Side-chain pairing preferences in the parallel coiled-coil dimer motif: Insight on ion pairing between core and flanking sites

Article abstract of DOI:10.1021/ja100080q

A new strategy for rapid evaluation of sequence-stability relationships in the parallel coiled-coil motif is described. The experimental design relies upon thiol-thioester exchange equilibria, an approach that is particularly well suited to examination of heterodimeric systems. Our model system has been benchmarked by demonstrating that it can quantitatively reproduce previously reported trends in interhelical a-a′ side-chain pairing preferences at the coiled-coil interface. This new tool has been used to explore the role of Coulombic interactions between a core position on one helix and a flanking position on the other helix (a-g′). This type of interhelical contact has received relatively little attention to date. Our results indicate that such interactions can influence coiled-coil partner preferences.

Full text of DOI:10.1021/ja100080q

Published on Web 05/14/2010
Side-Chain Pairing Preferences in the Parallel Coiled-Coil Dimer Motif: Insight
on Ion Pairing between Core and Flanking Sites
Jay D. Steinkruger, Derek N. Woolfson,* and Samuel H. Gellman*
Department of Chemistry, UniVersity of Wisconsin, Madison, Wisconsin 53706, School of Chemistry, UniVersity of
Bristol, Bristol, BS8 1TS U.K., and Department of Biochemistry, UniVersity of Bristol, Bristol, BS8 1TD U.K.
Received January 5, 2010; E-mail: gellman@chem.wisc.edu; D.N.Woolfson@bristol.ac.uk
Proteins collectively display a broad array of tertiary and
quaternary structures, with many different modes of packing
between neighboring secondary structure elements. Among the
possibilities, the R-helical coiled coil is unusual in that it is both
common and regular.1 In the simplest case, two R-helices associate
side-by-side, wrapping around one another with a slight left-handed
superhelical twist. A characteristic “knobs-into-holes” interdigitation
of side chains is observed at the helix-helix interface, whether
the helices are parallel or antiparallel.2 The relative simplicity of
this architecture has led to extensive exploration of sequence-stability
relationships,3 motivated by the prospects of predicting coiled-coil
structure from sequence information alone, refining computational
tools, and using coiled coils as building blocks in rational protein
design and synthetic biology.4 Although some principles that govern
coiled-coil stability have been elucidated, our understanding remains
incomplete.
Here we introduce a heterodimeric parallel coiled-coil model
system designed to provide new insights on the origins of stability
and helix-pairing preferences. Our system employs relatively short
peptide segments (20 or 21 residues), which facilitates broad
exploration of sequence variations. Parallel, two-helix assembly is
promoted by a thioester linkage between the C-terminus of one
segment and the side chain of a C-terminal Cys residue on the other;
this design enables us to monitor coiled-coil stability under native
conditions via thiol-thioester exchange equilibration.5 Mutation
and thermodynamic analysis generate hydrophobic side-chain
pairing preferences at the helix-helix interface that agree well with
results previously obtained by Vinson et al. with larger heterodimers
comprising 96-mer proteins.3a Furthermore, an analysis of ionizable
Figure 1. (a) Primary sequence of CT-C′ (X ) Ψ ) Ile). (b) Helical
side chains at the interface offers new insight on contributions of
wheel diagram showing the proposed helical regions of CT-C′. Guest sites
Coulombic interactions to coiled-coil stability and pairing preferences.
Our experimental design (Figure 1) is based on well-known
characteristics of sequences that form coiled coils. The segments
intended to adopt R-helical conformations feature a heptad sequence
repeat pattern (abcdefg), in which side chains at a and d dominate
the helix-helix contacts. Two-helix stoichiometry (rather than
alternate three- or four-helix assemblies) is directed by placing Leu
at the d sites, Ile at the N-terminal a positions, and Asn at the a
sites closest to the covalent connection.6,7 The remaining (central)
a positions of each segment (designated X and Ψ) are “guest” sites
for substitutions that allow us to probe the impact of paired
mutations on coiled-coil stability. The positions that flank the
helix-helix interface, e and g, are occupied exclusively by Arg in
one segment and by Glu in the other. Upon intramolecular coiled-
coil formation (Figure 1B), these side chains can form interhelical
ion pairs (esg′ and e′sg; the primes indicate sites on different
helices). The remaining positions (b, c, and f) are occupied by
nonionizable residues with high helical propensity8 but low
hydrophobicity.
are highlighted in dashed boxes. (c) Cartoon illustrating the thiol-thioester
exchange process.
Our heterodimeric coiled-coil design was characterized physically
for X ) Ψ ) Ile using a disulfide-linked analogue (CS-SC′),
because the thioester version (CT-C′) suffered hydrolysis over the
time period required for measurements (e.g., multiple days for
sedimentation equilibrium analytical ultracentrifugation (AUC)).
C
S-SC′ was generated by replacing the final Gly residue of the
basic segment with Cys and then forming the heterodisulfide.
Sedimentation equilibrium AUC and circular dichroism (CD)
spectra recorded for different concentrations for CS-SC′ show that
this molecule does not self-associate under conditions used for
thioester exchange.9 The far-UV CD spectrum for CS-SC′ is
consistent with extensive R-helicity, which is disrupted upon
heating.9 CD spectra for peptides corresponding to either the basic
or acidic segment in CS-SC′ indicate that these fragments are largely
unfolded in isolation.9
Thiol-thioester exchange (TE) was initiated for X ) Ψ ) Ile
by mixing CT-C′ and HSY, or by mixing CT-Y and HSC′ (Figure
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C O M M U N I C A T I O N S
1C), in aqueous buffer (pH 7). Equilibrium is achieved in ∼2 h,
with KTE ) 158. This value does not change when the starting
component concentrations are varied between 50 and 300 µM. We
have shown previously that the folding equilibrium constant for
intramolecular coiled-coil formation (KCC) should be equal to
KTE - 1 if there is no significant packing interaction between the
Tyr residue of the HSY-derived fragment and the remainder of the
molecule in CT-Y and if the thioester bonds in CT-Y and CT-C′
are isoenergetic. Control experiments suggest that these conditions
are approached in the present system.9 KTE ) 158 translates to a
favorable free energy (∆GCC) of -3.0 kcal/mol for intramolecular
coiled-coil formation in CT-C′, relative to an unfolded state that
lacks tertiary contacts.
provides quantitative insight regarding sequence-stability relation-
ships for parallel coiled-coil structure.
We applied the TE approach to examine a factor that has recently
been suggested to influence coiled-coil stability and selectivity.10
Based on extensive evaluation and modeling of natural and designed
coiled-coil pairs, Keating et al. drew the unexpected conclusion
that a-g′ and a′-g ion pairs may exert a substantial effect on
coiled-coil specificity. This suggestion is noteworthy because
interactions involving ion pairing at flanking sites (e-g′ and e′-g)
are widely understood to confer pairing specificity, but the
occurrence of ionic side chains at core positions (a or d) has
generally been regarded as simply destabilizing, although such
residues can be important in specifying the oligomer state.11
We explored the energetic significance of a-g′ ion pairing by
examining X ) Glu vs Arg for Ψ ) Ile. This mutational analysis
explores two unique cases. In the first case the intrahelical a-e
ion pair is attractive, while the interhelical a-g′ ion pair is repulsive
(Figure 3a). This side-chain arrangement should increase R-helicity
in the isolated helix-prone segment12 but discourage coiled-coil
formation due to the repulsive a-g′ ion pair. In the second case
the situation is reversed: the intrahelical a-e ion pair is repulsive,
while the interhelical a-g′ ion pair is attractive (Figure 3b). This
side-chain arrangement should encourage coiled-coil formation by
allowing the a side chain to ion pair with the g′ side chain.
Comparable possibilities occur for Ψ ) Glu vs Arg for X ) Ile,
as indicated in Figure 3c-d.
Figure 2. (a) Thermodynamic data (∆GCC) values determined from thioester
exchange of CT-C′ mutants. Assays were conducted in 50 mM sodium
phosphate buffer (pH 7) with 2 mM TCEP at 25 °C. (b) Correlation diagram
comparing ∆(∆G) data from the Vinson laboratory3a and our work using
thioester exchange. ∆(∆G) values are normalized to the Ala-Ala homotypic
pairing.
Upon intramolecular coiled-coil formation by CT-C′, the
residues at guest sites X and Ψ should be laterally paired at the
interhelical interface. We used our system to evaluate the energetics
of all 25 a-a′ pairings involving Ile, Leu, Val, Ala, and Asn (Figure
2a). Replicate analysis (at least duplicate) was conducted for each
mutant. Standard deviations were calculated to arrive at the reported
uncertainty of (0.1 kcal/mol, though the precision of the measure-
ment is higher ((0.03 kcal/mol) in many cases. In general, the
uncertainty associated with ∆GCC becomes larger when KTE
approaches unity, because KCC approaches zero (KCC ∼ KTE - 1).
Figure 3. Partial helical net diagrams and ∆GCC values for different a-g′
or a′-g ion pairs.
Compared to X ) Ψ ) Ile (∆GCC ) -3.0 kcal/mol), all four
variants indicated in Figure 3 are destabilized, as expected for
placement of an acidic or basic side chain at the helix-helix
interface.3a However, the data clearly show that stability is greater
when the interfacial side chain has the possibility to form an a-g′
or a′-g ion pair (X ) Arg or Ψ ) Glu) than when the a-g′ or
a′-g pairing leads to Coulombic repulsion (X ) Glu or Ψ ) Arg;
compare Figure 3a with 3c, and 3b with 3d). The ∆GCC values for
the mutants shown in Figure 3 are independent of concentration
when the starting components are varied between 50 and 300 µM.
Sedimentation equilibrium AUC of the heterodisulfide correspond-
Vinson et al. have reported an extensive evaluation of a-a′
pairing energetics, based on thermal denaturation of intermolecular
heterocoiled coils formed between designed 96-residue proteins.3a
Despite deriving from significantly different model system designs
(primary sequence, length, inter- vs intramolecular association) and
methods for extracting thermodynamic parameters (thermal dena-
turation vs thioester exchange), the two data sets appear to correlate
well (Figure 2b). This correlation indicates that our model system
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C O M M U N I C A T I O N S
ing to Figure 3b indicates that this molecule does not self-associate
under conditions used for thioester exchange.9 This observation is
consistent with other AUC studies on coiled coils containing a
charged residue at an a position.11,13 These control experiments
indicate that intermolecular interactions (i.e., self-association of the
coiled coil) do not influence the ∆GCC measured for this set of
mutants. Analogous stability trends were obtained for the examples
in which the nonpolar residue at either X or Ψ is Leu, rather than
Ile as shown in Figure 3.9 Overall, these data indicate that
interhelical Coulombic interactions between a and g′ influence
coiled-coil stability. Our findings support and amplify recent
conclusions that a-g′ charge complementarity can exert a sub-
stantial influence on coiled-coil pairing specificity.10
was recently suggested based on computational and combinatorial
design.10 We have extended the understanding of Coulombic
contributions to coiled-coil stability by showing that such a-g′
pairings can influence coiled-coil pairing specificity, at least for
Arg-Glu combinations. From a methodological perspective, it is
noteworthy that the thiol component in the new system presented
here (e.g., in HSC′) is provided by a Cys side chain rather than a
backbone-mimetic R-thioacid. This design was necessitated by the
parallel alignment of helical segments in the structure under study;
the success of this approach suggests that the thiol-thioester
exchange technique need not be limited to peptidic backbones. The
system introduced here should be useful for further delineation of
the factors that control parallel coiled-coil stability and pairing
selectivity.
To test this hypothesis further, we used the CC+ database14 to
identify a set of parallel dimeric coiled coils in the protein data
bank (PDB) that had either Arg or Glu at a. While the numbers of
examples are small, 24 and 19 respectively, inspection of the
structures is informative. A number of a-g′ interactions are
observed, but the data set reveals more generally that Coulombic
interactions of side chains can involve a variety of two- and three-
component combinations. In the case of Arg-Glu ion pairs in which
one partner resides at an a site, interhelical a-g′ and intrahelical
a-e Coulombic interactions are detected, along with more complex
salt-bridged networks.9 Comparing the Cꢀ-Cδ distances for these
Arg-Glu pairs gives averages of 4.76 Å for a-g′ pairs (n ) 14,
SD ) 0.69 Å) and 5.19 Å for a-e pairs (n ) 13, SD ) 0.77 Å).
The mean values of the distances might suggest that a-g′ pairs
lead to “better” interactions than the a-e alternatives, but for this
limited data set we can confidently conclude only that interhelical
a-g′ salt bridges are at least as good as intrahelical salt bridges
between a and e.
Acknowledgment. This research was supported by the NIH
(GM061238, to S.H.G.) and the BBSRC of the U.K. (D003016 to
D.N.W.). We thank Mr. Craig Armstrong for discussions, Mr. Tom
Vincent and Dr. Gail Bartlett for maintaining the CC+ database,
and Dr. Darrell R. McCaslin for assistance with AUC experiments.
Supporting Information Available: Experimental procedures, CD
and AUC data, HPLC chromatograms, and representative structures
uncovered using CC+ are included and discussed. This material is
available free of charge via the Internet at http://pubs.acs.org.
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