Flexible Conformation May be Key to Design of Potent Therapeutic Peptides for Diabetes

Research headed by scientists at the University of Wisconsin-Madison suggests that peptides, such as GLP-1, which are used to treat type 2 diabetes (T2D) may be more effective if they’re able to flexibly shape shift back and forth between different conformations. The discovery counters common wisdom that that molecular signaling in the body is based on having one ideal—and static—partner to activate cellular receptors, and instead indicates that such cellular mechanics might be more dynamic than previously thought. The researchers suggest that their findings could help improve in the future design of diabetes drugs and possibly other therapeutic peptides.

“I think most molecular scientists have an image of this peptide bound to the receptor as having a single ideal shape,” says Sam Gellman, PhD, professor of chemistry at the University of Wisconsin–Madison who supervised the new research. “And what we’re saying is that this vision of an ideal interaction between these two units is probably too simplistic. That in order to be effective, that peptide needs to remain mobile in certain ways.”

Gellman and an international collaboration of researchers published their findings in Nature Chemical Biology, in a paper titled, “Structural and functional diversity among agonist-bound states of the GLP-1 receptor.” The work was led by Brian Cary, PhD, when he was a doctoral student in the Gellman lab.

Many hormones, including insulin and GLP-1, are peptides. They bind to and activate specialized receptor proteinssuch as G-protein-coupled receptors (GPCRs) – which the triggers cellular function that ultimately impacts on metabolism, such as blood sugar control. “GPCRs are critical conduits for intercellular communication,” the authors wrote. “Understanding mechanisms governing agonist activation of GPCRs is integral to interrogation of physiological processes controlled by these receptors and offers a basis for developing therapeutic agents.” But while scientists now have molecular-level snapshots of GPCR structure, including ligand-induced changes in GPCR structure, and of interactions between GPCRs and intracellular partner proteins, static structures determined via X-ray crystallography or cryo-electron microscopy (cryo-EM) don’t give scientists a full understanding of the dynamic processes that occur.

Biologists often imagine the peptide as a key that fits into and turns the receptor’s lock. Just like for keys, the right shape is critical for a peptide to work properly. GLP-1R is a peptide hormone GPCR that plays a critical role in glucose metabolism. Synthetic agonists of this receptor are used to treat type 2 diabetes, the team further explained. GLP-1 had previously been known to adopt a rigidly helical, corkscrew shape. Drug development often involves trying to adjust the shape of a peptide to make it a better drug. And because GLP-1 adopts a corkscrew shape, the assumption was that forcing the peptide to be more helical might make GLP-1 better able to activate its target in the body. Yet when Cary engineered GLP-1-like peptides to better form this corkscrew shape, he discovered that they were less potent.

To dig into this unexpected finding, Cary designed and created a series of differently shaped GLP-1 varieties to test. “We use variants of the peptides GLP-1 and exendin-4 (Ex4) to explore the interplay between helical propensity near the agonist N terminus and the ability to bind to and activate the receptor,” the investigators noted. Using amino acids not normally found in natural peptides, Cary was able to produce two types of shape. One category was helical throughout its length, while the other one bent at a severe angle close to one end.

When the research team tested these different shapes, they uncovered a puzzle: helical peptides bound strongly to the receptor, but were terrible at activating it; kinked proteins bound weakly, but effectively activated the receptor when they finally docked in. Compared to a peptide locked into this helical shape, a peptide engineered to form a sudden kink near its end better activated its cellular target, which promotes insulin release from the pancreas. It’s thus likely that, in the body, GLP-1 is able to switch back and forth between these two forms, maximizing its potency.

Based on their findings, the team came up with a new model of how GLP-1 might work. In this model, GLP-1 binds to and activates its target as a helix—the correctly shaped key to fit in the lock. But then, GLP-1 is able to switch to a new shape with a kink near the end. The kink helps reset GLP-1’s cellular target, preparing it to send a new signal. The peptide can then switch back to a helix to fully dock again and activate the target once more.

This model was supported by the results of cryo-EM analysis, which showed a GLP-1-like peptide bound to its receptor in the two different shapes. This molecular-level imaging of the shapes of proteins helps scientists to see how biological machinery fits together to function. “The pleasure of seeing that cryo-EM structure and recognizing that there are two states was seeing strong evidence that there is a second state that plays a functional role here,” commented Gellman. “By going back and forth, but never completely popping off the receptor, you get to keep signaling and be more effective as a signal-inducing peptide. Only a peptide able to switch back and forth can accomplish this feat.”

The authors further concluded, “The combination of functional, structural and computational data presented here supports a new view of signal transduction via the GLP-1R in which two distinct states of the receptor–agonist complex play important roles in the transfer of information across the cell membrane … Our data are consistent with a model in which the completely helical agonist induces a GPCR conformation that activates an intracellular partner protein, but a distinct agonist conformation, lacking helicity in the N-terminal segment, is reversibly accessed to enable multiple rounds of partner protein activation from a single agonist–receptor engagement, which leads to high agonist efficacy.”

Going forward, Gellman suggests that drug makers should consider whether their peptides of choice may similarly benefit from being able to adopt multiple shapes. “We generally think of a single idealized structure that we are trying to obtain. But I would conclude from these results that actually the way to be most effective is to ensure you maintain particular modes of flexibility,” he says. “If you have that idea in mind, then you’re looking at the molecule in different ways.”

The team further stated, “Understanding the role of structural dynamics in the propagation of molecular information across the cell membrane is important in terms of elucidating GPCR function and developing improved therapeutic agents … A deeper understanding of the conformational possibilities available to GPCRs bound to flexible agonists and of relationships among conformational states and signal transduction will enhance prospects for elucidating signal-propagating mechanisms at the molecular level and optimizing therapeutic performance.”