Mars – robust automatic backbone assignment of proteins

Jung, Young-Sang; Zweckstetter, Markus

doi:10.1023/b:jnmr.0000042954.99056.ad

Cited by 290 publications

(274 citation statements)

References 42 publications

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“…Spectra were processed with NMRPipe (28) and analyzed with Sparky (29). Backbone assignment was done semiautomatically using MARS (30). For backbone and side chain assignment CBCA(CO)NH, CBCANH and (H)CCH-TOCSY spectra were recorded (31).…”

Section: Methodsmentioning

confidence: 99%

Structural Basis for Homodimerization of the Src-associated during Mitosis, 68-kDa Protein (Sam68) Qua1 Domain

Meyer

Tripsianes

Vincendeau

et al. 2010

Journal of Biological Chemistry

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Sam68 (Src-associated during mitosis, 68 kDa) is a prototypical member of the STAR (signal transducer and activator of RNA) family of RNA-binding proteins. STAR proteins bind mRNA targets and modulate cellular processes such as cell cycle regulation and tissue development in response to extracellular signals. Sam68 has been shown to modulate alternative splicing of the pre-mRNAs of CD44 and Bcl-xL, which are linked to tumor progression and apoptosis. Sam68 and other STAR proteins recognize bipartite RNA sequences and are thought to function as homodimers. However, the structural and functional roles of the self-association are not known. Here, we present the solution structure of the Sam68 Qua1 homodimerization domain. Each monomer consists of two antiparallel ␣-helices connected by a short loop. The two subunits are arranged perpendicular to each other in an unusual four-helix topology. Mutational analysis of Sam68 in vitro and in a cell-based assay revealed that the Qua1 domain and residues within the dimerization interface are essential for alternative splicing of a CD44 minigene. Together, our results indicate that the Qua1 homodimerization domain is required for regulation of alternative splicing by Sam68. Sam682 (Src-associated during mitosis, 68 kDa) (1) belongs to the STAR (signal transducer and activator of RNA) family of RNAbinding proteins, which also includes Qk1 (quaking 1), SF1 (splicing factor 1), and Gld-1 (germline development defective-1) (2). STAR family proteins link signaling pathways and many aspects of RNA metabolism (splicing, localization, and translation). They are regulated by post-translational modifications such as phosphorylation, acetylation, and arginine methylation (2).Sam68 acts in post-transcriptional regulation of pre-mRNA splicing in response to extracellular signals (3). It is involved in a variety of pathways, including insulin and T-cell receptor signaling (4), and plays a key role in cell cycle regulation (5). Sam68 exhibits binding specificity for homopolymeric poly(U) RNA and has been shown to recognize UAAA or UUUA sequences with high affinity as determined by Systematic Evolution of Ligands by EXponential Enrichment (SELEX) and in vivo crosslinking (6, 7). Post-translational modifications can regulate Sam68 function by critically affecting the accessibility to RNA (8, 9). Tyrosine phosphorylation by Src kinase during mitosis enhances the interaction of Sam68 with Ras-GAP (10) but prevents its association with RNA. On the other hand, acetylation of lysine residues by histone acetyltransferases enhances RNA binding (11). Finally, overexpression of Sam68 has been linked to prostate cancer, cell proliferation, and survival (12).Sam68 has been identified as a key determinant in the alternative splicing of various important RNA targets, like CD44 (13) and Bcl-x L (14), which are linked to apoptosis and cancer. In particular, alternative splicing of CD44 impacts embryonic development and immune response (15-18). Up to 10 variant exon sequences can be included in the mature C...

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Section: Methodsmentioning

confidence: 99%

Structural Basis for Homodimerization of the Src-associated during Mitosis, 68-kDa Protein (Sam68) Qua1 Domain

Meyer

Tripsianes

Vincendeau

et al. 2010

Journal of Biological Chemistry

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“…The spectra were processed using the software package NMRPipe (22) and analyzed using NMRView (23). A semiautomatic strategy was followed to assign the backbone chemical shifts using the software MARS (24). All backbone amide chemical shifts were obtained except Ile 13 that was invisible in the spectra.…”

Section: Methodsmentioning

confidence: 99%

The Solution Structure of Escherichia coli Wzb Reveals a Novel Substrate Recognition Mechanism of Prokaryotic Low Molecular Weight Protein-tyrosine Phosphatases

Lescop

Hu²,

Xu³

et al. 2006

Journal of Biological Chemistry

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Low molecular weight protein-tyrosine phosphatases (LMWPTPs) are small enzymes that ubiquitously exist in various organisms and play important roles in many biological processes. In Escherichia coli, the LMW-PTP Wzb dephosphorylates the autokinase Wzc, and the Wzc/Wzb pair regulates colanic acid production. However, the substrate recognition mechanism of Wzb is still poorly understood thus far. To elucidate the molecular basis of the catalytic mechanism, we have determined the solution structure of Wzb at high resolution by NMR spectroscopy. The Wzb structure highly resembles that of the typical LMW-PTP fold, suggesting that Wzb may adopt a similar catalytic mechanism with other LMW-PTPs. Nevertheless, in comparison with eukaryotic LMW-PTPs, the absence of an aromatic amino acid at the bottom of the active site significantly alters the molecular surface and implicates Wzb may adopt a novel substrate recognition mechanism. Furthermore, a structure-based multiple sequence alignment suggests that a class of the prokaryotic LMW-PTPs may share a similar substrate recognition mechanism with Wzb. The current studies provide the structural basis for rational drug design against the pathogenic bacteria.Low molecular weight protein-tyrosine phosphatases (LMW-PTPs) 3 are small cytoplasmic enzymes (ϳ18 kDa) that are widely distributed in prokaryotes and eukaryotes (1, 2). In eukaryotes, LMW-PTPs specifically dephosphorylate and down-regulate many tyrosine kinase receptors, such as platelet-derived growth factor receptor (3, 4), insulin receptor (5), or ephrin receptor (6). The reaction mechanism of eukaryotic LMW-PTPs has been structurally, thermodynamically, and kinetically characterized (7). In contrast, very limited knowledge is available for prokaryotic LMW-PTPs thus far. All LMW-PTPs share a highly conserved C(X) 5 RS motif, where X can be any amino acid. This motif adopts a loop structure, where the phosphate group of the substrate binds to, and is known as the P-loop (1). In addition, some amino acids required for the catalysis are also highly conserved, such as an aspartate residue that acts as a general acid (8). Based on the structures of mammalian LMW-PTPs in complex with exogenous substrates or serendipitous ligands, determinants for substrate specificity were proposed, including the ring stacking around the targeted phosphotyrosine provided by two aromatic side chains of the LMW-PTPs (9, 10).Many bacterial species harbor a layer of capsular polysaccharides and surface-associated exopolysaccharides (EPS). In Escherichia coli, the group 1 capsular polysaccharides and the colanic acid EPS are assembled in a Wzy-dependent polymerization system (11). The ca gene cluster contains one tyrosine autokinase (Wzc) and one LMW-PTP (Wzb) that function as a pair of kinase/phosphatase in the regulation of colanic acid production (12, 13). As a consequence, colanic acid is only produced after the dephosphorylation of the phosphorylated Wzc by Wzb (14). Wzc was identified to regulate the activity of UDPglucose dehydrogenase b...

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“…Kneller, University of California, San Francisco). H15 and H16 backbone assignment (SI Materials and Methods) was facilitated by the use of the program MARS (32). The program TALOS (33) allowed determination of the , angles on the basis of the analysis of the 13 C␣, 13 C␤, 13 CO, H␣, and 15 N chemical shifts.…”

Section: Nmr Spectroscopy and Structure Calculationmentioning

confidence: 99%

Structure of bacteriophage SPP1 head-to-tail connection reveals mechanism for viral DNA gating

Lhuillier

Gallopin

Gilquin

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

113

138

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In many bacterial viruses and in certain animal viruses, the doublestranded DNA genome enters and exits the capsid through a portal gatekeeper. We report a pseudoatomic structure of a complete portal system. The bacteriophage SPP1 gatekeeper is composed of dodecamers of the portal protein gp6, the adaptor gp15, and the stopper gp16. The solution structures of gp15 and gp16 were determined by NMR. They were then docked together with the X-ray structure of gp6 into the electron density of the Ϸ1-MDa SPP1 portal complex purified from DNA-filled capsids. The resulting structure reveals that gatekeeper assembly is accompanied by a large rearrangement of the gp15 structure and by folding of a flexible loop of gp16 to form an intersubunit parallel ␤-sheet that closes the portal channel. This stopper system prevents release of packaged DNA. Disulfide cross-linking between ␤-strands of the stopper blocks the key conformational changes that control genome ejection from the virus at the beginning of host infection.NMR ͉ structure docking ͉ unstructured proteins ͉ virus assembly ͉ virus infection

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Mars – robust automatic backbone assignment of proteins

Cited by 290 publications

References 42 publications

Structural Basis for Homodimerization of the Src-associated during Mitosis, 68-kDa Protein (Sam68) Qua1 Domain

Structural Basis for Homodimerization of the Src-associated during Mitosis, 68-kDa Protein (Sam68) Qua1 Domain

The Solution Structure of Escherichia coli Wzb Reveals a Novel Substrate Recognition Mechanism of Prokaryotic Low Molecular Weight Protein-tyrosine Phosphatases

Structure of bacteriophage SPP1 head-to-tail connection reveals mechanism for viral DNA gating

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