Rethinking Leucine Zipper ��� a ubiquitous signal transduction motif Yaroslav Nikolaev1* and Konstantin Pervushin1,2 1 Biozentrum of University Basel, Klingelbergstrasse 50/70, CH-4056, Basel, Switzerland 2 School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 * Correspondence: y.nikolaev@unibas.ch This work is supported by the Swiss National Science Foundation Grant 3100A0-118381. Nature Precedings : hdl:10101/npre.2009.3271.1 : Posted 21 May 2009

��� 2 ��� Table of contents = 1 = Introduction ...............................................................................................................................4 = 2 = Structure.....................................................................................................................................5 = 2.1 = Primary ��� heptad repeat ......................................................................................................................5 = 2.2 = Secondary and tertiary ��� stability and stoichiometry........................................................................6 = 2.3 = Quaternary ��� specificity......................................................................................................................8 = 3 = Stability and specificity ............................................................................................................8 = 3.1 = D-D��� interactions (stability)................................................................................................................9 = 3.2 = G-E��� interactions (specificity) ..........................................................................................................10 = 3.3 = A-A��� interactions (stability and specificity) ....................................................................................15 = 3.6 = Anti-parallel leucine zippers.............................................................................................................18 = 3.7 = LZ network design ............................................................................................................................20 = 3.9 = Conclusion .........................................................................................................................................20 = 4 = Folding ..................................................................................................................................... 21 = 4.1 = Overview............................................................................................................................................22 = 4.2 = Folding models..................................................................................................................................24 = 4.3 = Folding intermediates........................................................................................................................27 = 4.4 = Diffusion-Collision-Desolvation (DCD) model..............................................................................31 = 4.5 = Summary............................................................................................................................................34 = 4.6 = Conclusion .........................................................................................................................................36 = 5 = Functional diversity ............................................................................................................... 36 = 5.1 = Transcription factors ��� bZIP, bHLH-LZ, HD-ZIP..........................................................................37 = 5.2 = Immune response signalling ��� NF-kappaB pathway ......................................................................39 = 5.3 = More kinases ��� PKG, ZIPK, DAPK ................................................................................................43 = 5.4 = Ion channels ��� AKAP .......................................................................................................................43 = 5.5 = Transport vesicles ��� SNARE............................................................................................................44 = 5.6 = Viral envelopes and capsides............................................................................................................44 = 5.7 = Innate antiviral defense ��� interferon induced Mx proteins.............................................................45 = 6 = LZ in protein engineering ..................................................................................................... 46 = 7 = Conclusions and Outlook...................................................................................................... 46 = 8 = References................................................................................................................................ 48 Nature Precedings : hdl:10101/npre.2009.3271.1 : Posted 21 May 2009

��� 3 ��� Abstract In this essay we attempt to reconsider the concept of the ���Leucine Zipper��� (LZ) protein oligomerization motif. Reasoning on the wealth of existing data, we suggest that despite of the structural similarity with highly stable extended ���Coiled Coil��� motifs, on the functional level short and moderately stable ���Leucine Zippers��� might stand out as a distinct group. Namely, this family of oligomerization motifs facilitates combinatorial protein-protein recognition in the course of signal transduction events, thus going beyond the structural role of the extended ���Coiled Coils���. Summarizing the existing knowledge on stability, specificity and folding of Leucine Zippers we demonstrate how a simple set of rules, applied in the context of the universal coiled coil scaffold, creates a robust LZ interaction vocabulary. Owing to the high abundance of Leucine Zippers, this motif might account for coupling of distinct protein signalling pathways into a unified intracellular signalling network. In the last part of this essay we provide examples demonstrating prevalence of the LZ-mediated signal transduction and illustrate applicability of ���LZ code��� formalism to interpret evidences of couplings between cytoplasmic and nuclear signalling networks. Nature Precedings : hdl:10101/npre.2009.3271.1 : Posted 21 May 2009

��� 4 ��� = 1 = Introduction Decryption of protein one-dimensional sequence from the corresponding nucleic acid sequence provided one of the key advancements for the emergence of genetic engineering and molecular biology. Unfortunately, high complexity of protein three-dimensional structures defers the widespread advent of the protein engineering. Namely, decryption of protein 3D structure from its primary sequence is not accessible yet and remains one of the fundamental frontiers in modern biology, generally referred to as the ���protein folding��� problem. One of the main motivations for solving this problem is the desire to understand and accurately predict interactions between proteins and other biomolecules within the cells. This knowledge is vital for understanding of a wide range of cellular processes governed by protein signal transduction (for example transmittance of extracellular signals to the transcription machinery). As a rule, these interactions are defined by extended and often highly dynamic 3D protein interfaces, making ab initio prediction of these interactions a daunting task, which cannot be solved at the current state of science and technology. However, a small part of this problem appears solvable already today. Leucine Zippers (LZ) represent a family of abundant protein-protein interaction motifs. Being based on the well characterized coiled coil scaffold, Leucine Zippers allow to reduce the interaction prediction problem to a simple comparison of two linear LZ amino acid sequences. This does not bring us closer to solving the general ���protein folding��� problem, but omnipresence of Leucine Zipper-based protein interactions makes such ���LZ code��� formalism a useful tool for evaluation of protein interactions among plethora of LZ-mediated signalling pathways. Leucine zippers belong to the class of coiled coil structural motifs, arguably the simplest and the most ubiquitous mediators of protein-protein interactions (1, 2). The members of the LZ class exhibit extreme thermodynamic stability owing to the prevalence of leucine residues at the key positions of their hydrophobic interface. This allows reduction of a minimal peptide length required for oligomerization to three (3), sometimes even two (4, 5) heptad repeats. Based on this high stability per heptad the LZ motifs and fragments were proposed to serve as folding triggering sequences in the context of extended coiled coil structures (6, 7). Based on the data from genome sequencing projects, coiled coils are established as the most abundant protein motif and are predicted to be found in 5-10% of all proteins (1). Their importance and versatility both in vivo and in vitro is underscored by the amount of literature Nature Precedings : hdl:10101/npre.2009.3271.1 : Posted 21 May 2009

��� 5 ��� available on the topic, with a substantial number of valuable reviews appearing in the recent years (2, 8-11). Contrary to the ���elder��� members of the coiled coil class of proteins, which are ���obligatory oligomers��� and mainly participate as structural cores in macromolecular ensembles (filaments, extracellular matrices, cytoskeletal networks, spacers, stalks, etc), LZ motifs represent ���transient oligomers���, predominantly found in the signalling and regulatory proteins (receptors, kinases, transcription factors, etc), which reflects the transient nature of these interactions. The Leucine Zipper motif was originally discovered in 1988 in the family of transcription factors named bZIP (basic region leucine zippers) (12). Shortly after its discovery, their presence was revealed in a much broader array of proteins (13, 14). During the two past decades the LZ motif has been actively employed as a model for protein folding (15, 16) and protein engineering studies (17, 18) (and references therein). Here we review the existing data on the structure, interaction specificity and folding characteristics of LZ motifs, revealing the molecular mechanisms underlying LZ-enabled protein signalling. We discuss the omnipresence of LZ motifs and illustrate their ability to couple distinct protein signalling pathways. = 2 = Structure = 2.1 = Primary ��� heptad repeat Primary structure of leucine zippers, as coiled coils class of proteins, is defined by characteristic seven residue (heptad) sequence repeat ��� (a b c d e f g)n, where the pattern is formed by hydrophobic residues at the a and d positions, charged residues at the e and g positions, and generally polar residues elsewhere (19) (Figure 2.1). Nature Precedings : hdl:10101/npre.2009.3271.1 : Posted 21 May 2009

��� 6 ��� Figure 2.1. LZ structure and interactions. (A) Linear and wheel representation of coiled coil heptad repeat structure. (C) LZ core formed by hydrophobic d-d���, a-a��� and electrostatic g-e��� interactions. (D) LZ surface b, c and f positions generally do not affect stability and specificity of LZ structure. = 2.2 = Secondary and tertiary ��� stability and stoichiometry Regular amphiphatic primary sequence drives polypeptide assembly into a supercoiled structure, with the knobs-into-holes packing of hydrophobic a and d side chains at the interacting interface (20, 21). Charged residues at the e and g positions pack over the hydrophobic core effectively shielding it from the solvent, stabilizing the structure by inter- Nature Precedings : hdl:10101/npre.2009.3271.1 : Posted 21 May 2009

��� 7 ��� chain g-e��� electrostatic interactions and providing essential determinants for specificity of dimerization interface (22, 23) (more details below). Figure 2.2. Packing interactions in the coiled coil hydrophobic core (see text for details). The key structural difference of leucine zippers from other coiled coils is almost exclusive presence of leucine residues in the d positions of the hydrophobic core (12), which essentially defines their dimeric nature. As shown by Pehr Harbury and colleagues (24) stoichiometry of a coiled coil is mainly determined by side chain packing geometry of the hydrophobic residues in the a and d positions of the interface, which varies systematically between different oligomeric states (reviewed in (2)). Briefly ��� packing topology of coiled coil hydrophobic core is distinguished by the orientation of C��-C�� bond of the hydrophobic residues (a and d positions) relative to the peptide bond of the opposing helix (Figure 2.2). In parallel orientation the C��-C�� vector projects out of the dimer interior allowing more space between residues and thus favoring ��-branched side chains (Ile, Val, Thr), where methyls branching from C�� project back into the core, providing efficient Van der Waals interactions. Conversely, in perpendicular orientation C��-C�� vector projects directly into the core, limiting space available for the sidechains branched at C��, simultaneously providing excellent packing space for C��-branched Leucines. Knobs-into-holes folding topology of the dimeric coiled coils brings residues of the a-layer into parallel orientation, and d-layer ��� into perpendicular. Thus, sequences bearing Leucines in d positions, and beta-branched residues in a, are very likely to fold into dimers. The situation is reversed in the tetrameric coiled coils fold: a-layer adopts perpendicular orientation, and d ��� parallel. Therefore this fold is favored by the sequences containing (Ile, Val, Thr) in d positions, and Leu - in the a. Topology of trimeric coiled coil fold is less restrictive - it has an intermediate (���acute���) geometry in both a and d layers - thus allowing more versatile sequence patterns. Nature Precedings : hdl:10101/npre.2009.3271.1 : Posted 21 May 2009

��� 8 ��� = 2.3 = Quaternary ��� specificity Regular topology of interactions within the coiled coil motif together with a diverse set of available amino acid side-chains, provides LZ with a wide range of stabilities and specificities, allowing them to form both homodimeric and heterodimeric structures depending on the motif composition. Moreover, a significant fraction of natural LZ motifs exhibits a wide range of intrinsic specificity allowing them to form a variable set of heterodimeric pairs. This variability of specificities is a fundamental property that enables the LZ transcription factors to assemble combinatorial regulatory networks based on their LZ motifs. These networks - bZIP, bHLH-LZ, HD-ZIP - are amongst the most advanced regulatory networks developed by eukaryotic species (25), and have evolved as key regulators in many processes, ranging from cell metabolism to tissue differentiation (26). The rules governing interaction specificity within these networks have been thoroughly characterized during last two decades, and are mainly defined by electrostatics of g-e��� couplings and polar interactions of the a-a��� pairs, as discussed in more details below. = 3 = Stability and specificity Core packing at a and d positions, together with ionic interactions between e and g positions are the key factors influencing stability and specificity of the coiled coil assembly. Applying reductionist approach to the most widely studied family of LZ proteins ��� bZIP TFs, three main interactions can be distinguished for the analysis of thermodynamic contributions to stability and specificity of the LZ interface (Figure 2.1): 1) d-d��� interactions (primarily hydrophobic & VdW defining stability) 2) g-e��� interactions (primarily electrostatic & VdW defining specificity) 3) a-a��� interactions (mixed hydrophobic/VdW/electrostatic defining stability and specificity) Most of currently existing data on the weights of these contributions to the stability and specificity of leucine zipper motifs was produced by Charles Vinson group through application of double-mutant thermodynamic cycle analysis (27) in the context of LZ motif from bZIP factor VBP for d-d��� (28), g-e��� (23, 29) and a-a��� (7, 30) pairs. Obtained results were largely corroborated by studies performed by Robert Hodges group (31-33), who targeted predominantly homodimeric interactions in the context of engineered coiled coils stabilized by covalent cross-linking. However, highly convoluted oligomerization equilibrium exhibited by engineered peptides in the latter cases, in the absence of high- Nature Precedings : hdl:10101/npre.2009.3271.1 : Posted 21 May 2009

��� 9 ��� resolution structural data and double-mutant cycle free energy analysis urges to treat these data with caution when applied to canonical LZ motifs. Detailed review of bZIP LZ stability and specificity, as well as specificity-based classification of bZIP transcription factors can be found elsewhere (10). Herein we provide a general summary on the topic, along with some contextual re-evaluation of available data. = 3.1 = D-D��� interactions (stability) Hydrophobic d-d��� interactions are the key stabilizing component and the distinctive feature of the LZ family. Efficient packing of Leucine side chains in the d positions of the knobs-into-holes topology dramatically stabilizes the dimeric coiled coil interface (28), to a large extent defining the stoichiometry of the complex (24). Importantly, stability is conferred not only by the hydrophobic effect (burial of the hydrophobic side-chain in the protein interior, shielding it from the polar solvent) but also through Van der Waals interactions (efficient packing of the sidechain against neighboring residues). The latter contribution provides leucine with up to 5.2 ��� 5.9 kcal/mol/pair (contribution from one heptad) advantage in packing energy over similarly sized methionine and isoleucine pairs (28) (Table 3.1-A). 3D structure modeling suggests that the favorable rotamer conformations of beta-branched Ile and Val side-chains produce interhelical clashes between the C��2 methyls if placed into the d-position (28). Thus, energy required to compensate for the thermodynamically unfavorable rotamer conformation may account for a part of the remarkable stability difference between leucine and beta-branched residues. This stability compromise does not play a significant role in the case of long structural coiled coil proteins, where a variety of hydrophobic amino acids have been shown to occupy the d position of the amphipathic helix (34). However, stabilizing effect of the leucine side chain appears crucial for the short leucine zipper sequences involved in signal transduction, thus yielding near invariance of this residue in the d position of the interface (28, 33). Analyzed solely in the context of bZIP motifs, the role of d-position in determining the LZ interface specificity is apparently underestimated. For example in the Myc/Max/Mxd family of bHLH-LZ transcription factors, d-position histidine of Max protein forms a unique buried salt bridge with anionic sidechains in the heterodimerization partners, which defines the specificity of this network (35, 36). Thus, it is important to recognize that empirical dimerization rules discussed here provide only a part of the ���LZ code��� definition. Nature Precedings : hdl:10101/npre.2009.3271.1 : Posted 21 May 2009

��� 10 ��� Table 3.1. Free energy differences (������GA-A [kcal/mol/pair] ��� useful to compare between LZ interaction types) and coupling energies (���������Gint [kcal/mol/pair] ��� useful when comparing pairs within one LZ interaction type) of common LZ coupling relative to a pair of alanines. Values obtained from LZ dimer thermal stabilities in 12 mM PO4, 150 mM KCl, pH 7.4. Data reproduced from (A) d-d��� (28), (B) g-e��� (23, 29), (C) and (D) a-a��� (7). For g-e��� and a-a��� interactions individual pairs are sorted according to the coupling energy strengths, and grouped in four categories: �� 0.2 kcal/mol (neutral), ��� 0.2 kcal/mol (stabilizing), ��� 0.2 kcal/mol (destabilizing), ��� 2 kcal/mol (strongly destabilizing). Free and coupling energies for heterodimeric a-a��� interactions (D) are averaged according to the residue type full set of energies can be found in Table 3.2. = 3.2 = G-E��� interactions (specificity) G-E��� interactions primarily involve charged amino acids with long aliphatic side-chains (Arg, Lys, Glu, Gln) (22), which simultaneously brings electrostatic, VdW and hydrophobic effects into play. Nature Precedings : hdl:10101/npre.2009.3271.1 : Posted 21 May 2009

��� 11 ��� Compared to a pair of alanines, the most common bZIP g-e��� salt bridges stabilize the coiled coil dimer by 1.3-1.6 (ER-RE) and 1-1.4 (EK-KE) kcal/mol/pair (Table 3.1-B). Remarkably, even identically charged Arg-Arg and Lys-Lys g-e��� pairs have stabilizing effect, contributing respectively 0.1 and 0.34 kcal/mol/pair more energy than a pair of alanines. These repulsive electrostatic interactions are considered to be largely compensated by increased hydrophobic burial and favorable VdW interactions between the methylenes of g/e sidechains and hydrophobic core of the structure (21, 29, 35, 37, 38). Compared to alanine, the only destabilizing effect is shown by a pair of glutamates, which reduces the dimer stability by 0.38 kcal/mol/pair. Obviously two methylenes of a glutamate have less compensatory effect than three methylenes of an arginine and four methylenes of a lysine, with net energy differences markedly conforming ~0.5���1 kcal/mol protein stability gain commonly observed upon burial of additional methylene (39). The overall contribution of interhelical salt bridges to the stability of leucine zippers for a long time has been a matter of debate (23, 29, 38, 40-42). The issue has been recently resolved by Hans Rudolf Bosshard and Daniel Marti, showing that the net thermodynamic contribution of a salt bridge is balanced between favorable charge-charge interaction, unfavorable desolvation energy and background interactions (such as coupling with the dipole moment of the helix) (43, 44). As it is evident from the Table 3.1, the effect of ionic g- e��� couplings compared to hydrophobic core is rather moderate, and in the context of a canonical LZ heptade will be offset by energies of a-a��� and d-d��� couplings. Nevertheless, as will be discussed in the next section, the ionic interactions have a potential to regulate specificity of oligomerization by modulating kinetics of early steps of LZ folding process, when a-a��� and d-d��� interactions have not yet stabilized the structure. In this arrangement the long-range Coulombic interactions between charged side-chains shall be able to determine the specificity of coiled coil formation. The magnitude of these interactions for particular pairs of sidechains is most efficiently evaluated employing the concept of coupling energy, which is defined as the energy conveyed by the mutual interaction of two residues, devoid of energy contributions from isolated side-chains (23, 27) (Figure 3.1). For example coupling energy of E-R pair (���������Gint = ���0.45 kcal/mol) can be estimated as total E-R contribution to the dimer stability (������GA-A = ���1.3 kcal/mol) devoid of the stability contributions of individual E (������GE-A = ���0.15 kcal/mol) and R (������GA-R = ���0.7 kcal/mol) side-chains (���������Gint = ������GA-A ��� ������GE-A ��� ������GA-R) (23). Nature Precedings : hdl:10101/npre.2009.3271.1 : Posted 21 May 2009

��� 12 ��� Figure 3.1. Thermodynamic double-mutant cycle for the Glu-Arg interaction. Measurement of thermal stabilities of four dimers yields three energy differences relative to a pair of alanines. Coupling energy (���������Gint) of Glu-Arg ionic interaction is obtained by subtracting individual contributions of Glu and Arg sidechains from overall stability of the dimer. Employing this concept the g-e��� interactions can be arranged on a more reliable thermodynamic scale, defined by pure coupling energies devoid of stabilities conferred via interactions with the core of the molecule (Table 1, column ������Gint). On this scale the most stabilizing interhelical coupling energies, on the order of ���1 kcal/mol/pair, are shown by R-E and K-E pairs, while the most destabilizing, on the order of +0.8 kcal/mol/pair, by repulsive E-E and R-R couplings (23, 29). Importantly, coupling energies do not cluster and instead are uniformly distributed over the accessible energy scale. This diverse range of attractive, neutral and repulsive couplings available within common coiled coil scaffold, multiplied by the number of variable positions (8 in an average 4-heptad LZ motif) creates an efficient combinatorial key-lock mechanism for definition of interaction specificity. Distribution of specificity determinants along the whole leucine zipper sequence allows regulation of populations of different dimers in accordance with their composition (i.e. dimers with more attractive interactions and fewer repulsive interactions would be favored over dimers with fewer attractive and more repulsive interactions). This gives a potential for establishing a complex signalling node, capable of emitting a rich output signal instead of a simple on/off event. Moreover, as highlighted by differences in reciprocal K-E/E-K (���0.91 vs. ���0.25 kcal/mol) and R-E/E-R (���1.07 vs. ���0.45 kcal/mol) pairs (29), coupling energies of g-e��� interactions strongly depend on the context, extending the combinatorial nature of LZ interface even further. However this effect appears to step into place only when underlying a Nature Precedings : hdl:10101/npre.2009.3271.1 : Posted 21 May 2009

��� 13 ��� positions bear polar or charged side-chains, and is negligible in the case of purely aliphatic core (7). Figure 3.2. Schematic representation of interhelical g-e��� interactions in defining oligomerization specificity. (A) LZ with identically charged (i, i+5) g-e��� residues ��� favoring heterodimerization, disfavoring homodimerization. (B) LZ with oppositely charged (i, i+5) g-e��� residues ��� favor homodimerization. (C) LZ with non-ionic g-e��� residues are not discriminative in oligomerization. In the simplest case of homo- versus hetero-dimer formation, a pair of g-e��� residues with the same charge (acidic + acidic or basic + basic) would favor asymmetric oligomerization ��� favoring heterodimers and disfavoring homodimers (Figure 3.2-A). A g-e��� pair with alternating charges would favor symmetric oligomers (homodimers) and disfavor asymmetrical oligomers (heterodimers with mirrored charge allocation) (Figure 3.2-B). Non- charged side-chain would give the most liberal specificity range, allowing coupling with any type of residue (Figure 3.2-C). In vivo these selective specificity mechanisms are successfully employed to decouple LZ-TF networks that operate in different functional realms. For example, specific g-e��� electrostatic interactions define a subfamily of PAR factors, involved in regulation of circadian rhythms, precluding its cross-reactivity with other bZIP families (45). These considerations, together with the specificity rules conveyed by residues in a-positions, were successfully employed for classification of bZIP proteins based on their dimerization properties (46, 47). Nature Precedings : hdl:10101/npre.2009.3271.1 : Posted 21 May 2009

��� 14 ��� Figure 3.3. Dependence of LZ oligomer stoichiometry on the size of continuous hydrophobic core. (A) Canonical LZ dimer with (a+d) hydrophobic interface. (B) Extended hydrophobic interface (a+d+e) yields a tetramer (23, 48). (C) Four-residue hydrophobic interface (a+d+e+g) yields up to a heptameric ensemble (18). In addition to functional specificity (selection of dimerization partners), g-e��� ionic interactions contribute to the structural specificity of LZ motifs, modulating register and orientation of monomer chains in the oligomeric ensemble (18, 49, 50). Furthermore, in the context of in vitro engineering studies, e and g positions can be employed for generation of high-order oligomers by extending the hydrophobic interface of the monomer chain. As originally shown by Harbury (24) (see ���2.2 - secondary and tertiary structure��� section above) ��� the stoichiometry of the coiled coil oligomers is to a large extent defined by the packing geometry of the residues occupying a and d positions of the sequence. However, a simpler rule might also be of some value in this respect ��� an estimate of continuous hydrophobic surface area carried by the coiled coil monomer. For example extension of a dimer-favoring 2-pair (a+d) hydrophobic interface (Figure 3.3-A), to a 3-pair (a+d+e) hydrophobic patches induces formation of tetramers (Figure 3.3-B) (23, 48), replacement of 14 sidechains in a and d positions with bulky tryptophan residues results in pentameric bundle (51), and extension of a 2-pair interface (a+d) to a 4-pair (a+d+g+e) creates high-order oligomers (52) with a heptameric coiled coil being the most striking structurally characterized example (Figure 3.3- C)(18). Nature Precedings : hdl:10101/npre.2009.3271.1 : Posted 21 May 2009

��� 15 ��� = 3.3 = A-A��� interactions (stability and specificity) The nature of this interaction has the most complex effect on the stability and specificity of LZ interfaces. Similarly to the residues in d-positions, packing of aliphatic side chains in a-position affects the stability and stoichiometry of the complex, with prevalence of C��- branched amino acids (Ile, Val) (47) strongly favoring the dimeric structure of leucine zippers (24). Similarly to Leucine in d-positions, isoleucine exhibits uniquely efficient side-chain packing in a-position, providing 9.2 kcal/mol/pair more energy than homotypic Ala interaction, and ~4 kcal/mol/pair more energy compared to similarly sized Leu or Val sidechains (30). However, as opposed by the extreme conservation of leucines in d-positions of the interface, isoleucine is a relatively infrequent residue in the a-position, with its occurrence probability being twice less compared to that of either leucine, valine and even asparagine (7). Sidechain selection working against the interface stability can be explained by two evolutionary advantages. First, as will be discussed below, incorporation of destabilizing polar residues provides additional mechanism for control over transcription factor functional (defining appropriate partners) and structural (defining stoichiometry and orientation) specificities. Thus, high occurrence of asparagine in the a-positions of bZIP factors highlights specificity-driven rather than stability-driven evolutionary pressures acting on these motifs. Secondly, moderate stability of the interface defined by high abundance of leucine and valine sidechains in the a-positions, as discussed in more detail in the ���folding��� section, reduces the activation energy needed for LZ dissociation, decreasing lifetime of the folded coiled coil state and elevating sensitivity of the LZ network to changes in external stimuli. This aspect underscores the notion of leucine zippers being a transient motif for signal transduction, rather than a static structural motif, as in the case of extended coiled coils. A-A��� stability scale In addition to the ���default��� set of hydrophobic side chains, LZ factors often bear polar and charged residues in the a-positions of the interface. This creates an additional mechanism for control of specificity directing a wide range of homo- and hetero-dimerization events (7, 46, 47). Thermodynamic contribution of different residues to homodimeric a-a��� interactions varies between stabilizing aliphatic, neutral polar and destabilizing charged sidechains (Table 3.1-C and diagonal in Table 3.2-D). This energy scale, relative to a pair of alanines, spans from ���9.2 kcal/mol/pair for isoleucine to +6 kcal/mol/pair for glutamate (���0.9 kcal/mol/pair and +2.1 kcal/mol/pair in terms of coupling energies ��� Table 3.2-A), which signifies importance of individual a-a��� couplings to the overall stability of the interface. Thus a vast 15 kcal/mol energy range is employed in regulation of LZ homodimerization specificity. Nature Precedings : hdl:10101/npre.2009.3271.1 : Posted 21 May 2009