Looking for an Opening
For many immune cells, activation triggers the release of a flood of calcium ions from the endoplasmic reticulum (ER), which in turn drives a variety of downstream signaling events. As these calcium reservoirs run dry, protein channels in the plasma membrane belonging to the Orai family open and selectively allow influx of additional calcium from the extracellular environment, enabling signaling to proceed uninterrupted.
Humans have three Orai proteins, and defects in Orai1 in particular have been linked to immunodeficiency. However, relatively little is known about their structure except that they bear little resemblance to other ion channels, and so Hou et al. embarked on a thorough crystallographic analysis of Drosophila Orai, which shows strong sequence identity with Orai1.
The researchers determined that channels are composed of six Orai monomers, with each subunit's transmembrane helices forming a pattern of concentric circles surrounding the central pore. The pore itself features an outward-facing ring of glutamate residues, followed by a hydrophobic stretch of residues and, finally, a stretch of basic residues that leads to a wide opening into the cytosol.
Close examination of their structure led the researchers to conclude that they had crystallized a ‘closed’ configuration of the pore, in which calcium can only bind the outer glutamate ring but does not penetrate the pore. In fact, they determined that the inner pore is likely to be physically occluded by binding of anions associated with the basic region. Orai opening is normally triggered by the ER protein STIM, and the authors propose a model whereby cytosolic interactions between STIM and Orai could physically widen the pore, enabling displacement of the bound anion and allowing calcium to stream through the membrane. – Michael Eisenstein
Hou, X. et al. Science, 2012, 338, 1308–1313.
Stuck in the Middle
The various members of the Hsp70 family of molecular chaperones play a critical role in cellular ‘quality control’, preventing aggregation of nascent or misfolded proteins among other functions. Hsp70 proteins feature a nucleotide-binding domain (NBD) and a polypeptide substrate-binding domain (SBD); ATP binding at the NBD shifts Hsp70 into a conformational state that favors substrate release, whereas substrate interaction with the SBD of an ATP-bound Hsp70 molecule triggers ATP hydrolysis and results in strengthened substrate binding. 1
The transition between these two distinct conformations occurs via an allosteric intermediate state, which Zhuravleva et al. were recently able to characterize in a series of nuclear magnetic resonance (NMR) experiments. They captured this transient state by working with a mutant version of DnaK, the Escherichia coli homologue of Hsp70, which was capable of binding but not hydrolyzing ATP and essentially remained trapped in a substrate- and ATP-bound conformation.
While in this allosteric state, DnaK is subject to competing forces that favor two possible rearrangements. On the one hand, ATP binding promotes interactions between the NBD and the linker that connects it to the SBD, resulting in a compact ‘docked’ configuration. Conversely, substrate binding destabilizes SBD-NBD interaction, instead favoring interaction between the SBD and a separate ‘lid’ domain, yielding an extended ‘undocked’ configuration. The allosteric intermediate is subject to fluctuations that are markedly affected by factors such as mutations, interactions with co-chaperones or specific substrate characteristics, which in turn influence whether DnaK preferentially shifts to a state favoring ATP hydrolysis or substrate release. The authors propose that a stronger understanding of these mechanistic determinants could inform the design of effective chemical modulators for both research and therapeutic applications. – Michael Eisenstein
Zhuravleva, A. et al. Cell, 2012, 151, 1296–1307.