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At 10 one-millionths of a meter wide, a single human cell is tiny. But something even smaller exerts an enormous influence on everything a cell does: proton concentration, or pH. On the microscopic level, pH-dependent structures regulate cell movement and division. Altered pH response can accelerate the development of cancers and neurodegenerative diseases such as Alzheimer's and Huntington's.

Researchers hope that pinpointing pH-sensitive structures in proteins would help them determine how proteins respond to pH changes in normal and diseased cells alike and, ultimately, to design drugs to treat these diseases.

Now, in a appearing in Science Signaling, researchers at the University of Notre Dame present a computational process that can scan hundreds of proteins in a few days, screening for pH-sensitive protein structures.

"Before even picking up a pipette or running a single experiment, we can predict which proteins are sensitive to these pH changes, which proteins actually drive these critical processes like division, migration, cancer development and neurodegenerative disease development," said Katharine White, the Clare Boothe Luce Assistant Professor in the Department of Chemistry and Biochemistry. "No more searching for the needle in the haystack."

Traditional challenges in identifying pH-sensitive proteins

Determining exactly how pH changes affect the behavior-driving proteins on a molecular level has been a challenge because researchers must laboriously test individual proteins in a signaling pathway for pH sensitivity one by one.

For example, actin cytoskeleton remodeling, a key aspect of cell migration, was shown to be pH dependent in 1993. Since then, scientists in the field have characterized only four pH-sensitive proteins among the many suspected to be involved in the process. Across biology, only 70 cytoplasmic proteins have been confirmed as pH-sensitive—though researchers hypothesize that there are many, many more—and of those, the molecular mechanisms of only 20 are known.

How the new computational pipeline works

The new study developed and validated a modular, computational pipeline that predicts the location of pH-sensitive structures based on existing structural and experimental data.

"Their interior pH is an important feature of cells, but it has been largely ignored because of difficulties in measuring and manipulating it," said Daniel DiMaio, deputy director of the Yale Cancer Center. "Because Dr. White has developed a viable path to analyze the role of intracellular pH, her work provides the incentive to search for more cellular activities that respond to this feature—and I predict that there will be many."

In the process of developing the pipeline, White's research group predicted and validated the pH sensitivity of a distinctive binding module known as the Src homology 2 (SH2) domain, which appears in proteins crucial for cell signaling, immune response and development, as well as the pH-dependent function of c-Src, an intensively studied enzyme that is activated in many cancers.

"These proteins are central to cell regulation in addition to being mutated in certain cancers, and in addition to showing that they are pH-sensitive, we've also found exactly where on the protein the pH regulation is occurring," explained Papa Kobina Van Dyck, the lead study author and a recent doctoral graduate in biophysics. "We've managed to condense 25 years of work into a few weeks."

Gathering data and modeling protein charge interactions

Researchers obtained structural data from the RCSB Protein Data Bank, a global repository that stores and enables access to three-dimensional structural data of proteins, nucleic acids and other biological macromolecules.

The program then integrates experimental pKa values, which estimate the pH value at which a specific amino acid—the building blocks of proteins—might gain or lose a proton. These values permit the program to predict the electric charge of sites throughout the protein and model how interactions between charges affect the protein's shape. Of particular interest for pH sensitivity studies are those building blocks that are likely to have a charge in the narrow window of normal physiological pH, around 7.2 to 7.6.

"We're able to look at how all of these charges are connected to each other," said White, who is a faculty affiliate of the Harper Cancer Research Institute. "Since opposite charges attract and like charges repel, one of these motifs switching charges has the potential to affect neighboring charges. The amino acids we zeroed in on were those where a flip to the opposite charge caused a cascade of charge-flipping across the whole network."

Uncovering allosteric mechanisms and protein regulation

The consequences of the charge-flipping of just one amino acid can have an outsized impact, as in the case of SHP2, a phosphatase signaling protein investigated in the new study. While SHP2 was shown to be pH-sensitive in 2005, no specific molecular mechanism had been elucidated—until now. With the binding of protons to two key residues, the conformation of the entire, 593 amino-acid protein changes from closed to open.

"This is a mechanism in biochemistry that's called allostery," explained White. "Allostery is an indirect effect, something that happens away from the protein active site but regulates activity. It's hard to identify structurally and it's hard to identify the mechanisms of allostery, but our pipeline can do it."

While studying SHP2, an SH2-domain containing protein, the research team noticed that the two sites flagged as pH-sensitive by the computational pipeline were located at the interface between two key structures, including the regulatory SH2 domain. Functioning as a molecular bridge, SH2 domains facilitate the assembly of protein complexes that activate cellular responses such as growth, differentiation, survival, and immune activation.

"To identify one pH sensitive protein, that's very exciting, because it's very hard to identify pH sensitive proteins in general," Van Dyck said. "But then we got to thinking, what if this is a mechanism that's common in all SH2 domain-containing proteins?"

Implications for cancer and targeted therapies

The pipeline identified an assortment of SH2 signaling proteins that contain the same pH-sensitive sites at the SH2 interface as SHP2. Present in the medley was c-Src, a highly studied enzyme that is more active than normal in many human cancers. The computational analysis of Src flagged four potential pH-sensitive sites, which were then confirmed experimentally by Van Dyck and colleagues.

The researchers found that normal, healthy Src has high activity at low pH and low activity at high pH. Cancer-associated mutations at the pH-sensitive sites flagged by the computer render the protein insensitive to changing pH, abolishing the regulation of Src activity and contributing to the unchecked cell proliferation characteristic of aggressive cancers.

But now that White and colleagues have mapped and validated the exact molecular mechanism of pH regulation in Src, the door is opened for the development of targeted drugs that mimic key allosteric sites and restore native pH sensitivity.

"This is one of the grand challenges in our understanding of human biology: If you can understand the molecular mechanism, then you can target it and you can perturb it, and reduce the negative effects of these mutations in patients," White said.

In the case of Src and SHP2, such drugs would be selective for only the mutant protein, leaving the normal protein untouched and preserving activity in a patient's healthy cells. The benefit of such targeted treatments goes beyond cancer, stretching into disease biology more broadly.

"In addition to cancer and neurodegeneration, pH dynamics are associated with diabetes, autoimmune disorders and traumatic brain injury," White said. "Our pipeline is a powerful tool for understanding and, ultimately, designing treatments for these conditions, with the potential to transform the field."