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Chemical shift assignments of the dimerization domain of NF-KappaB c-Rel subunit

This paper reports the NMR backbone chemical shift assignments for the 23.2 kDa homodimer of the NF-κB c-Rel subunit, a hematopoietic-specific transcription factor whose dysregulation is linked to immune dysfunction and blood cancer, revealing a secondary structure consistent with existing DNA-bound crystal structures.

Original authors: Sayan Pal, Mrunal Nanda, Sulakshana P. Mukherjee

Published 2026-09-02

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CC BY 4.0

Original authors: Sayan Pal, Mrunal Nanda, Sulakshana P. Mukherjee

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

Inside the cells of vertebrates, from the simplest fish to humans, a complex family of proteins acts as a master control system for life. This family, known as NF-κB, directs critical processes such as how the body fights infection, manages inflammation, and decides whether a cell lives or dies. These proteins do not work alone; they must pair up, joining two halves together to form a functional unit called a dimer. Once paired, they travel to the cell's nucleus to switch specific genes on or off, acting like a team of editors deciding which instructions in the genetic code get read. While most of these proteins are found throughout the body, one specific member, called c-Rel, operates primarily within the blood and immune system. When c-Rel goes out of control, it can lead to chronic inflammation or blood cancers, making it a vital subject for medical research.

To understand how these proteins work, scientists must first understand their shape and how they hold together. The part of the c-Rel protein responsible for sticking to its partner is called the dimerization domain. While scientists have previously taken static pictures of this domain frozen in a crystal, they have lacked a clear map of how it behaves while moving freely in a liquid environment, which is how it exists inside a living cell. Without this map, it is difficult to understand how c-Rel swaps partners or changes its shape to perform its duties. Researchers at the Indian Institute of Science Education and Research Berhampur set out to fill this gap by creating a detailed chemical map of the c-Rel dimerization domain.

The team began by building a version of the c-Rel protein that contained only the specific section needed for pairing, a segment consisting of 101 amino acids. They inserted the instructions for this piece into bacteria, which then acted as tiny factories to produce the protein. To make the protein visible to their instruments, they fed the bacteria special nutrients containing heavy versions of carbon and nitrogen atoms. Once the bacteria had produced enough of the protein, the scientists harvested them and purified the c-Rel pieces until they had a pure sample ready for study.

To see the protein in action, the researchers placed the sample into a powerful machine called a nuclear magnetic resonance spectrometer. Unlike a camera that takes a picture of a still object, this machine listens to the magnetic signals emitted by the atoms inside the protein as they vibrate. By analyzing these signals, the team could determine exactly where every single atom in the protein chain was located and how it was oriented. They successfully mapped the positions of nearly every atom in the chain, achieving a level of detail that covers 99 percent of the protein's structure. This map serves as a precise coordinate system, allowing scientists to see the protein's shape as it exists in solution.

The resulting picture revealed that the protein is built from a series of flat, sheet-like structures that fold together to form a rigid core. This core matches the shape seen in previous crystal images, confirming that the basic architecture of the protein is consistent. However, the new study also showed something the crystal pictures could not: the ends of the protein and the loops connecting the sheets are flexible and move around. In the crystal, these parts were locked in place by the tight packing of the crystal itself, but in the liquid environment of the cell, they are dynamic and fluid. The researchers also noticed that one specific part of the protein, a residue near the end, sits very close to a ring-shaped structure in the middle, causing its signal to shift in a unique way. This observation aligns perfectly with the known crystal structure, validating the accuracy of their new map.

This work provides the first complete set of coordinates for the c-Rel dimerization domain as it moves in a liquid state. By establishing this baseline, the researchers have created a reference point that others can use to study how c-Rel interacts with other proteins or how it might change shape when it binds to DNA. While the study does not yet explain the full mechanism of how these proteins exchange partners, it removes a major obstacle by defining the starting shape of the molecule. With this chemical map in hand, scientists can now begin to investigate the specific movements and interactions that allow c-Rel to regulate the immune system and, when necessary, how its malfunction leads to disease.

Technical Summary: Chemical Shift Assignments of the Dimerization Domain of NF-κB c-Rel Subunit

Problem and Biological Context
The NF-κB family of transcription factors acts as a master regulator for diverse cellular processes in vertebrates, including immune activation, inflammation, and cell survival. This family comprises five members (NF-κB1/p50, NF-κB2/p52, RelA/p65, c-Rel, and RelB) that assemble into various dimers to recognize specific DNA sequences. While the DNA-binding domain (Rel Homology Region, RHR) is well-characterized, the specific mechanisms driving the formation of distinct NF-κB dimers remain poorly understood, even with existing crystal structures.

The c-Rel subunit is distinct from ubiquitously expressed members like RelA and p50; its expression is largely confined to hematopoietic lineages, particularly B and T lymphocytes. Dysregulated c-Rel activity is linked to immune dysfunction, chronic inflammation, and blood cancers. Although c-Rel functions as a homodimer or heterodimer with partners like p50, the structural dynamics of its dimerization domain (DD) in solution are not fully resolved. Understanding the resonance assignments of the c-Rel homodimer is a necessary prerequisite for investigating the mechanism of subunit exchange during heterodimer formation.

Methodology
The study focused on the murine c-Rel dimerization domain (residues 182–282), which forms a 23.2 kDa homodimer. The protein was expressed in Escherichia coli BL-21(DE3) cells using M9 minimal media supplemented with $^{13}$ C-labeled glucose and $^{15}$ N-labeled ammonium chloride to facilitate NMR analysis. The protein was purified via anion-exchange chromatography (Q-Sepharose) followed by size-exclusion chromatography (Superdex-75).

NMR experiments were conducted at 30°C on a Bruker Avance 700 MHz spectrometer equipped with a cryogenic probe. The dataset included standard triple-resonance experiments (HNCA, HNcoCA, HNCACB, CBCAcoNH, HNCO, HNcaCO) for backbone assignment, a sensitivity-enhanced 2D [ $^{15}$ N, $^{1}$ H]-HSQC, and a [ $^{15}$ N- $^{1}$ H]-NOESY-HSQC with a 100 ms mixing time. Data processing was performed using NMRPipe, and analysis was conducted using CARA. Secondary structure and dynamics were assessed using the TALOS-N web server to estimate backbone dihedral angles and RCI order parameters ( $S^2$ ).

Key Results and Assignments
The authors report the backbone chemical shift assignments for the c-Rel DD homodimer, achieving a 99% completeness across all assigned nuclei ( $^{1}$ H $^N$ , $^{15}$ N, $^{13}$ C $\alpha$ , $^{1}$ H $\alpha$ , $^{13}$ C $\beta$ , and $^{13}$ CO). The resonance assignments have been deposited in the BioMagResBank (accession number 53211).

Structural Consistency: The chemical shift-derived secondary structure largely aligns with existing DNA-bound crystal structures of the protein. Specifically, eight of the nine identified $\beta$ -strands ( $\beta$ 1 through $\beta$ 8) correspond to those observed in the chicken c-Rel homodimer crystal structure (PDB ID: 1GJI).
Solution Dynamics vs. Crystal State: A notable discrepancy was observed between the NMR solution structure and the X-ray crystal structure. The NMR data lacks the second $\beta$ -strand (residues 195–197) and the first helix (residues 212–216) found in the crystal structure. The authors suggest these elements are dynamic in solution and stabilized primarily by crystal-packing interactions in the crystalline state.
Flexibility: RCI order parameters ( $S^2$ ) indicate a rigid core structure (values close to 1), with increased flexibility observed at the N- and C-termini and in the loops connecting the $\beta$ -strands ( $S^2 < 0.8$ ).
Specific Resonance Features: The amide proton of residue C250 exhibits an unusually up-field shifted chemical shift in the HSQC spectrum. This is attributed to its spatial proximity to the aromatic ring of the conserved W225 residue, consistent with the chicken crystal structure.

Significance and Claims
The paper positions these backbone resonance assignments as a foundational step toward understanding the mechanism of subunit exchange during the formation of heterodimers between c-Rel and other NF-κB partners. By providing a high-resolution NMR map of the c-Rel DD, the study offers a tool to investigate how this domain behaves in solution compared to its crystallographic state, particularly regarding the dynamic regions that may be critical for dimerization specificity. The work does not claim to solve the full mechanism of dimer formation but establishes the necessary spectroscopic baseline for future investigations into the structural dynamics of NF-κB subunit interactions.

Technical Summary: Chemical Shift Assignments of the Dimerization Domain of NF-κB c-Rel Subunit

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