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Figure 3.
Figure 3. Formation of the Asymmetric
hMukE–hMukF(M+C)–(hMukBhd^EQ–ATPγS)[2] Dimer
(A)
Structure of the asymmetric dimer in the Form II crystal. Only
one C-WHD of hMukF is bound to dimerized MukB heads. In the
inset, the 2F[o]-F[c] map calculated with the final refined
model highlights the electron density for the linker segment
preceding α1.
(B) Structural superposition. The C-WHDs of
MukF in the asymmetric dimer (red) and the symmetric dimer
(pink) in the Form I crystal are superposed. A circle highlights
that α1 in the asymmetric dimer is longer than that in the
symmetric dimer. The linker segment in the asymmetric dimer
overlaps with one MukF C-WHD in the symmetric dimer.
(C)
Detachment of hMukE–hMukF(M+C) from dimerized MukB heads in
solution. A sample containing 4 μM
hMukE–hMukF(M+C)–hMukBhd^EQ (band a) was reacted with 2 mM
ATP and visualized on a native gel. The mixture was partially
separated with a Superdex 200 column, and the chromatogram is
shown along with the size marker positions. The eluted
fractions, indicated by a double-headed arrow, were visualized
on a native and a denaturing gel, showing that bands b and c
correspond to high- and low-molecular weight species,
respectively. Band c is assigned to detached hMukE–hMukF(M+C)
according to the same band position of purified
hMukE–hMukF(M+C) (labeled as “Con”). By subtraction, band
b is assigned to hMukE–hMukF(M+C)–(hMukBhd^EQ–ATP)[2],
which was confirmed by quantification of the band intensities
for lane 1 of the denaturing gel ([hMukBhd^EQ]/[hMukE dimer] =
 vert,
similar 1.7). Accordingly, the minor species present in the
unreacted sample is identified as copurified asymmetric dimer
resulting from very low catalytic activity of hMukBhd^EQ. The
observed conversion reaction is schematically illustrated.
(D) Control experiments. Triple complexes between MukE,
MukF(M+C) and the indicated head domain of MukB were analyzed by
native gel electrophoresis. Left: Catalytically active
MukE–MukF(M+C)–MukBhd in reacting with 2 mM AMPPNP produced
the conversion products observed in C. Middle: In the presence
of 2 mM ATP, the conversion reaction was observed with the
MukBhd^EQ-containing complex, but not with the
MukBhd^SR-containing complex. Right: Conversion of the triple
complexes containing the indicated MukB head did not take place,
when incubated without ATP.
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