Control of the stability of a protein-RNA complex by the position of fluorine in a base analogue

Article abstract of DOI:10.1021/ja102601h

The effects of modifying the electronic characteristics of nonpolar base analogues substituted at positions involved in stacking interactions between SL2 RNA and the U1A protein are described. A surprisingly large difference in the stability between complexes formed with base analogues that differ only in the position of substitution of a single fluorine atom is observed. The results of high-level ab initio calculations of the interactions between the nonpolar base analogue and the amino acid side chain correlate with the experimentally observed trends in complex stability, which suggests that changes in stacking interactions that result from varying the position and degree of fluorine substitution contribute to the effects of fluorine substitution on the stability of the U1A-SL2 RNA complex.

Full text of DOI:10.1021/ja102601h

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Control of the Stability of a Protein-RNA Complex by the Position
of Fluorine in a Base Analogue
Yulia Benitex† and Anne M. Baranger*,†,‡
†Department of Chemistry, Wesleyan University, Middletown, Connecticut 06459, United States
‡University of Illinois, Department of Chemistry, 600 South Mathews Avenue, Urbana, Illinois 61801, United States
S Supporting Information
b
ABSTRACT: The effects of modifying the electronic
characteristics of nonpolar base analogues substituted at
positions involved in stacking interactions between SL2
RNA and the U1A protein are described. A surprisingly
large difference in the stability between complexes formed
with base analogues that differ only in the position of
substitution of a single fluorine atom is observed. The
results of high-level ab initio calculations of the interactions
between the nonpolar base analogue and the amino acid side
chain correlate with the experimentally observed trends in
complex stability, which suggests that changes in stacking
interactions that result from varying the position and degree
Figure 1. (a) The U1A-SL2 RNA complex from the X-ray cocrystal
of fluorine substitution contribute to the effects of fluorine
substitution on the stability of the U1A-SL2 RNA complex.
structure.5f (b) SL2 RNA. (c) An expanded view of the structure.
conserved stacking interaction formed between Tyr13 of the
U1A protein and C5 of SL2 RNA (Figure 1). Hydrogen bonds
are formed between the NH2 of C5 and the side chain of Gln85
and the amide backbone of Tyr86 and between O2 and N3 of C5
and the amide backbone of Lys88.5f The substitution of Tyr13
with Phe or Thr results in a 3.6 or 4.6 kcal/mol destabilization of
the complex, respectively,5g while the substitution of C5 with U
results in a 2.3 kcal/mol destabilization of the complex.5h
tacking interactions between aromatic amino acids and
S
nucleic acid bases are common in complexes formed with
single-stranded nucleic acids, including RNA-protein com-
plexes.1 Investigations involving nonpolar base analogues have
revealed the ability of dispersive forces and solvation effects to
stabilize stacking interactions in helical structures and nucleic
acid-protein complexes.2 A useful characteristic of aromatic
groups is the ability to vary their electronic properties. Although
investigations with small molecules suggest that varying electro-
nic properties of aromatic groups affects the strength of stacking
interactions,3 such correlations in biological systems have been
unclear,2a,4 and studies with RNA-protein complexes have not
been conducted.
The RNA recognition motif (RRM) is the most common
RNA binding domain in higher vertebrates and is found in all
organisms.5a The most conserved interactions between RRMs
and RNA are stacking interactions. Thus, we have used an
RRM-RNA complex to vary the electronic properties of
aromatic groups involved in stacking interactions. We have found
that variation of the pattern of fluorination of a base analogue in
stem loop 2 (SL2) of U1 snRNA changes the stability of the
complex formed with the N-terminal RRM of the U1A protein by
4.3 kcal/mol. High-level calculations suggest a correlation
between the strength of the stacking interaction and the stability
of the U1A-SL2 RNA complex.
To investigate whether varying the electronic character of the
aromatic rings involved in stacking interactions could be used to
modulate the stability of the U1A-RNA complex, we replaced
C5 with a series of phenyl base analogues containing different
fluorine substitution patterns (Figure 2). Fluorine reduces elec-
tron density in the π system, but is small and forms considerably
weaker hydrogen bonds than the natural bases.4a,6 These altera-
tions may perturb the structure of the free RNA or the complex
and thus affect binding affinity independently of the stacking
interaction with Tyr13. However, structural studies have shown
little perturbation of helical structure upon incorporation of
phenyl-derived base analogues.7 We reasoned that structural
perturbations would be likely to be relatively constant across
the series of phenyl base analogues, allowing an evaluation of the
sensitivity of complex stability to varying electronic properties.
Phosphoramidites of the phenyl base analogues were synthe-
sized and incorporated into SL2 RNA.6c,7b,8 The synthesis of
the phosphoramidite of the 2,4,6-trifluorophenyl base analogue
has not previously been reported. The stability of SL2 RNA,
measured by temperature melt analyses, was not altered by
these base analogues. SL2 RNAs substituted with phenyl and
U1A is a component of the U1 snRNP, a subunit of the
spliceosome, and regulates polyadenylation of U1A and other
pre-mRNAs.5b-5e The focus of these investigations is the
Received: March 28, 2010
Published: March 01, 2011
r
2011 American Chemical Society
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dx.doi.org/10.1021/ja102601h J. Am. Chem. Soc. 2011, 133, 3687–3689
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4-fluorophenyl base analogues bound with similar weak affinities
to U1A protein (Table 1). Substitution of C5 with 2,4-difluor-
ophenyl or 2-fluorophenyl groups resulted in a large gain of
binding energy compared to phenyl substitution, 4.3 and 3.7
kcal/mol, respectively. 2- And 4-fluorophenyl base analogues
have similar polarizability, size, and hydrophobicity, yet the
stabilities of complexes containing these two analogues differ
by 2.8 kcal/mol. It is unlikely that a hydrogen bond formed
between 2-F and the amide backbone of Lys88 or another residue
is responsible for the large difference in stability of the complexes
because hydrogen bonds with F are weak.4a,6 It is also unlikely
that unfavorable interactions involving 4-F are responsible for the
decreased stability of this complex because the most stable
complex in the series contains the 2,4-difluorophenyl base
analogue.
We calculated properties of the fluoroaryl groups that often
affect stacking interactions, including log P,2b,9 polarizability,
surface area, quadrupole moment, and dipole moment, to reveal
whether the origins of the effects of fluorophenyl substitutions on
complex stability may be related to effects on stacking interac-
tions (Table 1). The changes in binding affinities do not correlate
well with any single parameter, even when taking into account
the direction of the dipole moment.
To evaluate stacking interactions between the base analogues
and tyrosine, while effectively including many of the parameters
in Table 1 simultaneously, we performed high-level ab initio
calculations using the polarizable continuum model (PCM) with
Gaussian 03 using MP2 6-31G*(0.25). These types of calcula-
tions have been found to be accurate in reproducing trends in
stacking affinities of nucleic acid bases.10 The calculations were
performed on the isolated tyrosine side chain and fluorophenyl
group interacting within a geometry similar to that found in the
X-ray structure of the complex. The calculated energies (ΔΔE’s)
reproduce the significant differences in stability between com-
plexes containing phenyl or 4-fluorophenyl base analogues and
those containing 2-fluorophenyl or 2,4-difluorophenyl base
analogues (Table 2). Not surprisingly, the magnitudes of the
experimentally determined ΔΔG’s and the calculated ΔΔE’s
Figure 2. Phenyl and fluorinated phenyl base analogues. Below each
structure is shown calculated electrostatic surface potentials for each
base or base analogue. The electrostatic surface potentials were calcu-
lated for structures in which R = Me, using MP2 6-31G*(0.25)//B3LYP6-
31G(d).
Table 1. Binding Affinity of Wild-Type and Modified RNAs to U1A and Calculated Properties of the Free Base Analogues
polarizability
(Å3) d
surface area
(Å2)e
dipole moment
(D) f
quadrupole
SL2 RNA
Kd (M)a
ΔG (kcal/mol) ΔΔG (kcal/mol)b
-13.1 ( 0.3
log Pc(water-octanol)
moment (B)
wild type
P
3 ( 2 ꢀ 10-10
4 ( 3 ꢀ 10-6
9 ( 1 ꢀ 10-7
8 ( 6 ꢀ 10-9
3 ( 1 ꢀ 10-9
1.5 ( 0.4 ꢀ 10-7
-7.4 ( 0.3
-8.3 ( 0.1
-11.1 ( 0.4
-11.7 ( 0.2
-9.3 ( 0.2
5.7
4.8
2.0
1.4
3.8
2.20
2.34
2.34
2.48
2.62
9.5
9.5
9.5
9.5
9.5
222.83
223.69
223.68
225.52
228.27
0.34
2.76
2.16
2.63
0.52
-10.03
-6.27
-7.75
-3.10
0.82
4-PF
2-PF
2,4-PF2
2,4,6-PF3
a Kd values were measured by gel mobility shift assays and are the average of at least three independent experiments. b ΔΔG is the difference in affinity
between the indicated complex and the wild-type complex. c Ghose-Crippen method.2b,9 d RHF/6-31G**//RHF6-31G** was used with methylated
base analogues. e Base analogues contained a hydrogen in place of ribose. f Calculations used methylated bases in water using the PCM with Gaussian 03
using MP2 6-31G* (0.25)//B3LYP 6-31G(d).
Table 2. Comparison of Measured Binding Affinities and Calculated Stacking Energies with Tyr13
RNA
ΔG (kcal/mol)
ΔΔG (kcal/mol)a
stacking energy (kcal/mol)b,c
ΔΔE (kcal/mol)a
P
-7.4 ( 0.3
-8.3 ( 0.1
-11.1 ( 0.4
-11.7 ( 0.2
-9.3 ( 0.2
0
-1.84
-2.57
-3.43
-4.18
-4.09
0
4-PF
-0.9
-3.7
-4.3
-1.9
-0.7
-1.6
-2.3
-2.2
2-PF
2,4-PF2
2,4,6-PF3
a ΔΔG and ΔΔE are the differences in energy of the complexes containing P and the indicated base analogue. b Calculations were performed in water
using the PCM with Gaussian 03 using MP2 6-31G*(0.25)//B3LYP6-31G(d). c The stacking energy was obtained by subtracting the basis set
superposition error (BSSE) energy calculated in vacuum from the stacking energy in water. For these calculations the methylene of tyrosine and the
ribose were replaced with methyl groups.
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COMMUNICATION
differ, which could result from changes in the structure of the
complex or RNA upon incorporation of the base analogues and/or
entropic effects that are not included in the calculations. Never-
theless, the calculations suggest that differences in stacking inter-
actions may contribute to the sensitivity of complex stability to
fluorination position.
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Previous investigations of fluorinated phenyl base analogues in
DNA and RNA helices have suggested that stacking interactions
can be modulated by the position of fluorination. Incorporation
of a series of fluorinated phenyl base analogues into the 50-end of
a DNA helix resulted in a maximum stabilization of the duplex of
1.3 kcal/mol.2a Similar to the results discussed in this paper,
correlations between quadrupole moment, dipole moment,
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consistent with those reported here on the U1A-RNA complex,
and taken together, they reveal a complex relationship between
fluorination and the stability of biological complexes.
In conclusion, we have shown that the stability of an RRM-
RNA complex is surprisingly sensitive to the position of fluorine
substitution of a phenyl base analogue in the RNA target site.
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the complex by 2.8 kcal/mol. The correlation between experi-
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’ ASSOCIATED CONTENT
S
Supporting Information. Description of syntheses, bind-
b
ing reactions, and calculations. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
baranger@illinois.edu
’ ACKNOWLEDGMENT
We are grateful to Prof. Petersson and Prof. Novick for
discussions on the calculations and to Prof. K. Nagai for the
expression vector for U1A. Funding was provided by NSF
(MCB-1019958), NIH (GM-056857), and the Petroleum Re-
search Fund.
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