STIM1 and Orai1 are key components of the Ca2+-release activated Ca2+ (CRAC) current. Orai1, which represents the subunit forming the CRAC channel complex, is activated by the ER resident Ca2+ sensor STIM1. The genetically inherited Stormorken syndrome disease has been associated with the STIM1 single point R304W mutant. The resulting constitutive activation of Orai1 mainly involves the CRAC-activating domain CAD/SOAR of STIM1, the exposure of which is regulated by the molecular interplay between three cytosolic STIM1 coiled-coil (CC) domains. Here we present a dual mechanism by which STIM1 R304W attains the pathophysiological, constitutive activity eliciting the Stormorken syndrome. The R304W mutation induces a helical elongation within the CC1 domain, which together with an increased CC1 homomerization, destabilize the resting state of STIM1. This culminates, even in the absence of store depletion, in structural extension and CAD/SOAR exposure of STIM1 R304W leading to constitutive CRAC channel activation and Stormorken disease.

The calcium release activated calcium (CRAC) channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. Upon activation, STIM1 C-terminus changes from an inactive, tight to an active, extended conformation. A coiled-coil (CC) clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as key entity. The NMR-derived solution structure of the CC1 domain represents a three-helix bundle stabilized by interhelical contacts, which are absent in the Stormorken disease-related STIM1 R304W mutant. Two interhelical sites between CC1α 1 and CC1α 2 helices are key in controlling STIM1 activation, affecting the balance between tight and extended conformations. NMR-directed mutations within these interhelical interactions restore the physiological, store-dependent activation behavior of the gain-of-function STIM1 R304W mutant. This study reveals the functional impact of interhelical interactions within the CC1 domain for modifying the CC1-CC3 clamp strength to control the activation of STIM1.

Developing novel compounds with antimicrobial properties can be an effective approach to decreasing the number of healthcare-associated infections, particularly in the context of medical devices and touch surfaces. A variety of molybdate powders (AgMoO, CaMoO, CuMoO and CuMoO) were synthesized and characterized, and Escherichia coli was used as a model gram-negative bacterium to demonstrate their antimicrobial properties. Optical density measurements, bacterial colony growth, and stained gel images for protein expression clearly showed that silver- and copper molybdates inhibit bacterial growth, whereas CaMoO exhibited no bactericidal effect. All tests were performed in both daylight and darkness to assess the possible contribution of a photocatalytic effect on the activity observed. The main mechanism responsible for the antibacterial effect observed for AgMoO is related to Ag release in combination with medium acidification, whereas for compounds containing copper, leaching of Cu ions is proposed. All these effects are known to cause damage at the cellular level. A photocatalytic contribution to the antibacterial activity was not clearly observable. Based on the pH and solubility measurements performed for powders in contact with various media (ultrapure water and bacterial growth medium), silver molybdate (AgMoO) was identified as the best antibacterial candidate. This compound has great potential for further use in hybrid powder-polymer/varnish systems for touch surfaces in healthcare settings.

Calmodulin (CaM) binds most of its targets by wrapping around an amphipathic a-helix. The N-terminus of Orai proteins contains ac onserved CaM-binding segment but the binding mechanismh as been only partially characterized. Here,m icroscale thermophoresis (MST), surface plasmon resonance (SPR), and atomic force microscopy( AFM) were employed to study the binding equilibria, the kinetics,a nd the single-molecule interaction forces involved in the binding of CaM to the conserved helical segments of Orai1 and Orai3. The results consistently indicated stepwise binding of two separate target peptides to the two lobes of CaM. An unparalleled high affinity was found when two Orai peptides were dimerized or immobilized at high lateral density,thereby mimicking the close proximity of the N-termini in native Orai oligomers.T he analogous experiments with smooth muscle myosin light chain kinase (smMLCK) showed only the expected 1:1b inding,c onfirming the validity of our methods. Orai1andOrai3areCa2+ channels in the plasma membrane of non-excitable cells which are activated by Ca 2+ depletion of the endoplasmic reticulum (ER);areduction of [Ca 2+ ]inthe ER causes the stromal interaction molecule (STIM) in the ER membrane to oligomerize,w hereupon it binds to Orai and activates its channel function. [1][2][3][4] Much less is known about the Ca 2+ -dependent inactivation (CDI) of Orai which is important in the regulation of the intracellular Ca 2+ level. In anumber of studies,calmodulin (CaM) was found to bind to ahighly conserved N-terminal segment of Orai1/3, and to act as an egative regulator of channel function, [5][6][7] although this was lately questioned.[8] Thec rystal structure of the complex between CaM and the isolated CaM-binding segment of Orai1 (aa69-88) revealed an unusual extended conformation of CaM with only its C-terminal lobe binding one Orai1 peptide.[9] Parallel pulldown and isothermal titration calorimetry (ITC) experiments suggested stepwise binding of two Orai1 69-88 peptides with different affinities,o ne on the Cterminal lobe of CaM (K d = 1.1 mm)a nd one on the Nterminal lobe (K d = 4.6 mm). Motivated by these findings,weaimed at acomprehensive characterization of this interaction mechanism, for the following reasons:( i) the measured K d values (1.1 and 4.6 mm)a re much higher than those usually reported for Ca 2+ -induced CaM binding (typically 10 À7 to 10 À11 m) [10] and were probably influenced by the high calmodulin concentration used in ITC,( ii)the transitions between monovalent and bivalent bond formation have not yet been addressed, and, most prominently,( iii)quantitative information about the kinetics of stepwise association and dissociation is still missing. Here,t hese questions were addressed by complementary in vitro methods.E quilibrium measurements by MST yielded the distinct affinities of both binding steps at equilibrium, SPR provided information about the kinetic rate constants,a nd AFM revealed the interaction forces of monovalent and bivalent binding,aswell as the...

We report a new NMR-scale purification procedure for two recombinant wild type fragments of the stromal interaction molecule 1 (STIM1). This protein acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol accumulating at ER - plasma membrane (PM) junctions upon calcium store depletion ultimately leading to activation of the Orai/CRAC channel. The functionally relevant cytosolic part of STIM1 consists of three coiled coil domains, which are mainly involved in intra- and inter-molecular homomeric interactions as well as coupling to and gating of CRAC channels. The optimized one-step rapid purification procedure for two N,C isotope-labeled cytosolic coiled coil fragments, which avoids the problems of previous approaches. The high yields of soluble well folded N,C isotope-labeled cytosolic coiled coil fragments followed by detergent screening provide for initial NMR characterization of these domains. The longer 30.5 kDa fragment represents the largest STIM1 wild type fragment that has been recombinantly prepared and characterized in solution without need for mutation or refolding.

<b><i>Introduction:</i></b> Epidermolysis bullosa (EB) describes a family of rare genetic blistering skin disorders. Various subtypes are clinically and genetically heterogeneous, and a lethal postpartum form of EB is the generalized severe junctional EB (gs-JEB). gs-JEB is mainly caused by premature termination codon (PTC) mutations in the skin anchor protein LAMB3 (laminin subunit beta-3) gene. The ribosome in majority of translational reads of LAMB3PTC mRNA aborts protein synthesis at the PTC signal, with production of a truncated, nonfunctional protein. This leaves an endogenous readthrough mechanism needed for production of functional full-length Lamb3 protein albeit at insufficient levels. Here, we report on the development of drugs targeting ribosomal protein L35 (rpL35), a ribosomal modifier for customized increase in production of full-length Lamb3 protein from a LAMB3PTC mRNA. <b><i>Methods:</i></b> Molecular docking studies were employed to identify small molecules binding to human rpL35. Molecular determinants of small molecule binding to rpL35 were further characterized by titration of the protein with these ligands as monitored by nuclear magnetic resonance (NMR) spectroscopy in solution. Changes in NMR chemical shifts were used to map the docking sites for small molecules onto the 3D structure of the rpL35. <b><i>Results:</i></b> Molecular docking studies identified 2 FDA-approved drugs, atazanavir and artesunate, as candidate small-molecule binders of rpL35. Molecular interaction studies predicted several binding clusters for both compounds scattered throughout the rpL35 structure. NMR titration studies identified the amino acids participating in the ligand interaction. Combining docking predictions for atazanavir and artesunate with rpL35 and NMR analysis of rpL35 ligand interaction, one binding cluster located near the N-terminus of rpL35 was identified. In this region, the nonidentical binding sites for atazanavir and artesunate overlap and are accessible when rpL35 is integrated in its natural ribosomal environment. <b><i>Conclusion:</i></b> Atazanavir and artesunate were identified as candidate compounds binding to ribosomal protein rpL35 and may now be tested for their potential to trigger a rpL35 ribosomal switch to increase production of full-length Lamb3 protein from a LAMB3PTC mRNA for targeted systemic therapy in treating gs-JEB.

This communication is focused on the synthesis, characterization and experimental proof of the mechanism of antimicrobial activity of powders from the molybdenum–tungsten–oxygen (Mo–W–O) system. Materials with a discrete ratio of Mo/W ranging from 100% MoO3 to 100% WO3 with a stepwise increase of 5–10 wt % W were synthesized by the spray drying method following calcination. Spherical hollow particles with a broad size distribution were formed and the composition influenced the crystalline phases in such a way that either pure and/or mixed oxides (Mo0.6W0.4O3) were obtained. A good correlation between composition variation and phases present on the antimicrobial activity is obtained and provides a detailed screening of the activity efficiency versus compositional transition. Antimicrobial tests were performed against a model Gram-negative bacterium (Escherichia coli). Furthermore, the mechanism of antimicrobial activity is proven by correlating the medium acidification via pH measurements to the bacteria lifespan at low pH values. The mechanism is additionally supported by the bacterial growth when a buffered nutrient medium was used, together with the evidence that the powder particles have no disruptive effect on the cell wall. Consequently, an extended mechanism is proposed for the mixed oxide, relating both the structure and solubility results. Solubility measurements displayed a steep decrease in metal ions concentration with the addition of W. A narrow compositional range was identified (80 to 60 wt % Mo) where the antimicrobial activity was present, which is concurrent with a very strong decrease in solubility. Materials within this range show adequate features for being implemented into hybrid systems consisting of inorganic materials–polymers/varnishes that can be used for touch surfaces in healthcare settings.

PsbP (23 kDa) is an extrinsic eukaryotic protein of photosystem II found in the thylakoid membrane of higher plants and green algae. It has been proven to be indispensable for proper functioning of the oxygen evolving complex. By interaction with other extrinsic proteins (PsbQ, PsbO and PsbR), it modulates the concentration of two cofactors of the water splitting reaction, Ca2+ and Cl−. The crystallographic structure of PsbP from Spinacia oleracea lacks the N-terminal part as well as two inner regions which were modelled as loops. Those unresolved parts are believed to be functionally crucial for the binding of PsbP to the thylakoid membrane. In this NMR study we report 1H, 15N and 13C resonance assignments of the backbone and side chain atoms of the PsbP protein. Based on these data, an estimate of the secondary structure has been made. The structural motifs found fit the resolved parts of the crystallographic structure very well. In addition, the complete assignment set provides preliminary insight into the dynamic regions.