Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions.

Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. Here, we report the structures of four domains of Regnase-1 from Mus musculus-the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity. We found that the NTD associates with the PIN domain and significantly enhances its RNase activity. The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions.

Identification of a Regulatory Acidic Motif as the Determinant of Membrane Localization of TICAM-2.

TLR4 triggers LPS signaling through the adaptors Toll/IL-1R domain-containing adaptor molecule (TICAM)-2 (also called TRAM) and TICAM-1 (also called TRIF), together with Toll/IL-1R domain-containing adaptor protein (TIRAP) and MyD88. The MyD88 pathway mediates early phase responses to LPS on the plasma membrane, whereas the TICAM pathway mediates late-phase responses, which induce the production of type I IFN and activation of inflammasomes. TICAM-2 bridges TLR4 and TICAM-1 for LPS signaling in the endosome. Recently, we identified an acidic motif, E87/D88/D89 in TICAM-2, that provides the interaction surfaces between TICAM-2 and TICAM-1. In the present study, we found additional D91/E92 in TICAM-2, conserved across species, that is crucial for TICAM-1 activation. The D91A/E92A mutant protein was distributed largely to the cytosol, despite myristoylation, suggesting its importance for assistance of membrane localization of TICAM-2. An ectopically expressed D91A/E92A mutant per se failed to activate TICAM-1, unlike its wild-type counterpart that forms self-aggregation, but it still retained the ability to pass LPS-mediated IFN regulatory factor (IRF)3 activation. In a TICAM-2 knockout human cell line expressing TLR4/MD-2 with or without CD14, overexpression of the D91A/E92A mutant did not activate IRF3, but upon LPS stimulation, it induced sufficient TLR4-mediated IRF3 activation with high coefficient colocalization. Hence, the D91/E92 motif guides TICAM-2 membrane localization and self-activation for signaling. Our results suggest the presence of two distinct steps underlying endosomal LPS signaling on TICAM-2 for TICAM-1 activation: TICAM-2 assembling in TLR4 and/or TICAM-2 self-activation. D91A/E92A of TICAM-2 selectively associates the TLR4-dependent TICAM-2 assembling, but not cytosolic TICAM-2 self-aggregation, to activate TICAM-1.

Structures and interface mapping of the TIR domain-containing adaptor molecules involved in interferon signaling.

Homotypic and heterotypic interactions between Toll/interleukin-1 receptor (TIR) domains in Toll-like receptors (TLRs) and downstream adaptors are essential to evoke innate immune responses. However, such oligomerization properties present intrinsic difficulties in structural studies of TIR domains. Here, using BB-loop mutations that disrupt homotypic interactions, we determined the structures of the monomeric TIR domain-containing adaptor molecule (TICAM)-1 and TICAM-2 TIR domains. Docking of the monomeric structures, together with yeast two hybrid-based mutagenesis assays, reveals that the homotypic interaction between TICAM-2 TIR is indispensable to present a scaffold for recruiting the monomeric moiety of the TICAM-1 TIR dimer. This result proposes a unique idea that oligomerization of upstream TIR domains is crucial for binding of downstream TIR domains. Furthermore, the bivalent nature of each TIR domain dimer can generate a large signaling complex under the activated TLRs, which would recruit downstream signaling molecules efficiently. This model is consistent with previous reports that BB-loop mutants completely abrogate downstream signaling.

Unfolding of higher DNA structures formed by the d(CGG) triplet repeat by UP1 protein.

Fragile X syndrome is caused by expansion of a d(CGG) triplet repeat in the 5'-untranslated region of the first exon of the FMR1 gene resulting in silencing of the gene. The d(CGG) repeat has been reported to form hairpin and quadruplex structures in vitro, and formation of these higher structures could be responsible for its unstable expansion in the syndrome, although molecular mechanisms underlying the repeat expansion still remain elusive. We have previously proved that UP1, a proteolytic product of hnRNP A1, unfolds the intramolecular quadruplex structures of d(GGCAG)5 and d(TTAGGG)4 and abrogates the arrest of DNA synthesis at d(GGG)n sites. Here, we demonstrate that the d(CGG) repeat forms a peculiar DNA structure, which deviates from the canonical B-form structure. In addition, UP1 was demonstrated by CD spectrum analysis to unfold this characteristic higher structure of the d(CGG) repeat and to abrogate the arrest of DNA synthesis at the site. This ability of UP1 suggests that unfolding of unusual DNA structures of a triplet repeat is required for DNA synthesis processes.

Structure of hnRNP D complexed with single-stranded telomere DNA and unfolding of the quadruplex by heterogeneous nuclear ribonucleoprotein D.

Heterogeneous nuclear ribonucleoprotein D, also known as AUF1, has two DNA/RNA-binding domains, each of which can specifically bind to single-stranded d(TTAGGG)n, the human telomeric repeat. Here, the structure of the C-terminal-binding domain (BD2) complexed with single-stranded d(TTAGGG) determined by NMR is presented. The structure has revealed that each residue of the d(TAG) segment is recognized by BD2 in a base-specific manner. The interactions deduced from the structure have been confirmed by gel retardation experiments with mutant BD2 and DNA. It is known that single-stranded DNA with the telomeric repeat tends to form a quadruplex and that the quadruplex has an inhibitory effect on telomere elongation by telomerase. This time it is revealed that BD2 unfolds the quadruplex of such DNA upon binding. Moreover, the effect of BD2 on the elongation by telomerase was examined in vitro. These results suggest the possible involvement of heterogeneous nuclear ribonucleoprotein D in maintenance of the telomere 3'-overhang either through protection of a single-stranded DNA or destabilization of the potentially deleterious quadruplex structure for the elongation by telomerase.

Destruction of quadruplex by proteins, and its biological implications in replication and telomere maintenance.

The minisatellite DNA Pc-1 consists of tandem repeats of d(GGCAG). We previously reported that a d(GGCAG)n strand folds into an intramolecular quadruplex under physiological conditions and that during replication the progression of DNA polymerase is blocked by the quadruplex in vitro. Therefore, the formation of the quadruplex was supposed to be responsible for the hypermutable features of Pc-1. Then, we have identified proteins that bind to Pc-1, one of which is hnRNP A1. Here, we have demonstrated that hnRNP A1 destroys the quadruplex of Pc-1 on binding and abrogates the arrest of DNA polymerase at the repeat. Thus, hnRNP A1 functions as if it is a chaperon to assist Pc-1 DNA to form the proper folding suitable for replication. We have also found that hnRNP A1 and a related protein, hnRNP D, destroy the quadruplex of telomere DNA, which suggests the involvement of these proteins in telomere maintenance as DNA chaperons.

Unfolding of quadruplex structure in the G-rich strand of the minisatellite repeat by the binding protein UP1.

The mouse hypervariable minisatellite (MN) Pc-1 consists of tandem repeats of d(GGCAG) and flanked sequences. We have previously demonstrated that single-stranded d(GGCAG)(n) folds into the intramolecular folded-back quadruplex structure under physiological conditions. Because DNA polymerase progression in vitro is blocked at the repeat, the characteristic intramolecular quadruplex structure of the repeat, at least in part, could be responsible for the hypermutable feature of Pc-1 and other MNs with similar repetitive units. On the other hand, we have isolated six MN Pc-1 binding proteins (MNBPs) from nuclear extracts of NIH 3T3 cells. Here, we describe one of those MNBPs, MNBP-B, that binds to the single-stranded d(GGCAG)(n). Amino acid sequences of seven proteolytic peptide fragments of MNBP-B were determined, and the cDNA clones were isolated. MNBP-B was proven identical to the single-stranded DNA-binding protein, UP1. Recombinant UP1 bound to single-stranded d(GGCAG)(n) and other G-rich repetitive sequences, such as d(GTCAGG)(n) and d(GTTAGG)(n). In addition, UP1 was demonstrated by CD spectrum analysis to unfold the intramolecular quadruplex structure of d(GGCAG)(5) and d(TTAGGG)(4) and to abrogate the arrest of DNA synthesis at the d(GGG)(n) site. This ability of UP1 suggests that unfolding of quadruplex DNA is required for DNA synthesis processes.