a natural receptor for g3p, TolA-C, competitively interferes with Aβ binding to g3p. NMR studies show that g3p binding to Aβ fibers is predominantly through middle and C-terminal residues of the Aβ subunit, indicating β strand-g3p interactions. A recombinant bivalent g3p molecule, an immunoglobulin Fc (Ig) fusion of the two N-terminal g3p domains, (1) potently binds Aβ fibers (fAβ) (KD=9.4nM); (2); blocks fAβ assembly (IC50~50nM) and (3) dissociates fAβ (EC50=40-100nM). The binding of g3p to misfolded protein assemblies is generic, and amyloid-targeted activities can be demonstrated using other misfolded protein systems. Taken together, our studies show that g3p(N1N2) acts as a general amyloid interaction motif.
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http://dx.doi.org/10.1016/j.jmb.2014.04.015DOI Listing
June 2014

Biophysical fragment screening of the β1-adrenergic receptor: identification of high affinity arylpiperazine leads using structure-based drug design.

J Med Chem 2013 May 9;56(9):3446-55. Epub 2013 Apr 9.

Heptares Therapeutics Ltd. , BioPark, Welwyn Garden City, Hertfordshire, AL7 3AX, U.K.

Biophysical fragment screening of a thermostabilized β1-adrenergic receptor (β1AR) using surface plasmon resonance (SPR) enabled the identification of moderate affinity, high ligand efficiency (LE) arylpiperazine hits 7 and 8. Subsequent hit to lead follow-up confirmed the activity of the chemotype, and a structure-based design approach using protein-ligand crystal structures of the β1AR resulted in the identification of several fragments that bound with higher affinity, including indole 19 and quinoline 20. In the first example of GPCR crystallography with ligands derived from fragment screening, structures of the stabilized β1AR complexed with 19 and 20 were determined at resolutions of 2.8 and 2.7 Å, respectively.
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http://dx.doi.org/10.1021/jm400140qDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3654563PMC
May 2013

CCR5 is a receptor for Staphylococcus aureus leukotoxin ED.

Nature 2013 Jan 12;493(7430):51-5. Epub 2012 Dec 12.

Department of Microbiology, New York University School of Medicine, New York, New York 10016, USA.

Pore-forming toxins are critical virulence factors for many bacterial pathogens and are central to Staphylococcus aureus-mediated killing of host cells. S. aureus encodes pore-forming bi-component leukotoxins that are toxic towards neutrophils, but also specifically target other immune cells. Despite decades since the first description of staphylococcal leukocidal activity, the host factors responsible for the selectivity of leukotoxins towards different immune cells remain unknown. Here we identify the human immunodeficiency virus (HIV) co-receptor CCR5 as a cellular determinant required for cytotoxic targeting of subsets of myeloid cells and T lymphocytes by the S. aureus leukotoxin ED (LukED). We further demonstrate that LukED-dependent cell killing is blocked by CCR5 receptor antagonists, including the HIV drug maraviroc. Remarkably, CCR5-deficient mice are largely resistant to lethal S. aureus infection, highlighting the importance of CCR5 targeting in S. aureus pathogenesis. Thus, depletion of CCR5(+) leukocytes by LukED suggests a new immune evasion mechanism of S. aureus that can be therapeutically targeted.
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http://dx.doi.org/10.1038/nature11724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536884PMC
January 2013

Interactions of the human LIP5 regulatory protein with endosomal sorting complexes required for transport.

J Biol Chem 2012 Dec 26;287(52):43910-26. Epub 2012 Oct 26.

Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84112-5650, USA.

The endosomal sorting complex required for transport (ESCRT) pathway remodels membranes during multivesicular body biogenesis, the abscission stage of cytokinesis, and enveloped virus budding. The ESCRT-III and VPS4 ATPase complexes catalyze the membrane fission events associated with these processes, and the LIP5 protein helps regulate their interactions by binding directly to a subset of ESCRT-III proteins and to VPS4. We have investigated the biochemical and structural basis for different LIP5-ligand interactions and show that the first microtubule-interacting and trafficking (MIT) module of the tandem LIP5 MIT domain binds CHMP1B (and other ESCRT-III proteins) through canonical type 1 MIT-interacting motif (MIM1) interactions. In contrast, the second LIP5 MIT module binds with unusually high affinity to a novel MIM element within the ESCRT-III protein CHMP5. A solution structure of the relevant LIP5-CHMP5 complex reveals that CHMP5 helices 5 and 6 and adjacent linkers form an amphipathic "leucine collar" that wraps almost completely around the second LIP5 MIT module but makes only limited contacts with the first MIT module. LIP5 binds MIM1-containing ESCRT-III proteins and CHMP5 and VPS4 ligands independently in vitro, but these interactions are coupled within cells because formation of stable VPS4 complexes with both LIP5 and CHMP5 requires LIP5 to bind both a MIM1-containing ESCRT-III protein and CHMP5. Our studies thus reveal how the tandem MIT domain of LIP5 binds different types of ESCRT-III proteins, promoting assembly of active VPS4 enzymes on the polymeric ESCRT-III substrate.
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http://dx.doi.org/10.1074/jbc.M112.417899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3527974PMC
December 2012

Structure of a proteasome Pba1-Pba2 complex: implications for proteasome assembly, activation, and biological function.

J Biol Chem 2012 Oct 28;287(44):37371-82. Epub 2012 Aug 28.

Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84112-5650, USA.

The 20S proteasome is an essential, 28-subunit protease that sequesters proteolytic sites within a central chamber, thereby repressing substrate degradation until proteasome activators open the entrance/exit gate. Two established activators, Blm10 and PAN/19S, induce gate opening by binding to the pockets between proteasome α-subunits using C-terminal HbYX (hydrophobic-tyrosine-any residue) motifs. Equivalent HbYX motifs have been identified in Pba1 and Pba2, which function in proteasome assembly. Here, we demonstrate that Pba1-Pba2 proteins form a stable heterodimer that utilizes its HbYX motifs to bind mature 20S proteasomes in vitro and that the Pba1-Pba2 HbYX motifs are important for a physiological function of proteasomes, the maintenance of mitochondrial function. Other factors that contribute to proteasome assembly or function also act in the maintenance of mitochondrial function and display complex genetic interactions with one another, possibly revealing an unexpected pathway of mitochondrial regulation involving the Pba1-Pba2 proteasome interaction. Our determination of a proteasome Pba1-Pba2 crystal structure reveals a Pba1 HbYX interaction that is superimposable with those of known activators, a Pba2 HbYX interaction that is different from those reported previously, and a gate structure that is disrupted but not sufficiently open to allow entry of even small peptides. These findings extend understanding of proteasome interactions with HbYX motifs and suggest multiple roles for Pba1-Pba2 interactions throughout proteasome assembly and function.
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http://dx.doi.org/10.1074/jbc.M112.367003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3481334PMC
October 2012

Survey of the 2009 commercial optical biosensor literature.

J Mol Recognit 2011 Nov-Dec;24(6):892-914

Center for Biomolecular Interaction Analysis, University of Utah, Salt Lake City, UT, USA.

We took a different approach to reviewing the commercial biosensor literature this year by inviting 22 biosensor users to serve as a review committee. They set the criteria for what to expect in a publication and ultimately decided to use a pass/fail system for selecting which papers to include in this year's reference list. Of the 1514 publications in 2009 that reported using commercially available optical biosensor technology, only 20% passed their cutoff. The most common criticism the reviewers had with the literature was that "the biosensor experiments could have been done better." They selected 10 papers to highlight good experimental technique, data presentation, and unique applications of the technology. This communal review process was educational for everyone involved and one we will not soon forget.
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http://dx.doi.org/10.1002/jmr.1138DOI Listing
February 2012

Fragment screening of stabilized G-protein-coupled receptors using biophysical methods.

Methods Enzymol 2011 ;493:115-36

Heptares Therapeutics, Biopark, Welwyn Garden City, Hertfordshire, United Kingdom.

Biophysical studies with G-protein-coupled receptors (GPCRs) are typically very challenging due to the poor stability of these receptors when solubilized from the cell membrane into detergent solutions. However, the stability of a GPCR can be greatly improved by introducing a number of point mutations into the protein sequence to give a stabilized receptor or StaR®. Here, we present the utility of StaRs for biophysical studies and the screening of fragment libraries. Two case studies are used to illustrate the methods: first, the screening of a library of fragments by surface plasmon resonance against the adenosine A(2A) receptor StaR, demonstrating how very small and weakly active xanthine fragments can be detected binding to the protein on chips; second, the screening and detection of fragment hits of a larger fragment library in an NMR format called TINS (target-immobilized NMR screening) against the β(1) adrenergic StaR.
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http://dx.doi.org/10.1016/B978-0-12-381274-2.00005-4DOI Listing
June 2011

Biacore analysis with stabilized G-protein-coupled receptors.

Anal Biochem 2011 Feb 20;409(2):267-72. Epub 2010 Oct 20.

Center for Biomolecular Interaction Analysis, University of Utah School of Medicine, Salt Lake City, 84132, USA.

Using stabilized forms of β₁ adrenergic and A₂(A) adenosine G-protein-coupled receptors, we applied Biacore to monitor receptor activity and characterize binding constants of small-molecule antagonists spanning more than 20,000-fold in affinity. We also illustrate an improved method for tethering His-tagged receptors on NTA (carboxymethylated dextran preimmobilized with nitrilotriacetic acid) chips to yield stable, high-capacity, high-activity surfaces as well as a novel approach to regenerate receptor binding sites. Based on our success with this approach, we expect that the combination of stabilized receptors with biosensor technology will become a common method for characterizing members of this receptor family.
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http://dx.doi.org/10.1016/j.ab.2010.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3010267PMC
February 2011

Two independent histidines, one in human prolactin and one in its receptor, are critical for pH-dependent receptor recognition and activation.

J Biol Chem 2010 Dec 30;285(49):38524-33. Epub 2010 Sep 30.

Department of Laboratory Medicine, Yale School of Medicine, New Haven, Connecticut 06520, USA.

Human prolactin (hPRL), a member of the family of hematopoietic cytokines, functions as both an endocrine hormone and autocrine/paracrine growth factor. We have previously demonstrated that recognition of the hPRL·receptor depends strongly on solution acidity over the physiologic range from pH 6 to pH 8. The hPRL·receptor binding interface contains four histidines whose protonation is hypothesized to regulate pH-dependent receptor recognition. Here, we systematically dissect its molecular origin by characterizing the consequences of His to Ala mutations on pH-dependent receptor binding kinetics, site-specific histidine protonation, and high resolution structures of the intermolecular interface. Thermodynamic modeling of the pH dependence to receptor binding affinity reveals large changes in site-specific protonation constants for a majority of interface histidines upon complexation. Removal of individual His imidazoles reduces these perturbations in protonation constants, which is most likely explained by the introduction of solvent-filled, buried cavities in the crystallographic structures without inducing significant conformational rearrangements.
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http://dx.doi.org/10.1074/jbc.M110.172072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2992285PMC
December 2010

Biosensor-based fragment screening using FastStep injections.

Anal Biochem 2010 Dec 25;407(2):270-7. Epub 2010 Aug 25.

Center for Biomolecular Interaction Analysis, University of Utah School of Medicine, Salt Lake City, UT 84132, USA.

We have developed a novel analyte injection method for the SensíQ Pioneer surface plasmon resonance-based biosensor referred to as "FastStep." By merging buffer and sample streams immediately prior to the reaction flow cells, the instrument is capable of automatically generating a two- or threefold dilution series (of seven or five concentrations, respectively) from a single analyte sample. Using sucrose injections, we demonstrate that the production of each concentration within the step gradient is highly reproducible. For kinetic studies, we developed analysis software that utilizes the sucrose responses to automatically define the concentration of analyte at any point during the association phase. To validate this new approach, we compared the results of standard and FastStep injections for ADP binding to a target kinase and a panel of compounds binding to carbonic anhydrase II. Finally, we illustrate how FastStep can be used in a primary screening mode to obtain a full concentration series of each compound in a fragment library.
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http://dx.doi.org/10.1016/j.ab.2010.08.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2949542PMC
December 2010

Kinetic analysis and fragment screening with Fujifilm AP-3000.

Anal Biochem 2010 Jul 3;402(2):170-8. Epub 2010 Apr 3.

Center for Biomolecular Interaction Analysis, University of Utah School of Medicine, Salt Lake City, 84132, USA.

We evaluated the performance of Fujifilm's new AP-3000 surface plasmon resonance biosensor for kinetic analysis and fragment screening. Using carbonic anhydrase II as a model system, we characterized a set of 10 sulfonamide-based inhibitors that range in molecular mass from 98 to 341Da and approximately 10,000-fold in affinity (0.4mM to 20nM). Although the data collected from the AP-3000 were generally similar to those collected using a Biacore T100, the AP-3000's stop-flow analyte delivery system complicated the shapes of the association- and dissociation-phase binding responses. We illustrate how reasonable estimates of the kinetic rate constants can be extracted from AP-3000 data by limiting data analysis to only the regions of the responses collected during flow conditions. We also provide an example of the results obtained for a fragment-screening study with the AP-3000, which is the ideal application of this technology.
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http://dx.doi.org/10.1016/j.ab.2010.03.043DOI Listing
July 2010

Grading the commercial optical biosensor literature-Class of 2008: 'The Mighty Binders'.

J Mol Recognit 2010 Jan-Feb;23(1):1-64

Center for Biomolecular Interaction Analysis, University of Utah, Salt Lake City, UT 84132, USA.

Optical biosensor technology continues to be the method of choice for label-free, real-time interaction analysis. But when it comes to improving the quality of the biosensor literature, education should be fundamental. Of the 1413 articles published in 2008, less than 30% would pass the requirements for high-school chemistry. To teach by example, we spotlight 10 papers that illustrate how to implement the technology properly. Then we grade every paper published in 2008 on a scale from A to F and outline what features make a biosensor article fabulous, middling or abysmal. To help improve the quality of published data, we focus on a few experimental, analysis and presentation mistakes that are alarmingly common. With the literature as a guide, we want to ensure that no user is left behind.
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http://dx.doi.org/10.1002/jmr.1004DOI Listing
March 2010

Multivalent benzoboroxole functionalized polymers as gp120 glycan targeted microbicide entry inhibitors.

Mol Pharm 2010 Feb;7(1):116-29

Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112-5820, USA.

Microbicides are women-controlled prophylactics for sexually transmitted infections. The most important class of microbicides target HIV-1 and contain antiviral agents formulated for topical vaginal delivery. Identification of new viral entry inhibitors that target the HIV-1 envelope is important because they can inactivate HIV-1 in the vaginal lumen before virions can come in contact with CD4+ cells in the vaginal mucosa. Carbohydrate binding agents (CBAs) demonstrate the ability to act as entry inhibitors due to their ability to bind to glycans and prevent gp120 binding to CD4+ cells. However, as proteins they present significant challenges in regard to economical production and formulation for resource-poor environments. We have synthesized water-soluble polymer CBAs that contain multiple benzoboroxole moieties. A benzoboroxole-functionalized monomer was synthesized and incorporated into linear oligomers with 2-hydroxypropylmethacrylamide (HPMAm) at different feed ratios using free radical polymerization. The benzoboroxole small molecule analogue demonstrated weak affinity for HIV-1BaL gp120 by SPR; however, the 25 mol % functionalized benzoboroxole oligomer demonstrated a 10-fold decrease in the K(D) for gp120, suggesting an increased avidity for the multivalent polymer construct. High molecular weight polymers functionalized with 25, 50, and 75 mol % benzoboroxole were synthesized and tested for their ability to neutralize HIV-1 entry for two HIV-1 clades and both R5 and X4 coreceptor tropism. All three polymers demonstrated activity against all viral strains tested with EC(50)s that decrease from 15000 nM (1500 microg mL(-1)) for the 25 mol % functionalized polymers to 11 nM (1 microg mL(-1)) for the 75 mol % benzoboroxole-functionalized polymers. These polymers exhibited minimal cytotoxicity after 24 h exposure to a human vaginal cell line.
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http://dx.doi.org/10.1021/mp900159nDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2815106PMC
February 2010

Structural models for interactions between the 20S proteasome and its PAN/19S activators.

J Biol Chem 2010 Jan 4;285(1):13-7. Epub 2009 Nov 4.

Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84112, USA.

Proteasome activity is regulated by sequestration of its proteolytic centers in a barrel-shaped structure that limits substrate access. Substrates enter the proteasome by means of activator complexes that bind to the end rings of proteasome alpha subunits and induce opening of an axial entrance/exit pore. The PA26 activator binds in a pocket on the proteasome surface using main chain contacts of its C-terminal residues and uses an internal activation loop to trigger gate opening by repositioning the proteasome Pro-17 reverse turn. Subunits of the unrelated PAN/19S activators bind with their C termini in the same pockets but can induce proteasome gate opening entirely from interactions of their C-terminal peptides, which are reported to cause gate opening by inducing a rocking motion of proteasome alpha subunits rather than by directly contacting the Pro-17 turn. Here we report crystal structures and binding studies of proteasome complexes with PA26 constructs that display modified C-terminal residues, including those corresponding to PAN. These findings suggest that PA26 and PAN/19S C-terminal residues bind superimposably and that both classes of activator induce gate opening by using direct contacts to residues of the proteasome Pro-17 reverse turn. In the case of the PAN and 19S activators, a penultimate tyrosine/phenylalanine residue contacts the proteasome Gly-19 carbonyl oxygen to stabilize the open conformation.
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http://dx.doi.org/10.1074/jbc.C109.070425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2804157PMC
January 2010

Structural basis for ESCRT-III protein autoinhibition.

Nat Struct Mol Biol 2009 Jul 14;16(7):754-62. Epub 2009 Jun 14.

Department of Biochemistry, University of Utah, Salt Lake City, Utah, USA.

Endosomal sorting complexes required for transport-III (ESCRT-III) subunits cycle between two states: soluble monomers and higher-order assemblies that bind and remodel membranes during endosomal vesicle formation, midbody abscission and enveloped virus budding. Here we show that the N-terminal core domains of increased sodium tolerance-1 (IST1) and charged multivesicular body protein-3 (CHMP3) form equivalent four-helix bundles, revealing that IST1 is a previously unrecognized ESCRT-III family member. IST1 and its ESCRT-III binding partner, CHMP1B, both form higher-order helical structures in vitro, and IST1-CHMP1 interactions are required for abscission. The IST1 and CHMP3 structures also reveal that equivalent downstream alpha5 helices can fold back against the core domains. Mutations within the CHMP3 core-alpha5 interface stimulate the protein's in vitro assembly and HIV-inhibition activities, indicating that dissociation of the autoinhibitory alpha5 helix from the core activates ESCRT-III proteins for assembly at membranes.
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http://dx.doi.org/10.1038/nsmb.1621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2712734PMC
July 2009

E2 interaction and dimerization in the crystal structure of TRAF6.

Nat Struct Mol Biol 2009 Jun 24;16(6):658-66. Epub 2009 May 24.

Weill Medical College of Cornell University, New York, New York, USA.

Tumor necrosis factor (TNF) receptor-associated factor (TRAF)-6 mediates Lys63-linked polyubiquitination for NF-kappaB activation via its N-terminal RING and zinc finger domains. Here we report the crystal structures of TRAF6 and its complex with the ubiquitin-conjugating enzyme (E2) Ubc13. The RING and zinc fingers of TRAF6 assume a rigid, elongated structure. Interaction of TRAF6 with Ubc13 involves direct contacts of the RING and the preceding residues, and the first zinc finger has a structural role. Unexpectedly, this region of TRAF6 is dimeric both in the crystal and in solution, different from the trimeric C-terminal TRAF domain. Structure-based mutagenesis reveals that TRAF6 dimerization is crucial for polyubiquitin synthesis and autoubiquitination. Fluorescence resonance energy transfer analysis shows that TRAF6 dimerization induces higher-order oligomerization of full-length TRAF6. The mismatch of dimeric and trimeric symmetry may provide a mode of infinite oligomerization that facilitates ligand-dependent signal transduction of many immune receptors.
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http://dx.doi.org/10.1038/nsmb.1605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2834951PMC
June 2009

Inhibition and binding kinetics of the hepatitis C virus NS3 protease inhibitor ITMN-191 reveals tight binding and slow dissociative behavior.

Biochemistry 2009 Mar;48(11):2559-68

InterMune Inc., 3280 Bayshore Boulevard, Brisbane, California 94005, USA.

The protease activity of hepatitis C virus nonstructural protein 3 (NS3) is essential for viral replication. ITMN-191, a macrocyclic inhibitor of the NS3 protease active site, promotes rapid, multilog viral load reductions in chronic HCV patients. Here, ITMN-191 is shown to be a potent inhibitor of NS3 with a two-step binding mechanism. Progress curves are consistent with the formation of an initial collision complex (EI) that isomerizes to a highly stable complex (EI*) from which ITMN-191 dissociates very slowly. K(i), the dissociation constant of EI, is 100 nM, and the rate constant for conversion of EI to EI* is 6.2 x 10(-2) s(-1). Binding experiments using protein fluorescence confirm this isomerization rate. From progress curve analysis, the rate constant for dissociation of ITMN-191 from the EI* complex is 3.8 x 10(-5) s(-1) with a calculated complex half-life of approximately 5 h and a true biochemical potency (K(i)*) of approximately 62 pM. Surface plasmon resonance studies and assessment of enzyme reactivation following dilution of the EI* complex confirm slow dissociation and suggest that the half-life may be considerably longer. Abrogation of the tight binding and slow dissociative properties of ITMN-191 is observed with proteases that carry the R155K or D168A substitution, each of which is likely in drug resistant mutants. Slow dissociation is not observed with closely related macrocyclic inhibitors of NS3, suggesting that members of this class may display distinct binding kinetics.
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http://dx.doi.org/10.1021/bi900038pDOI Listing
March 2009

Detergent screening of a G-protein-coupled receptor using serial and array biosensor technologies.

Anal Biochem 2009 Mar 24;386(1):98-104. Epub 2008 Dec 24.

Center for Biomolecular Interaction Analysis, School of Medicine, University of Utah, Salt Lake City, 84132, USA.

We describe the benefits and limitations of two biosensor approaches for screening solubilization conditions for G-protein-coupled receptors (GPCRs). Assays designed for a serial processing instrument (Biacore 2000/3000/T100) and an array platform (Biacore Flexchip) were used to examine how effectively 96 different detergents solubilized the chemokine receptor CCR5 while maintaining its binding activity for a conformationally sensitive Fab (2D7). Using the serial processing instrument, we were able to analyze three samples in each 30-min binding cycle, thereby requiring approximately 24h to screen an entire 96-well plate of conditions. In-line capturing allowed us to normalize the 2D7 binding responses for different receptor capture levels. In contrast, with the array system, we could characterize the effects of all 96 detergents simultaneously, completing the assay in less than 1h. But the current array technology requires that we capture the GPCR preparations off-line, making it more challenging to normalize for receptor capture levels. Also, the array platform is less sensitive than the serial platforms, thereby limiting the size of the analyte to larger molecules (>5000Da). Overall, the two approaches proved to be highly complementary; both assays identified identical detergents that produced active solubilized CCR5 as well as those detergents that either were ineffective solubilizers or inactivated the receptor.
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http://dx.doi.org/10.1016/j.ab.2008.12.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2669081PMC
March 2009

A global benchmark study using affinity-based biosensors.

Anal Biochem 2009 Mar 27;386(2):194-216. Epub 2008 Nov 27.

Center for Biomolecular Interaction Analysis, School of Medicine, University of Utah, Salt Lake City, UT 84132, USA.

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.
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http://dx.doi.org/10.1016/j.ab.2008.11.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3793259PMC
March 2009

"Spot and hop": internal referencing for surface plasmon resonance imaging using a three-dimensional microfluidic flow cell array.

Anal Biochem 2009 Feb 20;385(2):309-13. Epub 2008 Nov 20.

Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA.

We have developed a novel referencing technique for surface plasmon resonance imaging systems referred to as "spot and hop." The technique enables internal referencing for individual flow cells in a parallel processing microfluidic network. Internal referencing provides the ability to correct for nonspecific binding and instrument drift, significantly improving data quality at each region of interest. The performance of a 48-flow-cell device was demonstrated through a series of studies, including "rise and fall" time, ligand preconcentration, ligand immobilization, analyte binding, and regeneration tests. Interfacing parallel processing fluidics with imaging systems will significantly expand the throughput and applications of array-based optical biosensors while retaining high data quality.
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http://dx.doi.org/10.1016/j.ab.2008.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6691735PMC
February 2009

Survey of the year 2007 commercial optical biosensor literature.

J Mol Recognit 2008 Nov-Dec;21(6):355-400

Center for Biomolecular Interaction Analysis, University of Utah, Salt Lake City, UT, USA.

In 2007, 1179 papers were published that involved the application of optical biosensors. Reported developments in instrument hardware, assay design, and immobilization chemistry continue to improve the technology's throughput, sensitivity, and utility. Compared to recent years, the widest range of platforms, both traditional format and array-based, were used. However, as in the past, we found a disappointingly low percentage of well-executed experiments and thoughtful data interpretation. We are alarmed by the high frequency of suboptimal data and over-interpreted results in the literature. Fortunately, learning to visually recognize good--and more importantly, bad--data is easy. Using examples from the literature, we outline several features of biosensor responses that indicate experimental artifacts versus actual binding events. Our goal is to have everyone, from benchtop scientists to project managers and manuscript reviewers, become astute judges of biosensor results using nothing more than their eyes.
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http://dx.doi.org/10.1002/jmr.928DOI Listing
January 2009

Optimal conditions for protein array deposition using continuous flow.

Anal Chem 2008 Nov 22;80(22):8561-7. Epub 2008 Oct 22.

Department of Chemical Engineering, University of Utah, Salt Lake City, Utah 84112, USA.

Optimal conditions for depositing protein microarrays using a continuous-flow microfluidic device, the continuous-flow microspotter (CFM), have been determined using a design of experiments approach. The amount of protein deposited on the surface depends on the rates of convective and diffusive transport to the surface and binding at the surface. These rates depend on parameters such as the flow rate, time, and capture mechanism at the surface. The process parameters were optimized, and uniform protein spots were obtained at a protein concentration of 10 microg/mL and even at 0.4 microg/mL. A 150-fold dilution in protein concentration in the sample solution decreased surface concentration by a factor of only 16. If the capture mechanism of the protein on the substrate is nonspecific, optimal deposition is obtained at higher flow rates for short periods of time. If the capture mechanism is specific, such as biotin-avidin, deposition is optimal at medium flow rates with little advantage beyond 30 min. The CFM can be used to deposit protein arrays with good spot morphology, spot-to-spot uniformity and enhanced surface concentration. The CFM was used to deposit an array of various antibodies, and their interactions with an antigen were studied using surface plasmon resonance (SPR). Affinity values were obtained at low antibody concentrations (5 microg/mL) with low coefficients of variation. Thus, the CFM can be used to effectively capture proteins and antibodies from dilute samples while depositing multiple spots, thereby increasing the quality of spots in protein microarrays and especially improving screening throughput of SPR.
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http://dx.doi.org/10.1021/ac8014609DOI Listing
November 2008

Thermodynamic characterization of pyrazole and azaindole derivatives binding to p38 mitogen-activated protein kinase using Biacore T100 technology and van't Hoff analysis.

Anal Biochem 2008 Dec 19;383(2):255-64. Epub 2008 Aug 19.

Center for Biomolecular Interaction Analysis, University of Utah, Salt Lake City, UT 84132, USA.

Biacore T100 technology was used in conjunction with a van't Hoff analysis to characterize the thermodynamic binding parameters of 85 small-molecule inhibitors of adenosine triphosphate (ATP) binding to p38 mitogen-activated protein (MAP) kinase. The compounds were selected from a large panel of azaindole and pyrazole derivatives for which IC(50) data exist. We showed a strong relationship between the K(D) and IC(50) of a compound, but only a modest relationship between k(off) and IC(50) was detected and an apparent relationship between a compound's k(on) and its IC(50) could not be discerned. Similarly, a correlation between a compound's IC(50) and its thermodynamic parameters DeltaH degrees and DeltaS degrees could not be established. The lack of a predominant kinetic or thermodynamic signature associated with the inhibitory potential of these compounds demonstrates that there exists, even within a single well-defined system, a library of kinetic routes or, alternatively, a library of initial and final enthalpic and entropic states from which to effect inhibition. As a complement to these studies, selected double mutant thermodynamic cycles were performed to probe the energetic coupling, if any, between common sites of fluorination in both the azaindole and pyrazole classes and two different substituents. Although both cycles indicated negligible coupling free energies, both revealed significant coupling enthalpies, an observation made in other similarly dissected systems. The possible significance and caveats associated with these findings along with the advantages of using Biacore technology to derive thermodynamic parameters in drug discovery efforts are discussed.
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http://dx.doi.org/10.1016/j.ab.2008.08.010DOI Listing
December 2008

Improved continuous-flow print head for micro-array deposition.

Anal Biochem 2008 Nov 3;382(1):55-9. Epub 2008 Aug 3.

Department of Bioengineering, University of Utah, Salt Lake City, UT 84132, USA.

Limitations in depositing ligands using conventional micro-array pin spotting have hindered the application of surface plasmon resonance imaging (SPRi) technology. To address these challenges we introduce a modification to our continuous-flow micro-spotting technology that improves ligand deposition. Using Flexchip protein A/G and neutravidin capturing surfaces, we demonstrate that our new microfluidic spotter requires 1000 times less concentrated antibodies and biotinylated ligands than is required for pin spotting. By varying the deposition flow rate, we show that the design of our tip overlay flow cell is efficient at delivering sample to the substrate surface. Finally, contact time studies show that it is possible to capture antibodies and biotinylated ligands at concentrations of less than 0.1 ug/ml and 100 pM, respectively. These improvements in spotting technology will help to expand the applications of SPRi systems in areas such as antibody screening, carbohydrate arrays, and biomarker detection.
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http://dx.doi.org/10.1016/j.ab.2008.07.031DOI Listing
November 2008

High-affinity interaction between IKKbeta and NEMO.

Biochemistry 2008 Mar 12;47(10):3109-16. Epub 2008 Feb 12.

Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021, USA.

The Ser/Thr-specific IkappaB kinase (IKK), which comprises IKKalpha or IKKbeta and the regulatory protein NEMO, is at the bottleneck for NF-kappaB activation. IKK activity relies on interaction between NEMO and IKKalpha or IKKbeta. A conserved region in the C-terminal tail of IKKbeta or IKKalpha (NEMO-binding domain, NBD, residues 734-745 of IKKbeta) is important for interaction with NEMO. Here we show that the NBD peptide of IKKbeta is not sufficient for interaction with NEMO. Instead, a longer region of the IKKbeta C-terminal region provides high affinity for NEMO. Quantitative measurements using surface plasmon resonance and isothermal titration calorimetry confirm the differential affinities of these interactions and provide insight into the kinetic and thermodynamic behaviors of the interactions. Biochemical characterization using multiangle light scattering (MALS) coupled with refractive index shows that the longer IKKbeta C-terminal region forms a 2:2 stoichiometirc complex with NEMO.
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http://dx.doi.org/10.1021/bi702312cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3673718PMC
March 2008

Survey of the year 2006 commercial optical biosensor literature.

J Mol Recognit 2007 Sep-Oct;20(5):300-66

Center for Biomolecular Interaction Analysis, University of Utah, Salt Lake City, Utah 84132, USA.

We identified 1219 articles published in 2006 that described work performed using commercial optical biosensor platforms. It is interesting to witness how the biosensor market is maturing with an increased number of instrument manufacturers offering a wider variety of platforms. However, it is clear from a review of the results presented that the advances in technology are outpacing the skill level of the average biosensor user. While we can track a gradual improvement in the quality of the published work, we clearly have a long way to go before we capitalize on the full potential of biosensor technology. To illustrate what is right with the biosensor literature, we highlight the work of 10 groups who have their eye on the ball. To help out the rest of us who have the lights on but nobody home, we use the literature to address common myths about biosensor technology.
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http://dx.doi.org/10.1002/jmr.862DOI Listing
June 2008

Structural and functional studies of ALIX interactions with YPX(n)L late domains of HIV-1 and EIAV.

Nat Struct Mol Biol 2008 Jan 9;15(1):43-9. Epub 2007 Dec 9.

Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84112-5650, USA.

Retrovirus budding requires short peptide motifs (late domains) located within the viral Gag protein that function by recruiting cellular factors. The YPX(n)L late domains of HIV and other lentiviruses recruit the protein ALIX (also known as AIP1), which also functions in vesicle formation at the multivesicular body and in the abscission stage of cytokinesis. Here, we report the crystal structures of ALIX in complex with the YPX(n)L late domains from HIV-1 and EIAV. The two distinct late domains bind at the same site on the ALIX V domain but adopt different conformations that allow them to make equivalent contacts. Binding studies and functional assays verified the importance of key interface residues and revealed that binding affinities are tuned by context-dependent effects. These results reveal how YPX(n)L late domains recruit ALIX to facilitate virus budding and how ALIX can bind YPX(n)L sequences with both n = 1 and n = 3.
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http://dx.doi.org/10.1038/nsmb1319DOI Listing
January 2008

Molecular basis for passive immunotherapy of Alzheimer's disease.

Proc Natl Acad Sci U S A 2007 Oct 25;104(40):15659-64. Epub 2007 Sep 25.

Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA.

Amyloid aggregates of the amyloid-beta (Abeta) peptide are implicated in the pathology of Alzheimer's disease. Anti-Abeta monoclonal antibodies (mAbs) have been shown to reduce amyloid plaques in vitro and in animal studies. Consequently, passive immunization is being considered for treating Alzheimer's, and anti-Abeta mAbs are now in phase II trials. We report the isolation of two mAbs (PFA1 and PFA2) that recognize Abeta monomers, protofibrils, and fibrils and the structures of their antigen binding fragments (Fabs) in complex with the Abeta(1-8) peptide DAEFRHDS. The immunodominant EFRHD sequence forms salt bridges, hydrogen bonds, and hydrophobic contacts, including interactions with a striking WWDDD motif of the antigen binding fragments. We also show that a similar sequence (AKFRHD) derived from the human protein GRIP1 is able to cross-react with both PFA1 and PFA2 and, when cocrystallized with PFA1, binds in an identical conformation to Abeta(1-8). Because such cross-reactivity has implications for potential side effects of immunotherapy, our structures provide a template for designing derivative mAbs that target Abeta with improved specificity and higher affinity.
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http://dx.doi.org/10.1073/pnas.0705888104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1994138PMC
October 2007
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