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.

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.

NMR evidence of charge-dependent interaction between various PND V3 and CCR5 N-terminal peptides.

The third variable (V3) loop is an important region of glycoprotein 120 (gp120) for many biological processes, as it contains the highly conserved GPGR sequence and it represents the binding site for human immunodeficiency virus 1 (HIV-1) antibodies and for CCR5 and CXCR4 host cell coreceptors. The interaction of the principal neutralizing determinant (PND) V3 with the chemokine receptor CCR5 N-terminal region has been reported to be crucial for HIV-1 infection. The goal of this study is to characterize the solution structures of three HIV-1 gp120 V3 subtype B peptides and their interaction with a nonsulfated N-terminal CCR5 peptide. NMR titration experiments revealed that the CCR5Nt-PND V3 interaction is dependent on the number of the positively charged V3 residues, which is in agreement with the observation that increase in positive charge in the V3 sequence correlates with the augmentation of the interaction. As expected for free peptides in solution, the peptides representing the PND V3 region of gp120 exhibit conformational flexibility, but they also exhibit a large number of NOEs which allowed convergence to a dominant conformation. The PND V3 peptides retain the U-turn conformation observed in the crystal structures of gp120 complexes independently of CCR5 presence. The interaction of different regions of the CCR5Nt peptide is gradually increasing proportionally to the positive charge increase in the V3 peptides. The data demonstrate that the PND V3 and CCR5Nt peptide sequences have propensities for interaction even in the absence of sulfated tyrosines and that their binding and selectivity is determined by simple electrostatic attraction mechanisms.

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.

The delivery of copper for thylakoid import observed by NMR.

The thylakoid compartments of plant chloroplasts are a vital destination for copper. Copper is needed to form holo-plastocyanin, which must shuttle electrons between photosystems to convert light into biologically useful chemical energy. Copper can bind tightly to proteins, so it has been hypothesized that copper partitions onto ligand-exchange pathways to reach intracellular locations without inflicting damage en route. The copper metallochaperone Atx1 of chloroplast-related cyanobacteria (ScAtx1) engages in bacterial two-hybrid interactions with N-terminal domains of copper-transporting ATPases CtaA (cell import) and PacS (thylakoid import). Here we visualize copper delivery. The N-terminal domain PacS(N) has a ferredoxin-like fold that forms copper-dependent heterodimers with ScAtx1. Removal of copper, by the addition of the cuprous-ion chelator bathocuproine disulfonate, disrupts this heterodimer, as shown from a reduction of the overall tumbling rate of the protein mixture. The NMR spectral changes of the heterodimer versus the separate proteins reveal that loops 1, 3, and 5 (the carboxyl tail) of the ScAtx1 Cu(I) site switch to an apo-like configuration in the heterodimer. NMR data ((2)J(NH) couplings in the imidazole ring of (15)N ScAtx1 His-61) also show that His-61, bound to copper(I) in [Cu(I)ScAtx1](2), is not coordinated to copper in the heterodimer. A model for the PacS(N)/Cu(I)/ScAtx1 complex is presented. Contact with PacS(N) induces change to the ScAtx1 copper-coordination sphere that drives copper release for thylakoid import. These data also elaborate on the mechanism to keep copper(I) out of the ZiaA(N) ATPase zinc sites.