• About COILCHECK+
  • Background
  • Methodology
  • Citation

About COILCHECK+ server

COILCHECK + is a webserver that can be used to measure the strength of interactions between helices involved in coiled-coils. The interactions are measured using standard energy calculations involving non-bonded and electrostatic interactions and for the presence of hydrogen bonds and salt bridges. The sum of these interactions is expressed as psuedoenergy, whose ranges have been standardized using known structural entries that contain coiled coils. Such an energy measure is quite useful for assessing the energy of dimers that are likely to form coiled-coils, can help to rationalise point mutations and can also help to design experiments.

Features

COILCHECK+ being an updated version of COILCHECK program there are few features that are new and interesting.

H — bond fixing and H — bond energy calculations:
The method utilized for fixing hydrogens is as delineated here. The position of hydrogen with respect to the connecting atom has been determined/fixed geometrically using standard bond lengths, angles and torsion angles for all types of atoms, methyl, methylene, tertiary groups and considering sp3, sp2, sp atomic states of hybridization following the published method (M. Nardelli.,1982). The hydrogen bond energies for a given system is calculated using the Kabsch & Sander's equation as given in DSSP paper (Kabsch et at 1983). Refer methodology page for details on H-bond energy equation.
Electrostatic energy calculations:
The electrostatic interaction energy of the system is calculated using Coulomb's equation. Instead of a constant dielectric we had used the distant dependent dielectric (DDD) constant in the calculation of electrostatic energies. The current usage of DDD constant would yeild an appropriate energy value depending upon the effect of interaction between the residues involved. The application of this improvised method of electrostatic energy calculation has shown considerable changes in the electrostic energy component. Refer methodology page for details on the electrostatic energy equations and the various parameters been used.
van der Waals energy calculations:
The van der Waals energy component includes the hydrophobic interactions and the energy contributed by the van der Waals pairs. Here we fix the hydrogens to the given system and then use the H-fixed structure for the calculation of energy. Due to the fixing of hydrogen atoms the van der Waals pair would increase in number, leading to a change in this energy component when compared with the earlier version.
Standardization:
Thus the updated method COILCHECK+ would show changes in all three energy components. Hence the normalized Energy/residue values had been standardized using new structural entries and ranges of stabilizing and destabilizing zones are identified. This method will act as a promising tool in recognizing the stability of a coiled coil dimer and validate the structure based on the energy parameter.

Background

Coiled coils (Pauling and Corey, 1953) are structural motifs found in a diverse array of proteins, from structural proteins such as collagen, keratin and myosin, which are the main components of bone, skin and hair, to transcription factors such as Jun and Fos, involved in cell growth and proliferation (Burkhard et al., 2001; Lupas, 1996). Sequence based predictions suggest that 3-4% of all protein residues form coiled coils. They consist of two or more right-handed amphipathic alpha helices, wrapped around each other in a parallel or an antiparallel orientation to form a left-handed supercoil (Fig. 1). They exist as homooligomers or heterooligomers. However, there are also single chains versions, wherein consecutive helices from the same polypeptide chain form an antiparallel coiled coil (Fig. 1c).


Figure 1: Gallery of coiled coil proteins. a) Parallel: dimerisation domain of transcription factor GCN4 bound to DNA (PDB: 1YSA). b) Antiparallel: oligomerisation domain of hepatitis delta antigen (PDB: 1A92). c) Single chain antiparallel: coiled coil region of apolipoprotein (PDB: 1BZ4).

In coiled coils the right-handed alpha helices are given a left-handed twist, thereby reducing the periodicity from 3.6 to 3.5 residues with respect to the supercoil axis. This results in a seven-residue periodicity (the heptad repeat), allowing residues to occupy an equivalent position on the helix surface after two turns (or 7 residues). The seven structural positions are denoted by: a, b, c, d, e, f, and g. The positions a and d form the dimerisation interface between the helices and are occupied by hydrophobic (apolar) amino acids, whereas e and g are solvent-exposed, polar residues that give specificity between the two helices through electrostatic interactions.

Knobs—into—holes packing
Coiled coils are characterized by a distinctive knobs—into—holes packing (Crick, 1952; Crick, 1953), in which a residue from one helix (knob) packs into the cavity formed by four side chains of the facing helix (hole). Hydrophobic side chains on one helix fits into a diamond of four side chains (i - 3, i, i + 1, i + 4) on the surface of the other helix. This geometry contrasts with the packing of helices in globular protein, referred to as ridges—into—grooves (Chothia et al., 1977), in which a residue packs above or beneath the equivalent residue from the facing helix.
Figure 2: Knobs-into-holes packing shown in a parallel coiled coil (1KQL). a packs into the hole formed by the side chains of residues at positions d-1, g-1, a, and d; d packs into the hole formed by a, d, e, a+1.

In parallel coiled coils residues at position a pack against those at a and d against d on the opposite strand (Fig. 3a). This leads to two geometrically distinct layers within the hydrophobic core. However, in anti-parallel coiled coils a residues pack against d and d residues against a residues (Fig. 3b); hence forming a single layer in the hydrophobic core.
Figure 3: Front and top views showing the packing of residues at a and d positions in parallel and antiparallel coiled coils; a residues are colored in blue and d in red. (a) Tropomyosin (1KQL): parallel dimer (b) Oligomerisation domain of hepatitis delta antigen (1A92): antiparallel oligomer.

References

Burkhard, P., Stetefeld, J. and Strelkov, S.V. (2001) Coiled coils: a highly versatile protein folding motif. Trends Cell Biol, 11, 82-88.
Chothia, C., Levitt, M. and Richardson, D. (1977) Structure of proteins: packing of alpha-helices and pleated sheets. Proc Natl Acad Sci U S A, 74, 4130-4134.
Crick, F.H. (1952) Is alpha-keratin a coiled coil? Nature, 170, 882-883.
Crick, F.H.C. (1953) The packing of alpha-helices: simple coiled-coils. Acta Crystallographica, 6, 689-697.
Lupas, A. (1996) Coiled coils: new structures and new functions. Trends Biochem Sci, 21, 375-382.
Pauling, L. and Corey, R.B. (1953) Compound helical configurations of polypeptide chains: structure of proteins of the alpha-keratin type. Nature, 171, 59-61.

Methodology

The key properties that provide stability to a coiled coil are helical propensity, hydrophobicity of the core, and electrostatic interactions. The hydrophobic residues at positions a and d exist on the same face of the helix (Fig. 1b), creating a hydrophobic interface between the two helices and providing the major force in stabilizing of the coiled coil. Interhelical surface electrostatic interactions (Fig. 1c) also contribute to the stability of coiled coils. In comparison to hydrophobic interactions, interhelical electrostatic interactions on the surface of a protein are usually weak and contribute little to protein stability. However, a large number of these interactions can contribute substantially to the stability of coiled coils. In some coiled coils polar residues frequent the dimer interface and in such cases the coiled coil is stabilized by interhelical hydrogen bonds. Hydrogen bonds also provide stability through intrahelical i to i + 4 bonding (Fig. 1a).

Figure 1: Illustration of noncovalent interactions in Tropomyosin (1KQL). (a) Hydrogen bonds (b) Hydrophobic interactions: residues at positions a are colored in blue and d in red. (c) Favorable electrostatic interaction: basic amino acids are shown in yellow and acidic in green. (d) Unfavorable electrostatic interactions.

We have developed a method for accessing the strength of interactions between two helices involved in coiled-coils on the basis of non-covalent interactions. The energies, which stabilize a coiled coil dimer, can be grouped into the following three classes:
 •Intra— and interchain Hydrogen bonding energy — Ehyd
 •Interchain Van der Waals interactions — Evw (hydrophobic interactions and short contacts)
 •Interchain electrostatic interactions — Eele (favorable and unfavorable)

For any given coiled coil, we calculate the energy values for the above listed interactions and sum them up to obtain the total interprotomer stabilizing energy (Et), which is divided by the total number of residues to obtain energy per residue (Ent).

Figure 2: Flowchart

Hydrogen bonds
The hydrogen atoms were fixed geometrically (M.Nardelli.,1982) and the Hydrogen bond energy are calculated using the following equation:
E = q1q2 [1/r(ON) + 1/r(CH) - 1/r(OH) - 1/r(CN)] * 332 * 4.184
Where q1 and q2 are partial atomic charges, r( ) is the inter-atomic distance between the corresponding atoms. The values for different parameters are as in DSSP (Kabsch et al., 1983).
Van der Waals interactions
The Van der Waals interaction energies are calculated using the following equation:
E = 4.184(EiEj)*[((Ri + Rj)/r)12 - 2((Ri + Rj)/r)6] kJ/mol
ATOM R E
H 1.2 0.02
O 1.4 0.20
N 1.5 0.16
C 1.7 0.12
S 1.8 0.20
P 1.9 0.20
Where R is the Van der Waals radius for an atom, E is the Van der Waals well depth, r is the distance between the atoms. All interprotomer atomic pairs 7 Å or less apart are considered for energy calculation. The Van der Waals interaction accounts for hydrophobic interactions and also for shorts contacts (Ramachandran & Sasisekaran, 1968 and Novotny et al., 1997).
Electrostatic interactions
The energy of electrostatic interaction energies are calculated according to the Coulomb's law. All interprotomer charged atomic pairs 18 Å or less apart have been considered for energy calculation.
E = (4.184*332*q1*q2)/(D*r) kJ/mol
Amino Acid Atomic Charge Atomic
ASP OD1=-0.76 OD2=-0.76
GLU OE1=-0.76 OE2=-0.76
ARG NH1=0.80 NH1=0.80
LYS NZ=0.30
HIS ND1=0.38 NE2=0.57
Where q1 and q2 are the partial atomic charges, r is the distance between the atoms and D is the dielectric constant of the medium, distance dependent diletric (DDD, where D=2r) is been used for the energy calculation. The values for the different parameters are as in CHARMM (Brooks et al., 1983 & Brooks et al., 2009).
Short contacts
Considered all non-bonded interprotomer atomic pairs.
D = r - (R - 0.40)
where R is the sum of the Van der Waals radii of the two atoms and r is the distance between the atoms.If D < 0, the atomic interaction is marked as a short contact as described in Ramachandran et al., 1963 and Lomize et al., 2002.
Hydrophobic interactions
All interprotomer interactions between hydrophobic amino acids (ALA, LEU, ILE, VAL, TRY, TYR, PHE) with CB-CB distance equal to or less than 7 Å have been considered and reported.
Salt Bridges and charge interactions
All interprotomer charged amino acid residue pairs with charged atomic pair distance equal to or less than 18Å have been considered. Both types of interactions, favourable (opposite charges) as well as unfavorable (similar) electrostatic interactions, within this distance limit, are reported. A salt bridge is formed if the side-chain nitrogen and oxygen atoms of two oppositely charged residues are within a 4Å distance.

References

Brooks, B.R., Bruccoleri, R.E., Olafson, B.D., States, D.J., Swaminathan, S. and Karplus, M. (1983) CHARMM: A Program for Macromolecular Energy, Minimization, and Dynamics Calculations, J. Comp. Chem. 4, 187-217.

M. Nardelli (1982) A calculation program for calculating hydrogen atom coordinates. Computers and Chemistry. 6,3,139-152.

Kabsch, W. and Sander, C. (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 22, 2577-637.

Lomize, A.L., Reibarkh, M.Y. and Pogozheva, I.D. (2002) Interatomic potentials and solvation parameters from protein engineering data for buried residues. Protein Sci., 11, 19842000.

K. Mizuguchi, C.M. Deane, T.L. Blundell, M.S. Johnson and J.P. Overington. (1998) JOY: protein sequence-structure representation and analysis. Bioinformatics 14, 617-623.

Novotny,J., Bruccoleri,R.E., Davis,M. and Sharp,K.A. ( (1997) ) Empirical free energy calculations: a blind test and further improvements to the method. J. Mol. Biol., 268, 401411.

Ramchandran, G.N., Ramakrishnan, C. and Sasisekharan, V. (1963) Stereochemistry of polypeptide chain configurations. J Mol Biol. 7, 95-9.
Ramachandran, G.N. and Sasisekharan, V. (1968). Conformation of polypeptides and proteins. Adv Protein Chem. 23, 283-438.
Brooks BR, Brooks CL 3rd, Mackerell AD Jr, Nilsson L, Petrella RJ, Roux B, Won Y, Archontis G, Bartels C, Boresch S, Caflisch A, Caves L, Cui Q, Dinner AR, Feig M, Fischer S, Gao J, Hodoscek M, Im W, Kuczera K, Lazaridis T, Ma J, Ovchinnikov V, Paci E, Pastor RW, Post CB, Pu JZ, Schaefer M, Tidor B, Venable RM, Woodcock HL, Wu X, Yang W, York DM and Karplus M. (2009) CHARMM: the biomolecular simulation program. J Comput Chem. 30, 1545-614.

Darrin M York, Thomas A Darden and L G Pedersen. (1993) The effect of long-range electrostatic interactions in simulations of macromolecular crystals - a Comparison of the Ewald and truncated list methods. Journal of Chemical Physic. 99, 8345-8348.

Eyal Neria, Stefan Fischer and Martin Karplus. (1996) Simulation of activation free energies in molecular systems. J. Chem. Phys. 105, 1902-1921.

Sagui C and Darden TA. (1999) Molecular dynamics simulations of biomolecules: long-range electrostatic effects. Annu Rev Biophys Biomol Struct. 28, 155-79.

Annick Thomas, Alain Milon and Robert Brasseur. (2004) Partial atomic charges of amino acids in proteins. Proteins: Structure, Function, and Bioinformatics. 56, 102-109.

Citing Coilcheck+ & Coilcheck+ Credits

In citing Coilcheck server please refer to
—Alva V, Syamala Devi DP, Sowdhamini R.
COILCHECK: an interactive server for the analysis of interface regions in coiled coils.
Protein Pept Lett. 2008;15(1):33-8. PMID: 18221010

In citing Coilcheck+ server (an updated version of Coilcheck) please refer to
—Margaret S Sunitha, Anu G Nair, Amol Charya, Kamalakar Jadhav, Sami Mukhopadhyay and Ramanathan Sowdhamini
Structural attributes for the recognition of weak and anomalous regions in coiled-coils of myosins and other motor proteins.
BMC Research Notes 2012;5(1):530.

Acknowledgements
— This study was supported by grants from the Human Frontier Science Program (HFSP).
— We would like to thank AnuNair for his technical assistance.