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Ruxolitinib (Jakavi) is a potent JAK1/JAK2 specific inhibitor, approved for the treatment of myeloproliferative neoplasia (MPN) such as primary myelofibrosis (PMF) and polycythemia Vera (PV). The primary clinical benefit of ruxolitinib in MPN patients is reduction in spleen size and alleviation of constitutional symptoms and significant improvement in the quality of life. However, the effect of ruxolitinib on bone marrow fibrosis and JAK2V617F allelic burden is modest. In addition, JAK2V617F negative MPNs also demonstrated clinical benefits, thus, is not clear whether the ruxolitinib primarily blocks the proliferation of the malignant clone or exerts its effects by targeting non-malignant cells. To gain the mechanism of ruxolitinib action in MPNs, we developed two JAK2V617F-driven MPN mouse models harboring ruxolitinib resistant mutations JAK2V617F+L902Q and JAK2V617F+L983F in the malignant clone. Similar to JAK2V617F recipients, JAK2V617F+L902Q and JAK2V617F+L983F transplanted mice showed an increase in WBC, HCT, RBC, HB and reticulocyte values. Histopathological analysis revealed that the myeloproliferative phenotype resembles PV with a proliferation of all three lineages albeit with only a moderate increase in megakaryopoeisis. Grade II bone marrow fibrosis was observed in JAK2V617F+L902Q and JAK2V617F+L983F after 2-3 months similar to JAK2V617F mice. Surprisingly, these mice respond to ruxolitinib treatment similar to ruxolitinib sensitive JAK2V617F mice, indicated by reduction of spleen size, leukocyte count and proinflammatory cytokines in the serum. Mechanistically, we could identify a direct role of ruxolitinib in the impairment of inflammatory processes in the bone marrow microenvironment, particularly on the cytokine production of mesenchymal stem cells and endothelial cells. These results suggest that the ruxolitinib mediated effects in MPN patients are mainly due to abrogation of inflammatory signaling processes in non-malignant cells. Therefore, combination treatment of JAK1/2 inhibitors with novel inhibitors, which specifically act on malignant cells might improve the clinical benefit for MPN patients to JAK family inhibitors. Citation Format: Sivahari Prasad Gorantla, Michael Rassner Rassner, Tony Andreas Müller, Teresa Poggio, Kirstyn Anne Crossley, Anna-Lena Denecke, Kornelia R Fritsch, Geoffroy Andrieux, Helen Kleinfelder, Shifa Khaja Saleem, Sudheer Madan Mohan Gambheer, Irene Gonzalez Menendez, Detlef Bentrop, Rainer Trittler, Svetlana Rylova, Dietmar Pfeifer, Leticia Quintanilla Martinez, Christine Dierks, Robert Zeiser, Anna Lena Illert, Nikolas von Bubnoff, Justus Duyster. Efficacy of Jak1/2 inhibition in murine myeloproliferative neoplasms is mediated by targeting nonmalignant cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4497.

Macro domains are conserved protein domains found in eukaryotic organisms, bacteria, and archaea as well as in certain viruses. They consist of 130–190 amino acids and can bind ADP-ribose. Although the exact role of these domains is not fully understood, the conserved binding affinity for ADP-ribose indicates that this ligand is important for the function of the domain. Such a macro domain is also present in the non-structural protein 3 (nsP3) of Chikungunya Alphavirus (CHIKV) and consists of 160 amino acids. In this study we describe the high yield expression of the macro domain from CHIKV and its preliminary structural analysis via solution NMR spectroscopy. The macro domain seems to be folded in solution and an almost complete backbone assignment was achieved. In addition, the α/β/α sandwich topology with 4 α-helices and 6 β-strands was predicted by TALOS+.

Mayaro virus (MAYV) is a member of Togaviridae family, which also includes Chikungunya virus as a notorious member. MAYV recently emerged in urban areas of the Americas, and this emergence emphasized the current paucity of knowledge about its replication cycle. The macro domain (MD) of MAYV belongs to the N-terminal region of its non-structural protein 3, part of the replication complex. Here, we report the first structural and dynamical characterization of a previously unexplored Alphavirus MD investigated through high-resolution NMR spectroscopy, along with data on its ligand selectivity and binding properties. The structural analysis of MAYV MD reveals a typical “macro” (ββαββαβαβα) fold for this polypeptide, while NMR-driven interaction studies provide in-depth insights into MAYV MD–ligand adducts. NMR data in concert with thermodynamics and biochemical studies provide convincing experimental evidence for preferential binding of adenosine diphosphate ribose (ADP‐r) and adenine-rich RNAs to MAYV MD, thus shedding light on the structure–function relationship of a previously unexplored viral MD. The emerging differences with any other related MD are expected to enlighten distinct functions., Graphical abstract Unlabelled Image, Highlights • First conformational and dynamics study of a previously unexplored Alphavirus macro domain (Mayaro virus macro domain, or MAYV MD) • MAYV MD selectivity and binding of ADP-ribose revealed by NMR spectroscopy and ITC • Identification of critical residues for the ADP-ribose binding • MAYV MD preference for binding of RNAs rich in adenine observed by NMR spectroscopy • Binding studies provide new insights for the design of novel ligands for MAYV and other Alphaviruses MDs.

Graphical abstract, Highlights • ADPr’s binding triggers conformational changes to the whole VEEV macro domain. • High flexibility of the loops β5-α3 and α3-β6 assist the ADPr’s binding. • Loops around ADPr site undergo a transition pathway between apo and complex state., Venezuelan equine encephalitis virus (VEEV) is a new world alphavirus which can be involved in several central nervous system disorders such as encephalitis and meningitis. The VEEV genome codes for 4 non-structural proteins (nsP), of which nsP3 contains a Macro domain. Macro domains (MD) can be found as stand-alone proteins or embedded within larger proteins in viruses, bacteria and eukaryotes. Their most common feature is the binding of ADP-ribose (ADPr), while several macro domains act as ribosylation writers, erasers or readers. Alphavirus MD erase ribosylation but their precise contribution in viral replication is still under investigation. NMR-driven titration experiments of ADPr in solution with the VEEV macro domain (in apo- and complex state) show that it adopts a suitable conformation for ADPr binding. Specific experiments indicate that the flexibility of the loops β5-α3 and α3-β6 is critical for formation of the complex and assists a wrapping mechanism for ADPr binding. Furthermore, along with this sequence of events, the VEEV MD undergoes a conformational exchange process between the apo state and a low-populated “dark” conformational state.

Macro domains are conserved protein domains found in eukaryotic organisms, bacteria, and archaea as well as in certain viruses. They consist of 130-190 amino acids and can bind ADP-ribose. Although the exact role of these domains is not fully understood, the conserved binding affinity for ADP-ribose indicates that this ligand is important for the function of the domain. Such a macro domain is also present in the non-structural protein 3 (nsP3) of Chikungunya Alphavirus (CHIKV) and consists of 160 amino acids. In this study we describe the high yield expression of the macro domain from CHIKV and its preliminary structural analysis via solution NMR spectroscopy. The macro domain seems to be folded in solution and an almost complete backbone assignment was achieved. In addition, the α/β/α sandwich topology with 4 α-helices and 6 β-strands was predicted by TALOS+.

Arkadia (Rnf111) is an E3 ubiquitin ligase that plays a central role in the amplification of transforming growth factor beta (TGF-β) signaling responses by targeting for degradation the negative regulators of the pathway, Smad6 and Smad7, and the nuclear co-repressors Ski and Skil (SnoN). Arkadia's function in vivo depends on the really interesting new gene (RING)–H2 interaction with the E2 enzyme UbcH5b in order to ligate ubiquitin chains on its substrates. A conserved tryptophan (W972) in the C-terminal α-helix is widely accepted as essential for E2 recruitment and interaction and thus also for E3 enzymatic activity. The present NMR-driven study provides an atomic-level investigation of the structural and dynamical properties of two W972 Arkadia RING mutants, attempting to illuminate for the first time the differences between a functional and a nonfunctional mutant W972A and W972R, respectively. A TGF-β-responsive promoter driving luciferase was used to assay for Arkadia function in vivo. These experiments showed that the Arkadia W972A mutant has the same activity as wild-type (WT) Arkadia in enhancing TGF-β signaling responses, while W972R does not. Only minor structural differences exist between the W972A RING domain and WT-RING. In contrast, the W972R mutant hardly interacts with E2. The loss of function correlates with structural changes in the C-terminal α-helix and an increase in the distance between the Zn(II) ions. Our data show that the position occupied by W972 within WT Arkadia is critical for the function of RING and that it depends on the nature of the residue at this position.

Biosynthesis of RNA polymerase III transcripts requires binding of the La protein at their 3′ end. La is an abundant nuclear RNA-binding protein which protects the nascent transcripts from 3′ exonuclease degradation. Here, we report the high yield expression and preliminary structural analysis through NMR spectroscopy of two recombinant RNA binding domains (La motif and NRRM) from the La protein of Dictyostelium discoideum. Both recombinant protein constructs were well-folded and allowed for an almost complete sequence-specific assignment of the 15N and 13C labeled domains and their secondary structure prediction using PECAN online tool.

Arkadia (Rnf111), an E3 Ubiquitin (Ub) ligase, amplifies TGF-β signaling responses by targeting for degradation of the negative regulators Smad6/7 and the SnoN/Ski transcriptional repressors when they block the TGF-β effectors Smad2/3. The E3 ligase activity of Arkadia depends on its C-terminal RING-H2 domain that constitutes the docking site for the E2 Ub-conjugating enzyme carrying the activated Ub. We determined the nuclear magnetic resonance solution structure of Arkadia's RING-H2 domain and revealed a (β)ββα fold, fully consistent with the expected "cross-brace" mode of Zn(II)-ligation. In addition, the interaction of the Arkadia RING-H2 domain with its E2 partner enzyme (UbcH5b) was examined through chemical shift perturbation. Proteins 2012. © 2012 Wiley Periodicals, Inc.

Anthrax lethal toxin (LeTx) stands for the major virulence factor of the anthrax disease. It comprises a 90 kDa highly specific metalloprotease, the anthrax lethal factor (LF). LF possesses a catalytic Zn{sup 2+} binding site and is highly specific against MAPK kinases, thus representing the most potent native biomolecule to alter and inactivate MKK [MAPK (mitogen-activated protein kinase) kinases] signalling pathways. Given the importance of the interaction between LF and substrate for the development of anti-anthrax agents as well as the potential treatment of nascent tumours, the analysis of the structure and dynamic properties of the LF catalytic site are essential to elucidate its enzymatic properties. Here we report the recombinant expression and purification of a C-terminal part of LF (LF{sub 672-776}) that harbours the enzyme's core protease domain. The biophysical characterization and backbone assignments ({sup 1}H, {sup 13}C, {sup 15}N) of the polypeptide revealed a stable, well folded structure even in the absence of Zn{sup 2+}, suitable for high resolution structural analysis by NMR.

The oxidized Fe4S4 ferredoxin from the hyperthermophilic bacterium Thermotoga maritima has been investigated by one- and two-dimensional NMR in order to characterize its hyperfine-shifted resonances originating from the cysteinyl cluster ligands and to assign its resonances in the diamagnetic shift range. The chemical shift and relaxation time pattern of the hyperfine-shifted signals is very similar to other oxidized Fe4S4 ferredoxins. A tentative sequence-specific assignment of these resonances according to a general pattern of chemical shift of cysteine protons versus sequence position of cluster ligand is presented. Furthermore, sequence-specific assignments for 85% of the amino acid residues that were obtained without any guidance by known X-ray structures of ferredoxins are given. They reveal the formation of at least two elements of secondary structure by the polypeptide chain of T. maritima ferredoxin: an α-helix comprising residues C43–D49 and a double–stranded antiparallel β-sheet consisting of the N-and C-terminal parts of the protein. This folding pattern is very similar to that of the crystallographically characterized ferredoxin from the mesophile Desulfovibrio gigas [Kissinger, C. R., Sieker, L. C., Adman E. T. & Jensen, L. H. (1991) J. Mol. Biol. 219, 693–715] and therefore suggesting different mechanisms of stabilization for T. maritima ferredoxin and the ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus that was recently investigated by NMR [Teng, Q., Zhou, Z. H., Smith, E. T., Busse, S. C., Howard, J. B., Adams M. W. W. & La Mar, G. N. (1994) Biochemistry 33, 6316–6326].

Macro domains consist of 130-190 amino acid residues and appear to be highly conserved in all kingdoms of life. Intense research on this field has shown that macro domains bind ADP-ribose and other similar molecules, but their exact function still remains intangible. Macro domains are highly conserved in the Alphavirus genus and the Venezuelan equine encephalitis virus (VEEV) is a member of this genus that causes fatal encephalitis to equines and humans. In this study we report the high yield recombinant expression and preliminary solution NMR study of the macro domain of VEEV. An almost complete sequence-specific assignment of its (1)H, (15)N and (13)C resonances was obtained and its secondary structure predicted by TALOS+. The protein shows a unique mixed α/β-fold.

Macro domains are ADP-ribose-binding modules present in all eukaryotic organisms, bacteria and archaea. They are also found in non-structural proteins of several positive strand RNA viruses such as alphaviruses. Here, we report the high yield expression and preliminary structural analysis through solution NMR spectroscopy of the macro domain from New World Mayaro Alphavirus. The recombinant protein was well-folded and in a monomeric state. An almost complete sequence-specific assignment of its (1)H, (15)N and (13)C resonances was obtained and its secondary structure determined by TALOS+.

Cumulative inactivation of voltage-gated (Kv) K(+) channels shapes the presynaptic action potential and determines timing and strength of synaptic transmission. Kv1.4 channels exhibit rapid "ball-and-chain"-type inactivation gating. Different from all other Kvalpha subunits, Kv1.4 harbors two inactivation domains at its N terminus. Here we report the solution structure and function of this "tandem inactivation domain" using NMR spectroscopy and patch clamp recordings. Inactivation domain 1 (ID1, residues 1-38) consists of a flexible N terminus anchored at a 5-turn helix, whereas ID2 (residues 40-50) is a 2.5-turn helix made up of small hydrophobic amino acids. Functional analysis suggests that only ID1 may work as a pore-occluding ball domain, whereas ID2 most likely acts as a "docking domain" that attaches ID1 to the cytoplasmic face of the channel. Deletion of ID2 slows inactivation considerably and largely impairs cumulative inactivation. Together, the concerted action of ID1 and ID2 may promote rapid inactivation of Kv1.4 that is crucial for the channel function in short term plasticity.

The N-terminal half of La protein consists of two concatenated motifs: La motif (LAM) and the N-terminal RNA recognition motif (RRM1) both of which are responsible for poly(U) RNA binding. Here, we present the backbone and side-chain assignments of the (1)H, (13)C and (15)N resonances of the 191-residue LAM-RRM1 region of the La protein from the lower eukaryote Dictyostelium discoideum and its secondary structure prediction.

The La protein (Lupus antigen), a key mediator during biogenesis of RNA polymerase III transcripts, contains a characteristic La motif and one or two RNA recognition motif (RRM) domains, depending on the organism of origin. The RRM1 domain is conserved in higher eukaryotes and located in the N-terminal region, whereas the C-terminal RRM2 domain is absent in most lower eukaryotes and its specific role remains, so far, uncharacterized. Here, we present the backbone and side-chain assignment of the (1)H, (13)C and (15)N resonances of RRM2 of La protein from Dictyostelium discoideum. Interestingly, the La protein in this lower eukaryote, exhibits high homology to its human counterpart. Moreover, it contains two RRM domains, instead of one, raising questions on its evolutionary origin and the putative role of RRM2 in vivo. We also provide its secondary structure as predicted by the TALOS+ online tool.

NMR-based structural biology urgently needs cost- and time-effective methods to assist both in the process of acquiring high-resolution NMR spectra and their subsequent analysis. Especially for bigger proteins (>20 kDa) selective labeling is a frequently used means of sequence-specific assignment. In this work we present the successful overexpression of a polypeptide of 233 residues, corresponding to the structured part of the N-terminal domain of Anthrax Lethal Factor, using Escherichia coli expression system. The polypeptide was subsequently isolated in pure, soluble form and analyzed structurally by solution NMR spectroscopy. Due to the non-satisfying quality and resolution of the spectra of this 27 kDa protein, an almost complete backbone assignment became feasible only by the combination of uniform and novel amino acid-selective labeling schemes. Moreover, amino acid-type selective triple-resonance NMR experiments proved to be very helpful.

The auxiliary beta-subunit KCNMB2 (beta(2)) endows the non-inactivating large conductance Ca(2+)- and voltage-dependent potassium (BK) channel with fast inactivation. This process is mediated by the N terminus of KCNMB2 and closely resembles the "ball-and-chain"-type inactivation observed in voltage-gated potassium channels. Here we investigated the solution structure and function of the KCNMB2 N terminus (amino acids 1-45, BKbeta(2)N) using NMR spectroscopy and patch clamp recordings. BKbeta(2)N completely inactivated BK channels when applied to the cytoplasmic side; its interaction with the BK alpha-subunit is characterized by a particularly slow dissociation rate and an affinity in the upper nanomolar range. The BKbeta(2)N structure comprises two domains connected by a flexible linker: the pore-blocking "ball domain" (formed by residues 1-17) and the "chain domain" (between residues 20-45) linking it to the membrane segment of KCNMB2. The ball domain is made up of a flexible N terminus anchored at a well ordered loop-helix motif. The chain domain consists of a 4-turn helix with an unfolded linker at its C terminus. These structural properties explain the functional characteristics of BKbeta(2)N-mediated inactivation.

Anthrax lethal factor (LF) is a zinc-metalloprotease that together with the protective antigen constitutes anthrax lethal toxin, which is the most prominent virulence factor of the anthrax disease. The solution nuclear magnetic resonance and in silico conformational dynamics of the 105 C-terminal residues of the LF catalytic core domain in its apo form are described here. The polypeptide adopts a compact structure even in the absence of the Zn(2+) cofactor, while the 40 N-terminal residues comprising the metal ligands and residues that participate in substrate and inhibitor recognition exhibit more flexibility than the C-terminal region.

The PsaC subunit of Photosystem I (PS I) is a 9.3-kDa protein that binds two important cofactors in photosynthetic electron transfer: the [4Fe-4S] clusters FA and FB. The g-tensor orientation of FA − and FB − is believed to be correlated to the preferential localization of the mixed-valence and equal-valence (ferrous) iron pairs in each [4Fe-4S]+ cluster. The preferential position of the mixed-valence and equal-valence pairs, in turn, can be inferred from the study of the temperature dependence of contactshifted resonances by 1H NMR spectroscopy. For this, a sequence-specific assignment of these signals is required. The 1H NMR spectrum of reduced, unbound PsaC from Synechococcus sp. PCC 7002 at 280.4 K in 99% D2O solution shows 18 hyperfine-shifted resonances. The non-solvent-exchangeable, hyperfineshifted resonances of reduced PsaC are clearly identified as belonging to the cysteines coordinating the clusters FA − and FB − by their downfield chemical shifts, by their temperature dependencies, and by their short T 1 relaxation times. The usual fast method of assigning the 1H NMR spectra of reduced [4Fe-4S] proteins through magnetization transfer from the oxidized to the reduced state was not feasible in the case of reduced PsaC. Therefore, a de novo self-consistent sequence-specific assignment of the hyperfine-shifted resonances was obtained based on dipolar connectivities from 1D NOE difference spectra and on longitudinal relaxation times using the X-ray structure of Clostridium acidi urici 2[4Fe-4S] cluster ferredoxin at 0.94 A resolution as a model. The results clearly show the same sequence-specific distribution of Curie and anti-Curie cysteines for unbound, reduced PsaC as established for other [4Fe-4S]-containing proteins; therefore, the mixed-valence and equal-valence (ferrous) Fe-Fe pairs in FA − and FB − have the same preferential positions relative to the protein. The analysis reveals that the magnetic properties of the two [4Fe-4S] clusters are essentially indistinguishable in unbound PsaC, in contrast to the PsaC that is bound as a component of the PS I complex.

The solution structure of the dicerium(III) complex of the N-terminal domain of calmodulin (Ce2-TR1C hereafter) has been solved employing paramagnetic T1 relaxation enhancements and pseudocontact shifts introduced by the Ce3+ ions, together with conventional NOE constraints. The use of pseudocontact shift constraints constitutes the first attempt to locate metal ions within a protein structure by NMR. Like calcium(II), paramagnetic cerium(III) has been found to bind to the two metal binding sites of the TR1C fragment of calmodulin in a cooperative manner. Due to the presence of pseudocontact interactions between the Ce3+ ions and protons of the 76-residue protein, the 1H NMR spectra of the complex show resonances shifted between +22 and -9 ppm. Eighty percent of its proton resonances could be assigned through a standard approach using TOCSY/COSY and NOESY spectra and through 1D NOE difference spectra for the broad resonances of protons close to the paramagnetic ions. A family of structures was calculated by means of the torsion angle dynamics program DYANA [Guntert, P., Mumenthaler, C., & Wuthrich, K. (1996) XVIIthInternational Conference on Magnetic Resonance inBiological Systems (Abstract)] using 1012 NOEs. Longitudinal proton relaxation times helped to roughly define the position of the metal ions within the protein. A total of 381 pseudocontact shift constraints, whose evaluation and use are critically discussed, have then been added to further refine the metal coordinates within the protein frame and to improve the structure resolution. A dramatic resolution improvement of the metal coordinates together with a sizable resolution improvement in the regions close to the paramagnetic centers, where the number of NOEs is low, is observed. The good quality of the solution structure permitted a meaningful comparison with the solid-state structure of calcium-loaded calmodulin at 1.7 A resolution [Chattopadhyaya, R., Meador, W. E., Means, A. R., & Quiocho, F. A. (1992) J. Mol. Biol. 228, 1177]. The Ce2-TR1C complex is overall more compact than the Ca form.

The solution structure of the 60-residue 1[Fe4-S4] ferredoxin from the hyperthermophilic bacterium Thermotoga maritima was determined based on 683 distance and 35 dihedral angle restraints that were obtained from NMR data. In addition, data known from crystallographic studies of ferredoxins was used for modeling of the iron-sulfur cluster and its environment. The protein shows a globular fold very similar to the fold of the related 1[Fe4-S4] ferredoxins from Desulfovibrio gigas and Desulfovibrio africanus, and elements of regular secondary structure similar to those in other ferredoxins were found in the T. maritima protein. In particular, the T. maritima protein displayed a beta-sheet structure made up of strands located at the very NH(2) and COOH termini of the protein, and an internal alpha-helix. The internal beta-sheet observed in the D. gigas and D. africanus ferredoxins could not be confirmed in T. maritima ferredoxin and is thus suggested to be only weakly present or even absent in this protein. This result suggests that thermostability in ferredoxins is not necessarily correlated with the content of stable elements of regular secondary structure.

The oxidized Fe4S4 ferredoxin from the hyperthermophilic bacterium Thermotoga maritima has been investigated by one- and two-dimensional NMR in order to characterize its hyperfine-shifted resonances originating from the cysteinyl cluster ligands and to assign its resonances in the diamagnetic shift range. The chemical shift and relaxation time pattern of the hyperfine-shifted signals is very similar to other oxidized Fe4S4 ferredoxins. A tentative sequence-specific assignment of these resonances according to a general pattern of chemical shift of cysteine protons versus sequence position of cluster ligand is presented. Furthermore, sequence-specific assignments for 85% of the amino acid residues that were obtained without any guidance by known X-ray structures of ferredoxins are given. They reveal the formation of at least two elements of secondary structure by the polypeptide chain of T. maritima ferredoxin: an alpha-helix comprising residues C43-D49 and a double-stranded antiparallel beta-sheet consisting of the N- and C-terminal parts of the protein. This folding pattern is very similar to that of the crystallographically characterized ferredoxin from the mesophile Desulfovibrio gigas [Kissinger, C.R., Sieker, L.C., Adman E.T. & Jensen, L.H. (1991) J. Mol. Biol. 219, 693-715] and therefore suggesting different mechanisms of stabilization for T. maritima ferredoxin and the ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus that was recently investigated by NMR [Teng, Q., Zhou, Z.H., Smith, E.T., Busse, S. C., Howard, J.B., Adams M.W.W. & La Mar, G.N. (1994) Biochemistry 33, 6316-6326].

Biosynthesis of RNA polymerase III transcripts requires binding of the La protein at their 3' end. La is an abundant nuclear RNA-binding protein which protects the nascent transcripts from 3' exonuclease degradation. Here, we report the high yield expression and preliminary structural analysis through NMR spectroscopy of two recombinant RNA binding domains (La motif and NRRM) from the La protein of Dictyostelium discoideum. Both recombinant protein constructs were well-folded and allowed for an almost complete sequence-specific assignment of the (15)N and (13)C labeled domains and their secondary structure prediction using PECAN online tool.

Pentameric ligand-gated ion channels (pLGICs) of the Cys loop family are transmembrane glycoproteins implicated in a variety of biological functions. Here, we present a solution NMR study of the extracellular domain of a prokaryotic pLGIC homologue from the bacterium Gloeobacter violaceus that is found to be a monomer in solution.

E3 ubiquitin ligases play a key role in the recognition of target proteins and the degradation by 26S proteasomes. Arkadia is the first example of an E3 ubiquitin ligase that positively regulates TGF-β family signaling. It has been shown to induce ubiquitin-dependent degradation of negative regulators of TGF-β signaling through its C-terminal RING domain. Structural analysis of Arkadia RING domain is needed to elucidate its enzymatic properties. For such studies efficient production of pure and correctly folded Arkadia protein is required. Here we report the recombinant expression in Escherichia coli and purification of the C-terminal RING domain of Arkadia . NMR analysis of the soluble construct reveals a stable folded protein suitable for high resolution structural studies.

Potassium channel-interacting proteins (KChIPs) are EF-hand calcium-binding proteins of the recoverin/neuronal calcium sensor 1 family that co-assemble with the pore-forming Kv4 alpha-subunits and thus control surface trafficking of the voltage-gated potassium channels mediating the neuronal I(A) and cardiac I(to) currents. Different from the other KChIPs, KChIP4a largely reduces surface expression of the Kv4 channel complexes. Using solution NMR we show that the unique N terminus of KChIP4a forms a 6-turn alpha-helix that is connected to the highly conserved core of the KChIP protein via a solvent-exposed linker. As identified by chemical shift changes, N-terminal alpha-helix and core domain of KChIP4a interact with each other through the same hydrophobic surface pocket that is involved in intermolecular interaction between the N-terminal helix of Kv4alpha and KChIP in Kv4-KChIP complexes. Electrophysiological recordings and biochemical interaction assays of complexes formed by wild-type and mutant Kv4alpha and KChIP4a proteins suggest that competition of these two helical domains for the surface groove is responsible for the reduced trafficking of Kv4-KChIP4a complexes to the plasma membrane. Surface expression of Kv4 complexes may thus be controlled by an auto-inhibitory domain in the KChIP subunit.

JAK2 V617F can be identified in the majority of polycythemia vera cases and in 50% of essential thrombocythemia and idiopathic myelofibrosis patients. JAK2 inhibitors including ruxolitinib, fedratinib and lestaurtinib display clinical activity in trials for PV, ET and IMF, and ruxolitinib has recently been approved for the treatment of primary and secondary myelofibrosis. In other malignancies it has been demonstrated that acquired resistance to kinase inhibitors emerge due to secondary resistance mutations in the targeted kinase. To identify JAK2 point mutations mediating ruxolitinib resistance and predict alternatively treatment strategies, we exposed JAK2 V617F expressing Ba/F3 cells to ruxolitinib. Surprisingly, sublines resistant to ruxolitinib at 1000nM, 2000nM and even 4000nM did not harbor point mutations neither in the kinase nor in the pseudokinase domain of JAK2. However, western blot analysis of sublines resistant to ruxolitinib revealed a 45-kDa JAK2 variant together with full length JAK2 V617F protein in 87% of the cases. Sequencing of the short form in drug resistant clones revealed a novel JAK2 variant missing amino acids 76 to 820 resulting in the N-terminal FERM domain directly fused to the kinase domain of JAK2 (FERM-JAK2). FERM-JAK2 was highly resistant to the ATP-competitive JAK2 inhibitors ruxolitinib and fedratinib. Phospho-deficient mutant studies provided evidence that FERM-JAK2 preferentially exist in an inactive state. This would impede drug binding and explain the resistant phenotype of FERM-JAK2. However, in this study we could not identify other genetic TKI resistant JAK2 variants than FERM-JAK2. This is in remarkable contrast to BCR-ABL mediated CML, where more than 90 residues have been described as TKI resistant variants and might indicate that in case of JAK2, the residues which mediate the drug resistance might comprise the JAK2 kinase function. Therefore, we treated JAK2 V617F expressing Ba/F3 cells with a chemical mutagen (ENU) to predict point mutations in JAK2 that might cause resistance towards the ruxolitinib. Surprisingly, after ENU pretreatment the frequency of drug resistant clones increased and most of the drug resistant clones displayed point mutations in the JAK2 kinase domain. In this screen, we identified L902Q, Y931C, L983F as the most frequent mutations and L902Q+R938E, L902Q+N947Q, L902Q+E1028K and L983F+Q959H as minor mutations. All these mutations confer cross-resistance across a panel of different JAK2 kinase inhibitors except L983FJAK2. L983FJAK2 reduces the sensitivity of JAK2-dependent cells to ruxolitinib and JAK inhibitor-1 but does not reduce the sensitivity of cell proliferation against fedratinib, thereby indicated that we identified a compound specific resistant mutations which is not an ATP-competitor specific mutation. Structure based modeling studies suggest that ruxolitinib is held by numerous hydrophobic interactions with residues Leu 855, Val 863, Ala 880, Val 911, Met 929, Leu 932 and Leu 983 that line the binding pocket. The aromatic ring of Tyr 931 is close enough to the pyrimidine ring to have p-p interaction. Surprisingly, this important interaction does not seem to be critical for the orientation of the inhibitor in the binding pocket as the Y931C mutation does not lead to a different binding pose. Leu 902 does not directly interact with ruxolitinib, however, it is close to the binding pocket and its mutation to Gln with a polar side chain significantly disturbs the inhibitor binding, thus explaining the high resistance of this mutation to ruxolitinib. Mutation of Leu 983 to Phe disrupts important hydrophobic interactions (e.g. Ala 880, Val 911, Met 929) with the pyrrolopyrimidine moiety and induces aromatic-aromatic interaction between the new phenyl ring and the pyrrol and pyrazol rings. Finally, our study also provided evidence that HSP90 inhibitors 17-AAG and geldanamycin are highly potent against TKI resistant FERM-JAK2, L902Q, Y931C and L983FJAK2 through JAK2 degradation and inhibition of the JAK2-STAT5 signaling axis. Taken together, our study highlight HSP90 inhibitors as a promising therapeutic agent in drug resistant mutations against tyrosine kinase inhibitors in MPN. Disclosures No relevant conflicts of interest to declare.

Small conductance Ca(2+)-activated potassium (SK) channels underlie the afterhyperpolarization that follows the action potential in many types of central neurons. SK channels are voltage-independent and gated solely by intracellular Ca(2+) in the submicromolar range. This high affinity for Ca(2+) results from Ca(2+)-independent association of the SK alpha-subunit with calmodulin (CaM), a property unique among the large family of potassium channels. Here we report the solution structure of the calmodulin binding domain (CaMBD, residues 396-487 in rat SK2) of SK channels using NMR spectroscopy. The CaMBD exhibits a helical region between residues 423-437, whereas the rest of the molecule lacks stable overall folding. Disruption of the helical domain abolishes constitutive association of CaMBD with Ca(2+)-free CaM, and results in SK channels that are no longer gated by Ca(2+). The results show that the Ca(2+)-independent CaM-CaMBD interaction, which is crucial for channel function, is at least in part determined by a region different in sequence and structure from other CaM-interacting proteins.

This work presents the three-dimensional NMR solution structure of recombinant, oxidized, unbound PsaC from Synechococcus sp. PCC 7002. Constraints are derived from homo- and heteronuclear one-, two- and three-dimensional (1)H and (15)N NMR data. Significant differences are outlined between the unbound PsaC structure presented here and the available X-ray structure of bound PsaC as an integral part of the whole cyanobacterial PS I complex. These differences mainly concern the arrangement of the N- and C-termini with respect to the [4Fe-4S] core domain. In the NMR solution structure of PsaC the C-terminal region assumes a disordered helical conformation, and is clearly different from the extended coil conformation, which is one of the structural elements required to anchor PsaC to the PS I core heterodimer. In solution the N-terminus of PsaC is in contact with the pre-C-terminal region but slides in between the latter and the iron-sulfur core region of the protein. Together, these features result in a concerted movement of the N-terminus and pre-C-terminal region away from the F(A) binding site, accompanied by a bending of the N-terminus. In comparison, the same terminal regions are positioned much closer to F(A) and take up an anti-parallel beta-sheet arrangement in PsaC bound to PS I. The conformational changes between bound and unbound PsaC correlate with the differences reported earlier for the EPR spectra of reduced F(A) and F(B) in bound versus unbound PsaC. The observed different structural features in solution are highly relevant for unraveling the stepwise assembly process of the stromal PsaC, PsaD and PsaE subunits to the PS I core heterodimer. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0321-3.

Heteronuclear multidimensional NMR spectroscopy was used to investigate in detail the structural and dynamical properties of a partially unfolded intermediate of the reduced high-potential iron-sulfur protein (HiPIP) from Chromatium vinosum present in 4 M guanidinium chloride solution. After an extensive assignment of 15N and 1H resonances, NOE data, proton longitudinal relaxation times, and 3JHNHalpha coupling constants as well as 15N relaxation parameters (T1, T2, T1rho, and 1H-15N NOE) were obtained and used to build a structural model of the intermediate. The Fe4S4 cluster of the HiPIP plays a decisive role in determining the resulting structure, which is random in the N-terminal half of the protein and partially organized in the loops between the cysteines bound to the cluster. Consistent with the structural data, the backbone mobility is typical of folded proteins in the regions where there are elements of structure and increases with the structural indetermination.

The solution structure of the D13C variant of the thermostable Fe7S8 ferredoxin from Bacillus schlegelii has been determined by 1H-NMR spectroscopy in its oxidized form. In a variable-temperature NMR study the D13C variant was as thermostable (up to 90 degrees C) as the wild-type protein (WT). Seventy-five out of 77 amino acid residues and 81% of all theoretically expected proton resonances in the D13C Fe8S8 protein have been assigned. Its structure was determined through torsion angle dynamics calculations with the program DYANA, using 935 meaningful NOEs (from a total of 1251), hydrogen bond constraints, and NMR-derived dihedral angle constraints for the cluster-ligating cysteines. Afterwards, restrained energy minimization and restrained molecular dynamics were applied to each conformer of the family. The final family of 20 structures has RMSD values from the mean structure of 0.055 nm for the backbone atoms and of 0.099 nm for all heavy atoms. The overall folding of the WT is maintained in the mutant, except for the immediate vicinity of the new cysteine, which becomes much more similar to native Fe8S8 proteins. The two residues at positions 11 and 12, which constitute an insertion with respect to all known Fe8S8 proteins, assume a conformation that does not prevent the preceding and following residues from folding like in native Fe8S8 proteins. Clear evidence for the existence of two conformations involving almost half of the amino acid residues was found. The two conformations are structurally indistinguishable. Temperature-dependent NMR experiments show that one of them is thermodynamically more stable than the other.

The solution structure of the paramagnetic seven-iron ferredoxin from Bacillus schlegelii in its oxidized form has been determined by 1H NMR. The protein, which contains 77 amino acids, is thermostable. Seventy-two residues and 79% of all theoretically expected proton resonances have been assigned. The structure has been determined through torsion angle dynamics calculations with the program DYANA, using 966 meaningful NOEs (from a total of 1305), hydrogen bond constraints, and NMR derived dihedral angle constraints for the cluster ligating cysteines, and by using crystallographic information to build up the two clusters. Afterwards, restrained energy minimization and restrained molecular dynamics were applied to each conformer of the family. The final family of 20 structures has RMSD values from the mean structure of 0.68 A for the backbone atoms and of 1.16 A for all heavy atoms. The contributions to the thermal stability of the B. schlegelii ferredoxin are discussed by comparing the present structure to that of the less stable Azotobacter vinelandii ferredoxin I which is the only other available structure of a bacterial seven-iron ferredoxin. It is proposed that the hydrophobic interactions and the hydrogen bond network linking the N-terminus and the C-terminus together and a high number of salt bridges contribute to the stability.

Recombinant PsaC was reconstituted in vitro and investigated by UV/vis, EPR, and 1H NMR spectroscopy. Its UV/vis and EPR spectroscopic properties correspond to those of the wild-type protein. Fast repetition 1D and 2D 1H NMR spectra allowed the sequence-specific assignment of the hyperfine-shifted proton resonances of the cluster-ligating resonances, taking advantage also of chemical shift analogies with other 4 and 8 Fe ferredoxins and a structural model for PsaC. The Calpha-Cbeta-S-Fe dihedral angles of the cluster ligands could be estimated from the chemical shifts and relaxation properties of their betaCH2 protons. All NMR-derived structural information on PsaC confirms its similarity to smaller 8Fe ferredoxins serving as electron transfer proteins in solution. Partial reduction of PsaC leads to an intermediate species with strongly exchange broadened 1H NMR resonances. The intermolecular electron exchange rate is estimated to be in the 10(2)-10(4) s-1 range, the intramolecular electron exchange rate between the two [Fe4S4] clusters to be higher than 10(4) s-1. The consequences of these findings for the electron transfer in photosystem I are discussed.

The full 1H NMR assignment of the reduced C77S mutant of Chromatium vinosum high-potential iron-sulfur protein (HiPIP) was achieved by taking advantage of the assignment available for the wild-type protein. A total of 1565 nuclear Overhauser effect (NOE) spectroscopy cross peaks were integrated and converted into distance constraints, of which 497 were found to be irrelevant. An additional 24 dipolar constraints were obtained from one-dimensional NOE difference spectra by saturating hyperfine-shifted beta CH2 cysteine/serine protons. Forty-six 3JNH-H alpha coupling constants and eight hydrogen bonds provided further constraints. Through a distance geometry approach, a family of 15 structures was calculated, which was subsequently subjected to restrained energy minimization. The root mean square deviations of the minimized structures were 0.62 +/- 0.09 and 1.09 +/- 0.11 A for backbone and heavy atoms, respectively. The resulting solution structures are very similar to those of the reduced wild-type protein (WT). An analysis of the NOEs experienced by the protons of Ser-77 in both the reduced and oxidized forms reveals that they are very similar to those experienced by Cys-77 in WT. On the basis of the hyperfine shifts observed for the Ser-77 protons and of the present structural analysis, it is concluded that the serine O gamma atom is coordinated to the polymetallic center, thus confirming the strict analogy of the electronic structures of the polymetallic center in both proteins. Capillary electrophoresis experiments demonstrate coordination of Ser-77 as an anion. Serine versus cysteine coordination in iron-sulfur proteins is briefly discussed.

Surfactant-containing eluents are evaluated for the analysis of carbamazepine in serum with conventional reversed-phase columns. Bovine serum was quantitatively eluted at the column void volume using surfactant concentrations in conventional reversed-phase eluents. The effect of pH, guard columns and column switching was evaluated with respect to separating and detecting clinical levels of the drug and its primary metabolite. Column lifetime was also investigated.

The N-terminal cluster binding motif Cys8XXXXXXXCys16....Cys49 of Bacillus schlegelii 7Fe ferredoxin, which provides the ligands to the [Fe3S4]+ cluster, was modified by the mutation Asp13 --Cys. The mutant D13C is expressed in Escherichia coli as an 8Fe ferredoxin, with NMR properties similar to those of clostridial-type ferredoxins. The full assignment of the hyperfine shifted resonances indicates that Cys13 serves as ligand to the new fourth iron atom in the N-terminal cluster despite the atypical binding sequence CysXXXXCysXXCys....Cys. The C alpha-C beta-S-Fe dihedral angles of all cysteine ligands to the two [Fe4S4]2+ clusters of the D13C variant are similar to those observed in other 8Fe and 4Fe ferredoxins.

Graphical abstract, Highlights • Tick saliva proteins Salp15 and Iric-1 promote tick feeding and pathogen transmission. • We established the first bacterial expression system for soluble Salp15 and Iric-1. • Using this system we mapped monoclonal antibody epitopes on Salp15 and Iric-1. • We defined the interaction sites with Borrelia outer surface protein C (OspC). • We elucidated first secondary structure features in Iric-1 by NMR., Ticks transmit numerous pathogens, including borreliae, which cause Lyme disease. Tick saliva contains a complex mix of anti-host defense factors, including the immunosuppressive cysteine-rich secretory glycoprotein Salp15 from Ixodes scapularis ticks and orthologs like Iric-1 from Ixodesricinus. All tick-borne microbes benefit from the immunosuppression at the tick bite site; in addition, borreliae exploit the binding of Salp15 to their outer surface protein C (OspC) for enhanced transmission. Hence, Salp15 proteins are attractive targets for anti-tick vaccines that also target borreliae. However, recombinant Salp proteins are not accessible in sufficient quantity for either vaccine manufacturing or for structural characterization. As an alternative to low-yield eukaryotic systems, we investigated cytoplasmic expression in Escherichia coli, even though this would not result in glycosylation. His-tagged Salp15 was efficiently expressed but insoluble. Among the various solubility-enhancing protein tags tested, DsbA was superior, yielding milligram amounts of soluble, monomeric Salp15 and Iric-1 fusions. Easily accessible mutants enabled epitope mapping of two monoclonal antibodies that, importantly, cross-react with glycosylated Salp15, and revealed interaction sites with OspC. Free Salp15 and Iric-1 from protease-cleavable fusions, despite limited solubility, allowed the recording of 1H–15N 2D NMR spectra, suggesting partial folding of the wild-type proteins but not of Cys-free variants. Fusion to the NMR-compatible GB1 domain sufficiently enhanced solubility to reveal first secondary structure elements in 13C/15N double-labeled Iric-1. Together, E. coli expression of appropriately fused Salp15 proteins may be highly valuable for the molecular characterization of the function and eventually the 3D structure of these medically relevant tick proteins.

The seven-iron ferredoxin from the hyperthermophilic archaeon Desulfurolobus ambivalens has been investigated by one-dimensional and two-dimensional 1H-NMR in its oxidized and dithionite-reduced states. All iron atoms of both the three-iron and the four-iron cluster are bound to cysteine residues whose hyperfine-shifted resonances were characterized. The pattern of these resonances is similar to those from three-iron, four-iron and eight-iron ferredoxins previously described in the literature, but the four-iron cluster has a shift pattern different from that in other seven-iron proteins. A second set of hyperfine-shifted resonances clearly indicates sample heterogeneity, which possibly involves the four-iron cluster. The observation of interresidue NOEs between two different cysteine residues proves the existence of close spatial proximity of the two clusters in D. ambivalens ferredoxin and therefore indicates structural homology to other dicluster ferredoxins. Moreover, this feature is crucial for the sequence-specific assignment of the hyperfine-shifted resonances. The C alpha-C beta-S-Fe dihedral angles of the cysteine residues coordinating the four-iron cluster could be estimated, and the electronic structure of the three-iron cluster is discussed.