Research Initiation Award: Revealing the Structural Basis for Calprotectin-RAGE Signaling Axis

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Research Initiation Awards (RIAs) provide support for junior faculty at Historically Black Colleges and Universities (HBCUs) who are starting to build a research program, as well as for mid-career faculty who need to re-direct and re-build a research program. It is expected that the award helps to further the faculty member's research capability and effectiveness, improves research and teaching at the researcher's home institution, and involves undergraduate students in research experiences.Fisk University's RIA project utilizes well established experimental structural biology and biophysical techniques to establish a transformative new paradigm for the mechanism of regulation of protein-protein interactions in signal transduction. The results will have a significant impact on the fields of structural biology and signal transduction. Characterization of the CP-RAGE VC1 complex at atomic-resolution represents a significant advance forward for S100 and RAGE biology. This would be the first high resolution structure of the RAGE ligand binding extracellular domain in complex with a protein, and one of few S100 proteins in complex with its cognate binding partner. Moreover, the approaches employed in this investigation represent a general approach to addressing mechanism of receptor activation.This research addresses a critical gap in this knowledge by employing high resolution structural studies to reveal the atomic resolution details of the CP-RAGE complex. RAGE is a transmembrane protein comprised of three extracellular immunoglobulin domains (V, C1, and C2), a single transmembrane helix (TM), and a short intracellular peptide (IC). Like many other single pass receptors, activation is hypothesized to proceed through an oligomerization based conformational rearrangement that occurs during ligand binding. The outcomes of the research initiation project will further the diversification of the STEM workforce by providing unparalleled training to students that have been historically underrepresented in STEM. This interdisciplinary program provides integrated research training and didactic coursework at the interface of chemistry, biology, and physics illustrating to students the impact of transdisciplinary research and high impact discovery. Undergraduate students will present their findings at regional and national meetings and will co-author peer-reviewed publications. As an NSF Experimental Program to Stimulate Competitive Research (EPSCoR) jurisdiction, the project will build research capability and capacity in the state as well as within this minority-serving institution. Page AThis research initiation award proposes to understand the molecular basis of interactionsbetween the S100 calcium binding protein calprotectin (CP) and the receptor for advanced glycationendproducts (RAGE). CP binding to RAGE results in activation of the transcription factor NFkB,a molecule which plays a key role in inflammation. Despite the importance of this signalingaxis, little is known about the structural details of the protein-protein interactions whichgovern CP-RAGE binding. This proposal addresses a critical gap in this knowledge by employinghigh resolution structural studies to reveal the atomic resolution details of the CP-RAGEcomplex. RAGE is a transmembrane protein comprised of three extracellular immunoglobulin domains(V, C1, and C2), a single transmembrane helix (TM), and a short intracellular peptide (IC).Like many other single pass receptors, activation is hypothesized to proceed through an oligomerizationbased conformational rearrangement that occurs during ligand binding. The focus of this proposalis on the V and C1 extracellular domains of RAGE (RAGE VC1) and how they contribute to CPbinding and/or oligomerization during receptor activation. In order to address these questions,two complementary but independent objectives are proposed: Objective 1) Define the molecularbasis for CP interaction with RAGE. Nuclear magnetic resonance spectroscopy (NMR) will beused to identify amino acid residues in CP and RAGE VC1 that are critical for binding. TheNMR studies will be validated and used to guide strategies to create mutants in CP that aredefective in RAGE binding as assayed by isothermal titration calorimetry. Objective 2) Characterizethe oligomerization state of the CP-RAGE VC1 complex. X-ray crystallography will be used todetermine the structure of the CP-RAGE VC1 complex. This structure will reveal high resolutiondetails of the interaction between CP and RAGE, as well as interactions that define the oligomerizationbehavior of the overall complex.Intellectual Merit :The research initiation award utilizes well established experimental structural biology andbiophysical techniques to establish a transformative new paradigm for the mechanism of regulationof protein-protein interactions in signal transduction. The results will have a significantimpact on the fields of structural biology and signal transduction. Characterization of theCP-RAGE VC1 complex at atomic-resolution represents a significant advance forward for S100and RAGE biology. This would be the first high resolution structure of the RAGE ligand bindingextracellular domain in complex with a protein, and one of few S100 proteins in complex withits cognate binding partner. Moreover, the approaches employed in this proposal representa general approach to addressing mechanism of receptor activation.Broader Impacts :The outcomes of this research initiation award will further the diversification of the STEMworkforce by providing unparalleled training to students that have been traditionally underrepresentedin STEM. This interdisciplinary program provides integrated research training and didacticcoursework at the interface of chemistry, biology, and physics -illustrating to students theimpact of transdisciplinary research and high impact discovery. Undergraduate students willpresent their findings at regional and national meetings, and will co-author peer-reviewedpublications. Enhancements in research and curriculum infrastructure at Fisk University providedby this RIA will contribute to recruitment and retention of minorities in STEM disciplines,resulting in the development of early career scientists into discoverers and leaders in academicand research careers.