Many enzymes have their activities turned up or down by means of effectors—molecules that bind to enzymes and alter their shapes. At least one enzyme, however, responds to effectors by changing its specificity. This enzyme, ribonucleotide reductase (RNR), has an active site that can assume four different shapes depending on which effectors the enzyme binds.
The changeable active site accounts for RNR’s ability to catalyze four different reactions, each of which gives rise to a different DNA building block—deoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine. These deoxyribonucleosides are often abbreviated as A, G, C, and T.
Because a balanced pool of the four DNA building blocks is essential for cell survival, finding ways to create unbalanced pools—in cancer cells and in pathogens, for example—could lead to new therapeutic approaches to cancers and infections. Yet progress toward such therapies has been slowed because RNR’s ability to generate all four DNA building blocks (and also maintain a balance between them) was poorly understood.
RNR, however, has revealed at least some of its secrets to a research team at MIT led by Catherine Drennan, Ph.D., a professor of chemistry and biology. On January 12, Dr. Drennan and colleagues reported in the journal eLife (“Molecular basis for allosteric specificity regulation in class Ia ribonucleotide reductase from Escherichia coli”) that RNR's interactions with its downstream products via a special effector site cause the enzyme to change its shape, determining which of the four DNA building blocks it will generate.
“[We] have determined structures of Escherichia coli class Ia RNR with all four substrate/specificity effector-pairs bound (CDP/dATP, UDP/dATP, ADP/dGTP, GDP/TTP) that reveal the conformational rearrangements responsible for this remarkable allostery,” wrote the authors of the eLife article. “These structures delineate how RNR ‘reads’ the base of each effector and communicates substrate preference to the active site by forming differential hydrogen bonds, thereby maintaining the proper balance of deoxynucleotides in the cell.”
Previous studies have shown that RNR can take on different shapes, but it wasn't clear how those changes in configuration contributed to its specificity. In the new study, the MIT team took X-ray crystallographic images of the enzyme as it interacted with all four ribonucleotide substrates, allowing the researchers to determine how its structure changes.
They found that the enzyme's active site—the region that binds the substrate—changes shape depending on which effector molecule is bound to a distant site on the enzyme. For this enzyme, the effector molecules are deoxynucleoside trisphosphates such as deoxyadenosine triphosphate (dATP) or thymidine triphosphate (TTP).
Depending on which of these effectors is bound to the distant regulatory site, the active site can accommodate one of the four ribounucleotide substrates. Effector binding promotes closing of part of the protein over the active site like a latch to lock in the substrate. If the wrong base is in the active site, the latch can't close and the substrate will diffuse out.
“It's exquisitely designed so that if you have the wrong substrate in there, you can't close up the active site,” said Dr. Drennan. “It's a really elegant set of movements that allows for this kind of molecular screening process.”
The effectors can also shut off production completely by binding to a completely different site on the enzyme if the pool of building blocks is getting too big.
“[Our work] makes it possible to think about doing more rational drug design than was possible before,” Dr. Drennan asserted.