Structural analysis of a new carotenoid-binding protein: the C-terminal domain homolog of the OCP.

The Orange Carotenoid Protein (OCP) is a water-soluble protein that governs photoprotection in many cyanobacteria. The 35 kDa OCP is structurally and functionally modular, consisting of an N-terminal effector domain (NTD) and a C-terminal regulatory domain (CTD); a carotenoid spans the two domains. The CTD is a member of the ubiquitous Nuclear Transport Factor-2 (NTF2) superfamily (pfam02136). With the increasing availability of cyanobacterial genomes, bioinformatic analysis has revealed the existence of a new family of proteins, homologs to the CTD, the C-terminal domain-like carotenoid proteins (CCPs). Here we purify holo-CCP2 directly from cyanobacteria and establish that it natively binds canthaxanthin (CAN). We use small-angle X-ray scattering (SAXS) to characterize the structure of this carotenoprotein in two distinct oligomeric states. A single carotenoid molecule spans the two CCPs in the dimer. Our analysis with X-ray footprinting-mass spectrometry (XFMS) identifies critical residues for carotenoid binding that likely contribute to the extreme red shift (ca. 80 nm) of the absorption maximum of the carotenoid bound by the CCP2 dimer and a further 10 nm shift in the tetramer form. These data provide the first structural description of carotenoid binding by a protein consisting of only an NTF2 domain.

Structural insights into ubiquitin recognition and Ufd1 interaction of Npl4.

Npl4 is likely to be the most upstream factor recognizing Lys48-linked polyubiquitylated substrates in the proteasomal degradation pathway in yeast. Along with Ufd1, Npl4 forms a heterodimer (UN), and functions as a cofactor for the Cdc48 ATPase. Here, we report the crystal structures of yeast Npl4 in complex with Lys48-linked diubiquitin and with the Npl4-binding motif of Ufd1. The distal and proximal ubiquitin moieties of Lys48-linked diubiquitin primarily interact with the C-terminal helix and N-terminal loop of the Npl4 C-terminal domain (CTD), respectively. Mutational analysis suggests that the CTD contributes to linkage selectivity and initial binding of ubiquitin chains. Ufd1 occupies a hydrophobic groove of the Mpr1/Pad1 N-terminal (MPN) domain of Npl4, which corresponds to the catalytic groove of the MPN domain of JAB1/MPN/Mov34 metalloenzyme (JAMM)-family deubiquitylating enzyme. This study provides important structural insights into the polyubiquitin chain recognition by the Cdc48-UN complex and its assembly.

Crystal structure of importin-α3 bound to the nuclear localization signal of Ran-binding protein 3.

Ran-binding protein 3 (RanBP3) is a primarily nuclear Ran-binding protein that functions as an accessory factor in the Ran GTPase system. RanBP3 associates with Ran-specific nucleotide exchange factor RCC1 and enhances its catalytic activity towards Ran. RanBP3 also promotes CRM1-mediated nuclear export as well as CRM1-independent nuclear export of ß-catenin, Smad2, and Smad3. Nuclear import of RanBP3 is dependent on the nuclear import adaptor protein importin-α and, RanBP3 is imported more efficiently by importin-α3 than by other members of the importin-α family. Protein kinase signaling pathways control nucleocytoplasmic transport through phosphorylation of RanBP3 at Ser58, immediately C-terminal to the nuclear localization signal (NLS) in the N-terminal region of RanBP3. Here we report the crystal structure of human importin-α3 bound to an N-terminal fragment of human RanBP3 containing the NLS sequence that is necessary and sufficient for nuclear import. The structure reveals that RanBP3 binds to importin-α3 residues that are strictly conserved in all seven isoforms of human importin-α at the major NLS-binding site, indicating that the region of importin-α outside the NLS-binding site, possibly the autoinhibotory importin-ß1-binding domain, may be the key determinant for the preferential binding of RanBP3 to importin-α3. Computational docking simulation indicates that phosphorylation of RanBP3 at Ser58 could potentially stabilize the association of RanBP3 with importin-α through interactions between the phosphate moiety of phospho-Ser58 of RanBP3 and a cluster of basic residues (Arg96 and Lys97 in importin-α3) on armadillo repeat 1 of importin-α.

Structural Characterization of the Chaetomium thermophilum TREX-2 Complex and its Interaction with the mRNA Nuclear Export Factor Mex67:Mtr2.

The TREX-2 complex integrates mRNA nuclear export into the gene expression pathway and is based on a Sac3 scaffold to which Thp1, Sem1, Sus1, and Cdc31 bind. TREX-2 also binds the mRNA nuclear export factor, Mex67:Mtr2, through the Sac3 N-terminal region (Sac3N). Here, we characterize Chaetomium thermophilum TREX-2, show that the in vitro reconstituted complex has an annular structure, and define the structural basis for interactions between Sac3, Sus1, Cdc31, and Mex67:Mtr2. Crystal structures show that the binding of C. thermophilum Sac3N to the Mex67 NTF2-like domain (Mex67(NTF2L)) is mediated primarily through phenylalanine residues present in a series of repeating sequence motifs that resemble those seen in many nucleoporins, and Mlp1 also binds Mex67:Mtr2 using a similar motif. Deletion of Sac3N generated growth and mRNA export defects in Saccharomyces cerevisiae, and we propose TREX-2 and Mlp1 function to facilitate export by concentrating mature messenger ribonucleoparticles at the nuclear pore entrance.

Characterization of histone (H1B) oxalate binding protein in experimental urolithiasis and bioinformatics approach to study its oxalate interaction.

The rat kidney H1 oxalate binding protein was isolated and purified. Oxalate binds exclusively with H1B fraction of H1 histone. Oxalate binding activity is inhibited by lysine group modifiers such as 4',4'-diisothiostilbene-2,2-disulfonic acid (DIDS) and pyridoxal phosphate and reduced in presence of ATP and ADP. RNA has no effect on oxalate binding activity of H1B whereas DNA inhibits oxalate binding activity. Equilibrium dialysis method showed that H1B oxalate binding protein has two binding sites for oxalate, one with high affinity, other with low affinity. Histone H1B was modeled in silico using Modeller8v1 software tool since experimental structure is not available. In silico interaction studies predict that histone H1B-oxalate interaction take place through lysine121, lysine139, and leucine68. H1B oxalate binding protein is found to be a promoter of calcium oxalate crystal (CaOx) growth. A 10% increase in the promoting activity is observed in hyperoxaluric rat kidney H1B. Interaction of H1B oxalate binding protein with CaOx crystals favors the formation of intertwined calcium oxalate dehydrate (COD) crystals as studied by light microscopy. Intertwined COD crystals and aggregates of COD crystals were more pronounced in the presence of hyperoxalauric H1B.