 |
PDBsum entry 1swp
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Biotin-binding protein
|
PDB id
|
|
|
|
1swp
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
Contents |
 |
|
|
|
|
|
|
|
|
|
|
|
|
* Residue conservation analysis
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
Structural studies of binding site tryptophan mutants in the high-Affinity streptavidin-Biotin complex.
|
|
|
Authors
|
|
S.Freitag,
I.Le trong,
A.Chilkoti,
L.A.Klumb,
P.S.Stayton,
R.E.Stenkamp.
|
|
|
Ref.
|
|
J Mol Biol, 1998,
279,
211-221.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
Previous thermodynamic and computational studies have pointed to the important
energetic role of aromatic contacts in generating the exceptional binding free
energy of streptavidin-biotin association. We report here the crystallographic
characterization of single site tryptophan mutants in investigating structural
consequences of alterations in these aromatic contacts. Four tryptophan
residues, Trp79, Trp92, Trp108 and Trp120, play an important role in the
hydrophobic binding contributions, which along with a hydrogen bonding network
and a flexible binding loop give rise to tight ligand binding (Ka approximately
10(13) M-1). The crystal structures of ligand-free and biotin-bound mutants,
W79F, W108F, W120F and W120A, in the resolution range from 1.9 to 2.3 A were
determined. Nine data sets for these four different mutants were collected, and
structural models were refined to R-values ranging from 0.15 to 0.20. The major
question addressed here is how these mutations influence the streptavidin
binding site and in particular how they affect the binding mode of biotin in the
complex. The overall folding of streptavidin was not significantly altered in
any of the tryptophan mutants. With one exception, only minor deviations in the
unbound structures were observed. In one crystal form of unbound W79F, there is
a coupled shift in the side-chains of Phe29 and Tyr43 toward the mutation site,
although in a different crystal form these shifts are not observed. In the bound
structures, the orientation of biotin in the binding pocket was not
significantly altered in the mutant complex. Compared with the wild-type
streptavidin-biotin complex, there were no additional crystallographic water
molecules observed for any of the mutants in the binding pocket. These
structural studies thus suggest that the thermodynamic alterations can be
attributed to the local alterations in binding residue composition, rather than
a rearrangement of binding site architectures.
|
|
|
|
|
|
|
|
Figure 2.
Figure 2. Superposition of the
wild-type structure (grey) on a
|Fo|
-
|Fc| omit map (s
=
2.5)
and the refined new residue (black)
in the region of (a) W79F and (b)
W120A in one of the subunits.
|
|
Figure 3.
Figure 3. Least-squares fits of the biotin-bound and unbound mutant structures on the wild-type streptavidin.biotin
complex (blue); (a) W79F[MONO-1] (green), W79F (brown), W79F
+
biotin (red); (b) W108F (green), W108F
+
biotin
(red); (c) W120F (green), W120F
+
biotin (red); (d) W120A (green), W120A
+
biotin (red).
|
|
|
|
|
|
The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(1998,
279,
211-221)
copyright 1998.
|
|
|
Secondary reference #1
|
|
|
Title
|
|
Thermodynamic and structural consequences of flexible loop deletion by circular permutation in the streptavidin-Biotin system.
|
|
|
Authors
|
|
V.Chu,
S.Freitag,
I.Le trong,
R.E.Stenkamp,
P.S.Stayton.
|
|
|
Ref.
|
|
Protein Sci, 1998,
7,
848-859.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Figure 3.
Fig. 3. A: MOLSCRIPT (Kraulis, 991)stereoviewofthetetramericcircularlypermutedstreptavidin CP51/46. subunits2(green)
and3(cyan),theengineeredloopsextendawayfromtheglobulr structure and are stabilized by crystal packinginteractions.
B: MOLSCRIPTstereoview o themutant-biotincomplex. n subunit 3 (cyan)thenewconnectingloopadoptsadifferentconfor-
mationmoreintegratedinthe p barrelstructureand also stabilized y crystal packin.
|
|
Figure 8.
ig. 8. Superposition of thestreptavidinCP51/46unboundstructure(blue)andteCP51/46-biotincomplex(red)intheregion of the
iningsite (for ubunit 1 inthetetramer). A: Thetryptophan sidechainsthatmediatehydrophobicinteractionswithbitishowonly
inor deviatinsfromtheunboundstructure. B: Hydrogenbondinginteractionswithbiotin.
|
|
|
|
|
|
The above figures are
reproduced from the cited reference
which is an Open Access publication published by the Protein Society
|
|
|
Secondary reference #2
|
|
|
Title
|
|
Structural studies of the streptavidin binding loop.
|
|
|
Authors
|
|
S.Freitag,
I.Le trong,
L.Klumb,
P.S.Stayton,
R.E.Stenkamp.
|
|
|
Ref.
|
|
Protein Sci, 1997,
6,
1157-1166.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Figure 2.
Fig. 2. he four observed crystal forms of ligand-free (top) and biotin-
bound (bottom) streptavidin are depicted schematically to illustrate the
behavior f [he loop (residues 45 S2) relative lo the biotin
binding site. The circles represent the streptavidin tetramer subunits with
the binding sites (missing ectors). I and 2 . and 3 and 4. respec-
tively, build the dimer pairs. Subunit 1 and 4. and 2 and 3, respectively,
donate rp 120 to each others binding site. The curved lines over the
binding sites trace the loop with dotted lines representing
disordered conformations. Triangles in the binding sites symbolize biotin.
|
|
Figure 3.
Fig. 3. Ca representation of asuperposition of thebindinglooprgion in subunit 2 of structure4II(ligand free) on subunit 2
structure (biotin bound). Thi plotillustratestherelativeopen(red,unbound)and closed black,biotin-bound)conformations of te
inding loops.
|
|
|
|
|
|
The above figures are
reproduced from the cited reference
which is an Open Access publication published by the Protein Society
|
|
|
|
|
|
|
