Conformational plasticity of the VEEV macro domain is important for binding of ADP-ribose.

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 ß5-α3 and α3-ß6 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.

NMR conformational properties of an Anthrax Lethal Factor domain studied by multiple amino acid-selective labeling.

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.

NMR-based insights into the conformational and interaction properties of Arkadia RING-H2 E3 Ub ligase.

Arkadia (Rnf111), an E3 Ubiquitin (Ub) ligase, amplifies TGF-ß signaling responses by targeting for degradation of the negative regulators Smad6/7 and the SnoN/Ski transcriptional repressors when they block the TGF-ß 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 (ß)ßßα 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.

Impact of a Single Nucleotide Polymorphism on the 3D Protein Structure and Ubiquitination Activity of E3 Ubiquitin Ligase Arkadia.

Single nucleotide polymorphisms (SNPs) are genetic variations which can play a vital role in the study of human health. SNP studies are often used to identify point mutations that are associated with diseases. Arkadia (RNF111) is an E3 ubiquitin ligase that enhances transforming growth factor-beta (TGF-ß) signaling by targeting negative regulators for degradation. Dysregulation of the TGF-ß pathway is implicated in cancer because it exhibits tumor suppressive activity in normal cells while in tumor cells it promotes invasiveness and metastasis. Τhe SNP CGT > TGT generated an amino-acid (aa) substitution of Arginine 957 to Cysteine on the enzymatic RING domain of Arkadia. This was more prevalent in a tumor than in a normal tissue sample of a patient with colorectal cancer. This prompted us to investigate the effect of this mutation in the structure and activity of Arkadia RING. We used nuclear magnetic resonance (NMR) to analyze at an atomic-level the structural and dynamic properties of the R957C Arkadia RING domain, while ubiquitination and luciferase assays provided information about its enzymatic functionality. Our study showed that the R957C mutation changed the electrostatic properties of the RING domain however, without significant effects on the structure of its core region. However, the functional studies revealed that the R957C Arkadia exhibits significantly increased enzymatic activity supporting literature data that Arkadia within tumor cells promotes aggressive and metastatic behavior.

Unravelling the conformational plasticity of the extracellular domain of a prokaryotic nAChR homologue in solution by NMR.

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.

Conformational dynamics of the anthrax lethal factor catalytic center.

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.

Purification and biophysical characterization of the core protease domain of anthrax lethal factor.

Anthrax lethal toxin (LeTx) stands for the major virulence factor of the anthrax disease. It comprises a 90kDa highly specific metalloprotease, the anthrax lethal factor (LF). LF possesses a catalytic Zn(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(672-776)) that harbours the enzyme's core protease domain. The biophysical characterization and backbone assignments ((1)H, (13)C, (15)N) of the polypeptide revealed a stable, well folded structure even in the absence of Zn(2+), suitable for high resolution structural analysis by NMR.

High yield expression and NMR characterization of Arkadia E3 ubiquitin ligase RING-H2 finger domain.

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-beta family signaling. It has been shown to induce ubiquitin-dependent degradation of negative regulators of TGF-beta 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.

Deciphering the Nucleotide and RNA Binding Selectivity of the Mayaro Virus Macro Domain.

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" (ßßαßßαßαßα) 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.

Complex formation of APP with GABAB receptors links axonal trafficking to amyloidogenic processing.

GABAB receptors (GBRs) are key regulators of synaptic release but little is known about trafficking mechanisms that control their presynaptic abundance. We now show that sequence-related epitopes in APP, AJAP-1 and PIANP bind with nanomolar affinities to the N-terminal sushi-domain of presynaptic GBRs. Of the three interacting proteins, selectively the genetic loss of APP impaired GBR-mediated presynaptic inhibition and axonal GBR expression. Proteomic and functional analyses revealed that APP associates with JIP and calsyntenin proteins that link the APP/GBR complex in cargo vesicles to the axonal trafficking motor. Complex formation with GBRs stabilizes APP at the cell surface and reduces proteolysis of APP to Aß, a component of senile plaques in Alzheimer's disease patients. Thus, APP/GBR complex formation links presynaptic GBR trafficking to Aß formation. Our findings support that dysfunctional axonal trafficking and reduced GBR expression in Alzheimer's disease increases Aß formation.

NMR study of non-structural proteins-part III: 1H, 13C, 15N backbone and side-chain resonance assignment of macro domain from Chikungunya virus (CHIKV).

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/ß/α sandwich topology with 4 α-helices and 6 ß-strands was predicted by TALOS+.

A Residue Specific Insight into the Arkadia E3 Ubiquitin Ligase Activity and Conformational Plasticity.

Arkadia (Rnf111) is an E3 ubiquitin ligase that plays a central role in the amplification of transforming growth factor beta (TGF-ß) 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-ß-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-ß 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.

Soluble cysteine-rich tick saliva proteins Salp15 and Iric-1 from E. coli.

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 Ixodes ricinus. 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 (1)H-(15)N 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 (13)C/(15)N 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.

NMR study of non-structural proteins--part II: (1)H, (13)C, (15)N backbone and side-chain resonance assignment of macro domain from Venezuelan equine encephalitis virus (VEEV).

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/ß-fold.

Backbone and side chain NMR assignment, along with the secondary structure prediction of RRM2 domain of La protein from a lower eukaryote exhibiting identical structural organization with its human homolog.

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 study of non-structural proteins--part I: (1)H, (13)C, (15)N backbone and side-chain resonance assignment of macro domain from Mayaro virus (MAYV).

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+.

(1)H, (13)C and (15)N backbone and side-chain resonance assignment of the LAM-RRM1 N-terminal module of La protein from Dictyostelium discoideum.

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.

¹H, ¹5N, ¹³C assignment and secondary structure determination of two domains of La protein from D. discoideum.

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.

NMR analysis of KChIP4a reveals structural basis for control of surface expression of Kv4 channel complexes.

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.

Solution structure and function of the "tandem inactivation domain" of the neuronal A-type potassium channel Kv1.4.

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.