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

444 Cyclosporin Derivatives Complexed with CypA
Figure 6. Abu-pocket showing hydrogen bonding of conserved water molecules. Picture highlighting the conserved
water molecules W5, W6 and W7 in the binding pocket for Abu2. These water molecules are present in nine of
the complexes examined in this paper. Part of the CsA molecule is shown in magenta, water molecules are yellow
and hydrogen bonds are drawn in green. W7-Ser110 ˆ 2.75 AÊ , W7-Gly74 ˆ 3.29 AÊ , W7-Asn111 ˆ 3.13 AÊ ,
W5-Asn111 ˆ 3.12 AÊ , W5-Gly109 ˆ 2.87 AÊ , W5-W6 ˆ 3.26 AÊ , W5-Ala101 ˆ 2.70 AÊ , W6-Thr107 ˆ 2.77 AÊ , W6-
W244 ˆ 2.75 AÊ .
59 , 53 for Abu2, Val2 and Thr2, which places
Val2:C g0
and Thr2:O g in the trans and gauche conformations,
respectively. The conformation of Thr-
2 is stabilised by a strong 2.7 AÊ intramolecular
hydrogen bond from the g OH to the main-chain
carbonyl oxygen atom. This conformation is clearly
disfavoured in the Val2 structure because of steric
interaction between the C g methyl group and the
carbonyl oxygen atom. The alternative trans conformation
adopted by Val2 results in a number of
rather short van der Waals contacts to residues lining
the Abu-binding pocket. In particular there is a
short Val2/CG . . . Gln/NE2 contact of 3.56 AÊ .
There is a signi®cant effect of this repulsive
interaction on the conformation of CypA (Figure 7),
which results in a shift in position of the 70s loop
of CypA. A comparison with the native CypA/
CsA structure shows a shift in this loop which is
greater than 1.2 AÊ for each of the main-chain
atoms of residues 69 through 73. This compares
with an average displacement of less than 0.2 AÊ
for the corresponding region in all other derivative
structures. These changes, along with the presence
of the second C g group in the Abu-binding pocket,
prevent the formation of the hydrogen-bonded
water network found in the native structure
(Figure 6). An alternative water hydrogen-bonding
pattern is formed with the Val2C/S complex in
which the water molecule corresponding to W6 in
the native structure moved relative to the native
structure by more than 0.5 AÊ .
The threonine hydroxyl group from Thr2-CS
group in the Abu-binding pocket is involved in
hydrogen bonds to two water molecules, one of
which is 0.8 AÊ from the conserved W6 position.
Despite these additional hydrogen bonds and the
lack of steric clash of the Thr side-chain in the
Abu-pocket the IC50 increases by a factor of 1.4
(Table 1). This corresponds to a small reduction in
binding strength that may be explained by a loss
of entropy (rotational freedom) of the Thr sidechain.
The Val2 analogue (Figure 7) shows a sixfold
reduction in binding to CypA and a corresponding
threefold drop in biological activity in vitro. Again
the entropy loss of the Val side-chain on binding
may play a role. In addition, the distortion in the
70s loop found in the Val2/CS complex may also
provide a negative energy term for ligand binding.
The precision of the atomic positions of these structures
is about 0.2 AÊ and in many cases small
(0.5 AÊ ) movements of protein loops may be signi®cant.
The X-ray structure of the CypA/224698 complex
which was designed with a modi®cation at
position 2 to enhance speci®city for CypB-binding
(Mikol et al., 1995) shows that the hydroxypropyl
side chain penetrates deep into the Abu-pocket