'Computer-chemistry' Yields New Insight Into A Puzzle Of Cell Division

Significantly, their insights arose not just from meticulous biochemical studies, but also from using sophisticated simulation techniques to perform "chemistry in the computer."

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.