Electrost
atic inter
actions between the thrombin
anion-binding exosite-I (ABE-I)
and the hirudinC-termin
al t
ail pl
ay
an import
ant role in the form
ation of the thrombin-hirudin inhibitor complex
andserves
as
a model for the inter
actions of thrombin with its m
any other lig
ands. The role of e
ach solventexposed b
asic residue in ABE-I (Arg
35, Lys
36, Arg
67, Arg
73, Arg
75, Arg
77a, Lys
81, Lys
109, Lys
110,
and Lys
149e)in electrost
atic steering
and ionic tethering in the form
ation of thrombin-hirudin inhibitor complexesw
as explored by site directed mut
agenesis. The contribution to the binding energy (
![](/isubscribe/journ<font color=)
als/bich
aw/40/i16/eqn/bi0023549e10001.gif">) by e
ach residuev
aried from 1.9 kJ mol
-1 (Lys
110) to 15.3 kJ mol
-1 (Arg
73)
and were in gener
al
agreement to their observedinter
actions with hirudin residues in the thrombin-hirudin cryst
al structure [Rydel, T. J.,
Tulinsky, A.,Bode, W.,
and Huber, R. (1991)
J. Mol. Biol. 221, 583-601]. Coupling energies (
![](/isubscribe/journ<font color=)
als/bich
aw/40/i16/eqn/bi0023549e10002.gif">) werec
alcul
ated for the m
ajor ion-p
air inter
actions involved in ionic tethering using complement
ary hirudinmut
ants (h-D55N, h-E57Q,
and h-E58Q). Cooper
ativity w
as seen for the h-Asp
55/Arg
73 ion p
air (
![](/isubscribe/journ<font color=)
als/bich
aw/40/i16/eqn/bi0023549e10003.gif">2.4 kJ mol
-1); however, low coupling energies for h-Asp
55/Lys
149e (
![](/isubscribe/journ<font color=)
als/bich
aw/40/i16/eqn/bi0023549e10004.gif"> 0.6 kJ mol
-1)
and h-Glu
58/Arg
77a (
![](/isubscribe/journ<font color=)
als/bich
aw/40/i16/eqn/bi0023549e10005.gif"> 0.9 kJ mol
-1) suggest these
are not m
ajor inter
actions,
as
anticip
ated by the cryst
alstructure. Interestingly, high coupling energies were seen for the intermolecul
ar ion-p
air h-Glu
57/Arg
75 (
![](/isubscribe/journ<font color=)
als/bich
aw/40/i16/eqn/bi0023549e10006.gif"> 2.3 kJ mol
-1)
and for the solvent bridge h-Glu
57/Arg
77a (
![](/isubscribe/journ<font color=)
als/bich
aw/40/i16/eqn/bi0023549e10007.gif"> 2.7 kJ mol
-1) indic
ating th
ath-Glu
57 inter
acts directly with both Arg
75 and Arg
77a in the thrombin-hirudin inhibitor complex. Therem
aining ABE-I residues th
at do not form m
ajor cont
acts in tethering the C-termin
al t
ail of hirudinm
ake sm
all but collectively import
ant contributions to the over
all positive electrost
atic field gener
ated byABE-I import
ant in electrost
atic steering.