Investigating Innate Immune Response: The Effect of Charges on the Peptide-Binding of GRP94

This study explores the effect of charged residues around the N-terminal domain on the peptide-binding activity of Grp94. The ER chaperone Grp94 is a key part of the cell's innate immune response, as it helps prevent protein aggregation and chaperones several viral proteins to T-cells. Previous studies have found that the majority of Grp94’s binding activity occurs at the N-terminal domain (Gidalevitz et al., Journal of Biological Chemistry, 2004). By studying mutant Grp94 proteins in which charged residues around the N-domain have been changed to neutral amino acid residues, we hope to learn more about the binding mechanism of the protein. The wild type [Grp94 WT] and mutant proteins [Grp94 H146A and Grp94 E141A-K142A] were expressed in E. coli cells and purified using affinity and size-exclusion chromatography. To determine the binding affinity of these proteins for the peptide VSV8, a fluorescent polarization assay was performed, and the Kd values were calculated for both the WT and mutant proteins. Results found that the Grp94 WT had a higher affinity for the peptide than both the Grp94 H146A and Grp94 E141A-K142A mutants. In addition, the Grp94 E141-K142A mutant showed a higher binding affinity than the H146A mutant. This indicates that overall, the removal of charged residues in the N-terminal domain decreased the binding activity of the protein. Furthermore, this shows that the removal of a charged amino acid at the 146 residue of the protein had a more significant impact on binding affinity than changes in the 141 and 142 residues.

• Site-Directed Mutagenesis

  • Mutants: H146A and E141A-K142A

• Protein Production and Purification

  • Cell Lysis

  • Centrifugation and Precipitation

  • Column Chromatography

  • Dialysis

  • Gel Filtration

  • SDS-PAGE

  • Streptavidin Pulldown Assay

• Fluorescence Polarization Assay

  • Peptide Labeling using Fluorescein-5-Maleimide

• These findings indicate that the N-terminal domain is important for the binding of client peptides to Grp94

  • Changes in the residues around the N-domain cause a decrease in binding affinity

  • Charged amino acid residues around the N-domain cause an even lower binding affinity than that of neutral residues

• Current and Future Research

  • Studying the effects of charged residues around the N-terminal domain when Grp94 interacts with peptides of differing characteristics

    • VSVG (immunogenic)

    • Peptide A (non-immunogenic)

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(2)Amankwah, Y. S.; Yasmeen Fleifil; Unruh, E.; Collins, P.; Wang, Y.; Vitou, K.; Bates, A.; Ikponwmosa Obaseki; Meghana Sugoor; John Paul Alao; McCarrick, R. M.; Gewirth, D. T.; Sahu, I. D.; Li, Z.; Lorigan, G. A.; Kravats, A. N. Structural Transitions Modulate the Chaperone Activities of Grp94. Proceedings of the National Academy of Sciences of the United States of America 2024, 121 (12). https://doi.org/10.1073/pnas.2309326121.

(3)Marzec, M.; Eletto, D.; Argon, Y. GRP94: An HSP90-like Protein Specialized for Protein Folding and Quality Control in the Endoplasmic Reticulum. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2012, 1823 (3), 774–787. https://doi.org/10.1016/j.bbamcr.2011.10.013.

(4)Fluorescence Polarization Detection | BMG LABTECH. www.bmglabtech.com. https://www.bmglabtech.com/en/fluorescence-polarization/.

(5)Fluorescence Polarization (FP). Molecular Devices. https://www.moleculardevices.com/technology/fluorescence-polarization.‌