Johns Hopkins Medicine scientists report that they have probed the atomic structure of proteins to add evidence to the notion that the wobbles, shakes and quivers of proteins play a critical role in their ability to function. The research findings, the team says, may help scientists design new drugs that can modify or disrupt the intricate “dances” of proteins to alter their functions.

Dominique Frueh, PhD, associate professor of biophysics and biophysical chemistry at the Johns Hopkins University School of Medicine, and colleagues, reported on their findings in Science Advances, in a paper titled “Global protein dynamics as communication sensors in peptide synthetase domains.” In their report, the team concluded “We demonstrated that structural fluctuations within a protein enable molecular discrimination by sensing post-translational modifications of binding partners to promote interactions accompanied by remodeling of distant sites.”

Proteins are organic compounds with blueprints that are found in DNA, and which function as the “business ends” of biology, making up the structural components of tissues, along with enzymes, which orchestrate chemical changes within cells.

It has long been known that proteins wiggle and move, but scientists have debated the significance of this “dancing” act, said Frueh. “The way proteins engage with the right partner at the right time—essentially, how they communicate—is very important for understanding their function,” he noted, “and we have found that protein wiggles are critical for this communication.”

In a bid to further such understanding, Frueh’s team studied the wiggling action of the HMWP2 protein, a type of enzyme called a nonribosomal peptide synthetase (NRPS). These enzymes are made of several domains, or distinct regions, that work together like an assembly line to make complex natural products from small chemicals. These natural products often have pharmaceutical properties, such as bacitracin, found in topical antibiotic ointments. Frueh suggests that understanding how protein domains work together could enable scientists to modify the domains to make them produce new chemicals.

“NRPSs are microbial molecular factories that use a dynamic multidomain architecture to assemble simple substrates into complex natural products, including pharmaceuticals such as antibiotics (bacitracin), anticancer agents (epothilones), or immunosuppressants (cyclosporins), the team continued. In the case of HMWP2, its product is yersiniabactin, a molecule that scavenges iron molecules for bacteria, including Escherichia coli, found in urinary tract infections, and Yersinia pestis, the bacterium that causes bubonic plague.

To determine the importance of protein movement, the scientists tracked the motion of one of HMWP2’s domains down to each individual atom in the molecule using nuclear magnetic resonance (NMR) spectroscopy, which uses applies powerful magnetic fields to probe the molecular environments of nuclei within the center of atoms.

Although NMR is often used to determine small protein structures, tracking the motions within large proteins with the device is difficult. To overcome this challenge, Frueh’s team, including NMR scientist Subrata Mishra, PhD, graduate student Kenneth Marincin, and postdoctoral fellow Aswani Kancherla, PhD, used nitrogen-15 and carbon-13—naturally occurring forms of nitrogen and carbon—to flag two domains of the HMWP2 enzyme and track the change in motion of one domain when a second domain was modified, as occurs when the enzyme makes its natural product. The study results found that the widespread wiggling of one domain in the HMWP2 enzyme kick starts a process that enables the domain to connect with several partner domains at one time.

“We found that the two domains would only bind to one another as the second domain gets modified, which means they would only engage as needed for making the product and avoid wasting time together when the second domain is not modified,” noted Frueh. “Somehow, the first domain is able to sense when the second domain is modified, and we sought to investigate whether motions played a role in this recognition process.”

The investigators also found changes in motion across the entire domain flagged with carbon-13c, not only where it binds to the second domain but also at a second, remote binding site used by a third domain. On an atomic level these two sites on HMWP2 could be considered “far” apart—about 40 billionths of a meter, Frueh notes. And how they interact, despite their distance, was particularly intriguing to the scientists.

To show that movements facilitated the interaction with the remote site and the sensing of the second domain modification, they genetically engineered HMWP2 proteins with a mutation that occurred in a location on the domain far from the two sites the scientists had identified. Thus, the mutation did not directly block the sites’ ability to interact with other domains. “Using a nuclear magnetic resonance (NMR) atomic-level readout

of biochemical transformations, we demonstrate that global structural fluctuations help promote substrate-dependent communication and allosteric responses, and impeding these global dynamics by a point-site mutation hampers allostery and molecular recognition,” they wrote.

Frueh added, “We found that the protein domain was structurally stable, but all of its movement was hindered. The mutated protein’s lack of movement damaged its ability to bind with other domains even when they were modified, according to the researchers, demonstrating that the motions within the protein were necessary for the domains to work together.

In their report, the researchers said, “Our studies establish global structural dynamics as sensors of molecular events and bring new perspectives to understanding molecular communication. We demonstrated that structural fluctuations within a protein enable molecular discrimination by sensing post-translational modifications of binding partners to promote interactions accompanied by remodeling of distant sites … Overall, our results establish global structural fluctuations as reporters of allostery and sensors of protein modifications that ensure timely protein interactions during biological activity.”

Frueh suggests that detailed knowledge of protein movement could be leveraged by scientists designing new drugs that don’t target a protein’s natural active site, but instead halt its movement to inactivate it. Such an approach could offer more leeway to design drugs with fewer unwanted side effects, he indicated. To this aim, Frueh added, researchers are studying how computation and artificial intelligence can improve the understanding and prediction of protein movement.

The authors suggested that their results “ … highlight challenges in interpreting outcomes of mutagenesis in dynamic proteins as a point mutation cannot be interpreted solely through local effects, e.g., by considering how it affects a binding site. Instead, the mutation throws sand into the gears of intricate dynamics and affects a protein globally … Our approach, applicable to other systems, illustrates how tracking changes in dynamics informs on mechanisms of allostery, as structural fluctuations within molecules bring about the conformational ensembles used to describe allostery.”

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