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We have solved, refined, and analyzed the 2.0-å resolution crystal structure of
a 1:1 complex between the bacterial ribonuclease, barnase, and a
Cys-->Ala(40,82) double mutant of its intracellular polypeptide inhibitor,
barstar. Barstar inhibits barnase by sterically blocking the active site with a
helix and adjacent loop segment. Almost half of the 14 hydrogen bonds between
barnase and barstar involve two charged residues, and a third involve one
charged partner. The electrostatic contribution to the overall binding energy is
considerably greater than for other protein-protein interactions. Consequently,
the very high rate constant for the barnase-barstar association (10(8) s-1 M-1)
is most likely due to electrostatic steering effects. The barnase active-site
residue His102 is located in a pocket on the surface of barstar, and its
hydrogen bonds with Asp39 and Gly31 residues of barstar are directly responsible
for the pH dependence of barnase-barstar binding. There is a high degree of
complementarity both of the shape and of the charge of the interacting surfaces,
but neither is perfect. The surface complementarity is slightly poorer than in
protease-inhibitor complexes but a little better than in antibody-antigen
interactions. However, since the burial of solvent in the barnase-barstar
interface improves the fit significantly by filling in the majority of gaps, as
well as stabilizing unfavorable electrostatic interactions, its role seems to be
more important than in other protein-protein complexes. The electrostatic
interactions between barnase and barstar are very similar to those between
barnase and the tetranucleotide d(CGAC). In the barnase-barstar complex, the two
phosphate-binding sites in the barnase active site are occupied by Asp39 and
Gly43 of barstar. However, barstar has no equivalent for a guanine base of an
RNA substrate, resulting in the occupation of the guanine recognition site in
the barnase-barstar complex by nine ordered water molecules. Upon
barnase-barstar binding, entropy losses resulting from the immobilization of
segments of the protein chain and the energetic costs of conformational changes
are minimized due to the essentially preformed active site of barnase. However,
a certain degree of flexibility within the barnase active site is required to
allow for the structural differences between barnase-barstar binding and
barnase-RNA binding. A comparison between the bound and the free barstar
structure shows that the overall structural response to barnase-binding is
significant. This response can be best described as outwardly oriented,
rigid-body movements of the four alpha-helices of barstar, resulting in the
structure of bound barstar being somewhat expanded.
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