Contribution of the tyrosines to the structure and function of the human U1A N‐terminal RNA binding domain

Kranz, James K.; Lu, Jirong; Hall, Kathleen B.

doi:10.1002/pro.5560050812

Cited by 34 publications

(25 citation statements)

References 52 publications

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“…Charged residue substitutions at these sites were always found to inactivate the protein. Six additional examples of effects of Asp substitutions of buried, noncharged residues were obtained from the Protherm database (http:͞͞gibk26.bse.kyutech.ac.jp͞jouhou͞ protherm͞protherm search.html): A52D and I53D in RNaseH (36,37), L133D in case of T4 lysozyme (38), S91D in hen egg white lysozyme (39), Y282D in case of Lac repressor (40), and Y78D in U1A protein (41). In all of the cases, there was a drastic decrease in protein thermodynamic stability relative to WT.…”

Section: Analysis Of Previous Mutational Studiesmentioning

confidence: 99%

Mutagenesis-based definitions and probes of residue burial in proteins

Bajaj

Chakrabarti²,

Varadarajan³

2005

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

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Every residue of the 101-aa Escherichia coli toxin CcdB was substituted with Ala, Asp, Glu, Lys, and Arg by using site-directed mutagenesis. The activity of each mutant in vivo was characterized as a function of Controller of Cell Division or Death B protein (CcdB) transcriptional level. The mutation data suggest that an accessibility value of 5% is an appropriate cutoff for definition of buried residues. At all buried positions, introduction of Asp results in an inactive phenotype at all CcdB transcriptional levels. The average amount of destabilization upon substitution at buried positions decreases in the order Asp>Glu>Lys>Arg>Ala. Asp substitutions at buried sites in two other proteins, maltose-binding protein and thioredoxin, also were shown to be severely destabilizing. Ala and Asp scanning mutagenesis, in combination with dose-dependent expression phenotypes, was shown to yield important information on protein structure and activity. These results also suggest that such scanning mutagenesis data can be used to rank order sequence alignments and their corresponding homology models, as well as to distinguish between correct and incorrect structural alignments. With continuous reductions in oligonucleotide costs and increasingly efficient site-directed mutagenesis procedures, comprehensive scanning mutagenesis experiments for small proteins͞domains are quite feasible.accessibility ͉ aspartate ͉ scanning mutagenesis ͉ phenotype

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Section: Analysis Of Previous Mutational Studiesmentioning

confidence: 99%

Mutagenesis-based definitions and probes of residue burial in proteins

Bajaj

Chakrabarti²,

Varadarajan³

2005

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

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“…Many structural, biochemical, and mechanistic studies have focused on defining the aspects of U1A and U1hpII that are important for their very high affinity interaction (Scherly et al 1989(Scherly et al , 1990LutzFreyermuth et al 1990;Hall and Stump 1992;Hall 1994;Oubridge et al 1994;Kranz et al 1996;Zeng and Hall 1997;Hall 1998, 1999;Katsamba et al 2001Katsamba et al , 2002a. In 1 vitro selection (SELEX) experiments demonstrated that U1A displays high specificity for a 7-nucleotide (nt) recognition sequence, AUUGCAC (Tsai et al 1991).…”

Section: Introductionmentioning

confidence: 99%

The role of RNA structure in the interaction of U1A protein with U1 hairpin II RNA

Law

Rice

Lin

et al. 2006

RNA

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The N-terminal RNA Recognition Motif (RRM1) of the spliceosomal protein U1A interacting with its target U1 hairpin II (U1hpII) has been used as a paradigm for RRM-containing proteins interacting with their RNA targets. U1A binds to U1hpII via direct interactions with a 7-nucleotide (nt) consensus binding sequence at the 59 end of a 10-nt loop, and via hydrogen bonds with the closing C-G base pair at the top of the RNA stem. Using surface plasmon resonance (Biacore), we have examined the role of structural features of U1hpII in binding to U1A RRM1. Mutational analysis of the closing base pair suggests it plays a minor role in binding and mainly prevents ''breathing'' of the loop. Lengthening the stem and nontarget part of the loop suggests that the increased negative charge of the RNA might slightly aid association. However, this is offset by an increase in dissociation, which may be caused by attraction of the RRM to nontarget parts of the RNA. Studies of a single stranded target and RNAs with untethered loops indicate that structure is not very relevant for association but is important for complex stability. In particular, breaking the link between the stem and the 59 side of the loop greatly increases complex dissociation, presumably by hindering simultaneous contacts between the RRM and stem and loop nucleotides. While binding of U1A to a single stranded target is much weaker than to U1hpII, it occurs with nanomolar affinity, supporting recent evidence that binding of unstructured RNA by U1A has physiological significance.

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“…The aspartic residue links the protein-RNA interface with the C-terminal domain. The C-terminal tail has been the subject of several studies, indicating its importance for RNA binding stability and specificity (12,14,18,22,(25)(26)(27). With a systematic truncation of the C-terminal tail, Hall (22) demonstrated a loss of 2 kcal͞mol of binding free energy when terminating the Cterminal at the position 95.…”

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confidence: 99%

A binding mechanism in protein–nucleotide interactions: Implication for U1A RNA binding

Guallar

Borrelli

2005

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

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We present a close electronic view of the protein-base interface for the N-terminal domain of the human protein U1A. Combining accurate mixed quantum mechanics͞molecular mechanics techniques and protein structure prediction methods, we provide a detailed electronic structure description of the protein-RNA stacking interactions. Our analysis indicates the evolution of the protein structure optimizing the interaction between Asp-92 and the RNA bases. The results show a direct coupling of the C-terminal tail and Asp-92, providing a direct rationalization of the experimentally determined role of the C-terminal domain in RNA binding. Here, we propose a mechanism where a protein side chain, with a delocalized electronic pi system, assists in the nucleotide binding. The binding mechanism involves a short-range interaction of the side chain with the nucleotide base and an electronic long-range interaction through a sandwich-stacking motif. The structural motif of the binding mechanism is observed in similar protein-RNA interactions and in various protein-ATP-binding sites.ATP binding ͉ quantum mechanics͞molecular mechanics ͉ stacking ͉ aromatic T he mechanisms of protein-nucleotide association constitute a central intermolecular interaction. Such mechanisms, for example, regulate DNA, RNA, and NTP recognition and binding. As pointed out recently by Williamson (1), there is a large complexity of induced fit processes, requiring conformational change in the proteins, in the ligands, or in both. These dynamical processes, responsible for the induced affinity and specificity, are far from being understood. A common feature in ssDNA, RNA, and NTP binding is the direct interaction of the protein with the nucleotides' bases. Recent structural analyses have underlined the main features involved in these protein-base interactions (2, 3). These studies revealed the importance of van der Waal interactions, aromatic stacking interactions in particular, in the stabilization of the protein-base interaction.The ribonucleoprotein domain, also known as the RNAbinding domain or the RNA-recognition motif, binds RNA by using extensive interactions through its characteristic fourstranded ␤-sheet structure (Fig. 1). Among the members of this motif, the N-terminal domain of the human protein U1A is the most studied (4-29). The isolated domain binds to a short RNA hairpin with high affinity and specificity. The importance of stacking interactions in RNA binding is confirmed by the aromatic nature of three of the most highly conserved residues in the RNA-recognition motif (30). Baranger and coworkers (11) recently obtained 5.5 kcal͞mol destabilization of the protein-RNA complex when mutating Phe-56 to alanine. Subsequent studies by the same group indicated that stacking (hydrophobic) interactions are able to compensate for the loss of hydrogenbonding-capable functional groups in the RNA bases (5). Recent kinetic data by Laird-Offringa and coworkers (6) supported a rapid initial association based on electrostatic interactions, followed by a subs...

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Contribution of the tyrosines to the structure and function of the human U1A N‐terminal RNA binding domain

Cited by 34 publications

References 52 publications

Mutagenesis-based definitions and probes of residue burial in proteins

Mutagenesis-based definitions and probes of residue burial in proteins

The role of RNA structure in the interaction of U1A protein with U1 hairpin II RNA

A binding mechanism in protein–nucleotide interactions: Implication for U1A RNA binding

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