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  • 1
    Language: English
    In: Science, 02 October 2012, Vol.337(8, 2012)
    Description: αβ-Tubulin is the polymerizing subunit of microtubules, which are dynamic polymers that have essential roles in cell division and intracellular organization. TOG domains are αβ-tubulin binding modules that occur in the evolutionarily conserved Stu2p/XMAP215 family of proteins and promote microtubule elongation. Ayaz et al. (p. 857) used crystallographic and biochemical experiments to reveal that the TOG1 domain interacts with guanosine triphosphate-bound αβ-tubulin in a conformation-selective manner, binding preferentially to a "curved," microtubule-incompatible conformation. The binding mode apparently excludes analogous binding of a second TOG domain to the same heterodimer and may help to ensure polarized growth of microtubules. [PUBLICATION ] Stu2p/XMAP215/Dis1 family proteins are evolutionarily conserved regulatory factors that use αβ-tubulin-interacting tumor overexpressed gene (TOG) domains to catalyze fast microtubule growth. Catalysis requires that these polymerases discriminate between unpolymerized and polymerized forms of αβ-tubulin, but the mechanism by which they do so has remained unclear. Here, we report the structure of the TOG1 domain from Stu2p bound to yeast αβ-tubulin. TOG1 binds αβ-tubulin in a way that excludes equivalent binding of a second TOG domain. Furthermore, TOG1 preferentially binds a curved conformation of αβ-tubulin that cannot be incorporated into microtubules, contacting α- and β-tubulin surfaces that do not participate in microtubule assembly. Conformation-selective interactions with αβ-tubulin explain how TOG-containing polymerases discriminate between unpolymerized and polymerized forms of αβ-tubulin and how they selectively recognize the growing end of the microtubule. [PUBLICATION ]
    Keywords: Polymerase Chain Reaction ; Cell Division ; Evolutionary Biology;
    ISSN: 00368075
    E-ISSN: 10959203
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  • 2
    Language: English
    In: Science, August 17, 2012, Vol.337(6096), p.857(4)
    Description: Stu2p/XMAP215/Dis1 family proteins are evolutionarily conserved regulatory factors that use [alpha][beta]-tubulin-interacting tumor overexpressed gene (TOG) domains to catalyze fast microtubule growth. Catalysis requires that these polymerases discriminate between unpolymerized and polymerized forms of [alpha][beta]-tubulin, but the mechanism by which they do so has remained unclear. Here, we report the structure of the TOG1 domain from Stu2p bound to yeast [alpha][beta]-tubulin. TOG1 binds [alpha][beta]-tubulin in a way that excludes equivalent binding of a second TOG domain. Furthermore, TOG1 preferentially binds a curved conformation of [alpha][beta]-tubulin that cannot be incorporated into microtubules, contacting [alpha]- and [beta]-tubulin surfaces that do not participate in microtubule assembly. Conformation-selective interactions with [alpha][beta]-tubulin explain how TOG-containing polymerases discriminate between unpolymerized and polymerized forms of [alpha][beta]-tubulin and how they selectively recognize the growing end of the microtubule. 10.1126/science.1221698
    Keywords: Tubulin -- Structure ; Protein Structure -- Analysis
    ISSN: 0036-8075
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  • 3
    Language: English
    In: Proc. Natl. Acad. Sci. USA, 04 February 2014, Vol.110((28) ; 07, 2013)
    Description: Cohesin, along with positive regulators, establishes sister-chromatid cohesion by forming a ring to circle chromatin. The wings apart-like protein (Wapl) is a key negative regulator of cohesin and forms a complex with precocious dissociation of sisters protein 5 (Pds5) to promote cohesin release from chromatin. Here we report the crystal structure and functional characterization of human Wapl. Wapl contains a flexible, variable N-terminal region (Wapl-N) and a conserved C-terminal domain (Wapl-C) consisting of eight HEAT (Huntingtin, Elongation factor 3, A subunit, and target of rapamycin) repeats. Wapl-C folds into an elongated structure with two lobes. Structure-based mutagenesis maps the functional surface of Wapl-C to two distinct patches (I and II) on the N lobe and a localized patch (111) on the C lobe. Mutating critical patch I residues weaken Wapl binding to cohesin and diminish sister-chromatid resolution and cohesin release from mitotic chromosomes in human cells and Xenopus egg extracts. Surprisingly, patch III on the C lobe does not contribute to Wapl binding to cohesin or its known regulators. Although patch I mutations reduce Wapl binding to intact cohesin, they do not affect Wapl-Pds5 binding to the cohesin sub-complex of sister chromatid cohesion protein 1 (Scc1) and stromal antigen 2 (SA2) in vitro, which is instead mediated by Wapl-N. Thus, Wapl-N forms extensive interactions with Pds5 and Scc1-SA2. Wapl-C interacts with other cohesin subunits and possibly unknown effectors to trigger cohesin release from chromatin. chromosome segregation | crystallography | genomic stability | mitosis | protein-protein interaction www.pnas.org/cgi/doi/ 10.1073/pnas.1304594110
    Keywords: Cells (Biology) -- Chemical Properties ; Cells (Biology) -- Research;
    ISSN: 00278424
    E-ISSN: 10916490
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  • 4
    Language: English
    In: Analytical chemistry, 05 June 2012, Vol.84(11), pp.5066-73
    Description: Isothermal titration calorimetry (ITC) is a powerful classical method that enables researchers in many fields to study the thermodynamics of molecular interactions. Primary ITC data comprise the temporal evolution of differential power reporting the heat of reaction during a series of injections of aliquots of a reactant into a sample cell. By integration of each injection peak, an isotherm can be constructed of total changes in enthalpy as a function of changes in solution composition, which is rich in thermodynamic information on the reaction. However, the signals from the injection peaks are superimposed by the stochastically varying time-course of the instrumental baseline power, limiting the precision of ITC isotherms. Here, we describe a method for automated peak assignment based on peak-shape analysis via singular value decomposition in combination with detailed least-squares modeling of local pre- and postinjection baselines. This approach can effectively filter out contributions of short-term noise and adventitious events in the power trace. This method also provides, for the first time, statistical error estimates for the individual isotherm data points. In turn, this results in improved detection limits for high-affinity or low-enthalpy binding reactions and significantly higher precision of the derived thermodynamic parameters.
    Keywords: Calorimetry -- Methods ; Flow Injection Analysis -- Statistics & Numerical Data
    ISSN: 00032700
    E-ISSN: 1520-6882
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  • 5
    Language: English
    In: J. Mol. Biol, 30 July 2013, Vol.420((1-2) ; 06, 2012)
    Description: To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.jmb.2012.04.001 Byline: Chad A. Brautigam, Ranjit K. Deka, Peter Schuck, Diana R. Tomchick, Michael V. Norgard Keywords: TRAP transporter; syphilis; Treponema pallidum; TPR motif; protein interactions Abbreviations: TPAT, tetratricopeptide repeat-protein associated TRAP transporter; TRAP-T, tripartite ATP-independent periplasmic transporter; OM, outer membrane; IM, inner membrane; SBP, substrate-binding protein; cTPR, cryptic tetratricopeptide repeat; CF, cleft finger; SV, sedimentation velocity; HA, hydrophobic antechamber; SEC, size-exclusion chromatography; [beta]-OG, n-octyl [beta]-d-glucopyranoside; PEG, polyethylene glycol Abstract: Tripartite ATP-independent periplasmic transporters (TRAP-Ts) are bacterial transport systems that have been implicated in the import of small molecules into the cytoplasm. A newly discovered subfamily of TRAP-Ts [tetratricopeptide repeat-protein associated TRAP transporters (TPATs)] has four components. Three are common to both TRAP-Ts and TPATs: the P component, a ligand-binding protein, and a transmembrane symporter apparatus comprising the M and Q components (M and Q are sometimes fused to form a single polypeptide). TPATs are distinguished from TRAP-Ts by the presence of a unique protein called the "T component". In Treponema pallidum, this protein (TatT) is a water-soluble trimer whose protomers are each perforated by a pore. Its respective P component (TatP.sub.T) interacts with the TatT in vitro and in vivo. In this work, we further characterized this interaction. Co-crystal structures of two complexes between the two proteins confirm that up to three monomers of TatP.sub.T can bind to the TatT trimer. A putative ligand-binding cleft of TatP.sub.T aligns with the pore of TatT, strongly suggesting ligand transfer between T and P.sub.T. We used a combination of site-directed mutagenesis and analytical ultracentrifugation to derive thermodynamic parameters for the interactions. These observations confirm that the observed crystallographic interface is recapitulated in solution. These results prompt a hypothesis of the molecular mechanism(s) of hydrophobic ligand transport by the TPATs. Article History: Received 10 February 2012; Revised 30 March 2012; Accepted 2 April 2012 Article Note: (miscellaneous) Edited by R. Huber
    Keywords: Proteins -- Chemical Properties ; Syphilis -- Chemical Properties ; Blood Lipids -- Chemical Properties ; Polyols -- Chemical Properties ; Thermodynamics -- Chemical Properties;
    ISSN: 00222836
    E-ISSN: 10898638
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  • 6
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 12 January 2016, Vol.112((48) ; 12, 2015)
    Description: PDZ domains are abundant protein interaction modules and typically recognize a short motif at the C terminus of their ligands, with a few residues in the motif endowing the binding specificity. The sequence-based rules, however, cannot fully account for the specificity between the vast number of PDZ domains and ligands in the cell. Plexins are transmembrane receptors that regulate processes such as axon guidance and angiogenesis. Two related guanine nucleotide exchange factors (GEFs), PDZ-RhoGEF and leukemia-associated RhoGEF (LARG), use their PDZ domains to bind class B plexins and play critical roles in signaling. Here, we present the crystal structure of the full-length cytoplasmic region of PlexinB2 in complex with the PDZ domain of PDZ-RhoGEF. The structure reveals that, in addition to the canonical C-terminal motif/PDZ interaction, the 3D domain of PlexinB2 forms a secondary interface with the PDZ domain. Our biophysical and cell-based assays show that the secondary interface contributes to the specific interaction between plexin and PDZ-RhoGEF and to signaling by plexin in the cell. Formation of secondary interfaces may be a general mechanism for increasing affinity and specificity of modular domain-mediated interactions. PDZ | plexin | signaling | protein interaction module | specificity
    Keywords: Sciences (General)
    ISSN: 0027-8424
    E-ISSN: 1091-6490
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  • 7
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 16 August 2011, Vol.108(33), pp.E472-9
    Description: Actin related protein 2/actin related protein 3 (Arp2/3) complex nucleates new actin filaments in eukaryotic cells in response to signals from proteins in the Wiskott-Aldrich syndrome protein (WASP) family. The conserved VCA domain of WASP proteins activates Arp2/3 complex by inducing conformational changes and delivering the first actin monomer of the daughter filament. Previous models of activation have invoked a single VCA acting at a single site on Arp2/3 complex. Here we show that activation most likely involves engagement of two distinct sites on Arp2/3 complex by two VCA molecules, each delivering an actin monomer. One site is on Arp3 and the second is on ARPC1 and Arp2. The VCAs at these sites have distinct roles in activation. Our findings reconcile apparently conflicting literature on VCA activation of Arp2/3 complex and lead to a new model for this process.
    Keywords: Polymerization ; Actin-Related Protein 2-3 Complex -- Metabolism ; Wiskott-Aldrich Syndrome Protein -- Metabolism
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 8
    Language: English
    In: Journal of Bacteriology, Dec, 2012, Vol.194(23-24), p.6771(11)
    Description: Metal ion homeostasis is a critical function of many integral and peripheral membrane proteins. The genome of the etiologic agent of syphilis, Treponema pallidum, is compact and devoid of many metabolic enzyme genes. Nevertheless, it harbors genes coding for homologs of several enzymes that typically require either iron or zinc. The product of the tp0971 gene of T. pallidum, designated Tp34, is a periplasmic lipoprotein that is thought to be tethered to the inner membrane of this organism. Previous work on a water-soluble (nonacylated) recombinant version of Tp34 established that this protein binds to [Zn.sup.2+], which, like other transition metal ions, stabilizes the dimeric form of the protein. In this study, we employed analytical ultracentrifugation to establish that four transition metal ions ([Ni.sup.2+], [Co.sup.2+], [Cu.sup.2+], and [Zn.sup.2+]) readily induce the dimerization of Tp34; [Cu.sup.2+] (50% effective concentration [EC50] = 1.7 ?M) and Zn2+ (EC50 = 6.2 ?M) were the most efficacious of these ions. Mutations of the crystallographically identified metal-binding residues hindered the ability of Tp34 to dimerize. X-ray crystallography performed on crystals of Tp34 that had been incubated with metal ions indicated that the binding site could accommodate the metals examined. The findings presented herein, coupled with bioinformatic analyses of related proteins, point to Tp34's likely role in metal ion homeostasis in T. pallidum.
    Keywords: Binding Sites (Biochemistry) -- Research ; Homeostasis -- Research ; Membrane Proteins -- Chemical Properties ; Transition Metal Compounds -- Chemical Properties ; Treponema Pallidum -- Physiological Aspects
    ISSN: 0021-9193
    Source: Cengage Learning, Inc.
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  • 9
    Language: English
    In: Journal of molecular biology, 09 March 2012, Vol.416(5), pp.678-96
    Description: Treponema pallidum, the bacterial agent of syphilis, is predicted to encode one tripartite ATP-independent periplasmic transporter (TRAP-T). TRAP-Ts typically employ a periplasmic substrate-binding protein (SBP) to deliver the cognate ligand to the transmembrane symporter. Herein, we demonstrate that the genes encoding the putative TRAP-T components from T. pallidum, tp0957 (the SBP), and tp0958 (the symporter), are in an operon with an uncharacterized third gene, tp0956. We determined the crystal structure of recombinant Tp0956; the protein is trimeric and perforated by a pore. Part of Tp0956 forms an assembly similar to those of "tetratricopeptide repeat" (TPR) motifs. The crystal structure of recombinant Tp0957 was also determined; like the SBPs of other TRAP-Ts, there are two lobes separated by a cleft. In these other SBPs, the cleft binds a negatively charged ligand. However, the cleft of Tp0957 has a strikingly hydrophobic chemical composition, indicating that its ligand may be substantially different and likely hydrophobic. Analytical ultracentrifugation of the recombinant versions of Tp0956 and Tp0957 established that these proteins associate avidly. This unprecedented interaction was confirmed for the native molecules using in vivo cross-linking experiments. Finally, bioinformatic analyses suggested that this transporter exemplifies a new subfamily of TPATs (TPR-protein-associated TRAP-Ts) that require the action of a TPR-containing accessory protein for the periplasmic transport of a potentially hydrophobic ligand(s).
    Keywords: Lipoproteins -- Genetics ; Periplasmic Binding Proteins -- Genetics ; Treponema Pallidum -- Genetics
    ISSN: 00222836
    E-ISSN: 1089-8638
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  • 10
    Language: English
    In: Analytical Biochemistry, 01 June 2013, Vol.437(1), pp.104-108
    Description: We report systematic and large inaccuracies in the recorded elapsed time in data files from the analytical ultracentrifuge, leading to overestimates of the sedimentation coefficients of up to 10%. This far exceeds previously considered factors contributing to the uncertainty in this parameter and has significant ramifications for derived parameters such as hydrodynamic shape and molar mass estimates. The source of this error is currently unknown, but we found it to be quantitatively consistent across different instruments, increasing with rotor speed. Furthermore, its occurrence appears to correlate with the use of the latest data acquisition software from the manufacturer, in use in some of our laboratories for nearly 2 years. Many of the recently published sedimentation coefficients may need to be reexamined. The problem can be easily recognized by comparing the file timestamps provided by the operating system with the elapsed scan times recorded within the data files. Therefore, we implemented a routine in SEDFIT that can automatically examine the data files, alert the user to significant discrepancies, and correct the scan times accordingly. This eliminates errors in the recorded scan times.
    Keywords: Sedimentation Velocity ; Hydrodynamic Modeling ; Chemistry ; Anatomy & Physiology
    ISSN: 0003-2697
    E-ISSN: 1096-0309
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