Authors interview: Sudha, with a master’s in Botany, and Annu, with a background in neuroscience, joined the PhD program at IIT Kanpur in January 2022. Their research focuses on exploring the signaling mechanisms and structural dynamics of chemokine and complement receptors.
Twitter/X – @SudhsM7, @ANNUDALAL7
Lab: Prof. Arun Kumar Shukla, Indian Institute of Technology, Kanpur
Research Summary: We have determined the molecular basis of CXCR2 promiscuity, utilizing cryo-electron microscopy. This research has uncovered key structural motifs that facilitate flexible chemokine binding, paving the way for targeted drug development for inflammation and cancer.
- What was the core problem you aimed to solve with this research?
G-protein-coupled receptors (GPCRs), the largest family of cell surface receptors, are distinguished by their high ligand specificity. In contrast, chemokine receptors display a notable degree of promiscuous ligand binding, and the molecular determinants of this flexibility remain unclear. Employing CXCR2 as a model, we have identified the molecular basis for flexible chemokine engagement, revealing how selectivity is maintained. These insights will facilitate the development of targeted therapies for inflammatory diseases.
A schematic representation of the R-D and E-L-R motif interaction, C-X-C chemokines harboring the E-L-R motif interact with the R-D motif in the CXCR2, while others lacking this motif do not interact with the receptor. The crosstalk of these two structural motifs in the C-X-C chemokines and CXCR2 provides a molecular mechanism underlying promiscuous binding.
- How did you go about solving this problem?
To elucidate the mechanisms involved, we employed a multidisciplinary strategy encompassing biochemical, pharmacological, and structural analyses. We initiated our study with global chemokine profiling, utilizing G-protein and β-arrestin recruitment assays to quantify the interactions between all C-X-C chemokines and C-X-C receptors. Subsequently, we employed cryo-electron microscopy (cryo-EM) to resolve high-resolution structures of CXCR2 in complex with various chemokines, providing detailed visualization of ligand-receptor interactions. To corroborate our structural observations, we performed site-directed mutagenesis of critical residues within CXCR2 and the chemokines, focusing on the R-D and E-L-R motifs, and assessed their impact on promiscuous binding and subsequent receptor activation.
This study provides the molecular basis of promiscuous chemokine engagement at CXCR2, paving the way for the discovery of novel therapeutics with reduced off-target activity. — Prof. Arun K Shukla
- How would you explain your research outcomes (Key findings) to the non-scientific community?
Picture CXCR2 as a lock that can be opened by many keys, unlike most locks built for just one. We found that CXCR2 has a special structure that lets it recognize and be activated by different chemokines. A key “lock-and-key” interaction, involving the R-D motif of CXCR2 and the E-L-R motif of the chemokines, makes this flexibility possible. When we changed these key parts, the keys no longer fit, and CXCR2 stopped working. Interestingly, most chemokines bind as two-part keys (dimers), except for CXCL6, which binds as a single key. These findings are crucial for creating drugs that specifically block CXCR2, preventing overactive immune responses that cause inflammation and cancer.
- What are the potential implications of your findings for the field and society?
The implications of our research extend broadly across drug discovery, disease therapy, and immunological studies. An enhanced understanding of CXCR2 promiscuity would facilitate the development of selective CXCR2 inhibitors for targeting inflammatory diseases, including asthma, psoriasis, and chronic obstructive pulmonary disease (COPD). By modulating CXCR2 activity, we can potentially impede tumor progression and metastatic dissemination, offering novel strategies for cancer intervention. This study also contributes to our fundamental knowledge of GPCR biology, particularly regarding the molecular mechanisms governing multi-ligand recognition, and provides a framework for future investigations. Notably, our results pave the way for precision medicine approaches, where therapeutic interventions can be tailored to precisely regulate CXCR2 signaling, rather than relying solely on complete receptor blockade, thus reducing the likelihood of adverse effects.
- What was the exciting moment during your research?
One of the most rewarding moments in our research was when we successfully resolved the cryo-EM structures of CXCR2 bound to multiple chemokines. This gave us a clear molecular picture of how CXCR2 interacts with different ligands, finally answering a question that had been puzzling researchers for a long time. Discovering the R-D and E-L-R motifs was especially exciting, as it explained how CXCR2 can bind many chemokines while still maintaining a level of selectivity. What made it even better was when our mutagenesis experiments confirmed these findings. By changing key amino acid residues, we could directly see how important they were for activating the receptor. It was a fascinating surprise when we saw that most chemokines bind as dimers, but only CXCL6 binds as a monomer. This structural difference hinted at new levels of control in chemokine signaling that we hadn’t seen before. These discoveries not only helped us understand CXCR2 better but also provided us with a solid base that can now be leveraged for designing more targeted drugs for inflammation and cancer. Seeing our ideas come to life through structural biology was incredibly fulfilling and underscored the potential impact of our work in the development of novel therapeutics.
Reference: Saha S, Sano FK, Sharma S, et al. Molecular basis of promiscuous chemokine binding and structural mimicry at the C-X-C chemokine receptor, CXCR2. Mol Cell. Published online February 11, 2025. doi:10.1016/j.molcel.2025.01.024 (https://www.cell.com/molecular-cell/abstract/S1097-2765(25)00058-9)
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