# Coupled transitions of FK506-binding domains in molecular design and signaling

> **NIH NIH R01** · WADSWORTH CENTER · 2020 · $300,550

## Abstract

This proposal will structurally characterize the conformational transitions of five homologous FKBP domains
by a combination of NMR relaxation, NOE analysis, quantitative amide hydrogen exchange analysis, X-ray
crystallography, and molecular dynamics simulations. As these five FKBP proteins participate in regulating
distinctly different sets of signaling pathways, development of pharmacological selectivity among these
proteins is of significant interest. However, the marked similarity among their crystal structures has
appreciably impeded this process. We have observed a substantial degree of diversity in the conformational
dynamics of these five domains. We are identifying mutations which modulate these transitions that can then
assist both in structurally characterizing these transitions and in the stepwise optimization of drug leads. This
systematic mutational modulation of the intrinsic conformational transitions of the FKBP domains will facilitate
the analysis of the potential role of these transitions in coupling to the larger scale signaling processes of the
biological complexes in which these proteins function as will be examined regarding their roles in regulating
cardiac Ca2+ and Na+ membrane channels and in the transcription factor complexes of the glucocorticoid and
PPAR receptors.
 Specific Aim 1: The structure of transient conformations sampled by the FKBP domains of FKBP51,
FKBP52 and FKBP38 and the interactions between these transitions will be characterized by a combination
of NMR, crystallographic and molecular simulation studies. Variants of FKBP51/52, designed to modulate
these transitions, will be used to test a structural model for regulatory selectivity in steroid receptor
interactions. Similar structural studies will be applied to both human FKBP38 isoforms. Models of transient
conformations and known catalytic site-binding molecular scaffolds will be used for in silico ligand design.
 Specific Aim 2: This set of experimental and modeling approaches will be applied to FKBP12 and
FKBP12.6 to understand the structural basis of the energetic coupling between two distantly separated
conformational transitions centered on either side of the catalytic cleft. Structural insight into the comparative
inefficiency of this long range coupling in FKBP12.6 will be obtained.
 Specific Aim 3: Murine embryonic fibroblast knockout cell lines will be transfected with genes for the
FKBP51/FKBP52 variants. Glucocorticoid and PPAR receptors will be tested for competitive FKBP binding,
cytoplasmic vs. nuclear localization, transcription activity, and p38 kinase-mediated phosphorylation.
Cardiomyocyte Ca2+ spark frequency and voltage-gated sodium current will be measured in FKBP12.6-KO
and FKBP12cKO mouse cells, respectively, with FKBP12/12.6 variant proteins being introduced by dialysis.

## Key facts

- **NIH application ID:** 9844953
- **Project number:** 5R01GM119152-04
- **Recipient organization:** WADSWORTH CENTER
- **Principal Investigator:** Griselda Hernandez
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $300,550
- **Award type:** 5
- **Project period:** 2017-01-15 → 2022-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9844953

## Citation

> US National Institutes of Health, RePORTER application 9844953, Coupled transitions of FK506-binding domains in molecular design and signaling (5R01GM119152-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9844953. Licensed CC0.

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