Symplekin (Pta1 in yeast) is a scaffold in the large protein complex that is required for 3′-end cleavage and polyadenylation of eukaryotic messenger RNA precursors (pre-mRNAs) 1–4, and also participates in transcription initiation and termination by RNA polymerase II (Pol II) 5,6. Symplekin mediates interactions among many different proteins in this machinery 1,2,7–9, although the molecular basis for its function is not known. Here we report the crystal structure at 2.4 Å resolution of the N-terminal domain (residues 30–340) of human symplekin (Symp-N) in a ternary complex with the Pol II C-terminal domain (CTD) Ser5 phosphatase Ssu72 7,10–17 and a CTD Ser5 phosphopeptide. The N-terminal domain of symplekin has the ARM or HEAT fold, with seven pairs of anti-parallel α-helices arranged in the shape of an arc. The structure of Ssu72 has some similarity to that of low-molecular-weight phosphotyrosine protein phosphatase 18,19, although Ssu72 has a unique active site landscape as well as extra structural features at the C-terminus that is important for interaction with symplekin. Ssu72 is bound to the concave face of symplekin, and engineered mutations in this interface can abolish interactions between the two proteins. The CTD peptide is bound in the active site of Ssu72, unexpectedly with the pSer5-Pro6 peptide bond in the cis configuration, which contrasts with all other known CTD peptide conformations 20,21. While the active site of Ssu72 is about 25 Å away from the interface with symplekin, we found that the symplekin N-terminal domain stimulates Ssu72 CTD phosphatase activity in vitro. Furthermore, the N-terminal domain of symplekin inhibits polyadenylation in vitro, but importantly only when coupled to transcription. As catalytically active Ssu72 overcomes this inhibition, our results demonstrate a role for mammalian Ssu72 in transcription-coupled pre-mRNA 3′-end processing.

SUMMARY We recently reported that two homologous yeast proteins, Rai1 and Dxo1, function in a quality control mechanism to clear cells of incompletely 5′-end capped mRNAs. Here we report that their mammalian homolog, Dom3Z, possesses pyrophosphohydrolase, decapping and 5′-3′ exoribonuclease activities, and will be referred to as DXO. Surprisingly, we find that DXO preferentially degrades defectively capped pre-mRNAs in cells. Further studies show that incompletely capped pre-mRNAs are inefficiently spliced at all introns, in contrast to current understanding, and poorly cleaved for polyadenylation. Crystal structures of DXO in complex with substrate mimic and products at up to 1.5Å resolution provide elegant insights into the catalytic mechanism and molecular basis for its three apparently distinct activities. Our data reveal a pre-mRNA 5′-end capping quality control mechanism in mammalian cells, with DXO as the central player for this mechanism, and demonstrate an unexpected intimate link between proper 5′-end capping and subsequent pre-mRNA processing.

A recently developed human norovirus cell culture system revealed that the presence of bile enhanced or was an essential requirement for the growth of certain genotypes. Before this discovery, histo-blood group antigens (HBGAs) were the only well-studied cofactor known for human noroviruses, and there was evidence that several genotypes poorly bound HBGAs. Therefore, the purpose of this study was to investigate how human norovirus capsids interact with bile acids. We found that bile acids had low-micromolar affinities for GII.1, GII.10, and GII.19 capsids but did not bind GI.1, GII.3, GII.4, or GII.17. We showed that bile acid bound at a partially conserved pocket on the norovirus capsid-protruding (P) domain using X-ray crystallography. Amino acid sequence alignment and structural analysis delivered an explanation of selective bile acid binding. Intriguingly, we discovered that binding of the bile acid was the critical step to stabilize several P domain loops that optimally placed an essential amino acid side chain (Asp375) to bind HBGAs in an otherwise HBGA nonbinder (GII.1). Furthermore, bile acid enhanced HBGA binding for a known HBGA binder (GII.10). Altogether, these new data suggest that bile acid functions as a loop-stabilizing regulator and enhancer of HBGA binding for certain norovirus genotypes. IMPORTANCE Given that human norovirus virions likely interact with bile acid during a natural infection, our evidence that an HBGA nonbinder (GII.1) can be converted to an HBGA binder after bile acid binding is of major significance. Our data provide direct evidence that, like HBGAs, bile acid interaction on the capsid is an important cofactor for certain genotypes. However, more unanswered questions seem to arise from these new discoveries. For example, is there an association between the bile acid requirement and the prevalence of certain genotypes? That is, the GII.1 and GII.10 (bile acid binders) genotypes rarely caused outbreaks, whereas the GII.4 and GII.17 genotypes (bile acid nonbinders) were responsible for large epidemics. Therefore, it seems plausible that certain genotypes require bile acids, whereas others have modified their bile acid requirements on the capsid.

Human noroviruses are the leading cause of acute gastroenteritis in humans. Noroviruses also infect animals, such as cows, mice, cats, and dogs. How noroviruses bind and enter host cells is still incompletely understood. Recently, the type I transmembrane protein CD300lf was identified as the murine norovirus receptor, yet it is unclear how the virus capsid and receptor interact at the molecular level. In this study, we determined the X-ray crystal structure of the soluble CD300lf (sCD300lf) and the murine norovirus capsid protruding domain complex at a 2.05-Å resolution. We found that the sCD300lf-binding site is located on the topside of the protruding domain and involves a network of hydrophilic and hydrophobic interactions. sCD300lf locked nicely into a complementary cavity on the protruding domain that is additionally coordinated with a positive surface charge on sCD300lf and a negative surface charge on the protruding domain. Five of six protruding domain residues interacting with sCD300lf were maintained between different murine norovirus strains, suggesting that sCD300lf was capable of binding to a highly conserved pocket. Moreover, a sequence alignment with other CD300 paralogs showed that the sCD300lf-interacting residues were partially conserved in CD300ld but variable in other CD300 family members, consistent with previously reported infection selectivity. Overall, these data provide insights into how a norovirus engages a protein receptor and will be important for a better understanding of selective recognition and norovirus attachment and entry mechanisms. Noroviruses exhibit exquisite host range specificity due to species-specific interactions between the norovirus capsid protein and host molecules. Given this strict host range restriction, it has been unclear how the viruses are maintained within a species between relatively sporadic epidemics. While much data demonstrate that noroviruses can interact with carbohydrates, recent work has shown that expression of the protein CD300lf is both necessary and sufficient for murine norovirus infection of mice and binding of the virus to permissive cells. Importantly, the expression of this murine protein by human cells renders them fully permissive for murine norovirus infection, indicating that at least in this case, host range restriction is determined by molecular events that control receptor binding and entry. Defining the atomic-resolution interactions between the norovirus capsid protein and its cognate receptor is essential for a molecular understanding of host-range restriction and norovirus tropism.

Human norovirus infections are a major disease burden. In this study, we analyzed three new norovirus-specific Nanobodies that interacted with the prototype human norovirus (i.e., genogroup I genotype 1 [GI.1]). We showed that the Nanobodies bound on the side (Nano-7 and Nano-62) and top (Nano-94) of the capsid-protruding (P) domain using X-ray crystallography. Nano-7 and Nano-62 bound at a similar region on the P domain, but the orientations of these two Nanobodies clashed with the shell (S) domain and neighboring P domains on intact particles. This finding suggested that the P domains on the particles should shift in order for Nano-7 and Nano-62 to bind to intact particles. Interestingly, both Nano-7 and Nano-94 were capable of blocking norovirus virus-like particles (VLPs) from binding to histo-blood group antigens (HBGAs), which are important cofactors for norovirus infection. Previously, we showed that the GI.1 HBGA pocket could be blocked with the soluble human milk oligosaccharide 2-fucosyllactose (2′FL). In the current study, we showed that a combined treatment of Nano-7 or Nano-94 with 2′FL enhanced the blocking potential with an additive (Nano-7) or synergistic (Nano-94) effect. We also found that GII Nanobodies with 2′FL also enhanced inhibition. The Nanobody inhibition likely occurred by different mechanisms, including particle aggregation or particle disassembly, whereas 2′FL blocked the HBGA binding site. Overall, these new data showed that the positive effect of the addition of 2′FL was not limited to a single mode of action of Nanobodies or to a single norovirus genogroup. IMPORTANCE The discovery of vulnerable regions on norovirus particles is instrumental in the development of effective inhibitors, particularly for GI noroviruses that are genetically diverse. Analysis of these GI.1-specific Nanobodies has shown that similar to GII norovirus particles, the GI particles have vulnerable regions. The only known cofactor region, the HBGA binding pocket, represents the main target for inhibition. With a combination treatment, i.e., the addition of Nano-7 or Nano-94 with 2′FL, the effect of inhibition was increased. Therefore, combination drug treatments might offer a better approach to combat norovirus infections, especially since the GI genotypes are highly diverse and are continually changing the capsid landscape, and few conserved epitopes have so far been identified.

Norovirus infection is the major cause of nonbacterial gastroenteritis in humans and has been the subject of numerous studies investigating the virus's biophysical properties and biochemical function with the aim of deriving novel and highly potent entry inhibitors to prevent infection. Recently, it has been shown that the protruding P domain dimer (P-dimer) of a GII.10 Norovirus strain exhibits two new binding sites for l-fucose in addition to the canonical binding sites. Thus, these sites provide a novel target for the design of multivalent fucose ligands as entry inhibitors of norovirus infections. In this current study, a first generation of multivalent fucose-functionalized glycomacromolecules was synthesized and applied as model structures to investigate the potential targeting of fucose binding sites in human norovirus P-dimer. Following previously established solid phase polymer synthesis, eight precision glycomacromolecules varying in number and position of fucose ligands along an oligo(amidoamine) backbone were obtained and then used in a series of binding studies applying native MS, NMR, and X-ray crystallography. We observed only one fucose per glycomacromolecule binding to one P-dimer resulting in similar binding affinities for all fucose-functionalized glycomacromolecules, which based on our current findings we attribute to the overall size of macromolecular ligands and possibly to steric hindrance.

No abstract

As part of a functional analysis of archaeal Sm-related proteins, we have studied the oligomerization behavior of the Sm-2 type protein from the euryarchaeon Archaeoglobus fulgidus using gel filtration chromatography and noncovalent mass spectrometry. Our experiments show that the oligomeric state of the protein depends on the pH and presence of RNA. The protein forms a hexamer at acidic pH in the absence of RNA. The addition of RNA (oligo U 10 ) induces the formation of a heptamer over the whole pH range studied. The stability of both the hexamer and the RNA-bound heptamer increases at lower pH.Keywords: archaea; Sm-like protein; Sm fold; RNA binding; heptamer; hexamer; noncovalent mass spectrometry The Sm protein family is characterized by a conserved bipartite sequence motif of ;70 amino acids, which is called the Sm domain. The domain contains two conserved sequence segments, known as the Sm1 and Sm2 motifs, separated by a loop region which differs in length and sequence among the family (Séraphin 1995; SalgadoGarrido et al. 1999). b-Strands 1, 2, and 3 constitute the first motif, and b-strands 4 and 5 constitute the second motif of the Sm domain. The overall architecture of the Sm monomer, a barrel-type OB-fold structure, is completed by the N-terminal a-helix (Fig. 1). In eukaryotes, seven distinct Sm or Sm-like (Lsm) proteins, together with various RNAs that contain the short, single-stranded, uridine-rich Sm site, form the core domain of the small nuclear ribonucleoprotein particles (snRNPs), which are essential for several RNA-processing events such as (Mattaj et al. 1993;Hermann et al. 1995;Will and Lührmann 2001), and telomerase activity (Seto et al. 1999). Sm-related proteins have also been identified in archaea and eubacteria through database searches (Salgado-Garrido et al. 1999;Møller et al. 2002). The in vivo function of the archaeal Sm proteins is widely unknown; however, experimental results from our laboratory suggest their potential involvement in transfer RNA (tRNA) maturation (Törö et al. 2001). Archaeal Sm proteins share the common Sm fold, bind to U-rich RNA sequences, and form ringshaped homo-hexameric or -heptameric complexes. The assembly of the protein complexes is primarily maintained through interactions between the b4 and b5 strands of adjacent monomers (Collins et al. 2001;Törö et al. 2002). The maximum number of distinct Sm proteins found in archaeal species is currently three. At present, there are only four archaeal species known (Pyrobaculum aerophilum, Sulfolobus acidocaldarius, Sulfolobus solfataricus, and Sulfolobus tokodaii) that contain all three Sm-type proteins. The Sm1-type is most abundant among Reprint requests to: Dietrich Suck, Structural and Computational Biology Programme, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany; e-mail: suck@embl.de; fax: 49-6221-387306.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi