Biochemical And NMR Studies Of The Conformational Changes Of STIM1-TM As Well As The Specific Interaction Between STIMATE-CT And Membrane

STIM1（stromal interaction molecule 1）is a key molecular component of SOCE(store-operated Ca²⁺ entry)pathway,it is an ER（endoplasmic reticulum）-resident membrane protein sensing the change of Ca²⁺ content within ER.Upon ER Ca²⁺depletion,STIM1 is activated and migrates towards ER-PM（ER-plasma membrane）junction,where it recruits and gates ORAI1 Ca²⁺ channels,thus causing Ca²⁺ influx.STIM1 is a single-pass transmembrane protein,during the inside-out Ca²⁺ signalling,structural changes of its luminal domain lead to a dramatic conformational change in its cytoplasmic domain,but whether there exist conformational changes within its transmembrane domain（TM）remains to been addressed.In chapter 3,our collaborators identified a unique gain-of-function mutation within the STIM1-TM,which can ideally match the function of activating STIM1.We then carried out high-resolution NMR（nuclear magnetic resonance）studies on bicelles-reconstituted STIM1-TM of wild-type and mutant C227W.By comparing their HSQC（heteronuclear single quantum coherence）spectra,we observed the significant differences reflected in the chemical shift perturbation of certain residues,and further identified the key residues that undergo substantial structural changes between the two states.Moreover,the results of the nanodiscs-based FRET/LRET（fluorescence/luminescence resonance energy transfer）experiments suggest that the structural rearrangement of STIM1-TM will further lead to the conformational changes of STIM1-CC1（coiled-coil 1）,demonstrating the availability of STIM1-TM as a conformational switch to control signal transduction.Finally,in order to resolve the NMR structure of STIM1-TM,we optimized the constructs and reconstituting methods,and ultimately obtained an HSQC spectrum displaying high quality.STIMATE（stim-activating enhancer）was recently identified to regulate SOCE by interacting with STIM1.Additionally,it also participates in the regulation of the dynamic formation of ER-PM junction.STIMATE contains multiple putative transmembrane domains with a polybasic C tail（STIMATE-CT）that directly interacts with STIM1 to promote STIM1 conformational switch as well as regulates the acute rearrangement of ER-PM junction.Therefore,STIMATE-CT is closely related to the function of biological membranes,but whether there is a direct interaction between STIMATE-CT and membranes and the related interaction mechanism remains to been defined.In chapter 4,we focus on the interaction between STIMATE-CT and membrane.Using liposome pulldown assay,we show that STIMATE-CT can specifically interact with PI（4,5）P2 or PI（3,4,5）P3-containing membrane.NMR analysis indicates that STIMATE-CT is intrinsically disordered.Furthermore,NMR titration with bicelles and mutation analysis reveal that the regions of ²⁴²VRYR²⁴⁵ and 284KKKK287 in STIMATE-CT are both essential for its membrane binding.In the supplementary study,we introduced Ca²⁺into the interacting system of STIMATE-CT and membrane.Based on the results of NMR titration experiment and liposome pulldown assay,we draw a preliminary conclusion that Ca²⁺may influence the affinity of STIMATE-CT binding to membrane.In addition,we also investigated the interaction between STIMATE-CT and STIM1-CC1 through NMR titration experiments,laying a foundation for further study related to the molecular mechanism of their interaction.Our work demonstrates for the first time that the conformation of STIM1-TM is significantly different between its resting and activated states,and the structural rearrangement of STIM1-TM will further cause the conformational change of the cytoplasmic domain CC1.These results reveal the biological significance of TM domain within single-pass membrane proteins in transmitting cellular signal across membrane.In addition,we also demonstrated that STIMATE-CT can specifically bind to membranes,and Ca²⁺ can regulate the binding between STIMATE-CT and membranes,which revealed the potential biological functions of STIMATE-CT,that is,specifically binding to the plasma membrane.Taken together,for STIM1 activation model,our study enriched the information about STIM1-TM conformational changes and STIMATE-CT associating with membranes.