In a major breakthrough for rational drug design, a Texas A&M AgriLife team has described several protein structures of a crucial player in cellular processes. This advance could bring new ideas for the treatment of diseases such as Alzheimer’s disease, AIDS, cancer and others.
Artist’s rendering of a domain of protein kinase C C1 (copper), its ligand diacylglycerol (blue), and its detergent (cyan). Image courtesy of Sachin Katti.
Specifically, the work describes the C1 domain of protein kinase C, PKC, which helps regulate protein activity in organisms. In structures, the C1 domain wraps around different molecules of intense therapeutic interest, providing the first reliable atomic-resolution guide for the design of drug candidates.
Published on May 16 in Nature Communication, the research was led by Tatyana Igumenova, Ph.D., associate professor in the Department of Biochemistry and Biophysics at Texas A&M College of Agriculture and Life Sciences. The project’s lead author is Sachin Katti, Ph.D., a postdoctoral fellow working with Igumenova.
The study involved collaboration with Inna Krieger, Ph.D., assistant research professor, and James Sacchettini, Ph.D., professor, both in the Department of Biochemistry and Biophysics.
Grants from the National Institutes of Health and the Welch Foundation supported the work.
One of the most researched protein structures
A healthy cell responds to chemical signals in precise and complex ways. Receiving chemical inputs from the cell’s environment and transmitting them to central control systems within the cell nucleus is the task of specialized proteins such as PKC.
Inappropriate PKC activity appears in many human diseases. Accordingly, there is great interest in finding ways to fine-tune PKC activity with drugs. The design of such drugs will provide new approaches for the treatment of Alzheimer’s disease, AIDS, cancer and more.
“Protein kinase C is one of the most studied proteins in cell biology and pharmacology,” Igumenova said. “A major hurdle has been the lack of accurate structural information to guide drug design efforts.”
A complication for drug design is that the PKC family has 11 members. Different members of the PKC family can have opposing physiological effects, so a successful drug candidate must be selective as to which PKC it targets.
To do this, drug candidates must fit a target PKC like the key to a lock. But determining the 3D structure of a PKC “switch” – the C1 domain – linked to PKC activators has not been easy.
Protein structures are usually solved using X-ray crystallography. The technique involves using X-rays to determine the position of atoms in a crystal. For this method, researchers must create conditions in which the protein of interest crystallizes. Yet intense efforts in many research laboratories over the past three decades have failed to produce crystals of C1 domains bound to relevant ligands. Because of this lack of progress, several researchers have declared the task impossible, Igumenova said.
Solve a 30 year old problem
Crystals of a protein kinase C domain formed spontaneously in Katti’s NMR sample tube. Photo courtesy of Sachin Katti.
Accepting the problem as a challenge, Katti and Igumenova instead decided to study the molecules in solution using nuclear magnetic resonance, NMR, spectroscopy. This involved finding the right components to mimic cell membranes, where the C1 domain would encounter ligands.
“Then one fine day, Sachin discovered crystals forming in an old NMR tube,” Igumenova said. “I give full credit to Sachin, who basically said, ‘I’m going to go test them and see if they’re actually the protein. And he was right. This gave us confidence that crystallization is possible.”
In turn, Katti gives credit to the information obtained through NMR, and a bit of luck.
“I think that’s the beauty of doing research where you have to use multiple approaches,” he said. “You never know when one approach is going to be useful for doing something with other approaches.”
Overviews of NMR and X-ray crystallography
The new protein structures, together with the team’s NMR results, have already provided some interesting insights. A long-standing mystery is how C1 domains can accommodate ligands that have very different chemical structures, Igumenova said.
“Our previous NMR work indicated that the C1 domain loops that bind ligands are very dynamic,” Igumenova said. “This C1 domain is like a PAC-man. It binds to the membrane, then it searches for a ligand. Once it finds the ligand, it latches onto it.”
In addition, the structure shows that the ligand-binding groove has a “water-loving”, or hydrophilic, surface at the bottom and a “hydrophobic”, or hydrophobic, surface at the top.
“If you think of a lipid molecule, the head group is hydrophilic and the tail is hydrophobic,” Igumenova said. “So when the C1 domains bind to lipid ligands, the patterns match.”
The team’s findings include the structure of a C1 domain bound to its natural ligand, diacylglycerol. Additionally, the team describes several other structures of C1 that include different compounds of pharmacological interest.
The work also provides a method for testing different drug candidates, Katti said.
“If you want to study fish, you want to study them in water,” Katti said. “We now know how to create a membrane-like environment where these highly hydrophobic compounds can be tested for C1 binding.”
Next, Katti and Igumenova plan to explore the C1 domains of other members of the PKC family.
“It is important for us to focus on C1 domains because they have inherent differences that can be exploited to achieve selectivity,” Igumenova said. “What we’re seeing now is that not all C1 domains are created equal.”