The disposition of the LZCC protein residues in wenxiang diagram provides new insights into the protein-protein interaction mechanism.

Wenxiang diagram is a new two-dimensional representation that characterizes the disposition of hydrophobic and hydrophilic residues in α-helices. In this research, the hydrophobic and hydrophilic residues of two leucine zipper coiled-coil (LZCC) structural proteins, cGKIα(1-59) and MBS(CT35) are dispositioned on the wenxiang diagrams according to heptad repeat pattern (abcdefg)(n), respectively. Their wenxiang diagrams clearly demonstrate that the residues with same repeat letters are laid on same side of the spiral diagrams, where most hydrophobic residues are positioned at a and d, and most hydrophilic residues are localized on b, c, e, f and g polar position regions. The wenxiang diagrams of a dimetric LZCC can be represented by the combination of two monomeric wenxiang diagrams, and the wenxiang diagrams of the two LZCC (tetramer) complex structures can also be assembled by using two pairs of their wenxiang diagrams. Furthermore, by comparing the wenxiang diagrams of cGKIα(1-59) and MBS(CT35), the interaction between cGKIα(1-59) and MBS(CT35) is suggested to be weaker. By analyzing the wenxiang diagram of the cGKIα(1-59.)·MBS(CT42) complex structure, most affected residues of cGKIα(1-59) by the interaction with MBS(CT42) are proposed at positions d, a, e and g of the LZCC structure. These findings are consistent with our previous NMR results. Incorporating NMR spectroscopy, the wenxiang diagrams of LZCC structures may provide novel insights into the interaction mechanisms between dimeric, trimeric, tetrameric coiled-coil structures.

Introduction

Coiled coil (CC) structural proteins usually contain a repeated pattern of nonpolar and charged amino-acid residues, referred to as a heptad repeat, which is denoted (abcdefg). Where the positions for a and d are predominantly hydrophobic, often being occupied by leucine, isoleucine, or valine, and the positions at b, c, e and g are typically charged or polar (Chambers et al., 1990, Crick, 1952, Hartmann et al., 2009, Lupas and Gruber, 2005, Surks et al., 1999). It has been known that many CC type proteins are involved in important biological functions such as the regulation of gene expression. Representive examples include the oncoproteins c-fos and jun, and the muscle protein tropomyosin (Hartshorne, 1997, Hartshorne and Hirano, 1999, O’Shea et al., 1989, Surks and Mendelsohn, 2003).

Usually, a leucine zipper (LZ) structure is made up of two α-helical segments of a protein; it has leucines and/or isoleucines facing each other along the length of the helices, allowing them to dimerize and form a symmetric interface that can bind to the DNA on both sides of the double helix (Lupas and Gruber, 2005) or other proteins. As a three-dimensional structural motif, LZ structures are widely found in both eukaryotic and prokaryotic regulatory proteins (Baker et al., 2005, Rybalkin et al., 2002, Schlossmann et al., 2000, Surks et al., 1999). The protein’s domain possessing LZ motif usually is a CC structure. Thus this kind of protein is also referred as leucine zipper coiled-coil (LZCC) structural proteins. Evidences that the proteins of Leucine Zipper sequences belong to coiled-coil structures have been reported by many workers (O’Shea et al., 1989, Surks et al., 1999, Surks and Mendelsohn, 2003).

Previous studies have shown that both cGMP-dependent protein kinase I, cGKIα1−59 and the C-terminal 180 amino acids (residues 929–970) of the myosin binding subunit (MBS) of the myosin light-chain phosphatase (PPlM), MBSCT180 contain LZCC domains. cGKIα1−59 functions in the nitric oxide (NO) mediated relaxation of vascular smooth muscle (Lincoln, 1994). The cyclic GMP-mediated vascular smooth muscle cell relaxation is characterized by both a reduction of intracellular calcium concentration and by activation of PP1M, and thus results in sensitivity reduction of the contractile apparatus to intracellular calcium (Lincoln, 1994, Morgan and Morgan, 1984, Surks et al., 1999). The state of contraction or relaxation of vascular smooth muscle cells is closely coupled to phosphorylation and dephosphorylation of the regulatory myosin light chain, which is in part regulated by the binding of cGK1α to MBS of the PP1M. The disruption of the cGKIα-MBS interaction impairs cGMP-mediated dephosphorylation of myosin light chain, the critical determinant of smooth muscle cell contractile state (Nakamura et al., 2007). More recently, the specific interaction between cGKIα1−59 and MBSCT42 (residues 929–1070), the C-terminal 42 amino acids of MBS protein has been further confirmed by NMR, biochemical and other biophysical methods such as glutathione S-transferase pulldown experiments, chemical cross-linking, size exclusion chromatography, circular dichroism, and isothermal titration calorimetry (Sharma et al., 2008; Zhou, 2011). These results further supported that the interaction between the cGKIα and MBS proteins actually is that between the LZCC of cGKIα1−59 and the LZCC of MBSCT42.

In view of this, the present study was initiated in an attempt to utilize wenxiang diagram to characterize the LZCC domains of both cGKIα1−59 and the LZCC of MBSCT42. It has been known that many α-helices in proteins are amphiphilic, i.e., formed by the hydrophobic and hydrophilic amino acids according to a special order along the helix chain (Mercier et al., 1998, Schnell et al., 2005; Zhou, 2011). Besides, it has also been known that different types of proteins have different amphiphilic features, corresponding to different hydrophobic and hydrophilic order patterns (Chou, 2005, Kurochkina, 2010). As summarized in an article in Wikipedia (http://en.wikipedia.org/wiki/Alpha_helix, 2010), the wenxiang diagram (Chou et al., 1997) has the following advantages: (1) able to show the relative locations of the amino acids in an alpha-helix regardless how long it is; (2) able to indicate the direction of an alpha-helix; and (3) having the capacity to provide more information about each of the constituent amino acid residues in an α-helix. With these features, the wenxiang diagram can provide an easily visualizable picture in a 2D space for clearly characterizing the disposition of amphiphilic helices in proteins.

Using graphical approaches to study biological problems can provide an intuitive picture or useful insights for helping analyzing complicated mechanisms in these systems, as demonstrated by many previous studies on a series of important biological topics, such as enzyme-catalyzed reactions (Andraos, 2008, Chou, 1980, Chou, 1981, Chou, 1989, Chou and Forsen, 1980, Cornish-Bowden, 1979, King and Altman, 1956, Myers and Palmer, 1985, Zhou and Deng, 1984), protein folding kinetics and folding rates (Chou, 1990, Chou and Shen, 2009), inhibition of HIV-1 reverse transcriptase (Althaus et al., 1993a, Althaus et al., 1993b, Althaus et al., 1993c), inhibition kinetics of processive nucleic acid polymerases and nucleases (Chou et al., 1994), drug metabolism systems (Chou, 2010), analysis of DNA sequence (Xie and Mo, 2011, Yu et al., 2009), and protein sequence evolution (Wu et al., 2010). Moreover, graphical methods have been utilized to deal with complicated network systems (Gonzalez-Diaz et al., 2009, Munteanu et al., 2009) and identify the hub proteins from complicated network systems (Shen et al., 2010). Recently, the “cellular automaton image” (Wolfram, 1984, Wolfram, 2002) has also been applied to study hepatitis B viral infections (Xiao et al., 2006), HBV virus gene missense mutation (Xiao et al., 2005a), and visual analysis of SARS-CoV (Wang et al., 2005), as well as representing complicated biological sequences (Xiao et al., 2005b) and helping to identify various protein attributes (Xiao et al., 2009, Xiao et al., 2010). The present study was initiated in an attempt to use the elegant wenxiang diagram (Chou et al., 1997) to investigate protein–protein interactions in hope to gain useful insights for understanding some of their subtle action mechanisms. Because LZ and CC proteins have amphiphilic α-helical feature, it may be rewarding to use wenxiang diagram to investigate the disposition of their hydrophobic and hydrophilic residues. Our results will indicate that the heptad repeat and disposition of all residues in both cGKIα1−59 and MBSCT42 can be clearly displayed using the wenxiang diagrams. Furthermore, the specialties of the interaction between cGKIα1−59 and MBSCT42, could also be analyzed qualitatively by the wenxiang diagrams, and the dispositions of some key residues of cGKIα1−59 in the wenxiang diagrams are basically consistent with our previous NMR experimental results (Sharma et al., 2008).

Materials and methods

The sequences of cGKIα1−59 (residues 12–59) and MBSCT42 (residues 936–970) proteins

The sequence of cGKIα1−59 is

T1SELEEDFAK10ILMLKEERIK20ELEKRLSEKE30EEIQELKRKL40HKCQSVLPVP50STHIGPRTT59.

The sequence of MBSCT42 protein is

S929TD931FKKLY936EQILA941ENEKLKAQLHDTNMELTDL KLQ LEKATQR970.

The coiled-coil predictions of the heptad repeat region were made using the programs COILS (Lupas et al., 1991, Berger et al., 1995).

Ensemble principle of wenxiang diagram of a coiled-coil dimmer

It has been clearly known that all amphiphilic helical proteins, with most hydrophobic residues being distinctly distributed in one-half of each wenxiang diagram, and most hydrophilic residues being distributed in the other half (Hartshorne, 1997, Sharma et al., 2008). Thus, the wenxiang diagrams of any LZCC structures should have similar characteristics. The hydrophobicity-weighed (w +(i)) and hydrophilicity-weighed (w−(i)) contributions of each residue, and hydrophobic and hydrophilic centroids of each α-helix can be determined by the wenxiang diagram coordinate system (Hartshorne, 1997). Furthermore, the inclination angle Ω, which is defined as the angle formed by the axis of the helix and its projected line on the plane S ( Fig. 1) can be calculated using the following equation:where C 1 ⁎, C 2 ⁎, and C 3 ⁎ are directly related to the hydrophobic atom coordinate system (x +, y +, z +), hydrophilic atom coordinate system (x −, y −, z −), hydrophobicity-weighed w +(i), and hydrophilicity-weighed w −(i) (Hartshorne, 1997).

Fig. 1

Schematic drawing showing the inclination angle Ω and the intercept T of the interfacial plane S. Q is the intersection point of the helix axis z with the interfacial plane S. As shown in the figure, Ω is the angle between the helix axis Z and its projection on the plane S, whereas T is the distance from the projection of the frst Cα on the helix axis to Q (Hartshorne, 1997).

According to the ensemble principle of wenxiang diagram, an α-helix can be viewed as a two-dimensional diagram generated by its conical projection ( Fig. 2), where the projected image of a hollow cylinder Φ onto a plane perpendicular to it by conical projection will become a ring ψ. The outer and inner circles of the ring ψ correspond to the top and bottom circles of the hollow cylinder, respectively. Thus, the projected image of a helix could be viewed as a planar spiral with a continuous varying radius. If a helical COOH-terminal is close to its projected plane, and NH2-terminal is far away the plane, the COOH-terminal of the helix lies near the center of the projected plane, and NH2-terminal lies at the outer rim of the diagram. The disposition of a coiled-coil dimmer could be expressed by a pair of two-dimensional diagrams, i.e., two same monomer wenxiang diagrams.

Fig. 2

Conical projection of a hollow cylinder Φ onto a plane perpendicular to the cylinder. The dot-dash lines (―∙―∙―) represent the radiating lines from the apex §. The distance § from the projection plane is arbitrary and is chosen to ensure that the resulting projection graph clearly portrays the relevant information. The image of the hollow cylinder on the projection plane is the ring ψ.

Results and discussion

Distribution characteristics of the residues of cGKIα1−59 and MBSCT35 in the wenxiang diagrams

In previous studies, we have used programs COILS (Lupas, et al., 1991) to predict the coiled-coil region in cGKIα1−59 domain, which is from the residue L12 through residue H53. This result has been confirmed by the analysis of our NMR relaxation data and RDC data (Schnell et al., 2005). This domain contains a repeating pattern of amino acids a, b, c, d, e, f, and g ( Fig. 3A). Thus, the monomer structure of cGKIα1−59 could be characterized by a wenxiang diagram. Where each residue of the denoted repeating amino acids (abcdefg) in Fig. 3B is represented by a circle and repeated letters a, b, c, d, e, f, and g, respectively. It is clearly shown with the wenxiang diagram that the residues with same letters are laid on same side of the spiral diagram, and most hydrophobic residues are positioned on a/d region except Lysine 15 and 29 (Fig. 3B).

Fig. 3

The Sequences containing the heaptad repeat pattern (abcdefg)n in LZCC domains of cGKIα1−59 (A), and the monomer wenxiang diagram of cGKIα1−59 (B). The each residue position in a heptad repeat was labeled using a, b, c, d, e, f, and g, respectively. In B, all positions a/d are connected by the solid line, and a hydrophobic residues is denoted by filled circle with its code symbol in white, whereas a hydrophilic residue is denoted by an open circle with its code symbol in black.

Because a LZ domain must be a typical amphiphilic helix, i.e., part of the helix flank is dominated by hydrophobic residues and the other part by hydrophilic residues, their inclination angles are all very close to 0°, which is fully consistent with the requirement of minimum free energy in a amphiphilic helical system (Hartshorne, 1997).

It has been suggested that a critical region responsible for interacting with cGKIα1−59 is another LZ coiled-coil domain, which is located within the C-terminal 100 amino acids (residues 930–1030) of the MBS (Baker et al., 2005, Lincoln, 1994). This region has been further confirmed to be MBSCT42, the C-terminal 42 amino acids (residues 929–970) of the MBS protein by our NMR data (Sharma et al., 2008). Actually, according to our pair coil analysis, the LZCC domain with the highest probability (1.0) should be the C-terminal 35 amino acids of the MBS (MBSCT35), which contains a repeating pattern of amino acids a, b, c, d, e, f, and g from residues 936 to 970 ( Fig. 4A). Because all residues of MBSCT35 are included in MBSCT42 domain, the interaction between it and cGKIα1−59 has been confirmed by our biochemical and NMR data (Lincoln., 1994). According to this information, we obtained a wenxiang diagram of the monomer of MBSCT35, where all residues with same letter are also distributed on the same side of the spiral wenxiang diagram (Fig. 4B). It can be seen from both Fig. 3, Fig. 4B and that most nonpolar residues are localized on the hydrophobic a/d regions.

Fig. 4

The Sequences containing the heaptad repeat pattern (abcdefg)n in LZCC domains of MBSCT35 (A), and the monomer wenxiang diagram of MBSCT35 (B). The each residue position in a heptad repeat was labeled using a, b, c, d, e, f, and g, respectively. In B, all positions a/d are connected by the solid line, and a hydrophobic residues is denoted by filled circle with its code symbol in white, whereas a hydrophilic residue is denoted by an open circle with its code symbol in black. Only the residues of position a/d and hydrophobic residues are labeled by the sequence number. The larger the radius an amino acid residue has in the spiral, the farther above the projection plane is the corresponding amino acid in the helix and vice versa.

According to the calculated results for a number of proteins, the inclination angles for most helices therein are small (|Ω|<25°). This suggests that driven by the free energy an amphiphilic helix will tend to seek its own arrangement in a protein such that approximately half of its face is buried in the protein, whereas the other half is exposed to the aqueous environment that surrounds the protein (Hartshorne, 1997). Thus, a coiled-coil dimmer structure can be further expressed by a combination of two identical two-dimensional wenxiang diagrams. The ensemble principle of such wenxiang diagram should observe the following rule: a/d region of one monomer wenxiang diagram must be faced to the a/d region of another identical monomer wenxiang diagram. Because most hydrophobic residues are localized at the a/d positions, the approach to each other between the two monomer’s a/d regions should be helpful for the formation of a stable coiled-coil dimmer structure due to the hydrophobic interactions of the nonpolar residues between the two a/d regions. According to such an ensemble rule, the wenxiang diagrams of the cGKIα1−59 dimmer were assembled in Fig. 5A. Where most hydrophobic side chains of leucine and isoleucine in positions a and d, and two side chains of lysine (K15 and K29) in position d should be clearly displayed on the nearest neighbor region between the two wenxiang diagrams. This observation has been verified by our 3D structure of cGKIα1−59 ( Fig. 6) based on the previous NMR experimental data (Surks and Mendelsohn, 2003).

Fig. 5

The Wenxiang diagrams of two LZCC dimmers. (A) cGKIα1−59 dimer; (B) MBSCT35 dimer. All position a/d are connected by the solid line. The name and sequence numbers of all hydrophobic residues are labeled by blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

3D structure of LZCC domains of cGKIα1−59. (A) Bundle of 20 refined parallel coiled-coil dimer structures with lowest RDC energies superimposed on the backbone heavy atoms (blue). The hydrophobic side chains of leucine and isoleucine in positions an and d are shown in green, and the two side chains of lysine which are in position d are shown in red. (B) End-on view from the N-terminus of a representative refined dimer. The backbone structure is shown as a ribbon diagram, and side chains are colored as in (A). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

As shown in Fig. 5, the hydrophobic side chains of leucine and isoleucine in positions a and d are shown in green, and the two side chains of K15 and K29 are in position d are shown in red. Similarly, the wensiang diagrams of MBSCT35 dimer structure were also obtained according to the above the ensemble principle. As shown in Fig. 5B, the a/d position region of a monomer faces to the a/d position region of another monomer on the wenxiang diagrams of MBSCT35 LZCC structure. In Fig. 5A, although the seven apolar residues (M13, L14, Y46, P48, V49, G55, and P56) of cGKIα1−59 localize the outside of the a/d position region, most of them are still close to the a/d region except residues M13 and L14. However, for the wenxiang diagrams of MBSCT35, an interesting feature is that most apolar residues of outside the hydrophobic a/d position region such as 941 A, 948 A, 955 M and 962 L are located at position f (where usually are occupied by the hydrophilic residues) and are little far away from the hydrophobic a/d position region (Fig. 5B). These differences between Figs. 5A and B suggest that the either hydrophobic or hydrophilic interactions between cGKIα1−59 and MBSCT35 may be weaker due to the lack of the contributions of the more apolar residues near the a/d position region of MBSCT35, or lack of the contributions of polar residues from g, b, c, and e positions of cGKIα1−59 This may explain why the molar ratio of the interaction between MBSCT42 and cGKIα1−59 has to be 2:1(Lincoln, 1994).

Analysis of the interaction between cGKIα1−59 and MBSCT35 incorporating wenxiang diagrams and NMR data

In order to determine whether an interaction exists between cGKIα1−59 and MBSCT42, Figs. 5A and B were combined to form the wenxiang diagrams of a hexamer complex structure according to the molar ratio of cGKIα1−59 and MBSCT35 (1:2) ( Fig. 7).

Fig. 7

The wenxiang diagrams of the hexamer complex structure of cGKIα1−59 (middle pair diagrams) and two MBSCT35 (the topper and lower pair diagrams). Where, the a–e region of one molecule is close to the d–g region of another molecule.

The ensemble of these combined wenxiang diagrams should be reasonable. Because most hydrophobic residues (position a/d regions, or nearby a/d regions) are buried inside the bundle and most hydrophilic residues are exposed to the aqueous environment that surrounds the cGKIα1−59 ·MBSCT42 hexamer complex. As shown in Fig. 7, the a–e position region of cGKIα1−59 is close to the d–g position region of MBSCT35, and the d–g position regions of cGKIα1−59 is close to the a–e position regions of MBSCT35. The all residues (E16, E23, E30, K37, Q44 and S51) at position e of cGKIα1−59 are all polar residues. In the other hand, all residues (942E, 949Q, 956E, 963Q and 970R) at position g of MBSCT35 are also polar residues. According to the previous studies (Adamian and Liang, 2002), the salt bridge residue pairs of E–R may be formed between E16, E23, or E30 of cGKIα1−59 and 970R of MBSCT35, and the salt bridge residue pairs of R–E may be formed between R37 of cGKIα1−59 and 942E or 956E of MBSCT35. These salt bridges have high propensity for the interhelical polar-polar atomic contacts. In addition, other ionizable-polar residue pairs such as Q–R or R–Q (Q44–970R, or R37–949Q, or R37–963Q), and S–R (S51–970R) between cGKIα1−59 and MBSCT35 may also have high propensity to form the interhelical polar-polar interactions (Adamian, and Liang, 2002), Similarly, the all residues (R18, R25, E32, K39, and H53) at position g of cGKIα1−59 are all polar residues, and most residues (947K, 954N, 961K, and 968T) at position e of MBSCT35 are also polar residues. The ionzable-polar residue pairs such as R–N (R18–954N), or K-N (K39–954N), or H–T (H53–968T) between cGKIα1−59 and MBSCT35 may also be formed to contribute the interhelical polar-polar interactions.

Such ensemble of the wenxiang diagrams suggests that most affected residues of cGKIα1−59 and MBSCT35 might be at positions d, a, e and g of these two LZCC structures. This prediction has been supported by the previous NMR data (Lincoln, 1994; Sharma et al., 2008).

NMR spectroscopy is the preferred method for characterizing the structural details of protein–protein and protein-ligand interaction interfaces following complex formation in an aqueous environment (Chou et al., 1997). Our 15N-1H-HSQC titration data in which unlabeled MBSCT42 (not observable by 15N-1H-HSQC experiment) was titrated into 2H/13C/15N-labeled cGKIα1−59 support an interaction between cGKIα1−59 and MBSCT42, which is illustrated by the changes in chemical shifts of the 5 residues (Leu36, Lys37, Leu40, Ile54, and Gly55) as well as a significant decrease in peak intensity of the 8 residues (Leu22, Ile33, Leu36, Lys37, Lys39 , Leu40, Cys43, and Gln44) of cGKIα1−59 (Lincoln, 1994). Taken together, these data identify that these 10 residues (Leu22, Ile 33, Leu36, Lys37, Lys 39, Leu40, Cys43, Gln44, Ile54, and Gly55) of cGKIα1−59 are most affected by the interaction with MBSCT42 (Lincoln, 1994). The seven residues of them are hydrophobic and only three for hydrophilic residues (Lys 37, Lys 39 and Gln44). Among these 10 residues, the three residues (I33, L40 and I54) at position a, the three residues (L22, L36 and C43) at position d, the two residues (K37 and Q44) at position e, the one residue (K39) at the position g, and the one residue (G55) at position b of cGKIα1−59 are most affected residues by the interaction with MBSCT42 (Lincoln, 1994). Thus, the previous NMR experimental data have supported above prediction based on above combined wensiang diagrams that most affected residues of cGKIα1−59 by the interaction with MBSCT35 might be at positions d, a, e and g of these two LZCC structures.

In addition, the residues which are localized at positions c and f (particularly for f) region of cGKIα1−59, are far away from the all position regions of MBSCT35 according to the wenxiang diagrams of cGKIα1−59. ·MBSCT35 complex. This means that most residues at position c and f regions are more exposed the outer of the complex, or exposed to the aqueous environment that surrounds the protein complex. These results suggest that the interactions between cGKIα1−59 and MBSCT35 come from the contribution of their hydrophobic residues at positions a and d, as well as the hydrophilic residues at position g.

Interestingly, these residues are localized on the same side of the helix or close to each other in the wenxiang diagram (Fig. 7). These characteristics further support our previous suggestion that these 10 residues are reasonably either within close proximity to and/or involved in the formation of the interaction interface (Lincoln, 1994). Based on similar data obtained in studies of F-actin interacting with skeletal myosin light chain 1 and ubiquitin hydrolase interacting with its native substrate, it is likely that these residues are within the interaction interface between these proteins although additional experiments evaluating the on/off rate of the interaction may are necessary (Naik et al., 2001, Sakamoto et al., 2005, Hutchings et al., 2003).

As has been previously identified for the GCN4 LZ, upon tetramer formation (pairing of two dimers) there is an increase in packing density within the intermolecular interface, which results in additional perturbation of residues within the a, d, e, b and g positions that is unlike the typical packing of the a and d side chains of isolated helices (Harbury et al., 1994). Interestingly several of the residues of cGKIα1−59 that are conformationally perturbed following the addition of MBS are within a, d, e, g and b positions of the repeating heptads layer (Fig. 7). In addition, the previous studies about the GCN4 tetramer LZ, an isoleucine zipper trimer of GCN4, the asialoglycoprotein receptor heterotetramer, and an extensive study of Crick’s knobs-into-holes packing for 3-, 4- and 5-stranded structures have provided additional geometric, conformational packing information for generating this model (Harbury et al., 1993, Harbury et al., 1994;1995; Walshaw and Woolfson, 2003). Moreover, in the crystal structure of the GCN4 trimer and tetramer the authors observe significant packing and numerous interactions between residues in the a/d hydrophobic interface and the hydrophilic residues at positions e and g, which and our cGKIα1−59. ·MBSCT42 complex wenxiang diagrams may be consistent and helpful for a better understanding the packing mechanism that present between the cGKIα1−59 LZCC domain and MBSCT42 LZCC motif.

Conclusion

Although many leucine zipper domains of proteins have been characterized by a heptad repeat of leucine residues in a helical wheel (Jones et al., 1992), the information thus provided about the α-helices and their lengths are very limited. If the helical wheel diagrams of a LZCC structure are used to represent two α-helices longer than 20 residues, they must crowd into a very limited space or overlap with each another, and will be difficult to be distincted from each other. It is particularly difficult to represent a LZCC-LZCC tetramer complex using four helical wheel diagrams. In contrast, the wenxiang diagrams can be used to represent two α-helixes regardless how long they are. Furthermore, wenxiang diagrams of LZCC structure can provide much more information about the physico-chemical features of the constituent amino acids as well as their distribution even for four-alpha-helix bundle with heptad repeat residues. Furthermore, our results also suggest that the wenxiang diagrams of a LZCC structure can be used to identify the key residues that play the most important role for its interaction with another LZCC protein. It is demonstrated through this study that wenxiang diagram holds a great potential in providing more information about each of the constituent amino acid residues in a heptads’ repeat. Incorporated with NMR or other biological/biophysical experimental results, wenxiang diagram may provide useful insights into the 3D structural model of LZCC protein and the interaction mechanism between LZCC and LZCC proteins.