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PDBsum entry 2bdh
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References listed in PDB file
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Key reference
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Title
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Crystal structures of human tissue kallikrein 4: activity modulation by a specific zinc binding site.
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Authors
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M.Debela,
V.Magdolen,
V.Grimminger,
C.Sommerhoff,
A.Messerschmidt,
R.Huber,
R.Friedrich,
W.Bode,
P.Goettig.
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Ref.
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J Mol Biol, 2006,
362,
1094-1107.
[DOI no: ]
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PubMed id
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Abstract
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Human tissue kallikrein 4 (hK4) belongs to a 15-member family of closely related
serine proteinases. hK4 is predominantly expressed in prostate, activates
hK3/PSA, and is up-regulated in prostate and ovarian cancer. We have identified
active monomers of recombinant hK4 besides inactive oligomers in solution. hK4
crystallised in the presence of zinc, nickel, and cobalt ions in three crystal
forms containing cyclic tetramers and octamers. These structures display a novel
metal site between His25 and Glu77 that links the 70-80 loop with the N-terminal
segment. Micromolar zinc as present in prostatic fluid inhibits the enzymatic
activity of hK4 against fluorogenic substrates. In our measurements, wild-type
hK4 exhibited a zinc inhibition constant (IC50) of 16 microM including a
permanent residual activity, in contrast to the zinc-independent mutants H25A
and E77A. Since the Ile16 N terminus of wild-type hK4 becomes more accessible
for acetylating agents in the presence of zinc, we propose that zinc affects the
hK4 active site via the salt-bridge formed between the N terminus and Asp194
required for a functional active site. hK4 possesses an unusual 99-loop that
creates a groove-like acidic S2 subsite. These findings explain the observed
specificity of hK4 for the P1 to P4 substrate residues. Moreover, hK4 shows a
negatively charged surface patch, which may represent an exosite for prime-side
substrate recognition.
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Figure 3.
Figure 3. (a) Stereo view of the oligomeric architecture in
the P4[1]2[1]2 hK4-Zn form. One hK4 tetramer is located in the
asymmetric unit, highlighted by a transparent surface. This
arrangement is pseudo-symmetrical, in contrast to the
symmetrical tetramers of the P4 form. Only one zinc ion is
bound per tetramer, displayed as a pink ball with electron
density representing an anomalous Fourier map contoured at 3σ.
Molecules that are symmetry-related by a crystallographic 2-fold
axis are depicted in the same color. Both tetramers sit back to
back and contact each other with a flat interaction surface
resulting in an octameric assembly. (b) Top view of the octamer
from the P4[1]2[1]2 crystals rotated by 90° with respect to
(a). (c) Stereo representation of the asymmetric unit in the
hK4-Co P2[1] crystal form, which contains two hk4 octamers. One
cobalt ion is bound per pseudo-symmetrical tetramer, indicated
by an anomalous Fourier map.
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Figure 6.
Figure 6. Stereo view of the Ni^2+-binding site in the
70–80 loop of hK4, superimposed by the equivalent segments of
hK1 in grey. The Ni^2+ is shown as a cyan ball, bound by four
water molecules, Glu77 O^ε, and His25 N^ε2. The Ni^2+ ligand
contacts are depicted as dotted lines. The electron density
accounting for the coordinating water molecules is contoured at
1.0σ and that around nickel at 5.0σ. The most significant
difference between the compared loops is one helical turn in hK4.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2006,
362,
1094-1107)
copyright 2006.
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