Inherent flexibility of CLIC6 revealed by crystallographic and solution studies.

Chloride intracellular channels (CLICs) are a family of unique proteins, that were suggested to adopt both soluble and membrane-associated forms. Moreover, following this unusual metamorphic change, CLICs were shown to incorporate into membranes and mediate ion conduction in vitro, suggesting multimerization upon membrane insertion. Here, we present a 1.8 Å resolution crystal structure of the CLIC domain of mouse CLIC6 (mCLIC6). The structure reveals a monomeric arrangement and shows a high degree of structural conservation with other CLICs. Small-angle X-ray scattering (SAXS) analysis of mCLIC6 demonstrated that the overall solution structure is similar to the crystallographic conformation. Strikingly, further analysis of the SAXS data using ensemble optimization method unveiled additional elongated conformations, elucidating high structural plasticity as an inherent property of the protein. Moreover, structure-guided perturbation of the inter-domain interface by mutagenesis resulted in a population shift towards elongated conformations of mCLIC6. Additionally, we demonstrate that oxidative conditions induce an increase in mCLIC6 hydrophobicity along with mild oligomerization, which was enhanced by the presence of membrane mimetics. Together, these results provide mechanistic insights into the metamorphic nature of mCLIC6.

Trans-binding of UFM1 to UBA5 stimulates UBA5 homodimerization and ATP binding.

All ubiquitin-like proteins (UBLs) undergo an activation process before their conjugation to target proteins. Although the steps required for the activation of UBLs are conserved and common to all UBLs, we have previously shown that the activation of the UBL, ubiquitin fold modifier 1 (UFM1) by the E1, Ufm1 modifier-activating enzyme 5 (UBA5) is executed in a trans-binding mechanism, not observed in any other E1. In this study, we explored the necessity of that mechanism for UFM1 activation and found that it is needed not only for UFM1 binding to UBA5 but also for stabilizing the UBA5 homodimer. Although UBA5 functions as a dimer, in solution it behaves as a weak dimer. Dimerization of UBA5 is required for ATP binding; therefore, stabilization of the dimer by UFM1 enhances ATP binding. Our results make a connection between the binding of UFM1 to UBA5 and the latter's affinity to ATP, so we propose a novel mechanism for the regulation of ATP's binding to E1.-Mashahreh, B., Hassouna, F., Soudah, N., Cohen-Kfir, E., Strulovich, R., Haitin, Y., Wiener, R. Trans-binding of UFM1 to UBA5 stimulates UBA5 homodimerization and ATP binding.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Structure-based dynamic arrays in regulatory domains of sodium-calcium exchanger (NCX) isoforms.

Mammalian Na+/Ca2+ exchangers, NCX1 and NCX3, generate splice variants, whereas NCX2 does not. The CBD1 and CBD2 domains form a regulatory tandem (CBD12), where Ca2+ binding to CBD1 activates and Ca2+ binding to CBD2 (bearing the splicing segment) alleviates the Na+-induced inactivation. Here, the NCX2-CBD12, NCX3-CBD12-B, and NCX3-CBD12-AC proteins were analyzed by small-angle X-ray scattering (SAXS) and hydrogen-deuterium exchange mass-spectrometry (HDX-MS) to resolve regulatory variances in the NCX2 and NCX3 variants. SAXS revealed the unified model, according to which the Ca2+ binding to CBD12 shifts a dynamic equilibrium without generating new conformational states, and where more rigid conformational states become more populated without any global conformational changes. HDX-MS revealed the differential effects of the B and AC exons on the folding stability of apo CBD1 in NCX3-CBD12, where the dynamic differences become less noticeable in the Ca2+-bound state. Therefore, the apo forms predefine incremental changes in backbone dynamics upon Ca2+ binding. These observations may account for slower inactivation (caused by slower dissociation of occluded Ca2+ from CBD12) in the skeletal vs the brain-expressed NCX2 and NCX3 variants. This may have physiological relevance, since NCX must extrude much higher amounts of Ca2+ from the skeletal cell than from the neuron.

Purification and characterization of human dehydrodolychil diphosphate synthase (DHDDS) overexpressed in E. coli.

Protein asparagine (N)-linked glycosylation is a post-translational modification that occurs in the endoplasmic reticulum; it plays an important role in protein folding, oligomerization, quality control, sorting, and transport. Accordingly, disorders of glycosylation may affect practically every organ system. Dehydrodolichyl diphosphate synthase (DHDDS) is an eukaryotic cis prenyltransferase (cis-PT) that catalyzes chain elongation of farnesyl diphosphate via multiple condensations with isopentenyl diphosphate to form dehydrodolichyl diphosphate, a precursor for the glycosyl carrier dolichylpyrophophate involved in N-linked glycosylation. Mutations in DHDDS were shown to result in retinitis pigmentosa, ultimately leading to blindness, but the exact molecular mechanism by which the mutations affect DHDDS function remains elusive. In addition, bacterial cis-PT homologs are involved in bacterial wall synthesis and are therefore potential targets for new antibacterial agents. However, as eukaryotic cis-PT were not thoroughly characterized structurally and functionally, rational design of prokaryotic cis-PT specific drugs is currently impossible. Here, we present a simple protocol for purification of functionally active human DHDDS under non-denaturating conditions using a codon-optimized construct. The purified protein forms a stable homodimer, similar to its bacterial homologs, and shows time- and substrate-dependent activity. Purification of this protein requires the presence of a detergent for protein solubility. The protocol described here may be utilized for the overexpression of other eukaryotic cis-PT. Future structural and functional studies of the recombinant DHDDS may shed light on the mechanisms underlying DHDDS-related retinitis pigmentosa and lead to novel therapeutic approaches.

Structural Insights into the M-Channel Proximal C-Terminus/Calmodulin Complex.

The Kv7 (KCNQ) channel family, comprising voltage-gated potassium channels, plays major roles in fine-tuning cellular excitability by reducing firing frequency and controlling repolarization. Kv7 channels have a unique intracellular C-terminal (CT) domain bound constitutively by calmodulin (CaM). This domain plays key functions in channel tetramerization, trafficking, and gating. CaM binds to the proximal CT, comprising helices A and B. Kv7.2 and Kv7.3 are expressed in neural tissues. Together, they form the heterotetrameric M channel. We characterized Kv7.2, Kv7.3, and chimeric Kv7.3 helix A-Kv7.2 helix B (Q3A-Q2B) proximal CT/CaM complexes by solution methods at various Ca(2+)concentrations and determined them all to have a 1:1 stoichiometry. We then determined the crystal structure of the Q3A-Q2B/CaM complex at high Ca(2+) concentration to 2.0 Å resolution. CaM hugs the antiparallel coiled coil of helices A and B, braced together by an additional helix. The structure displays a hybrid apo-Ca(2+) CaM conformation even though four Ca(2+) ions are bound. Our results pinpoint unique interactions enabling the possible intersubunit pairing of Kv7.3 helix A and Kv7.2 helix B while underlining the potential importance of Kv7.3 helix A's role in stabilizing channel oligomerization. Also, the structure can be used to rationalize various channelopathic mutants. Functional testing of the chimeric channel found it to have a voltage-dependence similar to the M channel, thereby demonstrating helix A's importance in imparting gating properties.

Structural basis of a Kv7.1 potassium channel gating module: studies of the intracellular c-terminal domain in complex with calmodulin.

Kv7 channels tune neuronal and cardiomyocyte excitability. In addition to the channel membrane domain, they also have a unique intracellular C-terminal (CT) domain, bound constitutively to calmodulin (CaM). This CT domain regulates gating and tetramerization. We investigated the structure of the membrane proximal CT module in complex with CaM by X-ray crystallography. The results show how the CaM intimately hugs a two-helical bundle, explaining many channelopathic mutations. Structure-based mutagenesis of this module in the context of concatemeric tetramer channels and functional analysis along with in vitro data lead us to propose that one CaM binds to one individual protomer, without crosslinking subunits and that this configuration is required for proper channel expression and function. Molecular modeling of the CT/CaM complex in conjunction with small-angle X-ray scattering suggests that the membrane proximal region, having a rigid lever arm, is a critical gating regulator.

Long QT mutations at the interface between KCNQ1 helix C and KCNE1 disrupt I(KS) regulation by PKA and PIP2.

KCNQ1 and KCNE1 co-assembly generates the I(KS) K(+) current, which is crucial to the cardiac action potential repolarization. Mutations in their corresponding genes cause long QT syndrome (LQT) and atrial fibrillation. The A-kinase anchor protein, yotiao (also known as AKAP9), brings the I(KS) channel complex together with signaling proteins to achieve regulation upon ß1-adrenergic stimulation. Recently, we have shown that KCNQ1 helix C interacts with the KCNE1 distal C-terminus. We postulated that this interface is crucial for I(KS) channel modulation. Here, we examined the yet unknown molecular mechanisms of LQT mutations located at this intracellular intersubunit interface. All LQT mutations disrupted the internal KCNQ1-KCNE1 intersubunit interaction. LQT mutants in KCNQ1 helix C led to a decreased current density and a depolarizing shift of channel activation, mainly arising from impaired phosphatidylinositol-4,5-bisphosphate (PIP2) modulation. In the KCNE1 distal C-terminus, the LQT mutation P127T suppressed yotiao-dependent cAMP-mediated upregulation of the I(KS) current, which was caused by reduced KCNQ1 phosphorylation at S27. Thus, KCNQ1 helix C is important for channel modulation by PIP2, whereas the KCNE1 distal C-terminus appears essential for the regulation of IKS by yotiao-mediated PKA phosphorylation.

Regulation of neuronal M-channel gating in an isoform-specific manner: functional interplay between calmodulin and syntaxin 1A.

Whereas neuronal M-type K(+) channels composed of KCNQ2 and KCNQ3 subunits regulate firing properties of neurons, presynaptic KCNQ2 subunits were demonstrated to regulate neurotransmitter release by directly influencing presynaptic function. Two interaction partners of M-channels, syntaxin 1A and calmodulin, are known to act presynaptically, syntaxin serving as a major protein component of the membrane fusion machinery and calmodulin serving as regulator of several processes related to neurotransmitter release. Notably, both partners specifically modulate KCNQ2 but not KCNQ3 subunits, suggesting selective presynaptic targeting to directly regulate exocytosis without interference in neuronal firing properties. Here, having first demonstrated in Xenopus oocytes, using analysis of single-channel biophysics, that both modulators downregulate the open probability of KCNQ2 but not KCNQ3 homomers, we sought to resolve the channel structural determinants that confer the isoform-specific gating downregulation and to get insights into the molecular events underlying this mechanism. We show, using optical, biochemical, electrophysiological, and molecular biology analyses, the existence of constitutive interactions between the N and C termini in homomeric KCNQ2 and KCNQ3 channels in living cells. Furthermore, rearrangement in the relative orientation of the KCNQ2 termini that accompanies reduction in single-channel open probability is induced by both regulators, strongly suggesting that closer N-C termini proximity underlies gating downregulation. Different structural determinants, identified at the N and C termini of KCNQ3, prevent the effects by syntaxin 1A and calmodulin, respectively. Moreover, we show that the syntaxin 1A and calmodulin effects can be additive or blocked at different concentration ranges of calmodulin, bearing physiological significance with regard to presynaptic exocytosis.