X-ray Structures and Analysis of 11 Cyclosporin Derivatives ...

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442 Cyclosporin Derivatives Complexed with CypA Figure 4. Conserved intermolecular hydrogen bonds between CsAanalogues and CypA. The conformation of CsA in the CypA/CsA monomeric X-ray crystal structure is shown. Oxygen and nitrogen atoms of CsA are displayed in red and blue, respectively. All the CypA residues (dark grey bonds) which form hydrogen bonds (broken lines) and close van der Waals contacts (thin continuous lines) with the bound CsA (pale grey bonds) are shown in their positions in the crystal structure. Additional hydrogen bonds are mediated by water molecules which are also shown. Distances for these interactions for the different analogues are given in Table 4. water molecules were then de®ned such that each member of a cluster lies within a given radius of the centroid of the cluster. With a radius of 0.5 AÊ , there are 22 unique clusters containing a water molecule from each of the 11 structures. Water molecules are shown in Figure 3. Those water molecules belonging to the same cluster have been labeled identically in the deposited co-ordinate ®les. All of the 22 most conserved water positions form at least three hydrogen bonds. and two of the water molecules (W30 and W27) manage to form four hydrogen bonds to cyclophilin. The 22 con- Figure 5. Stereo picture of CsA/CypA with the most conserved water molecules. CsA is coloured magenta, CypA is coloured cyan and the 11 water molecules which occur in 10 or 11 of the 11 structures discussed in this paper are shown as labelled yellow spheres. The label corresponds to the number in Table 5 and also to the label of the water molecule in the deposited PDB ®le. The position of the sphere is the average position of the water molecules from the different structures which all re®ne to within 0.2 AÊ of each other.
Cyclosporin Derivatives Complexed with CypA 443 Table 5. Intermolecular distances for the 11 most conserved water molecules W11-2 Ala38 O 2.78 W11-2 Glu43 N 3.28 W11-2 Lys44 N 2.88 W11-2 Phe46 O 2.73 W11-3 Asn102 OD1 2.88 W11-3 Val127 N 3.23 W11-3 Asp85 OD2 3.05 W11-3 Asn108 OD1 2.80 W11-4 Ser51 OG 2.90 W11-4 Cys52 O 2.60 W11-4 Gly65 N 2.78 W10-8 Phe53 O 2.86 W10-8 Ile156 N 2.85 W10-8 W01-25 O1 2.93 W10-8 W01-604 O1 2.82 W10-8 Thr152 OG1 3.01 W10-9 Leu90 N 3.18 W10-9 W11-10 O1 2.88 W10-9 Asn87 OD1 2.83 W10-9 Val128 O 2.97 W11-10 Phe88 CA 3.22 W11-10 W01-521 O1 2.86 W11-10 Val128 N 2.91 W11-10 Leu90 O 2.75 W11-10 W10-43 O1 3.10 W11-10 W01-123 O1 3.20 W11-13 Asn106 N 3.22 W11-13 Asp85 OD2 2.73 W11-13 Thr107 N 2.77 W11-13 Gly104 O 2.75 W10-19 W05-121 O1 2.68 W10-19 W01-194 O1 2.52 W10-19 W02-46 O1 2.86 W10-19 W01-698 O1 2.64 W10-19 W01-474 O1 2.87 W10-19 Asp13 OD1 2.82 W10-19 His92 O 2.90 W11-23 Glu84 O 2.83 W11-23 Glu86 N 2.97 W11-23 Thr32 OG1 2.81 W11-23 Asn108 ND2 3.03 W10-25 Ala117 O 2.85 W10-25 Gly94 O 3.06 W10-25 His92 C 3.09 W10-25 Cys115 SG 3.30 W10-25 His92 O 3.23 W10-25 His92 CA 3.28 W10-25 His92 CB 3.22 W10-43 W01-153 O1 2.26 W10-43 Gly124 N 3.11 W10-43 Leu90 O 3.20 W10-43 W11-10 O1 3.10 W10-43 His126 O 2.81 Water molecules in the 11 re®ned structures were analysed and classi®ed in clusters as described in the text. Those water molecules re®ning to a position less than 0.2 AÊ from each other form a cluster. All distances of less than 3.2 AÊ from the averaged position of the 10 or 11 water molecules and atoms of the native CypA/CsA complex are given. Water molecules are labelled Wx y where x is the number of water molecules in the cluster (either 10 or 11) and y is the label of the water molecule shown in Figure 6; it was used to label the water molecule in the deposited PDB ®le. The positions of these water molecules are shown in Figure 4. served water molecules make a total of 53 hydrogen bonds directly with the protein, of which 15 are hydrogen bonds to side-chain atoms, 20 are hydrogen bonds to backbone carbonyl atoms and 17 are hydrogen bonds to main-chain amide nitrogen atoms. In general, protein structures show a greater frequency of hydration of main-chain CˆO groups compared with NH groups by a ratio of 3:1 (Baker & Hubbard, 1984). The unusually high proportion of hydrogen bonds to amide nitrogen atoms in the 22 conserved water sites in the CypA structures is explained by the fact that these water molecules tend to be buried and are likely to play a structural role (Williams et al., 1994; Baker & Hubbard, 1984). A more stringent criterion for a water cluster was made in which the cluster radius was reduced to 0.2 AÊ . Only 11 water molecules were found in ten or all 11 of the X-ray structures and these most strongly conserved water molecules are shown in Figure 5 with intermolecular distances given in Table 5. The average temperature factor for the water molecules in the different structures is approximately twice the amplitude of the CsA ligand (see Table 2). CypA-CsA complexes with modifications in CsA at position 2 The Abu-binding pocket In the CypA/CsA complex and indeed in all derivatives with Abu in position two, the ethyl side-chain ®ts snugly into the ``Abu-pocket'' and makes between 11 and 13 van der Waals contacts (C . . . C
Page 1 and 2: Article No. mb982108 J. Mol. Biol.
Page 3 and 4: Table 2. Crystallographic data for
Page 5 and 6: Cyclosporin Derivatives Complexed w
Page 7: All short contacts less than 3.7 A
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X-ray Structures and Analysis of 11 Cyclosporin Derivatives ...

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