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The protein stromal interaction molecule 1 (STIM1) plays a pivotal role in mediating store-operated calcium entry (SOCE) into cells, which is essential for adaptive immunity. It acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol, where it changes from an inactive (tight) to an active (extended) oligomeric form upon calcium store depletion. NMR studies of this protein are challenging due to its membrane-spanning and aggregation properties. Therefore follow the divide-and-conquer approach, focusing on individual domains first is in order. The cytosolic part is predicted to have a large content of coiled-coil (CC) structure. We report the 1H, 13C, 15N chemical shift assignments of the CC3 domain. This domain is crucial for the stabilisation of the tight quiescent form of STIM1 as well as for activating the ORAI calcium channel by direct contact, in the extended active form. Supplementary Information The online version contains supplementary material available at 10.1007/s12104-021-10042-7.

Decorin binding proteins (Dbps) mediate attachment of spirochetes in host organisms during the early stages of Lyme disease infection. Previously, different binding mechanisms of Dbps to glycosaminoglycans have been elucidated for the pathogenic species Borrelia burgdorferi sensu stricto and B. afzelii. We are investigating various European Borrelia spirochetes and their interactions at the atomic level using NMR. We report preparative scale recombinant expression of uniformly stable isotope enriched B. afzelii DbpA in Escherichia coli, its chromatographic purification, and solution NMR assignments of its backbone and sidechain 1H, 13C, and 15N atoms. This data was used to predict secondary structure propensity, which we compared to the North American B. burgdorferi sensu stricto and European B. garinii DbpA for which solution NMR structures had been determined previously. Backbone dynamics of DbpA from B. afzelii were elucidated from spin relaxation and heteronuclear NOE experiments. NMR-based secondary structure analysis together with the backbone dynamics characterization provided a first look into structural differences of B. afzelii DbpA compared to the North American species and will serve as the basis for further investigation of how these changes affect interactions with host components.

The calcium release activated calcium channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. On activation, STIM1 C terminus changes from an inactive, tight to an active, extended conformation. A coiled-coil clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as the key entity. The nuclear magnetic resonance-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 the CC1α1 and CC1α2 helices are key in controlling STIM1 activation, affecting the balance between tight and extended conformations. Nuclear magnetic resonance-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.

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% MoO

Introduction: 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. Methods: 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. Results: 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. Conclusion: 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.

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 15N,13C isotope-labeled cytosolic coiled coil fragments, which avoids the problems of previous approaches. The high yields of soluble well folded 15N,13C 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.

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 (Ag2MoO4, CaMoO4, CuMoO4 and Cu3Mo2O9) 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 CaMoO4 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 Ag2MoO4 is related to Ag+ release in combination with medium acidification, whereas for compounds containing copper, leaching of Cu2+ 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 (Ag2MoO4) 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.

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

Calmodulin (CaM) binds most of its targets by wrapping around an amphipathic α-helix. The N-terminus of Orai proteins contains a conserved CaM-binding segment but the binding mechanism has been only partially characterized. Here, microscale thermophoresis (MST), surface plasmon resonance (SPR), and atomic force microscopy (AFM) were employed to study the binding equilibria, the kinetics, and 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. The analogous experiments with smooth muscle myosin light chain kinase (smMLCK) showed only the expected 1:1 binding, confirming the validity of our methods.

Calcium is an important second messenger involved in many cellular processes such as cell proliferation, T-cell activation, muscle contraction, egg fertilization, or apoptosis. Calcium entry into non-excitable cells is mainly carried by store-operated Ca2+ release-activated Ca2+ (CRAC) channels, whereby Orai was found to be the calcium channel in the plasma membrane and the stromal interaction molecule (STIM) to act as a calcium sensor in the endoplasmatic reticulum (ER), and as an activator of Orai channels. A reduction of Ca2+ in the ER causes STIM to oligomerize, enabling its accumulation at the ER-plasma membrane junctions where it binds directly to Orai to open the Ca2+-channel. Compared with activation, much less is known about the mechanisms underlying Ca2+-dependent inactivation (CDI) processes. It has been proposed that binding of calmodulin (CaM) to a highly conserved N-terminal segment of Orai1 adjacent to its first transmembrane helix is critical for CDI. Hereby it is assumed that CaM acts in concert with STIM1 and the N terminus of Orai1 to evoke rapid CRAC channel inactivation. However, the molecular basis of these interactions remains unclear. In this study, the interaction between calmodulin and the N-terminal fragment of Orai1 and its homologue Orai3 was studied by Single Molecule Recognition Force Spectroscopy, Surface Plasmon Resonance, and Microscale Thermophoresis. Both Orai fragments showed a calcium-dependent and highly specific interaction with calmodulin, with slightly longer bond lifetimes and lower dissociation rate constants for Orai3 than for Orai1. Our data support a switch between a 1:1 and a 1:2 binding stoichiometry between CaM and Orai1/3. The latter occurs at low CaM concentration, has much higher affinity, and takes much longer to disscociate after removal of Ca2+ by EGTA. By comparison, only 1:1 binding with high affinity was found between CaM and smooth muscle myosin light chain kinase. This emphasizes the importance of the unique amino acid sequence of the conserved N-terminal Orai segment for high CaM affinity and CDI. This work was supported by the state of Upper Austria (project DK NanoCell).

The extrinsic proteins of photosystem II of higher plants and green algae PsbO, PsbP, PsbQ, and PsbR are essential for stable oxygen production in the oxygen evolving center. In the available X‐ray crystallographic structure of higher plant PsbQ residues S14‐Y33 are missing. Building on the backbone NMR assignment of PsbQ, which includes this “missing link”, we report the extended resonance assignment including side chain atoms. Based on nuclear Overhauser effect spectra a high resolution solution structure of PsbQ with a backbone RMSD of 0.81 Å was obtained from torsion angle dynamics. Within the N‐terminal residues 1–45 the solution structure deviates significantly from the X‐ray crystallographic one, while the four‐helix bundle core found previously is confirmed. A short α‐helix is observed in the solution structure at the location where a β‐strand had been proposed in the earlier crystallographic study. NMR relaxation data and unrestrained molecular dynamics simulations corroborate that the N‐terminal region behaves as a flexible tail with a persistent short local helical secondary structure, while no indications of forming a β‐strand are found. Proteins 2015; 83:1677–1686. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.