How Scientists Are Decoding a Key Protein to Save Lives
In the hidden world of microbiology, a silent crisis is unfolding. Invasive fungal infections are a leading cause of death in immunocompromised patients, from those undergoing chemotherapy to organ transplant recipients. Fighting these infections remains notoriously difficult. Since fungi, like us, are eukaryotic organisms, they share many of the same fundamental biological processes with humans. This makes developing drugs that kill the fungus without harming the patient a formidable challenge. Current antifungal therapies are often toxic, can interfere with human cellular pathways, and are increasingly losing their power as fungi develop resistance.
But where do we find a chink in the fungal armor? The answer may lie in the intricate, three-dimensional shape of a single protein. This article explores how structural biologists are using advanced nuclear magnetic resonance (NMR) spectroscopy to map the atomic structure of a fungal protein called FKBP12.
By understanding the subtle differences between the human and fungal versions of this protein, scientists are paving the way for a new generation of precision antifungal drugs that could save countless lives.
The core problem in treating fungal infections is evolutionary similarity. Because humans and fungi are both complex eukaryotes, a drug that disrupts an essential fungal process is very likely to disrupt a similar, crucial process in human cells. This is why many antifungal drugs have significant side effects and limited clinical utility. Compounding this problem, pathogens like Aspergillus fumigatus and Mucor circinelloides are developing increasing resistance to all major classes of existing drugs, pushing researchers to seek out novel targets 1 3 .
One of the most promising alternative targets is a signaling pathway inside fungal cells known as the calcium-calmodulin-calcineurin pathway. This pathway is critical for the virulence of a wide range of fungal pathogens—their ability to cause disease. Disrupting this pathway doesn't necessarily kill the fungus, but it severely cripples its ability to invade and damage human tissues 3 .
The immunosuppressant drug FK506 (tacrolimus) showed researchers the way. Inside a cell, FK506 acts like a molecular glue. It first binds tightly to the FKBP12 protein. This drug-protein complex then latches onto and inhibits calcineurin, the central enzyme in the pathway. It has been well established that calcineurin is essential for invasive fungal disease, and its inhibition via the FK506/FKBP12 complex blocks virulence 1 .
This is where the story takes a turn towards brilliant drug design. While calcineurin is highly conserved (over 80% identical) between humans and fungi, the FKBP12 protein shows much greater divergence. Human and fungal FKBP12 proteins share only 48-58% sequence identity 1 . This lower degree of similarity makes FKBP12 a much more attractive target. The goal is to design a drug that binds strongly to the fungal FKBP12 but weakly to the human version. Like a key that fits one lock but not another, such a drug could form the FK506/FKBP12-like complex in the fungus, inhibiting its calcineurin and virulence, while leaving the human immune system completely unaffected 1 3 .
Sequence Identity
Calcineurin Conservation
Drug Binding Potential
To design a selective drug, scientists first need a detailed blueprint of both the human and fungal FKBP12 proteins. They need to see the exact arrangement of every atom in the protein, both alone and when bound to a drug like FK506. This is where NMR spectroscopy comes in.
In a landmark 2019 study, researchers set out to solve the nuclear magnetic resonance (NMR) structures of the FKBP12 proteins from Mucor circinelloides and Aspergillus fumigatus, as well as the human FKBP12 for comparison. Their objective was to perform backbone and sidechain resonance assignments—the crucial first step in determining a protein's 3D structure and dynamic behavior in solution 1 .
Think of a protein as a complex, folded necklace with thousands of magnets (atomic nuclei) attached. In an NMR experiment, scientists place the protein in a powerful magnet and listen to the unique "radio stations" (resonance frequencies) that each magnet broadcasts. Assigning resonances is the process of figuring out which specific "radio station" belongs to which specific atom in the protein chain. Once every atom is identified and its signal recorded, scientists can calculate how the protein is folded in three dimensions 1 .
The researchers followed a meticulous process to achieve their goal 1 3 :
The genes for the fungal FKBP12 proteins were chemically synthesized and inserted into bacteria (E. coli), which were then used as tiny factories to overproduce large quantities of the pure proteins.
The bacteria were grown in a special "food" containing heavy isotopes of nitrogen (¹⁵N) and carbon (¹³C). The bacteria incorporated these isotopes into the proteins they produced, effectively making the proteins "NMR-active" and allowing scientists to track them in complex experiments.
The proteins were carefully extracted from the bacterial cells and purified to homogeneity to ensure that the NMR signals came only from FKBP12 and nothing else.
The purified, isotope-labeled proteins were placed in the high-field NMR spectrometer. A battery of sophisticated experiments with names like HNCO, HNCACB, and CCH-TOCSY was performed. These techniques allow researchers to walk through the protein's backbone and sidechains, correlating the signals of connected atoms to piece together the complete atomic network.
The complex spectral data was processed and analyzed using specialized software. Researchers systematically identified the unique NMR signature for nearly every hydrogen, nitrogen, and carbon atom in the protein, a process known as chemical shift assignment.
| Experiment Name | Primary Role in the Assignment Process |
|---|---|
| HNCO | Links the amide hydrogen & nitrogen of one amino acid to the carbon of the previous one; establishes the backbone chain. |
| HNCACB | Correlates the amide hydrogen & nitrogen with the alpha and beta carbons of the same and previous residue; identifies amino acid type. |
| CCH-TOCSY | "Spreads" magnetization through the sidechains of amino acids, allowing the assignment of complex sidechain networks. |
| 1H-15N HSQC | The foundational 2D "fingerprint" of the protein; each peak represents one amino acid (except proline). |
The research was a resounding success. The teams obtained nearly complete backbone and sidechain resonance assignments for the FKBP12 proteins from M. circinelloides and A. fumigatus 1 . They also did the same for the human FKBP12, and even gathered data on what happens when the drug FK506 binds to these proteins.
The assignments revealed well-dispersed, high-quality spectra, indicating that the proteins were properly folded and stable in solution. This high-quality data is the essential foundation for all subsequent structural and dynamic studies. Most importantly, it allows scientists to pinpoint, at a residue-specific level, the precise structural differences between human and fungal FKBP12. These differences, especially in and around the drug-binding pocket, are the golden tickets for designing a selective antifungal agent 1 3 .
| Pathogen | Primary Infection Site | Significance | Status of FKBP12 NMR Assignments |
|---|---|---|---|
| Aspergillus fumigatus | Lungs (Invasive aspergillosis) | Major cause of infection in immunocompromised individuals | Completed 1 |
| Mucor circinelloides | Sinuses, lungs, brain (Mucormycosis) | Serious, often fatal infection | Completed 1 |
| Candida auris | Bloodstream, wounds | Global urgent threat; highly drug-resistant & transmissible | Completed 3 |
| Candida glabrata | Bloodstream, urinary tract | Second most common Candida infection; rising resistance | Completed 3 |
| Reagent / Material | Function in the Research Process |
|---|---|
| pET11a Vector | A plasmid used to shuttle the FKBP12 gene into E. coli bacteria for high-level protein expression. |
| BL21(DE3) E. coli Cells | A specially engineered strain of bacteria optimized for producing large amounts of recombinant protein. |
| Isotope-Labeled Nutrients (¹⁵NH₄Cl, U-¹³C Glucose) | Critical for producing "NMR-visible" proteins; incorporated by the bacteria during growth. |
| Size Exclusion Chromatography Resins (Sephadex) | Used to purify the FKBP12 protein based on its size, separating it from other cellular components. |
| Ion Exchange Chromatography Resins (Q-/SP-Sepharose) | Further purifies the protein based on its electrical charge, yielding a sample of high purity for NMR. |
| Dithiothreitol (DTT) / Tris(2-carboxyethyl)phosphine (TCEP) | Reducing agents that keep cysteine residues in the protein from forming incorrect disulfide bonds, maintaining stability. |
| Sodium Phosphate Buffer | Provides a stable chemical environment (pH 6.5) that keeps the protein folded and functional during NMR analysis. |
The precise NMR resonance assignments of fungal and human FKBP12 proteins represent more than just an entry in a scientific database. They are a critical first step in a structure-based drug design pipeline that holds immense promise for combating devastating fungal infections. By providing a residue-by-residue map of the target, this work allows chemists to rationally design and optimize molecules that can discriminate between nearly identical locks.
Subsequent research has already built upon this foundation. Scientists have solved the crystal structure of Candida auris FKBP12 and are using these atomic maps to design modified versions of FK506 that are less immunosuppressive but retain their antifungal activity 5 .
The journey from understanding a protein's fundamental structure to developing a clinically approved drug is long and complex, but it always begins with a detailed map. The work to decode FKBP12 has provided that crucial map, charting a course toward a future where we can disarm fungal pathogens with precision and save the lives of the most vulnerable patients.
NMR and crystallography reveal atomic details of FKBP12 proteins
Key differences between human and fungal proteins identified
Computational and medicinal chemistry create selective inhibitors
Promising candidates move through preclinical and clinical trials