Proc Natl Acad Sci U S A 107(46): 19850-19855

PMC: PMC2993330

PMID: 21045134

Molecular basis of coiled-coil oligomerization-state specificity

Discussion

Detailed knowledge of the oligomerization state of coiled coils is crucial for exploiting their potential in a wide range of applications and understanding their functions in a large number of biological processes. Over the years, several oligomerization-state determinants have been identified and studied in detail; however, their presence does frequently not correlate with coiled-coil topology. Here, using three diverse coiled-coil model systems we addressed this discrepancy and demonstrated that oligomerization-state determinants specify the topology of a coiled coil when present in the trigger sequence. Furthermore, we show that multiple motifs can coexisit in the same trigger sequence and the resulting oligomerization state is controlled by the strength of the determinants relative to each other. The contribution of the relative strength of individual motifs to final coiled-coil topology remains to be elucidated.

Our findings on a unique and general link between oligomerization-state specificity and trigger sequence function explain some puzzling results reported in the literature. We previously showed that a chimera in which the trigger sequence of GCN4 was replaced by the one of cortexillin-1 (GCN4p-Cort T/L; note that Thr95 of Cort-Ir has been replaced by leucine to result by chance in a canonical trimerization motif) folds into a three-stranded coiled-coil structure despite the presence of Asn16(a) (5). This finding can now be rationalized by the presence of a trimerization motif (R-LAKLE) in the GCN4p-Cort T/L trigger sequence. Further, the coiled-coil domain of the human dystrophia myotonica kinase contains a motif (509-R-NRDLE), which is very similar to the R-hxxhE consensus. Although it is currently not known how mutations of the hydrophobic residues of the trimerization motif influence its function, the motif of the human dystrophia myotonica kinase is a good candidate to determine trimerization of the coiled coil by overruling the strong dimerization driving force of two asparagine residues at heptad a positions in the sequence (16). Another example is the coiled-coil domain of the transcription factor HY5 from Arabidopsis thaliana, which was reported to harbor two putative and overlapping consensus trigger sequences (6-SELENR-VKDLENK and 13-KDLENK-NSELEER) containing a trimerization motif (underlined in the first sequence stretch) and an asparagine residue at a heptad repeat a position (underlined in the second sequence stretch), respectively (17). A second asparagine residue, Asn33, is present at heptad repeat a position outside the predicted trigger sequences. The wild-type peptide is dimeric but substitution of one or both asparagine by leucine or valine resulted in a switch of the oligomerization state from a dimer to a trimer (17). A similar context-dependent effect of the trimerization motif (591-R-LNRVE) was recently also demonstrated for the K(V)7.1 (KCNQ1) A-domain tail coiled coil, where C-terminal truncation resulted in a switch of the oligomerization state from a tetramer to a trimer (18).

In conclusion, our study represents an important complementation and substantial extension of the sequence-to-structure rules outlined in the pioneering work of Alber, Kim, and colleagues (11, 12). These rules together with the characteristics of the heptad repeat have allowed the development of a variety of statistics- and pattern-based methods that predict the occurrence of coiled coils with a high degree of confidence (reviewed in ref. 19). However, despite the numerous efforts during the last three decades, a reliable prediction of the specific oligomerization state of many native coiled coils remains difficult (1). This highlights the need for improving existing algorithms by implementation of experimentally verified sequence-to-structure rules like the ones based on the present study. The inclusion of these parameters as well as the detailed dataset obtained in our study should therefore significantly improve and extend the kit toward predicting and engineering the structure and function of coiled-coil proteins, which should have major implications for all applications using these popular oligomerization domains.

Methods

Peptide Preparations and Crystal Structure Determination.

Standard recombinant peptide preparation and crystal structure determination is described in SI Text.

AUC, MALS, and CD Spectroscopy.

All solution studies were performed in 5 mM sodium phosphate, pH 7.4, supplemented with 150 mM PBS.

AUC was carried out on an Optima XLA analytical ultracentrifuge (Beckman) equipped with an adsorption optical system and an An-60Ti (GCN4-p1, ATF-p) or an An-50Ti (Cort-Ir) rotor. Sedimentation equilibrium data were collected at three different protein concentrations (0.14, 0.27, and 0.55 mg/mL for GCN4-p1 variants and 0.15, 0.3, and 0.6 mg/mL for ATF1-p variants) and at rotor speeds of 28,000, 35,000, and 41,000 rpm. The M_W reported in Table 1 is calculated at the highest sample concentration. Sedimentation equilibrium data of Cort-Ir were recorded at three different protein concentrations (0.25, 0.5, and 1.0 mg/mL) and at rotor speeds of 27,000, 31,000, and 35,000 rpm. The sedimentation profiles were monitored at two wavelengths (275 nm for tyrosine and 235 nm for peptide backbone absorption). The data were fitted globally using a single ideal species model. The partial specific volumes of the synthetic peptides were calculated from their amino acid sequence composition. Solvent density for PBS was taken as 1.004 g/mL.

Size exclusion chromatography coupled to MALS was performed on a DAWN EOS 18-angle detector connected to an Optilab Rex refractometer (Wyatt). Peptide solutions (100 μL of 3–5 mg/mL) were injected on a Superdex 75 10/30 size exclusion chromatography column equilibrated with PBS. Molecular weights were calculated by using the Wyatt ASTRA version 4.90.08 software package.

Far-UV CD spectroscopy was carried out on Jasco J-810 (Jasco Inc.; GCN4-p1 and ATF-p variants) or Chirascan-plus (Applied Photophysics; Cort-Ir variants) spectropolarimeters equipped with temperature-controlled quartz cells of 0.1-cm path length. A ramping rate of 1 °C per min was used to record the thermal unfolding profiles. Midpoints of the transitions, T_ms, were taken as the maximum of the derivative d[θ]₂₂₂/dT.

^{Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom; and}

^{Biomolecular Research, Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland}

^{To whom correspondence should be addressed. E-mail:}hc.isp@ztemniets.lehcim or hc.isp@reremmak.drahcir.

Edited* by Thomas D. Pollard, Yale University, New Haven, CT, and approved September 14, 2010 (received for review June 23, 2010)

Author contributions: B.C., S.B., S.H., H.J., M.O.S., and R.A.K. designed research; B.C., S.B., S.H., H.J., R.J., A.P., and T.J. performed research; B.C., S.B., S.H., H.J., T.J., M.O.S., and R.A.K. analyzed data; and M.O.S. and R.A.K. wrote the paper.

^{B.C., S.B., S.H., and H.J. contributed equally to this work.}

^{Present address: Department of Chemistry, University of Sheffield, Sheffield S3 7HF, United Kingdom.}

^{Present address: Novartis Institutes for Biomedical Research, Novartis Pharma AG, 4002, Basel, Switzerland.}

^{Present address: Roche Applied Science Customer Support Centre, Roche Diagnostics GmbH, D-68305 Mannheim, Germany.}

^{Present address: Biomolecular Research, Paul Scherrer Insititut, CH-5232 Villigen PSI, Switzerland.}

Edited* by Thomas D. Pollard, Yale University, New Haven, CT, and approved September 14, 2010 (received for review June 23, 2010)

Abstract

Coiled coils are extensively and successfully used nowadays to rationally design multistranded structures for applications, including basic research, biotechnology, nanotechnology, materials science, and medicine. The wide range of applications as well as the important functions these structures play in almost all biological processes highlight the need for a detailed understanding of the factors that control coiled-coil folding and oligomerization. Here, we address the important and unresolved question why the presence of particular oligomerization-state determinants within a coiled coil does frequently not correlate with its topology. We found an unexpected, general link between coiled-coil oligomerization-state specificity and trigger sequences, elements that are indispensable for coiled-coil formation. By using the archetype coiled-coil domain of the yeast transcriptional activator GCN4 as a model system, we show that well-established trimer-specific oligomerization-state determinants switch the peptide’s topology from a dimer to a trimer only when inserted into the trigger sequence. We successfully confirmed our results in two other, unrelated coiled-coil dimers, ATF1 and cortexillin-1. We furthermore show that multiple topology determinants can coexist in the same trigger sequence, revealing a delicate balance of the resulting oligomerization state by position-dependent forces. Our experimental results should significantly improve the prediction of the oligomerization state of coiled coils. They therefore should have major implications for the rational design of coiled coils and consequently many applications using these popular oligomerization domains.

Keywords: protein folding, protein structure, protein–protein interactions

Abstract

Because of its simplicity, the α-helical coiled coil has been used traditionally in a vast number of studies aimed at understanding the fundamental principles of protein stability, folding, and oligomerization (reviewed in refs. 1 and 2). As a result, coiled coils are exploited nowadays as multipurpose tools in a steadily increasing number of applications ranging from basic research to medicine (2). For example, designed two- and three-stranded coiled coils were successfully used as lead molecules to target the adenomatous polyposis coli tumor-suppressor protein that is implicated in colorectal cancers (3) or to inhibit HIV infection (4), respectively. For these and many other applications, knowledge of the factors that control the folding and oligomerization state appears essential to rationally engineer specific coiled-coil structures with the desired properties.

It is well established that so-called “trigger sequences,” short, distinct amino acid sequences, play an important role in controlling coiled-coil formation (reviewed in ref. 1). A characteristic feature of many trigger sequences is that they fold into reasonably stable monomeric helices before the coiled-coil structure forms (5 –7). We recently solved the NMR structure of a peptide spanning the GCN4 trigger sequence and showed that its structure is stabilized by a network of hydrogen bonds and electrostatic interactions (7). The helical conformation of the trigger sequence provides an effective local structural scaffold for the interaction of key residues of colliding chains. Accordingly, preformed and folding competent helices form a nucleation site, which promotes productive in-register chain association. Interacting helices then “zip up” along the molecule to direct formation of a stable coiled-coil structure. Trigger sequences of different proteins show considerable diversity, indicating that a consensus sequence is unlikely to exist (7). As a result, the prediction of trigger sequences on the basis of sequence information alone remains a challenge and still requires experimental verification.

With respect to coiled-coil topology, several determinants that control the oligomerization state of coiled coils have been identified and studied in detail (reviewed in refs. 1 and 2). It is generally acknowledged, for example, that the distribution of hydrophobic core residues, in particular isoleucine and leucine, correlates well with the oligomerization state of coiled coils. The occurrence of isoleucine and leucine residues at the heptad repeat a and d core positions, respectively, favors the formation of dimers whereas the reverse arrangement results in tetrameric structures. In contrast, a more even distribution of isoleucine at both the a and d positions facilitates trimer formation. Buried polar residues in hydrophobic interfaces also play an important role in determining the number of strands in coiled coils. Many two-stranded coiled-coil domains of transcription regulators, for example, frequently contain at least one conserved asparagine or lysine residue at an a position toward the center of the sequence (2).

Interhelical interactions between side chains of residues at the e and g positions as well as the packing of these amino acids against the hydrophobic a and d core residues have also been shown to contribute significantly to oligomerization-state specificity of coiled coils (reviewed in refs. 1 and 2). This is exemplified by the trimerization motif Arg(g)-h(a)-x-x-h(d)-Glu(e) (denoted R-hxxhE where h(a) = Ile, Leu, Val, Met; h(d) = Leu, Ile, Val; x = any amino acid residue) that specifies a three-stranded, parallel topology of coiled-coil domains (8). The trimerization driving force of the motif can be explained by optimal side chain–side chain interactions whereby the strictly conserved arginine and glutamate residues form a distinct bifurcated interhelical salt-bridge network and participate in the formation of the hydrophobic core by establishing tight packing interactions to the neighbouring residues at the a and d positions through their aliphatic moieties. Thus, similar to contacts stabilizing thermostable proteins, the trimerization motif encompasses both networks of surface salt bridges and optimal internal hydrophobic packing interactions (8).

An open issue is that the presence of a specific oligomerization-state determinant does frequently not correlate with the corresponding coiled-coil topology. Although the trimerization motif R-hxxhE is predominantly found in many diverse protein families harboring parallel three-stranded coiled-coil domains, it is also present in some dimers and antiparallel trimers, such as the transcriptional activators GCN4 (9) and ATF1 (10) or the actin-bundling protein cortexillin-1 from Dictyostelium discoideum (6). To understand the molecular basis of this discrepancy, we have carried out a detailed biophysical and structural study to systematically address how the topology of these two-stranded coiled-coil model systems is affected when particular oligomerization-state determinants are placed at different coiled-coil positions. We thus address the fundamental and still unresolved question how the position of specific motifs within a coiled coil defines its topology.

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Acknowledgments.

We are indebted to Ariel Lustig for performing initial AUC measurements. We thank Edward McKenzie, Sanjai Patel, Karolina Zielinska, and the Biomolecular Analysis and Crystallization Core Facilities (Faculty of Life Sciences, University of Manchester) for excellent technical assistance. M.O.S. and R.A.K. acknowledge support by grants from the Swiss National Science Foundation and the Wellcome Trust, respectively.

Acknowledgments.

Footnotes

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 3GJP and 2O7H).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1008502107/-/DCSupplemental.

Footnotes

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