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PDBsum entry 1qa2
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Viral protein/receptor
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PDB id
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1qa2
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References listed in PDB file
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Key reference
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Title
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Mutations improving the folding of phage p22 tailspike protein affect its receptor binding activity.
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Authors
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U.Baxa,
S.Steinbacher,
A.Weintraub,
R.Huber,
R.Seckler.
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Ref.
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J Mol Biol, 1999,
293,
693-701.
[DOI no: ]
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PubMed id
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Abstract
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Four previously isolated mutations in Salmonella phage P22 tailspike protein
were used to study the relationship between protein stability, folding, and
function. Tailspike protein binds and hydrolyzes the repetitive O-antigen
structure in Salmonella lipopolysaccharide. Four mutations (V331G, V331A, A334V,
A334I) are known to increase the folding efficiency, and two of them (at
position 331) also increase the thermal stability of the protein.
Octasaccharides comprising two repeating units of the O-antigens from two
different Salmonella strains were employed to analyze the receptor binding
function of the mutant proteins. Their endorhamnosidase enzymatic activity was
assayed with the aid of a fluorescence-labeled dodecasaccharide. Both V331A and
V331G were found to strongly affect O-antigen binding. Octasaccharide binding
affinities of the mutant proteins are reduced tenfold and 200-fold,
corresponding to a loss of 17% and 36% of the standard free energy of binding,
respectively. Both mutations at position 334 affected O-antigen binding only
slightly (DeltaDeltaG(0)B approximately 1 kJ/mol), but these mutations reduce
the thermal stability of the protein. The observed effects on the
endoglycosidase activity are fully explained by the changes in substrate
binding, suggesting that neither of the mutations affect the catalytic rate.
Crystal structures of all four mutants were determined to a resolution of 2.0 A.
Except for the partly or completely missing side-chain, no significant changes
compared to the wild-type protein structure were found for the mutants at
position 331, whereas a small but significant backbone displacement around the
mutation site in A334V and A334I may explain the observed thermal
destabilization.
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Figure 1.
Figure 1. Crystal structure of the
tailspike protein lacking the head-
binding domain complexed with
O-antigen octasaccharide. Stereo
diagrams of a side view (top) and
a cross-section through the b-
helices of the trimer (bottom) are
depicted. The binding site is shown
with octasaccharide only in one
subunit, for which only the C
a
-
trace is drawn. The other two
subunits are shown in an all atom
presentation. Note the solvent-
exposed groove in which the
saccharide is bound.
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Figure 3.
Figure 3. Superpositon of mutant structures with the wt crystal structure in the environment of the mutation site.
(a) Structure of A334I with the C
a
-trace in green and side-chains in magenta, and with the 2jF0
j - jF
C
j
electron den-
sity of residues 334 and 335 contoured at 1.5 s level in blue. The superposition of the wt backbone is shown in yel-
low with red side-chains. Note the bulging out of the backbone and the difference of Q335 in the mutant structure.
The structure of A334V is very similar to the structure of A334I shown here. (b) Superposition of wt (blue), V331A
(yellow), and V331G (red). Only residues near the mutation site are shown. The Figures were made using FRODO
(Jones et al., 1978) and MOLMOL (Koradi et al., 1996), respectively.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(1999,
293,
693-701)
copyright 1999.
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