Saturday, January 8, 2022

Desperately Seeking Calcium

How cells regulate internal calcium levels.

Now that we are getting a crash course in molecular biology and evolution courtesy of the pandemic, many will be familiar with the intricate and dynamic activities of some proteins. The SARS spike protein doesn't just dock at a particular receptor on our pulmonary epithelial surfaces, but goes through a gymnastic routine to facilitate membrane fusion as well. Many other proteins have dynamic behaviors as well- something that was not fully appreciated back when structural biology was in its infancy and knowing anything about the structure of a protein or DNA or RNA required it to be locked into crystaline form for X-ray diffraction studies.

Another example came up recently, involving calcium regulation within cells. Calcium is a hugely important ion and regulator, central to core signaling cascades in all eukaryotic cells- to neuronal function, and to muscle activation, among many other roles. Our blood levels of calcium are tightly regulated, (to within a 20% range), mostly by way of an axis of parathyroid hormone between the parathyroid gland and the kidney, with additional effects from factors such as vitamin D, calcitonin, and estrogen. So our cells can rely on having a constant level of calcium on the outside. How do they maintain their levels internally?

One way is to have a large store socked away, as we have in bones for the body generally. Within cells, the endoplasmic reticulum (ER) turns out to have far higher concentrations of calcium than the rest of the cytosol, up to 10,000 fold. In muscle cells, the ER gets a special name- as the sarcoplasmic reticulum. Many calcium regulatory events rely on calcium being released briefly from the ER, having some effect, and then gradually getting pumped back in. But what if the ER is short of calcium? That would be a crisis!  

It turns out that we have a sensor system for that, llinking an ER protein called STIM1, which senses levels of Ca++ in the ER with a plasma membrane channel called ORAI1, which can open to let in Ca++ from the outside. A recent paper, (review), in combination with much other past work, demonstrates how STIM1 works. The two proteins turn out to interact directly, thanks to the fact that the ER, which is a huge organelle that extends all over the cell, always has some spots that interact with the plasma membrane, called membrane contact sites. These are strucured by other proteins, so there is a set distance between the two membranes, which must never fuse together. This means that while STEM1 can get very close to ORAI1 in the plasma membrane, there will still be a gap between them. How to bridge it?

Overall model for how STIM1 works. The luminal side sticks into the ER and binds calcium (red dots). If levels are low, the protein dimerizes at the transmembrane and internal domains, causing extensive refolding of the external domains residing in the cytosol. This causes them to straighten out and span the space of the contact structure between the ER and the plasma membrane, where it activates the ORAI1 calcium channel protein by direct contact.


The STIM1 protein turns out to provide the bridge, in the form of a transformer-style mechanism that shifts it from a compact blob on the ER when calcium levels are high, to an extended rod that pokes into ORAI1, activating it, when calcium levels are low. Since it is the ER-internal level of calcium that needs to be sensed, it is the ER-internal (or luminal) portion of the STIM1 that does this sensing. It has about five calcium binding sites that, if filled, prevent its dimerization, but which if empty, promote it. Internal dimerization induces a dramatic refolding of the cytoplasmic portion of STIM1 into the active, extended rod. 

These authors were faced with a situation where the full STIM1 protein was apparently impossible to crystalize, so no full structure was available. Worse, some of the prior structural studies of fragments of STIM1 conflicted with each other. So they turned to very clever method to probe structural dimensions point by point, called fluorescence (or Förster) resonance energy transfer, (FRET). If by mutation or chemical modification one installs fluorescent molecules on a protein of interest, indeed installs two different ones, one of whose absorbtion spectrum overlaps with the emission spectrum of the other, one can measure quantitatively the distance between them.

How the FRET fluorescence method works. Different fluorophores are placed on the protein of interest, here the EFSAM luminal domain of STIM1. The absorption spectrum of one (acceptor) overlaps the emission spectrum of the other fluorophore (donor). In the first graph, the green graph shows that when the two are combined on the same molecule, emission from the acceptor goes up dramatically, due to its proximity-dependent absorbance of emissions from the donor fluorophore. The second graph shows how this protein responds to calcium, by increasing interaction (absorbance-emission intensity at 620 nm, reflecting the physical distance between the fluorescence probes) as Ca++ concentration goes down.
 

By placing fluorescence probe pairs all over the external regions of STIM1, these authors were able to definitively refute one of the prior structural models, and then outline the probable sequence of events by which STIM1 opens up into its active form. The image above ably summarizes their model, by which the ORAI1-interacting domain (CC2/CC3) is stored upside-down and inside out in the inactive conformation. It is quite a proposal, all carried out by domains which are alpha helixes hinged at strategic locations and obviously highly sensitive to slight changes in the structure, induced by the dimerization outlined above, in low calcium conditions.

Finally, they investigated a mutation which in humans causes Stormorken syndrome, a wide-ranging set of deficiencies including bleeding, dyslexia, muscle weakness, and hypocalcemia. In molecular terms it is a "gain of function" mutation. It weakens the interactions that keep STIM1 closed during high calcium conditions, so promotes its stimulation of ORAI1 and excess uptake by cells all over the body. The mutation changes argenine at position 304 in STIM1 to tryptophan, which has much different characteristics. It is genetically dominant, meaning that a single allele, combined with a wild-type allele on the other chromosome, gives the syndrome. Thus it is a powerful mutation, tweeking the sensitivity of this system just enough to screw up a lot of physiology. Deletions of this gene are not lethal, however, in part because there is also a STIM2 gene that encodes a similar function.

Analysis of the effect of the Stormorken mutation (R304W) on the physical proximities and overall shape of the STIM1 protein. The FRET graphs track different probe pairs that were placed all over the cytosolic (folding) portion of STIM1. In these graphs, degree of FRET relative frequency shift/communication is on the X axis, while photon counts are on the Y axis. They show noticeable shifts in distances, reflected in the structural model. The mutation significantly loosens up the high-calcium folded state, inducing more Ca++  influx when it is not needed.

So, we are just full of little machines, developed and refined over the billions of years in the ongoing race to live a little better, keep things humming, and to defend ourselves against all the other machines, such as parasitic viruses.


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