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PDBsum entry 1gi5
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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A novel serine protease inhibition motif involving a multi-Centered short hydrogen bonding network at the active site.
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Authors
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B.A.Katz,
K.Elrod,
C.Luong,
M.J.Rice,
R.L.Mackman,
P.A.Sprengeler,
J.Spencer,
J.Hataye,
J.Janc,
J.Link,
J.Litvak,
R.Rai,
K.Rice,
S.Sideris,
E.Verner,
W.Young.
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Ref.
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J Mol Biol, 2001,
307,
1451-1486.
[DOI no: ]
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PubMed id
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Abstract
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We describe a new serine protease inhibition motif in which binding is mediated
by a cluster of very short hydrogen bonds (<2.3 A) at the active site. This
protease-inhibitor binding paradigm is observed at high resolution in a large
set of crystal structures of trypsin, thrombin, and urokinase-type plasminogen
activator (uPA) bound with a series of small molecule inhibitors
(2-(2-phenol)indoles and 2-(2-phenol)benzimidazoles). In each complex there are
eight enzyme-inhibitor or enzyme-water-inhibitor hydrogen bonds at the active
site, three of which are very short. These short hydrogen bonds connect a
triangle of oxygen atoms comprising O(gamma)(Ser195), a water molecule co-bound
in the oxyanion hole (H(2)O(oxy)), and the phenolate oxygen atom of the
inhibitor (O6'). Two of the other hydrogen bonds between the inhibitor and
active site of the trypsin and uPA complexes become short in the thrombin
counterparts, extending the three-centered short hydrogen-bonding array into a
tetrahedral array of atoms (three oxygen and one nitrogen) involved in short
hydrogen bonds. In the uPA complexes, the extensive hydrogen-bonding
interactions at the active site prevent the inhibitor S1 amidine from forming
direct hydrogen bonds with Asp189 because the S1 site is deeper in uPA than in
trypsin or thrombin.Ionization equilibria at the active site associated with
inhibitor binding are probed through determination and comparison of structures
over a wide range of pH (3.5 to 11.4) of thrombin complexes and of trypsin
complexes in three different crystal forms. The high-pH trypsin-inhibitor
structures suggest that His57 is protonated at pH values as high as 9.5. The
pH-dependent inhibition of trypsin, thrombin, uPA and factor Xa by
2-(2-phenol)benzimidazole analogs in which the pK(a) of the phenol group is
modulated is shown to be consistent with a binding process involving ionization
of both the inhibitor and the enzyme. These data further suggest that the pK(a)
of His57 of each protease in the unbound state in solution is about the same,
approximately 6.8. By comparing inhibition constants (K(i) values), inhibitor
solubilities, inhibitor conformational energies and corresponding structures of
short and normal hydrogen bond-mediated complexes, we have estimated the
contribution of the short hydrogen bond networks to inhibitor affinity (
approximately 1.7 kcal/mol). The structures and K(i) values associated with the
short hydrogen-bonding motif are compared with those corresponding to an
alternate, Zn(2+)-mediated inhibition motif at the active site. Structural
differences among apo-enzymes, enzyme-inhibitor and enzyme-inhibitor-Zn(2+)
complexes are discussed in the context of affinity determinants, selectivity
development, and structure-based inhibitor design.
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Figure 4.
Figure 4. Chemical structures of serine protease inhibitor
analogs of APC-1144 that (A) make or (B) do not make short
hydrogen bonds at the active site.
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Figure 6.
Figure 6. (a) Structure and (2|F[o]| - |F[c]|), a[c] map
contoured at 1.0 and 2.4s for P2[1]2[1]2[1] (form b)
trypsin-APC-1144-Zn2+, pH 8.10, 1.37 Å resolution.
Coordinate bonds to the Zn2+ are cyan, and hydrogen bonds are
white. (b) Superposition of trypsin-APC-1144-Zn2+ (standard atom
coloring scheme) onto trypsin-APC-1144 (carbon, cyan; oxygen,
orange; and nitrogen, pink) shows a considerable change in the
position and orientation of the phenol ring of the inhibitor.
Coordinate and hydrogen bonds in the former are yellow, hydrogen
bonds in the latter are cyan.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2001,
307,
1451-1486)
copyright 2001.
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