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In a paper published Nov. 24, 2005 online in the
journal Biochemistry, members of the interdisciplinary collaboration
described how they discovered the probable orientation required for a
Cdc25B phosphatase enzyme to "dock" with and activate a
cyclin-dependent kinase protein complex that also functions as an
enzyme, known as Cdk2-pTpY--CycA. The work was funded by the National
Institutes of Health.
Detailed study of such docking is important because
uncontrolled overreaction of the Cdc25 family of enzymes has been
associated with the development of various cancers. Anti-cancer drugs
that jam the enzyme, preventing its docking with the kinase, could
halt cell over proliferation to treat such cancers. However,
developing such drugs has been hampered by lack of detailed
understanding of how the Cdc25s fit with their associated kinases.
"To me this is the culmination of my six years here
at Duke," said Johannes Rudolph, the Duke assistant professor of
chemistry and biochemistry who led the research. "It's very exciting.
I think it's a really hard problem."
A successful docking between the two enzymes not
only requires the "active sites" -- where chemical reactions occur
--on the phosphatase and the kinase to link precisely, Rudolph said.
The two molecules' component parts, or "residues," must also orient in
a tongue-and-groove fit at a few other special places, which the
researchers dubbed 'hot spots," on the irregular molecular surfaces.
Only when active sites and hot spots fit correctly
can this brief docking accomplish its role in the cell division cycle,
said Rudolph. That biochemical role is for the enzyme to remove the
phosphates from two phosphate-bearing amino acids on the protein.
Those removals alter electrical charges in a way
that allows the protein to pick up other phosphate-containing chemical
groups to pass along as part of a molecular bucket brigade.
Rudolph initially knew the kinase's and
phosphatase's general topographies as well as the locations of their
active sites. "But it was literally a guessing game trying to find
which residues might be important in this interaction," he said.
"Somehow these two large complicated molecules had
to also interact specifically somewhere other than the site where the
chemistry occurs."
Biochemists traditionally answer such questions by
laboriously making "mutant" versions of a protein in which a single
residue is altered and lab-testing whether the resulting subtle change
in the protein's shape or chemistry changes the way the molecules
interact with each other, he said. If there is no change, they then
move on to the next residue.
"So my students started to make these mutants
randomly and test their activities, one at a time," Rudolph said. "Each
of these experiments is pretty hard, and pretty tedious."
After this trial-and-error search remained
fruitless, Rudolph, his graduate students Jungsan Sohn, Kolbrun
Kristjansdottir and Alexias Safi and his post-doctoral investigator
Gregory Burhman began collaborating with a team led by computer
science and mathematics professor Herbert Edelsbrunner.
Edelsbrunner, who has developed techniques and
computational programs for modeling and analyzing complex molecular
shapes, used a large cluster of computers and custom software to
analyze about one thousand trillion different conceivable shape
match-ups between the molecules.
That initial mega-analysis reduced the potential
molecular combinations to about 1,000 possibilities, which Rudolph
called both "encouraging" and "discouraging."
Edelsbrunner's group, which included programmer
Paul Brown, then began narrowing that search further. They did so by
using a different software program that could identify the highest and
lowest places on the molecules' surfaces, and where "highest" on one
might fit into the "deepest" on the other. "That's not easy, because
there is no point of reference on those complicated shapes," Rudolph
said.
The researchers finally winnowed the possibilities
to what Rudolph called "one reasonable guess" by enlisting another
Duke group led by chemistry professor Waitao Yang.
Wang's team, including his graduate student Jerry
Parks, uses another bank of computers to calculate how components of
molecules behave in small spaces - in this case "how they wiggle,"
Rudolph said. By allowing both molecules to move - as they would in
the real world -- the researchers could evaluate whether match-ups
that looked right when motionless were actually off the mark.
"Tiny little shifts can change these things,"
Rudolph said.
The interdisciplinary group's Biochemistry paper,
whose first author was Rudolph's graduate student Sohn, confirmed the
calculations with extensive biochemical evaluations of the two hot
spot residues the study identified, one residue on the phosphatase and
the other on the kinase. Both hot spots are located some distance from
the molecules' active sites, Rudolph noted.
Overexpression of the Cdc25 group of enzymes has
been associated with the development of numerous cancers. But "drug
discovery targeting these phosphatases has been hampered by lack of
structural information about how Cdc25s interact with their native
protein substrates," the authors wrote in their Biochemistry paper.
With the study's results in hand, scientists can
now search for potential inhibiting drug molecules shaped so they can
overlap - and thus interfere - with the active sites as well as
outlying hot spots the research identified, Rudolph said.
He credited the study's success to the power of
interdisciplinary scientific collaborations, noting that he and
Edelsbrunner initially met "by coincidence" in Duke's Levine Science
Research Center building, where they both have separate labs in
separate wings. |
