REVEALING THE ORAI-STIM BINDING INTERFACE THROUGH THE USE OF NOVEL PEPTIDES
Restricted (Penn State Only)
- Author:
- Baraniak, James
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 22, 2021
- Committee Members:
- Salvatore Stella, Outside Unit Member
Thomas Spratt, Outside Field Member
Christopher Yengo, Major Field Member
Donald Gill, Chair & Dissertation Advisor
Ralph Keil, Program Head/Chair - Keywords:
- calcium
store-operated calcium entry
soce
calcium homeostasis
STIM
Orai
Calcium channels
M4x peptide
peptide modulation
Orai binding interface
SOAR
stormorken
signal transduction
NFAT
pan-specific inhibition
stim-orai interacting interface - Abstract:
- Store-operated Ca2+ entry (SOCE) is a crucial homeostatic and signaling process demonstrated in a wide variety of eukaryotic cells. This powerful process mediates a multitude of cellular activity including proliferation, apoptosis, cell motility, immune response, and gene expression. SOCE involves the work of two highly interconnected protein families, STIM and Orai. Ligand-induced depletion of sub-millimolar endoplasmic reticulum (ER) Ca2+ stores is sensed by the ER-resident single transmembrane STIM protein via its ER luminal EF hand motifs. This triggers a dynamic conformational change in the STIM molecule, allowing it to unfold and form tight junctions between the ER and plasma membrane (PM) through interaction of its polybasic C-terminus with overlying PM acidic phospholipids. Unfolding of STIM exposes a small, tetra-helical STIM-Orai activating region (SOAR), thus allowing it to interact and activate the highly Ca2+-selective Orai channels that reside within the PM. The reciprocating binding interface of Orai has been under much speculation, and better understanding of which has important physiological ramifications in a variety of both healthy and disease states. Prior crystallographic analysis of the Drosophila melanogaster Orai ortholog (dOrai) revealed a hexameric assembly of Orai proteins, whereupon the pore consists of each subunit’s first transmembrane region (TM1). This six-fold symmetry degenerates toward the peripheral into a three-fold arrangement involving a proposed ‘trimer of dimers’ consisting of asymmetrical, hydrophobic pairings of Orai’s cytosolic fourth transmembrane extension (M4x). The M4x, in addition to other cytosolic regions including the N-terminal first transmembrane extension (M1x) and intracellular second and third transmembrane loop (23L), have been suggested as an interacting interface. Our work involving a novel set of PM-tethering peptides can interrogate the molecular nature of the M4x region from any of the three major mammalian Orai subtypes (Orai1, Orai2, and Orai3). Strikingly, we’ve revealed that the M4x is necessary and sufficient to interact with STIM1. Our FRET studies exposed a pivotal set of leucine (and isoleucine) residues that served as a basis of a ‘leu-signature’ that directly mediates these interactions, and are not purely for stabilization of a dimeric, STIM binding pocket. Also quite excitingly, we have found that there are distinct differences in affinity of the M4x region between each Orai subtype. Orai1 is the least potent, Orai2 moderately avid, and the Orai3-M4x has very strong capability to bind with STIM1. This incredibly powerful Orai3-M4x peptide can be used as both a diagnostic tool and pharmacologic. Expression of this potent Orai3-M4x peptide in a variety of cell types blunt their SOCE activity, dramatically reducing physiological Ca2+ oscillations, and abolishes downstream translocation of NFAT1 that relies heavily on formation of these Ca2+ microdomains near Orai. Furthermore, our recent findings involving both peptides and full Orai also reveal that the weakly avid STIM2 counterpart may require more than just the M4x region alone to stabilize its otherwise very weak interaction. This work insinuates a new mechanistic paradigm, whereupon the leucine signature of the M4x helix predominates the Orai coupling interface with STIM, and that stabilization with the M1x may be necessary to facilitate much weaker interactions.