Isothermal Titration Calorimetry of Protein–Protein Interactions

Protein-protein interactions are interesting targets for various drug discovery campaigns. One such promising and therapeutically pertinent protein-protein complex is PfAMA1-PfRON2, which is involved in malarial parasite invasion into human red blood cells. A thorough understanding of the interactions between these macromolecular binding partners is absolutely necessary to design better therapeutics to fight against the age-old disease affecting mostly under-developed nations. Although crystal structures of several PfAMA1-PfRON2 complexes have been solved to understand the molecular interactions between these two proteins, the mechanistic aspects of the domain II loop-PfRON2 association is far from clear. The current work investigates a crucial part of the recognition event; i.e., how the domain II loop of PfAMA1 exerts its effect on the alpha helix of the PfRON2, thus influencing the overall kinetics of this intricate recognition phenomenon. To this end, we have conducted thorough computational investigation of the dynamics and free energetics of domain II loop closing processes using molecular dynamics simulation. The computational results are validated by systematic alanine substitutions of the PfRON2 peptide helix. The subsequent evaluation of the binding affinity of Ala-substituted PfRON2 peptide ligands by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) provides a rank of the relative importance of the residues in context. Our combined (computational and experimental) investigation has revealed that the domain II loop of PfAMA1 is in fact responsible for arresting the PfRON2 molecule from egress, K2027 and D2028 of PfRON2 being the determinant residues for the capturing event. Our study provides a comprehensive understanding of the molecular recognition event between PfAMA1 and PfRON2, specifically in the post binding stage, which potentially can be utilized for drug discovery against malaria.

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Characterization of Membrane Protein Interactions by Isothermal Titration Calorimetry

Journal of Molecular Biology ◽

10.1016/j.jmb.2014.08.020 ◽

2014 ◽

Vol 426 (21) ◽

pp. 3670-3680 ◽

Cited By ~ 16

Author(s):

Alan J. Situ ◽

Thomas Schmidt ◽

Parichita Mazumder ◽

Tobias S. Ulmer

Keyword(s):

Membrane Protein ◽

Isothermal Titration Calorimetry ◽

Protein Interactions ◽

Titration Calorimetry

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Isothermal Titration Calorimetry of Membrane Protein Interactions: FNR and the Cytochrome b6f Complex

Biophysical Journal ◽

10.1016/j.bpj.2021.12.014 ◽

2021 ◽

Author(s):

Stanislav D. Zakharov ◽

Sergei Savikhin ◽

Yuko Misumi ◽

Genji Kurisu ◽

William A. Cramer

Keyword(s):

Membrane Protein ◽

Isothermal Titration Calorimetry ◽

Protein Interactions ◽

Titration Calorimetry ◽

Cytochrome B6f Complex ◽

Cytochrome B6f ◽

B6f Complex

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UHM–ULM interactions in the RBM39–U2AF65 splicing-factor complex

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798316001248 ◽

2016 ◽

Vol 72 (4) ◽

pp. 497-511 ◽

Cited By ~ 17

Author(s):

Galina A. Stepanyuk ◽

Pedro Serrano ◽

Eigen Peralta ◽

Carol L. Farr ◽

Herbert L. Axelrod ◽

...

Keyword(s):

Protein Interactions ◽

Rna Binding ◽

Splicing Factor ◽

Structural Basis ◽

Titration Calorimetry ◽

Protein Protein Interactions ◽

Rrm Domain ◽

Nmr Structures ◽

Binding Interface ◽

Cell Extracts

RNA-binding protein 39 (RBM39) is a splicing factor and a transcriptional co-activator of estrogen receptors and Jun/AP-1, and its function has been associated with malignant progression in a number of cancers. The C-terminal RRM domain of RBM39 belongs to the U2AF homology motif family (UHM), which mediate protein–protein interactions through a short tryptophan-containing peptide known as the UHM-ligand motif (ULM). Here, crystal and solution NMR structures of the RBM39-UHM domain, and the crystal structure of its complex with U2AF65-ULM, are reported. The RBM39–U2AF65 interaction was confirmed by co-immunoprecipitation from human cell extracts, by isothermal titration calorimetry and by NMR chemical shift perturbation experiments with the purified proteins. When compared with related complexes, such as U2AF35–U2AF65 and RBM39–SF3b155, the RBM39-UHM–U2AF65-ULM complex reveals both common and discriminating recognition elements in the UHM–ULM binding interface, providing a rationale for the known specificity of UHM–ULM interactions. This study therefore establishes a structural basis for specific UHM–ULM interactions by splicing factors such as U2AF35, U2AF65, RBM39 and SF3b155, and a platform for continued studies of intermolecular interactions governing disease-related alternative splicing in eukaryotic cells.

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