TRIM16 Acts as an E3 Ubiquitin Ligase and Can Heterodimerize with Other TRIM Family Members

The TRIM family of proteins is distinguished by its tripartite motif (TRIM). Typically, TRIM proteins contain a RING finger domain, one or two B-box domains, a coiled-coil domain and the more variable C-terminal domains. TRIM16 does not have a RING domain but does harbour two B-box domains. Here we showed that TRIM16 homodimerized through its coiled-coil domain and heterodimerized with other TRIM family members; TRIM24, Promyelocytic leukaemia (PML) protein and Midline-1 (MID1). Although, TRIM16 has no classic RING domain, three-dimensional modelling of TRIM16 suggested that its B-box domains adopts RING-like folds leading to the hypothesis that TRIM16 acts as an ubiquitin ligase. Consistent with this hypothesis, we demonstrated that TRIM16, devoid of a classical RING domain had auto-polyubiquitination activity and acted as an E3 ubiquitin ligase in vivo and in vitro assays. Thus via its unique structure, TRIM16 possesses both heterodimerization function with other TRIM proteins and also has E3 ubiquitin ligase activity

NMR Studies of the C-Terminus of alpha4 Reveal Possible Mechanism of Its Interaction with MID1 and Protein Phosphatase 2A

Alpha4 is a regulatory subunit of the protein phosphatase family of enzymes and plays an essential role in regulating the catalytic subunit of PP2A (PP2Ac) within the rapamycin-sensitive signaling pathway. Alpha4 also interacts with MID1, a microtubule-associated ubiquitin E3 ligase that appears to regulate the function of PP2A. The C-terminal region of alpha4 plays a key role in the binding interaction of PP2Ac and MID1. Here we report on the solution structure of a 45-amino acid region derived from the C-terminus of alpha4 (alpha45) that binds tightly to MID1. In aqueous solution, alpha45 has properties of an intrinsically unstructured peptide although chemical shift index and dihedral angle estimation based on chemical shifts of backbone atoms indicate the presence of a transient α-helix. Alpha45 adopts a helix-turn-helix HEAT-like structure in 1% SDS micelles, which may mimic a negatively charged surface for which alpha45 could bind. Alpha45 binds tightly to the Bbox1 domain of MID1 in aqueous solution and adopts a structure consistent with the helix-turn-helix structure observed in 1% SDS. The structure of alpha45 reveals two distinct surfaces, one that can interact with a negatively charged surface, which is present on PP2A, and one that interacts with the Bbox1 domain of MID1

Deciphering ligand specificity of a Clostridium thermocellum family 35 carbohydrate binding module (CtCBM35) for Gluco- and Galacto- Substituted mannans and Its calcium induced stability

Articles in International JournalsThis study investigated the role of CBM35 from Clostridium thermocellum (CtCBM35) in polysaccharide recognition. CtCBM35 was cloned into pET28a (+) vector with an engineered His6 tag and expressed in Escherichia coli BL21 (DE3) cells. A homogenous 15 kDa protein was purified by immobilized metal ion chromatography (IMAC). Ligand binding analysis of CtCBM35 was carried out by affinity electrophoresis using various soluble ligands. CtCBM35 showed a manno-configured ligand specific binding displaying significant association with konjac glucomannan (Ka = 14.3×104 M−1), carob galactomannan (Ka = 12.4×104 M−1) and negligible association (Ka = 12 µM−1) with insoluble mannan. Binding of CtCBM35 with polysaccharides which was calcium dependent exhibited two fold higher association in presence of 10 mM Ca2+ ion with konjac glucomannan (Ka = 41×104 M−1) and carob galactomannan (Ka = 30×104 M−1). The polysaccharide binding was further investigated by fluorescence spectrophotometric studies. On binding with carob galactomannan and konjac glucomannan the conformation of CtCBM35 changed significantly with regular 21 nm peak shifts towards lower quantum yield. The degree of association (Ka) with konjac glucomannan and carob galactomannan, 14.3×104 M−1 and 11.4×104 M−1, respectively, corroborated the findings from affinity electrophoresis. The association of CtCBM35with konjac glucomannan led to higher free energy of binding (ΔG) −25 kJ mole−1 as compared to carob galactomannan (ΔG) −22 kJ mole−1. On binding CtCBM35 with konjac glucomannan and carob galactomannan the hydrodynamic radius (RH) as analysed by dynamic light scattering (DLS) study, increased to 8 nm and 6 nm, respectively, from 4.25 nm in absence of ligand. The presence of 10 mM Ca2+ ions imparted stiffer orientation of CtCBM35 particles with increased RH of 4.52 nm. Due to such stiffer orientation CtCBM35 became more thermostable and its melting temperature was shifted to 70°C from initial 50°C

SUMO-Interacting Motifs of Human TRIM5α are Important for Antiviral Activity

Human TRIM5α potently restricts particular strains of murine leukemia viruses (the so-called N-tropic strains) but not others (the B- or NB-tropic strains) during early stages of infection. We show that overexpression of SUMO-1 in human 293T cells, but not in mouse MDTF cells, profoundly blocks N-MLV infection. This block is dependent on the tropism of the incoming virus, as neither B-, NB-, nor the mutant R110E of N-MLV CA (a B-tropic switch) are affected by SUMO-1 overexpression. The block occurred prior to reverse transcription and could be abrogated by large amounts of restricted virus. Knockdown of TRIM5α in 293T SUMO-1-overexpressing cells resulted in ablation of the SUMO-1 antiviral effects, and this loss of restriction could be restored by expression of a human TRIM5α shRNA-resistant plasmid. Amino acid sequence analysis of human TRIM5α revealed a consensus SUMO conjugation site at the N-terminus and three putative SUMO interacting motifs (SIMs) in the B30.2 domain. Mutations of the TRIM5α consensus SUMO conjugation site did not affect the antiviral activity of TRIM5α in any of the cell types tested. Mutation of the SIM consensus sequences, however, abolished TRIM5α antiviral activity against N-MLV. Mutation of lysines at a potential site of SUMOylation in the CA region of the Gag gene reduced the SUMO-1 block and the TRIM5α restriction of N-MLV. Our data suggest a novel aspect of TRIM5α-mediated restriction, in which the presence of intact SIMs in TRIM5α, and also the SUMO conjugation of CA, are required for restriction. We propose that at least a portion of the antiviral activity of TRIM5α is mediated through the binding of its SIMs to SUMO-conjugated CA

The crystal structure of the Leishmania infantum Silent Information Regulator 2 related protein 1: implications to protein function and drug design.

The research leading to these results received funding from the European Community’s Seventh Framework Programme under grant agreement No.602773 (Project KINDRED).The de novo crystal structure of the Leishmania infantum Silent Information Regulator 2 related protein 1 (LiSir2rp1) has been solved at 1.99Å in complex with an acetyl-lysine peptide substrate. The structure is broadly commensurate with Hst2/SIRT2 proteins of yeast and human origin, reproducing many of the structural features common to these sirtuin deacetylases, including the characteristic small zinc-binding domain, and the larger Rossmann-fold domain involved in NAD+-binding interactions. The two domains are linked via a cofactor binding loop ordered in open conformation. The peptide substrate binds to the LiSir2rp1 protein via a cleft formed between the small and large domains, with the acetyl-lysine side chain inserting further into the resultant hydrophobic tunnel. Crystals were obtained only with recombinant LiSir2rp1 possessing an extensive internal deletion of a proteolytically-sensitive region unique to the sirtuins of kinetoplastid origin. Deletion of 51 internal amino acids (P253-E303) from LiSir2rp1 did not appear to alter peptide substrate interactions in deacetylation assays, but was indispensable to obtain crystals. Removal of this potentially flexible region, that otherwise extends from the classical structural elements of the Rossmann-fold, specifically the β8-β9 connector, appears to result in lower accumulation of the protein when expressed from episomal vectors in L. infantum SIR2rp1 single knockout promastigotes. The biological function of the large serine-rich insertion in kinetoplastid/trypanosomatid sirtuins, highlighted as a disordered region with strong potential for post-translational modification, remains unknown but may confer additional cellular functions that are distinct from their human counterparts. These unique molecular features, along with the resolution of the first kinetoplastid sirtuin deacetylase structure, present novel opportunities for drug design against a protein target previously established as essential to parasite survival and proliferation.Publisher PDFPeer reviewe

Paramagnetic effect on alpha45.

<p><b>A.</b> Superposition of the ¹H-¹⁵N HSQC spectra of alpha45 in 1% SDS (black) and in 1% SDS with 4× excess MnCl₂ (red). Amino acids that are solvent exposed and/or accessible to Mn²⁺ had their ¹H-¹⁵N signals broadened. Those amino acids are labeled. <b>B.</b> Ribbon representation of alpha45 shows the location of the basic residues that were most protected from the paramagnetic effects in 1% SDS. The regions colored in green represent the amino acids that were protected from paramagnetic effect only in SDS but not in aqueous solution. The residues whose NH signals were affected by Mn²⁺ in both SDS and aqueous solution are shown in red and found to be located on the outer surface of helix II <b>C.</b> Two views of the electrostatic map of alpha45. The blue represents basic patches and red indicates acid patches. The orientation of the left image is the same as that shown in ‘B’. The map was generated by PBEQ-solver <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028877#pone.0028877-Jo1" target="_blank">[59]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028877#pone.0028877-Im1" target="_blank">[60]</a>.</p

Interaction of alpha45 and the MID1 Bbox1 domain.

<p><b>A.</b> Superposition of the ¹H-¹⁵N HSQC spectra of free ¹⁵N-labeled alpha45 (black) and ¹⁵N-labeled alpha45 (red) in the presence of 1∶1 ratio of unlabeled Bbox1 in aqueous solution. Amino acids of alpha45 whose NH peaks showed chemical shift changes due to interaction are labeled. <b>B.</b> Ribbon representation of alpha45 indicates the location and residues that showed chemical shift changes when Bbox1 was added. <b>C.</b> Superposition of the ¹H-¹⁵N HSQC spectra of free ¹⁵N-labeled Bbox1 (black) and ¹⁵N-labeled Bbox1 with unlabeled alpha45 (red). The ratio of Bbox1 to alpha45 was 1∶0.5. In additions to peak shifts, new peaks with intensities corresponding to ∼0.5 that of the original peak were observed at different locations, indicating Bbox1 existed in two slow exchanging states: free and bound. <b>D.</b> Surface and ribbon representations of the MID1 Bbox1 domain showing the residues that underwent chemical shift changes when alpha45 was bound. Basic and acidic residues are colored blue and red, respectively, while hydrophobic residues are colored green. Polar residues are colored cyan.</p

Structure of alpha45.

<p><b>A.</b> Strip plots taken from the 2D ¹H-¹H projections of the ¹³C planes of the 3D-¹H-¹³C-edited HSQC-NOESY show long range NOEs between residues Val267 with Leu245 and Thr246, and between Thr246 and Tyr271, which were used in the tertiary structural calculation of alpha45. All intra-residue NOEs are labeled on the figure and arrows indicate inter-residues NOEs. In this modified version of the ¹³C-edited NOESY spectrum, the diagonal auto-peaks were suppressed. <b>B.</b> Superposition of the backbone Cα, C, N atoms for residues 243 to 277 of fourteen structures of alpha45 calculated based on NMR restraints acquired in 1% SDS. The backbone atoms of the two α-helices are colored red (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028877#pone-0028877-t001" target="_blank">Table 1</a>). <b>C.</b> Ribbon representation of the structure of alpha45 in 1% SDS. For clarity, two orientations of the structure are shown using the same color scheme noted above.</p

Secondary structure analysis of alpha45 in aqueous solution by NMR.

<p><b>A.</b> HSQC spectrum of alpha45 in aqueous solution reveals that the NH signals fall within 1 ppm of each other, suggesting a labile structure. The NH assignments were identified using the 3D NMR data. M* represents one of the three amino acids that is the result from TEV cleavage; this residue is not part of alpha4 sequence. <b>B.</b> Strip plots taken from the 2D ¹H-¹H projections from the ¹⁵N planes of the 3D ¹H-¹⁵N NOESY-HSQC spectrum showing NOE correlations between the NH_(i) to intra-residue and preceding Hα atoms (top panels) and sequential NH to NH atoms (bottom panels) for residues predicted to be helix II. An attempt to show NH-NH_(i,i±1) NOE correlations is indicated by lines, but the NOEs are weak, ambiguous and mostly missing. <b>C(i).</b> Analysis of the Cα and Hα atom chemical shift index (CSI) of alpha45 in aqueous solution. Upfield shifted Cα and simultaneous downfield shifted Hα values are indicative of α-helices. For the peptide in aqueous solution, helix I cannot be definitively characterized because while the Hα values are upfield shifted, the Cα values are closer to zero compared to those of helix II. <b>C(ii).</b> The phi (Φ, blue line) and psi (Ψ, red line) values and the order parameter (S², green line) are plotted for each amino acid. These values were predicted by TALOS+ based on the chemical shift data. Residues adopting helical structure will have similar Φ and Ψ values. The higher the S² values, the less mobile it is for that amino acid.</p