The N-terminal cavity of COMPcc is able to bind different single fatty acid molecules, with their charged carboxylate head group oriented towards the Gln54 ring system and the methylene tail oriented towards the N-terminus. The ability of COMPcc to bind various fatty acid molecules is directly related to its physicochemical properties. A key role in the electrostatic fixation of polarized ligands inside the aliphatic channel is played by the Gln54 ring system. The Gln54 residue belongs to a four amino acid motif (QVKE) that is conserved among the pentameric thrombospondins (TSP-3, TSP-4, and COMP) 
. Gln54 is situated at position d
of the characteristic heptad repeat (a−g
, which is unusual, since the a
positions are normally occupied by hydrophobic residues. The hydrogen bonds of the Gln54 ring are arranged into a funnel-like manner, such that the partial positive charges on the amide nitrogens are oriented towards the bottom of the funnel and the partial negative charges on the carbonyl oxygens towards the top. This creates a dipole, which is parallel to the dipole moment of the α-helices. The positively-charged bottom of the funnel can act as a trap for negatively-charged ions, as demonstrated in the native structure of COMPcc where a chloride ion is bound 
. Interestingly, it was shown that the melting point of COMPcc was increased from 73°C to 104°C when Gln54 was mutated to a Leu residue 
. This implies an evolutionary advantage of the less thermostable wild type COMPcc over the Q54L mutant and suggests an additional function of the glutamine residues inside the pentameric channel. This decrease in thermal stability can be compensated by ligand binding: the midpoint transition temperature (Tm
) of unfolding increased by 2°C with benzene or cyclohexane bound in the channel, by 8°C when vitamin D3
and by 10°C with 18:1 trans-
9 elaidic acid 
Two additional core residues, all at the d
position of the heptad repeat play a crucial role in the binding of diverse cargo elements (). Firstly, Met33 at the N-terminal opening of the COMPcc channel forms a gating pore with a diameter of 3.4 Å. The CH3
-moieties face each other and establish strong van der Waals contact forces (). In contrast the polarizable sulphur components of the thioether are oriented towards the inner core of the pentameric channel. Therefore, one can assume that in order for any ligand to enter the COMPcc channel, the gate has to open thereby permitting access. This assumption is underlined by changes within the helical backbone at the very N-terminus (data not shown). Secondly, Thr40, a subsequent residue in the next heptad repeat, forms interhelical hydrogen bonds between its β-hydroxyl group and the amide group of Asn41. Previous work has shown, that the side chains of the individual Thr40 residues undergo significant re-orientations during ligand binding 
. In addition to re-orientation, it has also been shown that between the concentric Thr40/Asn41 arrangement and Leu37, a water chamber is formed that contains up to five water molecules inside the pentameric channel (). Comparing wild type COMPcc (pdb-code 1VDF) with COMPcc in complex with vitamin D3
(pdb-code:1MZ9), myristic acid (pdb-code:3V2N), palmitic acid (pdb-code:3V2Q) and stearic acid (pdb-code:3V2P) reveals an interesting pattern (). Whereas apo-COMPcc has water molecules lined up along the full length of the channel, the complex structures only contain water in the water chamber (). An interesting result is observed in the structure of the COMPcc-palmitic acid complex. In this case, the water chamber is empty and instead a cloud of water molecules is surrounding a second bent palmitic acid ligand that is located outside the entrance to the channel (). This suggests that the release of channel waters plays a key role in facilitating the binding of fatty acids into the pentameric COMPcc channel. To summarize, our observations suggest that the core residues Met33 (gating pore), Thr40/Asn41 (water chamber) and Gln54 (electrostatic trap) are essential components for the binding of fatty acids by COMPcc.
The local environment of the aliphatic tail of the individual fatty acids is characterized by van der Waals contacts with β-branched side chains at a and d positions, pointing inside the channel of COMPcc ( and ). The binding site is fully extended, providing space for fatty acids up to ~22 Å in length (equivalent to C20:0). A careful comparisons of the crystallographic B-factors for the aliphatic tail carbons (C3 to C15) showed that they are similar in magnitude to those of the adjacent side chains of COMPcc. Whereas the methylene tail reveals an averaged B-factor of ~41 Å2, amino acid residues Leu37, Thr40, Leu44, Val47 and Leu51 show an averaged individual B-factor for their side chains of ~38 Å2. These finding suggests nearly fully occupancy of the fatty acid ligands inside the pentameric channel. However, the crystallographic studies on C16:0 at 2.2 Å resolution show a flattened electron density map for the ligand, missing the expected fine contouring for the individual CH2-groups (). This suggests that the ligand is rotating inside the channel. The role of hydrophobic interactions in the binding of nonpolar ligands to COMPcc can be assessed by analyzing how elongation of the fatty acid aliphatic chain affects the binding constant. For example, the binding data indicate that adding two carbons to myristic acid results in a decrease of ~0.26 kcal/mol in the binding energy (). This is only a fraction of ~0.8 kcal/mol, the free energy cost of hydrophobic solvation of the methyl group. A possible explanation for this smaller effect is that the binding of fatty acids to COMPcc is accompanied by a loss of conformational entropy in the aliphatic chain. In other words, when fatty acids bind to COMPcc, the aliphatic chain can not access all its conformational isomers, this entropic loss can partially cancel the gain in free energy due to the hydrophobic effect.
Because myristic acid has 14 carbon atoms, the contribution of the hydrophobic effect (this is equivalent to removing seven or eight pairs of carbon atoms) to the binding of myristic acid to COMPcc can be roughly estimated to be between 2.1 kcal/mol and 2.4 kcal/mol. From the Kd value, the free energy of binding of myristic acid to COMPcc can be estimated to be approximately 8.4 kcal/mol. This indicates that the hydrophobic effect contributes about a fourth of the interaction energy of the fatty acid binding.
It must also be emphasized that although COMPcc binding causes a loss of conformational entropy in the fatty acid ligands, the COMPcc binding pocket is still relatively flexible. This flexibility is shown by the fact that COMPcc can accommodate the unsaturated stearic, oleic and CPA molecules with only a modest change in the binding constant.
Coiled-coil proteins such as COMPcc are attractive candidates for the design of drug delivery systems 
. In this work we have studied the hydrophobic binding pocket of COMPcc and have characterized the various interactions that play an important role in the binding of hydrophobic ligands to the protein. The following is a summary of our findings:
- The COMPcc channel has been shown to be very flexible and this work demonstrates that the protein can accommodate a wide range of ligand geometric variations in its binding pocket. We suggest that a possible reason for this flexibility is the hydration of COMPcc channel in the apo-state. The presence of internal water molecules allows the coiled-coil to participate in “breathing motions” demonstrated by the dynamic opening of the COMPcc channel to accommodate spacious molecules. This remarkable capability is most dramtically illustrated in the COMPcc - vitamin D3 complex, in which the volume of the cavities increases by approximately 30 percent upon binding of the ligand . We intend to study the role of these internal water molecules on the dynamics of the COMPcc channel in future studies.
- The water chamber as defined by the residues Thr40 and Asn41, seems to play an important role in the ligand binding process. Our results indicate that disrupting the water chamber has an adverse effect on binding. Future work will determine the role of these two residues in establishing the water chamber and elucidate the role of the water chamber in ligand binding.
- We have quantified the contribution of hydrophobicity to the ligand binding process. In the case of the studied fatty acids, only approximately a fourth of the binding free energy is contributed by the hydrophobic effect and the rest is mostly due to interactions between the carboxylate head group and the Gln54 ring system. COMPcc is an attractive candidate for the design of a Carrier-Pathfinder-System . It combines unique storage properties for otherwise insoluble signalling molecules with the possibility that a targeting molecule can be attached in order to direct it to a specific location for delivery of a target cargo , .