A Novel Reduced-Dimensionality Triple-Resonance Experiment for Efficient Polypeptide Backbone Assignment, 3D HN N

Szyperski, Thomas; Braun, Doreen; Fernández, César Fernández; Bartels, Christian; Wüthrich, Kurt

doi:10.1006/jmrb.1995.1124

Cited by 39 publications

(80 citation statements)

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“…Interestingly, the phosphorylation of SIMs may be cross-regulated through acetylation of Lys 33 in SUMO-2 and Lys 37 in SUMO-1, although additional lysines near the SIM binding cleft may be involved (65)(66)(67). From a structural perspective, Lys 33 and Lys 35 are critical for electrostatic interactions with SIM or phosphorylated SIM; their acetylation abolishes SUMO-SIM binding, although not in all cases. In the case of RAP80, it will be of interest to determine whether the interaction with SUMO-2 is regulated by acetylation of Lys 33 and Lys 35 , linking regulation of SUMO acetylation with DNA repair by homologous recombination.…”

Section: Discussionmentioning

confidence: 99%

“…All samples were prepared in SHIGEMI microcell NMR tubes. Main chain resonance assignments for RAP80-(33-131) were accomplished at 25°C using a [U-13 C, 15 N] NMR sample in 50 mM Tris, 150 mM NaCl, 45 mM Tris(2-carboxyethyl)phosphine, pH 6.1, with a protein concentration of 0.3 mM, 0.1 mM DSS as an internal reference, and 10% D 2 O, and the three-dimensional HNCACB (30,31), CBCA-(CO)NNH (31,32), HNN(CA,CO) (33,34), HNCO (35), and HN(CA)CO (36) C ␣ , 13 CO, and 13 C ␤ chemical shifts were assigned manually using the SPARKY program (37) and verified using the automatic assignment feature of CARA (38). Main chain resonance assignments for SUMO-2 were obtained in a similar fashion using a [U-13 C/ 15 N] NMR sample containing 0.4 mM SUMO-2 in 50 mM Tris, 150 mM NaCl, 2 mM DTT, 10% D 2 O, and 0.1 mM DSS as an internal standard at pH 7.3.…”

Section: Methodsmentioning

confidence: 99%

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Molecular Basis for Phosphorylation-dependent SUMO Recognition by the DNA Repair Protein RAP80

Anamika

Spyracopoulos

2016

Journal of Biological Chemistry

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Recognition and repair of double-stranded DNA breaks (DSB) involves the targeted recruitment of BRCA tumor suppressors to damage foci through binding of both ubiquitin (Ub) and the Ub-like modifier SUMO. RAP80 is a component of the BRCA1 A complex, and plays a key role in the recruitment process through the binding of Lys 63 -linked poly-Ub chains by tandem Ub interacting motifs (UIM). RAP80 also contains a SUMO interacting motif (SIM) just upstream of the tandem UIMs that has been shown to specifically bind the SUMO-2 isoform. The RAP80 tandem UIMs and SIM function collectively for optimal recruitment of BRCA1 to DSBs, although the molecular basis of this process is not well understood. Using NMR spectroscopy, we demonstrate that the RAP80 SIM binds SUMO-2, and that both specificity and affinity are enhanced through phosphorylation of the canonical CK2 site within the SIM. The affinity increase results from an enhancement of electrostatic interactions between the phosphoserines of RAP80 and the SIM recognition module within SUMO-2. The NMR structure of the SUMO-2⅐phospho-RAP80 complex reveals that the molecular basis for SUMO-2 specificity is due to isoform-specific sequence differences in electrostatic SIM recognition modules.The DNA repair process in eukaryotic cells is an indispensible life process responsible for maintaining the fidelity of the genome (1, 2). The genomic information encoded within the molecular structure of DNA is relentlessly compromised as a result of factors that include free radicals arising from metabolic processes, radiation, and replication errors (1). By virtue of highly regulated and efficient DNA repair mechanisms, most DNA damage does not progress to viable malignant tumors (1). Among the different kinds of damage that alter DNA structure, double strand breaks are the most deleterious (1). Depending on the nature of DNA damage, checkpoint activation and cell cycle arrest accompany a number of repair pathways, including homologous recombination, non-homologous end joining, or alternative non-homologous end joining repair, which function to combat the damage (3). Similar to many life processes, homologous recombination is governed by the hierarchical and synergistic action of various post-translational modifications, such as phosphorylation, ubiquitination, and SUMOylation (4, 5). The severed ends of damaged DNA are sensed by the MRE11/Rad50/NBS1 (MRN) protein complex, followed by recruitment of ATM kinase and its concomitant activation (3,5,6). This results in the phosphorylation of nearby histones, which serves as a marker for initiation of repair (3, 5). Phosphorylated histones comprise the binding site for MDC1, which is also phosphorylated by ATM kinase; subsequently, phosphorylated MDC1 recruits the ubiquitination enzyme RNF8, which acts with the Ubc13/Mms2 heterodimer to attach Lys 63 -linked Ub chains at damaged sites, in combination with the Ub 2 ligase RNF168 (3,7,8). One of the biological functions for Lys 63 -linked poly-Ub chains is to serve as a signal for BRCA1 recru...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Molecular Basis for Phosphorylation-dependent SUMO Recognition by the DNA Repair Protein RAP80

Anamika

Spyracopoulos

2016

Journal of Biological Chemistry

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“…15 N-HSQC (Zhang et al 1994), 13 C-HSQC, CBCA(CO)NH , HNCACB , HACA(CO)CANH (Löhr and Rüterjans 1995), HNCO (Grzesiek and Bax 1992b;Muhandiram and Kay 1994), HN(CO)HB (Grzesiek et al 1992), and HNN(CO,CA) (Szyperski et al 1995;Panchal et al 2001). The main-chain 15 N and 1 H N atoms for His42 were not observed in 1 H N -detected spectra, presumably because of line broadening, and were unassigned.…”

Section: Chemical Shift Assignment For Traf6-rdmentioning

confidence: 99%

Structure, interactions, and dynamics of the RING domain from human TRAF6

et al. 2007

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A key step in the signaling cascade responsible for activation of the transcription factor NF-kB involves Lys63-linked polyubiquitination of TRAF6. Covalent attachment of ubiquitin (Ub) to TRAF6, and subsequent poly(Ub) chain synthesis, is catalyzed by the hUev1a-hUbc13 heterodimer. hUbc13 is a catalytically competent E2 enzyme, and hUev1a is an E2-like protein that binds substrate Ub. The hUev1a-hUbc13 heterodimer is targeted to TRAF6 through interactions between hUbc13 and the N-terminal RING domain from TRAF6. Nuclear magnetic resonance (NMR) spectroscopy was used to determine the solution state structure of the RING domain from human TRAF6, and the interaction between hUbc13 and TRAF6 was characterized using NMR chemical shift mapping. The main-chain dynamics of the RING domain from TRAF6 were studied using 15 N NMR relaxation. Analysis of the main-chain dynamics data indicates that residues within the a-helix and b-sheet of the RING domain are as rigid as regions of canonical secondary structure in larger proteins, consistent with the biological role of RING-domain E3 proteins, which requires that the E3 contain a recognition site for recruitment of E2 ubiquitin conjugation enzymes. The C-terminal MATH domain from TRAF2 and TRAF6 is involved in interactions with the cytoplasmic domains of TNF receptors (Wu and Arron 2003). The structure of the MATH or TRAF domain from TRAF6 has been determined using X-ray crystallography and forms an eight-stranded antiparallel b-sandwich (PDB: 1LB6) . The structure of the C-terminal region of TRAF2 is a trimer with a ''mushroom'' shape, whose self-association is due in part to the coiled-coil region, which forms the stalk of the mushroom (PDB: 1CA4) Reprint requests to: Leo Spyracopoulos, Department of Biochemistry, Room 416, Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada; e-mail: leo.spyracopoulos@ ualberta.ca; fax: (780) 492-0886.Abbreviations: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin protein ligase; HSQC, heteronuclear single quantum coherence; MATH domain, merpin and TRAF homology domain; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; RING, really interesting new gene; RMSD, root-mean-square deviation; R 1 , longitudinal relaxation rate; R 2 , transverse relaxation rate; TNF, tumor necrosis factor; TRAF, tumor necrosis receptor associated factor; TRAF6-RD, TRAF6-RING domain; Ub, ubiquitin.Article published online ahead of print. Article and publication date are at

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“…This is because each additional frequency labeling results in a twofold decrease in sensitivity, while a factor of p 2 is gained owing to the fact that phasesensitive detection for one shift is omitted. This loss in intrinsic sensitivity can be at least partially recovered by symmetrization 11,17,18,26 of subspectra about the position of (2) Scaling factors same as in (2) 4. (4,2)D PR-Intra-HNCACB 34 4.…”

Section: Sensitivity Considerationsmentioning

confidence: 99%

“…11,17 Other innovations that have been introduced until 1998 (see Ref. 1 for a recent review and additional references therein) include (i) the symmetrization of RD NMR spectra to increase signal-to-noise ratios, 11,17,18 (ii) the detection of 'central peaks' located at the center of the two components of the chemical shift doublets in order to restore the full potential of the parent ND NMR experiment (corresponding to projection angles of 0°and 90°), and (iii) the twofold application of the RD NMR approach yielding a total of four components resulting from a projection of an ND spectrum into an (N 2)D space. 19,20 Figure 1.…”

Section: Introductionmentioning

confidence: 99%

Principles and applications of GFT projection NMR spectroscopy

Szyperski

Atreya

2006

Magn. Reson. Chem.

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The two defining features of G-matrix Fourier transform (GFT) projection NMR spectroscopy are (i) repeated joint sampling of several indirect chemical shift evolution periods of a multidimensional NMR experiment so that transfer amplitudes are generated which are proportional to all possible permutations of cosine and sine modulations of the individual shifts, and (ii) linear combination of the subspectra resulting from such repeated joint sampling in the time or frequency domain which yields edited subspectra containing signals encoding phase-sensitively detected linear combinations of the jointly sampled shifts. This review sketches the underlying principles of GFT NMR and outlines its relation to further developments such as the reconstruction of multidimensional NMR spectra.

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A Novel Reduced-Dimensionality Triple-Resonance Experiment for Efficient Polypeptide Backbone Assignment, 3D HN N

Cited by 39 publications

References 0 publications

Molecular Basis for Phosphorylation-dependent SUMO Recognition by the DNA Repair Protein RAP80

Molecular Basis for Phosphorylation-dependent SUMO Recognition by the DNA Repair Protein RAP80

Structure, interactions, and dynamics of the RING domain from human TRAF6

Principles and applications of GFT projection NMR spectroscopy

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