Transient Electrostatic Interactions Dominate the Conformational Equilibrium Sampled by Multidomain Splicing Factor U2AF65: A Combined NMR and SAXS Study

Huang, Jie Rong; Warner, Lisa; Sanchez, C.; Gabel, Frank; Madl, Tobias; Mackereth, Cameron D.; Sattler, Michael; Blackledge, Martin

doi:10.1021/ja502030n

Cited by 78 publications

(95 citation statements)

References 75 publications

(130 reference statements)

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“…In particular, the conformations of the C-termini of GFP(À6) are the most distal ones with respect to the protein center and explain its slightly larger radius of gyration with respect to the two other constructs (Table 1). In conclusion, our results suggest that the conformational space of flexible parts is influenced by surface-charge distributions, a mechanism that has also been recently observed and quantified in flexible multidomain proteins and related to functionally important conformations (64).…”

Section: Discussionsupporting

confidence: 80%

SAXS/SANS on Supercharged Proteins Reveals Residue-Specific Modifications of the Hydration Shell

Kim

Martel

Girard

et al. 2016

Biophysical Journal

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Water molecules in the immediate vicinity of biomacromolecules, including proteins, constitute a hydration layer characterized by physicochemical properties different from those of bulk water and play a vital role in the activity and stability of these structures, as well as in intermolecular interactions. Previous studies using solution scattering, crystallography, and molecular dynamics simulations have provided valuable information about the properties of these hydration shells, including modifications in density and ionic concentration. Small-angle scattering of x-rays (SAXS) and neutrons (SANS) are particularly useful and complementary techniques to study biomacromolecular hydration shells due to their sensitivity to electronic and nuclear scattering-length density fluctuations, respectively. Although several sophisticated SAXS/SANS programs have been developed recently, the impact of physicochemical surface properties on the hydration layer remains controversial, and systematic experimental data from individual biomacromolecular systems are scarce. Here, we address the impact of physicochemical surface properties on the hydration shell by a systematic SAXS/SANS study using three mutants of a single protein, green fluorescent protein (GFP), with highly variable net charge (þ36, À6, and À29). The combined analysis of our data shows that the hydration shell is locally denser in the vicinity of acidic surface residues, whereas basic and hydrophilic/hydrophobic residues only mildly modify its density. Moreover, the data demonstrate that the density modifications result from the combined effect of residue-specific recruitment of ions from the bulk in combination with water structural rearrangements in their vicinity. Finally, we find that the specific surface-charge distributions of the different GFP mutants modulate the conformational space of flexible parts of the protein.

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Section: Discussionsupporting

confidence: 80%

SAXS/SANS on Supercharged Proteins Reveals Residue-Specific Modifications of the Hydration Shell

Kim

Martel

Girard

et al. 2016

Biophysical Journal

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“…Consequently, no single preferred orientation of the two domains with respect to each other could be defined by the NMR and SAXS data. This is often observed for dynamic multidomain proteins, which sample a diverse continuum of conformations in solution involving encounter-like interdomain contacts (57)(58)(59). Unexpectedly, the amino acid residues in the C-terminal domain involved in interactions with the N-terminal domain are similar to those amino acids involved in nisin binding.…”

Section: Discussionmentioning

confidence: 91%

The Solution Structure of the Lantibiotic Immunity Protein NisI and Its Interactions with Nisin

Hacker

Christ

Duchardt‐Ferner

et al. 2015

Journal of Biological Chemistry

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Background:The lipoprotein NisI confers immunity to Lactococcus lactis against its own lantibiotic nisin. Results: NisI contains a membrane and a nisin binding domain both with the same fold as SpaI from Bacillus subtilis. Conclusion: The C-terminal domain of NisI interacts directly and specifically with nisin. Significance: Our results help to understand the mechanism of the NisI mediated immunity against nisin on a structural level.

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“…N-HSQC spectra of URRM1,2/U2AF35(UHM) (black), URRM1,2-M144I/U2AF35(UHM) (red), and URRM1,2-L187V/U2AF35 (UHM) (green). Discussion RNA binding of U2AF65 is associated with an open domain configuration, whereas unbound U2AF65 represents an ensemble of closed conformations (28,29). When analyzing its conformational dynamics, we found that U2AF65 can adopt different conformational states, dynamically switching between a closed conformation with a FRET efficiency of ∼0.96 and an open state with a FRET efficiency of ∼0.43.…”

Section: U2af35 Enhances Py-tract Recognition By a Dynamic Populationmentioning

confidence: 82%

“…Moreover, based on a combined analysis of NMR and small angle X-ray scattering (SAXS) data, it has been shown that the unbound RRM1,2 protein adopts a range of closed and detached domain arrangements, which are not able to mediate high-affinity RNA binding (29). To understand the…”

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confidence: 99%

Recognition of the 3′ splice site RNA by the U2AF heterodimer involves a dynamic population shift

Voithenberg

Sánchez-Rico

Kang

et al. 2016

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

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An essential early step in the assembly of human spliceosomes onto pre-mRNA involves the recognition of regulatory RNA cis elements in the 3′ splice site by the U2 auxiliary factor (U2AF). The large (U2AF65) and small (U2AF35) subunits of the U2AF heterodimer contact the polypyrimidine tract (Py-tract) and the AG-dinucleotide, respectively. The tandem RNA recognition motif domains (RRM1,2) of U2AF65 adopt closed/inactive and open/active conformations in the free form and when bound to bona fide Py-tract RNA ligands. To investigate the molecular mechanism and dynamics of 3′ splice site recognition by U2AF65 and the role of U2AF35 in the U2AF heterodimer, we have combined single-pair FRET and NMR experiments. In the absence of RNA, the RRM1,2 domain arrangement is highly dynamic on a submillisecond time scale, switching between closed and open conformations. The addition of Py-tract RNA ligands with increasing binding affinity (strength) gradually shifts the equilibrium toward an open conformation. Notably, the protein-RNA complex is rigid in the presence of a strong Py-tract but exhibits internal motion with weak Py-tracts. Surprisingly, the presence of U2AF35, whose UHM domain interacts with U2AF65 RRM1, increases the population of the open arrangement of U2AF65 RRM1,2 in the absence and presence of a weak Py-tract. These data indicate that the U2AF heterodimer promotes spliceosome assembly by a dynamic population shift toward the open conformation of U2AF65 to facilitate the recognition of weak Py-tracts at the 3′ splice site. The structure and RNA binding of the heterodimer was unaffected by cancer-linked myelodysplastic syndrome mutants.uring gene expression, the removal of introns is essential for translation of mature mRNA. The splicing process involves a large number of splicing factors for the correct recognition of introns (1). Whereas U1 snRNP contacts the 5′ splice site, recognition of the 3′ splice site involves binding of SF1/BBP to the branch point sequence (BPS) (2-5) and binding of the U2 auxiliary factor (U2AF) heterodimer to the poly-pyrimidine-tract (Py-tract) that precedes the AG dinucleotide at the intron/exon junction. Binding of U2AF to the 3′ splice site during the early steps of spliceosome assembly recruits the U2 snRNP (6-9). The strength, i.e., splicing efficiency, of a 3′ splice site requires recognition of the BPS, Py-tract, and the AG dinucleotide. However, of these three RNA elements, the Py-tract exhibits the largest degree of variability, and thus, weak to strong splice sites are primarily classified depending on the composition of the Py-tract (7, 10).U2AF is a heterodimer consisting of a large (U2AF65) and a small (U2AF35) subunit. U2AF65 harbors two canonical RNA recognition motifs (RRM1,2) and an atypical C-terminal RRM domain, called the U2AF homology motif (UHM). U2AF35 has one RRM (which acts as an UHM), flanked N-and C-terminally by two CCCH-type zinc finger motifs, respectively (Fig. S1A) (9, 11, 12). The U2AF heterodimer is formed by recognition of a peptide motif, cal...

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Transient Electrostatic Interactions Dominate the Conformational Equilibrium Sampled by Multidomain Splicing Factor U2AF65: A Combined NMR and SAXS Study

Cited by 78 publications

References 75 publications

SAXS/SANS on Supercharged Proteins Reveals Residue-Specific Modifications of the Hydration Shell

SAXS/SANS on Supercharged Proteins Reveals Residue-Specific Modifications of the Hydration Shell

The Solution Structure of the Lantibiotic Immunity Protein NisI and Its Interactions with Nisin

Recognition of the 3′ splice site RNA by the U2AF heterodimer involves a dynamic population shift

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