Publications by authors named "Daoning Zhang"

22 Publications

  • Page 1 of 1

Polyubiquitin and ubiquitin-like signals share common recognition sites on proteasomal subunit Rpn1.

J Biol Chem 2021 Jan-Jun;296:100450. Epub 2021 Feb 20.

Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland, USA. Electronic address:

Proteasome-mediated substrate degradation is an essential process that relies on the coordinated actions of ubiquitin (Ub), shuttle proteins containing Ub-like (UBL) domains, and the proteasome. Proteinaceous substrates are tagged with polyUb and shuttle proteins, and these signals are then recognized by the proteasome, which subsequently degrades the substrate. To date, three proteasomal receptors have been identified, as well as multiple shuttle proteins and numerous types of polyUb chains that signal for degradation. While the components of this pathway are well-known, our understanding of their interplay is unclear-especially in the context of Rpn1, the largest proteasomal subunit. Here, using nuclear magnetic resonance (NMR) spectroscopy in combination with competition assays, we show that Rpn1 associates with UBL-containing proteins and polyUb chains, while exhibiting a preference for shuttle protein Rad23. Rpn1 appears to contain multiple Ub/UBL-binding sites, theoretically as many as one for each of its hallmark proteasome/cyclosome repeats. Remarkably, we also find that binding sites on Rpn1 can be shared among Ub and UBL species, while proteasomal receptors Rpn1 and Rpn10 can compete with each other for binding of shuttle protein Dsk2. Taken together, our results rule out the possibility of exclusive recognition sites on Rpn1 for individual Ub/UBL signals and further emphasize the complexity of the redundancy-laden proteasomal degradation pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbc.2021.100450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008175PMC
August 2021

A DNA G-quadruplex/i-motif hybrid.

Nucleic Acids Res 2019 12;47(22):11921-11930

Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, MD 20742, USA.

DNA can form many structures beyond the canonical Watson-Crick double helix. It is now clear that noncanonical structures are present in genomic DNA and have biological functions. G-rich G-quadruplexes and C-rich i-motifs are the most well-characterized noncanonical DNA motifs that have been detected in vivo with either proscribed or postulated biological roles. Because of their independent sequence requirements, these structures have largely been considered distinct types of quadruplexes. Here, we describe the crystal structure of the DNA oligonucleotide, d(CCAGGCTGCAA), that self-associates to form a quadruplex structure containing two central antiparallel G-tetrads and six i-motif C-C+ base pairs. Solution studies suggest a robust structural motif capable of assembling as a tetramer of individual strands or as a dimer when composed of tandem repeats. This hybrid structure highlights the growing structural diversity of DNA and suggests that biological systems may harbor many functionally important non-duplex structures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkz1008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145706PMC
December 2019

Crystal Structure of a Tetrameric DNA Fold-Back Quadruplex.

J Am Chem Soc 2018 11 13;140(47):16291-16298. Epub 2018 Nov 13.

Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization , University of Maryland , College Park , Maryland 20742 , United States.

DNA can adopt many structures beyond the Watson-Crick duplex. However, the bounds of DNA structural diversity and how these structures might regulate biological processes is only beginning to be understood. Here, we describe the 1.05 Å resolution crystal structure of a DNA oligonucleotide that self-associates to form a non-G-quadruplex fold-back structure. Distinct from previously described fold-back quadruplexes, two-fold-back dimers interact through noncanonical and Watson-Crick interactions to form a tetrameric assembly. These interactions include a hexad base pairing arrangement from two C-G-G base triples. The assembly is dependent on divalent cations, and the interface between the dimeric units creates a cavity in which a cation resides. This structure provides new sequence and structural contexts for the formation of fold-back quadruplexes, further highlighting the potential biological importance of this type of noncanonical DNA structure. This structure may also serve as the basis for designing new types of DNA nanoarchitectures or cation sensors based on the strong divalent cation dependence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jacs.8b10153DOI Listing
November 2018

Structural Basis for the Inhibitory Effects of Ubistatins in the Ubiquitin-Proteasome Pathway.

Structure 2017 12 16;25(12):1839-1855.e11. Epub 2017 Nov 16.

Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, MD 20742, USA. Electronic address:

The discovery of ubistatins, small molecules that impair proteasomal degradation of proteins by directly binding to polyubiquitin, makes ubiquitin itself a potential therapeutic target. Although ubistatins have the potential for drug development and clinical applications, the lack of structural details of ubiquitin-ubistatin interactions has impeded their development. Here, we characterized a panel of new ubistatin derivatives using functional and binding assays. The structures of ubiquitin complexes with ubistatin B and hemi-ubistatin revealed direct interactions with ubiquitin's hydrophobic surface patch and the basic/polar residues surrounding it. Ubistatin B binds ubiquitin and diubiquitin tighter than a high-affinity ubiquitin receptor and shows strong preference for K48 linkages over K11 and K63. Furthermore, ubistatin B shields ubiquitin conjugates from disassembly by a range of deubiquitinases and by the 26S proteasome. Finally, ubistatin B penetrates cancer cells and alters the cellular ubiquitin landscape. These findings highlight versatile properties of ubistatins and have implications for their future development and use in targeting ubiquitin-signaling pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2017.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5731780PMC
December 2017

Characterizing polyubiquitinated forms of the neurodegenerative ubiquitin mutant UBB.

FEBS Lett 2016 Dec 22;590(24):4573-4585. Epub 2016 Nov 22.

Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, MD, USA.

The ubiquitin mutant UBB has been identified as a hallmark of neurodegenerative diseases. In this study, we characterize polyubiquitinated forms of UBB in vitro and from patient samples. The ability of UBB to be readily ubiquitinated by several E2 enzymes provided a mechanism for the controlled synthesis and purification of defined conjugates. This allowed us to utilize polyUb-UBB conjugates for biochemical assays, as well as solution NMR. Coupled with our immunoassay for detection of ubiquitinated forms of UBB in patient blood samples, we gain a clearer picture of the molecular mechanisms underlying neurodegenerative diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/1873-3468.12484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5182158PMC
December 2016

Probing the role of sequence in the assembly of three-dimensional DNA crystals.

Biopolymers 2015 Nov;103(11):618-26

Department of Chemistry & Biochemistry, University of Maryland, College Park, MD, 20742.

DNA is a widely used biopolymer for the construction of nanometer-scale objects due to its programmability and structural predictability. One long-standing goal of the DNA nanotechnology field has been the construction of three-dimensional DNA crystals. We previously determined the X-ray crystal structure of a DNA 13-mer that forms a continuously hydrogen bonded three-dimensional lattice through Watson-Crick and non-canonical base pairs. Our current study sets out to understand how the sequence of the Watson-Crick duplex region influences crystallization of this 13-mer. We screened all possible self-complementary sequences in the hexameric duplex region and found 21 oligonucleotides that crystallized. Sequence analysis showed that one specific Watson-Crick pair influenced the crystallization propensity and the speed of crystal self-assembly. We determined X-ray crystal structures for 13 of these oligonucleotides and found sequence-specific structural changes that suggests that this base pair may serve as a structural anchor during crystal assembly. Finally, we explored the crystal self-assembly and nucleation process. Solution studies indicated that these oligonucleotides do not form base pairs in the absence of cations, but that the addition of divalent cations leads to rapid self-assembly to higher molecular weight complexes. We further demonstrate that crystals grown from mixtures of two different oligonucleotide sequences contain both oligonucleotides. These results suggest that crystal self-assembly is nucleated by the formation of the Watson-Crick duplexes initiated by a simple chemical trigger. This study provides new insight into the role of sequence for the assembly of periodic DNA structures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bip.22688DOI Listing
November 2015

DNA-damage-inducible 1 protein (Ddi1) contains an uncharacteristic ubiquitin-like domain that binds ubiquitin.

Structure 2015 Mar 19;23(3):542-557. Epub 2015 Feb 19.

Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, MD 20742, USA. Electronic address:

Ddi1 belongs to a family of shuttle proteins targeting polyubiquitinated substrates for proteasomal degradation. Unlike the other proteasomal shuttles, Rad23 and Dsk2, Ddi1 remains an enigma: its function is not fully understood and structural properties are poorly characterized. We determined the structure and binding properties of the ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains of Ddi1 from Saccharomyces cerevisiae. We found that while Ddi1UBA forms a characteristic UBA:ubiquitin complex, Ddi1UBL has entirely uncharacteristic binding preferences. Despite having a ubiquitin-like fold, Ddi1UBL does not interact with typical UBL receptors but unexpectedly binds ubiquitin, forming a unique interface mediated by hydrophobic contacts and by salt bridges between oppositely charged residues of Ddi1UBL and ubiquitin. In stark contrast to ubiquitin and other UBLs, the β-sheet surface of Ddi1UBL is negatively charged and therefore is recognized in a completely different way. The dual functionality of Ddi1UBL, capable of binding both ubiquitin and proteasome, suggests an intriguing mechanism for Ddi1 as a proteasomal shuttle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2015.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4448915PMC
March 2015

An intercalation-locked parallel-stranded DNA tetraplex.

Nucleic Acids Res 2015 Feb 27;43(3):1937-44. Epub 2015 Jan 27.

Department of Chemistry & Biochemistry, Center for Biomolecular Structure & Organization, Maryland Nanocenter, University of Maryland, College Park, MD 20742, USA

DNA has proved to be an excellent material for nanoscale construction because complementary DNA duplexes are programmable and structurally predictable. However, in the absence of Watson-Crick pairings, DNA can be structurally more diverse. Here, we describe the crystal structures of d(ACTCGGATGAT) and the brominated derivative, d(AC(Br)UCGGA(Br)UGAT). These oligonucleotides form parallel-stranded duplexes with a crystallographically equivalent strand, resulting in the first examples of DNA crystal structures that contains four different symmetric homo base pairs. Two of the parallel-stranded duplexes are coaxially stacked in opposite directions and locked together to form a tetraplex through intercalation of the 5'-most A-A base pairs between adjacent G-G pairs in the partner duplex. The intercalation region is a new type of DNA tertiary structural motif with similarities to the i-motif. (1)H-(1)H nuclear magnetic resonance and native gel electrophoresis confirmed the formation of a parallel-stranded duplex in solution. Finally, we modified specific nucleotide positions and added d(GAY) motifs to oligonucleotides and were readily able to obtain similar crystals. This suggests that this parallel-stranded DNA structure may be useful in the rational design of DNA crystals and nanostructures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkv033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4330391PMC
February 2015

Extended ubiquitin species are protein-based DUB inhibitors.

Nat Chem Biol 2014 Aug 6;10(8):664-70. Epub 2014 Jul 6.

Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.

A frameshift mutation in the transcript of the ubiquitin-B gene leads to a C-terminally extended ubiquitin (Ub), UBB(+1). UBB(+1) has been considered to inhibit proteasomes and as such to be the underlying cause for toxic protein buildup correlated with certain neuropathological conditions. We demonstrate that expression of extended Ub variants leads to accumulation of heterogeneously linked polyubiquitin conjugates, indicating a pervasive effect on Ub-dependent turnover. 20S proteasomes selectively proteolyzed Ub extensions, yet no evidence for inhibition of 26S holoenzymes was found. However, among susceptible targets for inhibition was Ubp6, the primary enzyme responsible for disassembly of Lys48 linkages at 26S proteasomes. Processing of Lys48 and Lys63 linkages by other deubiquitinating enzymes (DUBs) was also inhibited. Disruption of Ub-dependent degradation by extended Ub variants may therefore be attributed to their inhibitory effect on select DUBs, thus shifting research efforts related to protein accumulation in neurodegenerative processes from proteasomes to DUBs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nchembio.1574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4466224PMC
August 2014

Enhanced silencing and stabilization of siRNA polyplexes by histidine-mediated hydrogen bonds.

Biomaterials 2014 Jan 22;35(2):846-55. Epub 2013 Oct 22.

Department of Pathology, University of Maryland Baltimore, MSTF Building, 10 South Pine Street, Baltimore, MD 21201, United States; Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD 20742, United States. Electronic address:

Branched peptides containing histidines and lysines (HK) have been shown to be effective carriers for DNA and siRNA. We anticipate that elucidation of the binding mechanism of HK with siRNA will provide greater insight into the self-assembly and delivery of the HK:siRNA polyplex. Non-covalent bonds between histidine residues and nucleic acids may enhance the stability of siRNA polyplexes. We first compared the polyplex biophysical properties of a branched HK with those of branched asparagine-lysine peptide (NK). Consistent with siRNA silencing experiments, gel electrophoresis demonstrated that the HK siRNA polyplex maintained its integrity with prolonged incubation in serum, whereas siRNA in complex with NK was degraded in a time-dependent manner. Isothermal titration calorimetry of various peptides binding to siRNA at pH 7.3 showed that branched polylysine, interacted with siRNA was initially endothermic, whereas branched HK exhibited an exothermic reaction at initial binding. The exothermic interaction indicates formation of non-ionic bonds between histidines and siRNA; purely electrostatic interaction is entropy-driven and endothermic. To investigate the type of non-ionic bond, we studied the protonation state of imidazole rings of a selectively (15)N labeled branched HK by heteronuclear single quantum coherence NMR. The peak of Nδ1-H tautomers of imidazole shifted downfield (in the direction of deprotonation) by 0.5-1.0 ppm with addition of siRNA, providing direct evidence that histidines formed hydrogen bonds with siRNA at physiological pH. These results establish that histidine-rich peptides form hydrogen bonds with siRNA, thereby enhancing the stability and biological activity of the polyplex in vitro and in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2013.10.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3920840PMC
January 2014

Accurate measurements of the effects of deuteration at backbone amide positions on the chemical shifts of ¹⁵N, ¹³Cα, ¹³Cβ, ¹³CO and ¹Hα nuclei in proteins.

J Biomol NMR 2013 Jun 24;56(2):169-82. Epub 2013 Apr 24.

Department of Chemistry and Biochemistry, University of Maryland, Biomolecular Sci. Bldg./CBSO, College Park, MD 20742, USA.

An approach towards accurate NMR measurements of deuterium isotope effects on the chemical shifts of all backbone nuclei in proteins ((15)N, (13)Cα, (13)CO, (1)Hα) and (13)Cβ nuclei arising from (1)H-to-D substitutions at amide nitrogen positions is described. Isolation of molecular species with a defined protonation/deuteration pattern at successive backbone nitrogen positions in the polypeptide chain allows quantifying all deuterium isotope shifts of these nuclei from the first to the fourth order. Some of the deuterium isotope shifts measured in the proteins ubiquitin and GB1 can be interpreted in terms of backbone geometry via empirical relationships describing their dependence on (φ; ψ) backbone dihedral angles. Because of their relatively large variability and notable dependence on the protein secondary structure, the two- and three-bond (13)Cα isotope shifts, (2)ΔCα(NiD) and (3)ΔCα(Ni+1D), and three-bond (13)Cβ isotope shifts, (3)ΔCβ(NiD), are useful reporters of the local geometry of the protein backbone.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10858-013-9733-yDOI Listing
June 2013

Proton bridging in the interactions of thrombin with hirudin and its mimics.

Biochemistry 2013 Apr 1;52(14):2472-81. Epub 2013 Apr 1.

Department of Chemistry, The Catholic University of America, Washington, DC 20064, USA.

Thrombin is the pivotal serine protease enzyme in the blood cascade system and thus a target of drug design for control of its activity. The most efficient nonphysiologic inhibitor of thrombin is hirudin, a naturally occurring small protein. Hirudin and its synthetic mimics employ a range of hydrogen bonding, salt bridging, and hydrophobic interactions with thrombin to achieve tight binding with K(i) values in the nano- to femtomolar range. The one-dimensional (1)H nuclear magnetic resonance spectrum recorded at 600 MHz reveals a resonance 15.33 ppm downfield from silanes in complexes between human α-thrombin and r-hirudin in pH 5.6-8.8 buffers and between 5 and 35 °C. There is also a resonance between 15.17 and 15.54 ppm seen in complexes of human α-thrombin with hirunorm IV, hirunorm V, an Nα(Me)Arg peptide, RGD-hirudin, and Nα-2-naphthylsulfonyl-glycyl-DL-4-amidinophenylalanyl-piperidide acetate salt (NAPAP), while there is no such low-field resonance observed in a complex of porcine trypsin and NAPAP. The chemical shifts suggest that these resonances represent H-bonded environments. H-Donor-acceptor distances in the corresponding H-bonds are estimated to be <2.7 Å. Addition of Phe-Pro-Arg-chloromethylketone (PPACK) to a complex of human α-thrombin with r-hirudin results in an additional signal at 18.03 ppm, which is 0.10 ppm upfield from the observed signal [Kovach, I. M., et al. (2009) Biochemistry 48, 7296-7304] for thrombin covalently modified with PPACK. In contrast, the peak at 15.33 ppm remains unchanged. The fractionation factors for the thrombin-hirudin complexes are near 1.0 within 20% error. The most likely site of the short H-bond in complexes of thrombin with the hirudin family of inhibitors is in the hydrophobic patch of the C-terminus of hirudin where Glu(57') and Glu(58') are embedded and interact with Arg(75) and Arg(77) and their solvate water (on thrombin). Glu(57') and Glu(58') present in the hirudin family of inhibitors make up a key binding epitope of fibrinogen, thrombin's prime substrate, which lends substantial interest to the short hydrogen bond as a binding element at the fibrinogen recognition site.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi301625aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3750058PMC
April 2013

Structural and biochemical studies of the open state of Lys48-linked diubiquitin.

Biochim Biophys Acta 2012 Nov 16;1823(11):2046-56. Epub 2012 Apr 16.

Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.

Ubiquitin (Ub) is a small protein highly conserved among eukaryotes and involved in practically all aspects of eukaryotic cell biology. Polymeric chains assembled from covalently-linked Ub monomers function as molecular signals in the regulation of a host of cellular processes. Our previous studies have shown that the predominant state of Lys48-linked di- and tetra-Ub chains at near-physiological conditions is a closed conformation, in which the Ub-Ub interface is formed by the hydrophobic surface residues of the adjacent Ub units. Because these very residues are involved in (poly)Ub interactions with the majority of Ub-binding proteins, their sequestration at the Ub-Ub interface renders the closed conformation of polyUb binding incompetent. Thus the existence of open conformation(s) and the interdomain motions opening and closing the Ub-Ub interface is critical for the recognition of Lys48-linked polyUb by its receptors. Knowledge of the conformational properties of a polyUb signal is essential for our understanding of its specific recognition by various Ub-receptors. Despite their functional importance, open states of Lys48-linked chains are poorly characterized. Here we report a crystal structure of the open state of Lys48-linked di-Ub. Moreover, using NMR, we examined interactions of the open state of this chain (at pH4.5) with a Lys48-linkage-selective receptor, the UBA2 domain of a shuttle protein hHR23a. Our results show that di-Ub binds UBA2 in the same mode and with comparable affinity as the closed state. Our data suggest a mechanism for polyUb signal recognition, whereby Ub-binding proteins select specific conformations out of the available ensemble of polyUb chain conformations. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbamcr.2012.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3429157PMC
November 2012

Rpn1 and Rpn2 coordinate ubiquitin processing factors at proteasome.

J Biol Chem 2012 Apr 8;287(18):14659-71. Epub 2012 Feb 8.

Department of Biology, Technion Institute of Technology, 32000 Haifa, Israel.

Substrates tagged with (poly)ubiquitin for degradation can be targeted directly to the 26 S proteasome where they are proteolyzed. Independently, ubiquitin conjugates may also be delivered by bivalent shuttles. The majority of shuttles attach to the proteasome through a ubiquitin-like domain (UBL) while anchoring cargo at a C-terminal polyubiquitin-binding domain(s). We found that two shuttles of this class, Rad23 and Dsk2, dock at two different receptor sites embedded within a single subunit of the 19 S proteasome regulatory particle, Rpn1. Their association/dissociation constants and affinities for Rpn1 are similar. In contrast, another UBL-containing protein, the deubiquitinase Ubp6, is also anchored by Rpn1, yet it dissociates slower, thus behaving as an occasional proteasome subunit that is distinct from the transiently associated shuttles. Two neighboring subunits, Rpn10 and Rpn13, show a marked preference for polyubiquitin over UBLs. Rpn10 attaches to the central solenoid portion of Rpn1, although this association is stabilized by the presence of a third subunit, Rpn2. Rpn13 binds directly to Rpn2. These intrinsic polyubiquitin receptors may compete with substrate shuttles for their polyubiquitin-conjugate cargos, thereby aiding release of the emptied shuttles. By binding multiple ubiquitin-processing factors simultaneously, Rpn1 is uniquely suited to coordinate substrate recruitment, deubiquitination, and movement toward the catalytic core. The broad range of affinities for ubiquitin, ubiquitin-like, and non-ubiquitin signals by adjacent yet nonoverlapping sites all within the base represents a hub of activity that coordinates the intricate relay of substrates within the proteasome, and consequently it influences substrate residency time and commitment to degradation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M111.316323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3340268PMC
April 2012

Together, Rpn10 and Dsk2 can serve as a polyubiquitin chain-length sensor.

Mol Cell 2009 Dec;36(6):1018-33

Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, 20910, USA.

As a signal for substrate targeting, polyubiquitin meets various layers of receptors upstream to the 26S proteasome. We obtained structural information on two receptors, Rpn10 and Dsk2, alone and in complex with (poly)ubiquitin or with each other. A hierarchy of affinities emerges with Dsk2 binding monoubiquitin tighter than Rpn10 does, whereas Rpn10 prefers the ubiquitin-like domain of Dsk2 to monoubiquitin, with increasing affinities for longer polyubiquitin chains. We demonstrated the formation of ternary complexes of both receptors simultaneously with (poly)ubiquitin and found that, depending on the ubiquitin chain length, the orientation of the resulting complex is entirely different, providing for alternate signals. Dynamic rearrangement provides a chain-length sensor, possibly explaining how accessibility of Dsk2 to the proteasome is limited unless it carries a properly tagged cargo. We propose a mechanism for a malleable ubiquitin signal that depends both on chain length and combination of receptors to produce tetraubiquitin as an efficient signal threshold.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2009.11.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807407PMC
December 2009

1H, 13C, and 15N resonance assignment of the ubiquitin-like domain from Dsk2p.

Biomol NMR Assign 2008 Dec 29;2(2):147-9. Epub 2008 Aug 29.

Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, 1115 Biomolecular Sciences Building (#296), College Park, MD 20742-3360, USA.

The ubiquitin-like domain (UBL) of yeast protein Dsk2p is widely believed to recognize and bind to ubiquitin receptors on the proteasome and, as part of Dsk2p, to bridge polyubiquitinated substrates and proteasomal degradation machinery. Here we report NMR resonance assignment for (1)H, (15)N, and (13)C nuclei in the backbone and side chains of the UBL domain of Dsk2p. This assignment will aid in NMR studies focused on understanding of Dsk2's interactions with proteasomal receptors and its role as a polyubiquitin shuttle in the ubiquitin-dependent proteasomal degradation as well as other cellular pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12104-008-9107-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2892233PMC
December 2008

An NMR experiment for simultaneous TROSY-based detection of amide and methyl groups in large proteins.

J Am Chem Soc 2008 Aug 24;130(33):10872-3. Epub 2008 Jul 24.

Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA.

A sensitive 2D NMR experiment for simultaneous time-shared TROSY-type detection of amide and methyl groups in high-molecular-weight proteins is described. The pulse scheme is designed to preserve the slowly decaying components of both 1H-15N and methyl 13CH3 spin systems in the course of indirect evolution and acquisition periods. The proposed methodology is applied to the study of substrate binding to {U-[15N,2H]; Ile-[13CH3]; Leu,Val-[13CH3/12CD3]}-labeled 82-kDa enzyme Malate Synthase G and is expected to accelerate NMR-based screening of large proteins labeled with 15N and selectively labeled with 13CH3 at methyl sites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ja8036178DOI Listing
August 2008

Locating the rate-determining step(s) for three-step hydrolase-catalyzed reactions with DYNAFIT.

Biochim Biophys Acta 2008 May 10;1784(5):827-33. Epub 2008 Mar 10.

The Catholic University of America, Department of Chemistry, Washington DC 20064, USA.

Hydrolytic reactions of oligopeptide 4-nitroanilides catalyzed by human-alpha-thrombin, human activated protein C and human factor Xa were studied at pH 8.0-8.4 and 25.0+/-0.1 degrees C by the progress curve method and individual rate constants were calculated mostly within 10% internal error using DYNAFITV. A systematic strategy has been developed for fitting a three-step consecutive mechanism to eighteen hundred to six thousand time-course data points polled from two to four independent kinetic experiments. Enzyme and substrate concentrations were also calculated. Individual rate constants well reproduce published values obtained under comparable conditions and the Michaelis-Menten kinetic parameters calculated from these elementary rate constants are also within reasonable limits of published values. For comparison, the integrated Michaelis-Menten equation was also fitted to data from twelve sets. Both the k(cat) and k(cat)/K(m) values are within 15% agreement with those calculated using the elementary rate constants obtained with DYNAFITV. Rate constants for the second and third consecutive steps are within 3-4 fold indicating that both determine the overall rate. The Factor Xa-catalyzed hydrolysis of N-alpha-Z-D-Arg-Gly-Arg-pNA.2HCl at pH 8.4 in a series of buffers containing increasing fractions of deuterium at 25.0+/-0.1 degrees C shows a very strong dependence of k(3) and a moderate dependence of k(2) on D content in the buffer: the fractionation factors are: 0.49+/-0.03 for K(1,) 0.70+/-0.05 for k(2), and (0.32+/-0.03)(2) for k(3).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbapap.2008.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519142PMC
May 2008

Affinity makes the difference: nonselective interaction of the UBA domain of Ubiquilin-1 with monomeric ubiquitin and polyubiquitin chains.

J Mol Biol 2008 Mar 23;377(1):162-80. Epub 2007 Dec 23.

Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, MD 20910, USA.

Ubiquilin/PLIC proteins belong to the family of UBL-UBA proteins implicated in the regulation of the ubiquitin-dependent proteasomal degradation of cellular proteins. A human presenilin-interacting protein, ubiquilin-1, has been suggested as potential therapeutic target for treating Huntington's disease. Ubiquilin's interactions with mono- and polyubiquitins are mediated by its UBA domain, which is one of the tightest ubiquitin binders among known ubiquitin-binding domains. Here we report the three-dimensional structure of the UBA domain of ubiquilin-1 (UQ1-UBA) free in solution and in complex with ubiquitin. UQ1-UBA forms a compact three-helix bundle structurally similar to other known UBAs, and binds to the hydrophobic patch on ubiquitin with a K(d) of 20 microM. To gain structural insights into UQ1-UBA's interactions with polyubiquitin chains, we have mapped the binding interface between UQ1-UBA and Lys48- and Lys63-linked di-ubiquitins and characterized the strength of UQ1-UBA binding to these chains. Our NMR data show that UQ1-UBA interacts with the individual ubiquitin units in both chains in a mode similar to its interaction with mono-ubiquitin, although with an improved binding affinity for the chains. Our results indicate that, in contrast to UBA2 of hHR23A that has strong binding preference for Lys48-linked chains, UQ1-UBA shows little or no binding selectivity toward a particular chain linkage or between the two ubiquitin moieties in the same chain. The structural data obtained in this study provide insights into the possible structural reasons for the diversity of polyubiquitin chain recognition by UBA domains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2007.12.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2323583PMC
March 2008

Isolation and characterization of ribosome-inactivating proteins from Cucurbitaceae.

Chem Biodivers 2007 Mar;4(3):431-42

Center for Biomolecular Structure and Organization, Department of Chemistry & Biochemistry, University of Maryland, College Park, MD 57007, USA.

Due to their RNA-N-glycosidase activity, ribosome-inactivating proteins (RIPs) are attractive candidates as antitumor and antiviral agents in biomedical and agricultural research. We have isolated and characterized two such proteins, foetidissimin II and texanin, from two Cucurbitaceae species. Foetidissimin II, obtained from the roots of Cucurbita foetidissima, was identified as a type-2 RIP, with a molecular weight of 61 kDa, as estimated by gel electrophoresis. It is composed of two chains, a 29-kDa chain A, and a 32-kDa chain B. Texanin, isolated from the fruits of Cucurbita texana, is a type-I RIP, with a single chain of molecular weight 29.7 kDa, as estimated by MALDI-TOF-MS. Both proteins exhibit RNA-N-glycosidase activity, with aniline playing a critical role in rRNA cleavage. The IC50 value of foetidissimin II, determined by cell-free protein-synthesis inhibition, was 0.251 muM. In an in vitro cytotoxicity assay, foetidissimin II exhibited IC50 values of ca. 70 nM to both adenocarcinoma and erythroleukemia cells. Texanin exhibited a weaker anticancer activity against erythroleukemia cells, with an IC50 value of 95 microM, but no activity against adenocarcinoma cells. The N-terminal sequences of both proteins were compared with those of reported RIPs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbdv.200790035DOI Listing
March 2007

Deuterium solvent isotope effect and proton-inventory studies of factor Xa-catalyzed reactions.

Biochemistry 2006 Nov;45(47):14175-82

Department of Chemistry, The Catholic University of America, 620 Michigan Avenue, Washington, DC 20064, USA.

Kinetic solvent isotope effects (KSIEs) for the factor Xa (FXa)-catalyzed activation of prothrombin in the presence and absence of factor Va (FVa) and 5.0 x 10(-5) M phospholipid vesicles are slightly inverse, 0.82-0.93, when substrate concentrations are at 0.2 Km. This is consistent with the rate-determining association of the enzyme-prothrombin assembly, rather than the rate-limiting chemical transformation. FVa is known to effect a major conformational change to expose the first scissile bond in prothrombin, which is the likely event triggering a major solvent rearrangement. At prothrombin concentrations > 5 Km, the KSIE is 1.6 +/- 0.3, when FXa is in a 1:1 ratio with FVa but becomes increasingly inverse, 0.30 +/- 0.05 and 0.19 +/- 0.04, when FXa/FVa is 1:4, with an increasing FXa and substrate concentration. The rate-determining step changes with the conditions, but the chemical step is not limiting under any circumstance. This corroborates the proposed predominance of the meizothrombin pathway when FXa is well-saturated with the prothrombin complex. In contrast, the FXa-catalyzed hydrolysis of N-alpha-Z-D-Arg-Gly-Arg-pNA.2HCl (S-2765) and H-D-Ile-L-Pro-L-Arg-pNA.HCl (S-2288) is most consistent with two-proton bridges forming at the transition state between Ser195 OgammaH and His57 N(epsilon)2 and His57 Ndelta1 and Asp102 COObeta- at the active site, with transition-state fractionation factors of phi1 = phi2 = 0.57 +/- 0.07 and phiS = 0.78 +/- 0.16 for solvent rearrangement for S-2765 and phi1 = phi2 = 0.674 +/- 0.001 for S-2288 under enzyme saturation with the substrate at pH 8.40 and 25.0 +/- 0.1 degrees C. The rate-determining step(s) in these reactions is most likely the cleavage of the C-N bond and departure of the leaving group.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi061218mDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2535812PMC
November 2006

Full and partial deuterium solvent isotope effect studies of alpha-thrombin-catalyzed reactions of natural substrates.

J Am Chem Soc 2005 Mar;127(11):3760-6

Chemistry Department, The Catholic University of America, Washington, DC 20064, USA.

Proton inventory studies of the thrombin-catalyzed fibrinogen activation to fibrinopeptide A are most consistent with a two-proton bridge forming at the transition state probably between Ser195 OgammaH and His57 Nepsilon2 and His57 Ndelta1 and Asp102 COObeta- at the active site, with fractionation factors 0.66 +/- 0.03 under enzyme saturation with substrate and 0.64 +/- 0.03 at fibrinogen concentration at 0.2 Km, at pH 8.0, pD 8.6, and 25.0 +/- 0.1 degrees C. Strongly inverse solvent isotope effects (SIEs) result from inverse lag times and maximal slopes of blood clotting plots, which are also anion and cation dependent. The blood clot is much coarser in D2O, as indicated in clotting curves with 3-9 times shorter lag time and steeper slopes with respect to H2O. The finer the particles, the weaker the H-bonds interlocking the fibrin mesh and/or in water structure around fibrin. Proton inventories of inverse lag times and maximal slopes of blood clotting curves in buffers containing Na+ and Cl- ions give the best fit to an exponential dependence on deuterium content in the buffer and give fractionation factors 5.6 +/- 0.5 and 7.8 +/- 0.6 at pH 8.0 and 25.0 +/- 0.1 degrees C. The thrombin-catalyzed activation of protein C (PC) to APC is associated with inverse kinetic SIEs (KSIEs) of 0.75 +/- 0.09 and 1.02 +/- 0.06 in 0.3 M NaCl and 0.3 M choline chloride, respectively, at substrate concentrations = 0.2 Km. In comparison, thrombin-catalyzed hydrolysis of chromogenic substrates gives greater KSIEs (Enyedy, E. I.; Kovach. I. M J. Am. Chem. Soc. 2004, 126, 6017-6024) and more complex proton inventories than the ones reported here for the first time for natural substrates. The present study illuminates differences in the character of the rate-determining transition state for the initial phase of the two physiological reactions catalyzed by thrombin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ja043258oDOI Listing
March 2005
-->