Motif III in Superfamily 2 “Helicases” Helps Convert the Binding Energy of ATP into a High-Affinity RNA Binding Site in the Yeast DEAD-Box Protein Ded1

Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a “helicase” strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k_cat), but not the affinity for ATP (K_m). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of γ-PO₄ of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the β,γ-phosphoanhydride bond of ATP.     

Crystallographic Analysis of the Reaction Cycle of 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase,a Unique Member of the 2H Phosphoesterase Family

2H phosphoesterases catalyze reactions on nucleotide substrates and contain two conserved histidine residues in the active site. Very limited information is currently available on the details of the active site and substrate/product binding during the catalytic cycle of these enzymes. We performed a comprehensive X-ray crystallographic study of mouse 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), a membrane-associated enzyme present at high levels in the tetrapod myelin sheath. We determined crystal structures of the CNPase phosphodiesterase domain complexed with substrate, product, and phosphorothioate analogues. The data provide detailed information on the CNPase reaction mechanism, including substrate binding mode and coordination of the nucleophilic water molecule. Linked to the reaction, an open/close motion of the β5–α7 loop is observed. The role of the N terminus of helix α7—unique for CNPase in the 2H family—during the reaction indicates that 2H phosphoesterases differ in their respective reaction mechanisms despite the conserved catalytic residues. Furthermore, based on small-angle X-ray scattering, we present a model for the full-length enzyme, indicating that the two domains of CNPase form an elongated molecule. Finally, based on our structural data and a comprehensive bioinformatics study, we discuss the conservation of CNPase in various organisms.     

Structural Basis of Substrate Specificity and Selectivity of Murine Cytosolic 5′-Nucleotidase III

Cytosolic 5′-nucleotidase III (cN-III) is responsible for selective degradation of pyrimidine 5′-monoribonucleotides during maturation of reticulocytes to erythrocytes. The lack of this enzymatic activity due to genetic aberrations or lead poisoning results in a mild to moderate nonspherocytic hemolytic anemia. In affected individuals, pyrimidine nucleotides as well as their precursor polymers and their off-path metabolites accumulate in erythrocytes, interfering with their proper function in ways that are not yet fully understood. This report describes the first X-ray structure of a catalytically inactivated variant of murine cN-III with a natural substrate, uridine 5′-monophosphate, in the active site at 1.74 Å resolution. The structure captures in an atomic detail the closed conformation that cN-III adopts upon substrate binding. Structure and sequence analysis coupled with enzymatic characterization of several mutants confirmed that the aromatic ring of a nitrogenous base of substrate nucleotide is stabilized by parallel π-stacking interactions with conserved aromatic rings of Trp113 and His68. The nitrogenous base is further stabilized by T-shaped stacking with the conserved aromatic ring of Tyr114, as well as by polar contacts with side chains of Thr66 and Ser117. Two water molecules help to stabilize the nucleotide binding by bridging it to protein residues Asp72 and His68 via hydrogen bonds. Finally, fully conserved Glu96 is responsible for recognition of ribose ring via two hydrogen bonds. The presented substrate complex structure elucidates how cN-III achieves specificity for pyrimidine 5′-nucleotides and how it selects against purine 5′-nucleotides.     

Crystal Structure of Human RNA Helicase A (DHX9): Structural Basis for Unselective Nucleotide Base Binding in a DEAD-Box Variant Protein

RNA helicases of the DExD/H-box superfamily are critically involved in all RNA-related processes. No crystal structures of human DExH-box domains had been determined previously, and their structures were difficult to predict owing to the low level of homology among DExH-motif-containing proteins from diverse species. Here we present the crystal structures of the conserved domain 1 of the DEIH-motif-containing helicase DHX9 and of the DEAD-box helicase DDX20. Both contain a RecA-like core, but DHX9 differs from DEAD-box proteins in the arrangement of secondary structural elements and is more similar to viral helicases such as NS3. The N-terminus of the DHX9 core contains two long α-helices that reside on the surface of the core without contributing to nucleotide binding. The RNA-polymerase-II-interacting minimal transactivation domain sequence forms an extended loop structure that resides in a hydrophobic groove on the surface of the DEIH domain. DHX9 lacks base-selective contacts and forms an unspecific but important stacking interaction with the base of the bound nucleotide, and our biochemical analysis confirms that the protein can hydrolyze ATP, guanosine 5′-triphosphate, cytidine 5′-triphosphate, and uridine 5′-triphosphate. Together, these findings allow the localization of functional motifs within the three-dimensional structure of a human DEIH helicase and show how these enzymes can bind nucleotide with high affinity in the absence of a Q-motif.     

Cysteine-to-Serine Mutants Dramatically Reorder the Active Site of Human ABO(H) Blood Group B Glycosyltransferase without Affecting Activity: Structural Insights into Cooperative Substrate Binding

A common feature in the structures of GT-A-fold-type glycosyltransferases is a mobile polypeptide loop that has been observed to participate in substrate recognition and enclose the active site upon substrate binding. This is the case for the human ABO(H) blood group B glycosyltransferase GTB, where amino acid residues 177-195 display significantly higher levels of disorder in the unliganded state than in the fully liganded state. Structural studies of mutant enzymes GTB/C80S/C196S and GTB/C80S/C196S/C209S at resolutions ranging from 1.93 to 1.40 Å display the opposite trend, where the unliganded structures show nearly complete ordering of the mobile loop residues that is lost upon substrate binding. In the liganded states of the mutant structures, while the UDP moiety of the donor molecule is observed to bind in the expected location, the galactose moiety is observed to bind in a conformation significantly different from that observed for the wild-type chimeric structures. Although this would be expected to impede catalytic turnover, the kinetics of the transfer reaction are largely unaffected. These structures demonstrate that the enzymes bind the donor in a conformation more similar to the dominant solution rotamer and facilitate its gyration into the catalytically competent form. Further, by preventing active-site closure, these structures provide a basis for recently observed cooperativity in substrate binding. Finally, the mutation of C80S introduces a fully occupied UDP binding site at the enzyme dimer interface that is observed to be dependent on the binding of H antigen acceptor analog.     

Role of disulfide bonds in conformational stability and folding of 5′-deoxy-5′-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus

Sulfolobus solfataricus 5′-deoxy-5′-melthylthioadenosine phosphorylase II (SsMTAPII), is a hyperthermophilic hexameric protein with two intrasubunit disulfide bonds (C138–C205 and C200–C262) and a CXC motif (C259–C261). To get information on the role played by these covalent links in stability and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S mutants was studied by thermal and guanidinium chloride (GdmCl)-induced unfolding and analyzed by fluorescence spectroscopy, circular dichroism, and SDS-PAGE. No thermal unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while in the presence of the reducing agent Tris-(2-carboxyethyl) phosphine (TCEP), a Tm of 100 °C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Different from the wild-type, C262S and C259S/C261S show complete thermal denaturation curves with sigmoidal transitions centered at 102 °C and 99 °C respectively. Under reducing conditions these values decrease by 4 °C and 8 °C respectively, highlighting the important role exerted by the CXC disulfide on enzyme thermostability. The contribution of disulfide bonds to the conformational stability of SsMTAPII was further assessed by GdmCl-induced unfolding experiments carried out under reducing and nonreducing conditions. Thermal unfolding was found to be reversible if the protein was heated in the presence of TCEP up to 90 °C but irreversible above this temperature because of aggregation. In analogy, only chemical unfolding carried out in the presence of reducing agents resulted in a reversible process suggesting that disulfide bonds play a role in enzyme denaturation. Thermal and chemical unfolding of SsMTAPII occur with dissociation of the native hexameric state into denatured monomers, as indicated by SDS-PAGE.     

Repair efficiency of (5′S)-8,5′-cyclo-2′-deoxyguanosine and (5′S)-8,5′-cyclo-2′-deoxyadenosine depends on the complementary base

5′-R and 5′-S diastereoisomers of 8,5′-cyclo-2′-deoxyadenosine (cdA) and 8,5′-cyclo-2′-deoxyguanosine (cdG) containing a base-sugar covalent bond are formed by hydroxyl radicals. R-cdA and S-cdA are repaired by nucleotide excision repair (NER) in mammalian cellular extracts. Here, we have examined seven purified base excision repair enzymes for their ability to repair S-cdG or S-cdA. We could not detect either excision or binding of these enzymes on duplex oligonucleotide substrates containing these lesions. However, both lesions were repaired by HeLa cell extracts. Dual incisions by human NER on a 136-mer duplex generated 24–32 bp fragments. The time course of dual incisions were measured in comparison to cis-anti-B[a]P-N²-dG, an excellent substrate for human NER, which showed that cis-anti-B[a]P-N²-dG was repaired more efficiently than S-cdG, which, in turn, was repaired more efficiently than S-cdA. When NER efficiency of S-cdG with different complementary bases was investigated, the wobble pair S-cdG·dT was excised more efficiently than the S-cdG·dC pair that maintains nearly normal Watson-Crick base pairing. But S-cdG·dA mispair with no hydrogen bonds was excised less efficiently than the S-cdG·dC pair. Similar pattern was noted for S-cdA. The S-cdA·dC mispair was excised much more efficiently than the S-cdA·dT pair, whereas the S-cdA·dA pair was excised less efficiently. This result adds to complexity of human NER, which discriminates the damaged base pairs on the basis of multiple criteria.     

Structural insight into the mechanism of substrate specificity and catalytic activity of an HD-domain phosphohydrolase: the 5'-deoxyribonucleotidase YfbR from Escherichia coli

HD-domain phosphohydrolases have nucleotidase and phosphodiesterase activities and play important roles in the metabolism of nucleotides and in signaling. We present three 2.1-Å-resolution crystal structures (one in the free state and two complexed with natural substrates) of an HD-domain phosphohydrolase, the Escherichia coli 5′-nucleotidase YfbR. The free-state structure of YfbR contains a large cavity accommodating the metal-coordinating HD motif (H33, H68, D69, and D137) and other conserved residues (R18, E72, and D77). Alanine scanning mutagenesis confirms that these residues are important for activity. Two structures of the catalytically inactive mutant E72A complexed with Co²⁺ and either thymidine-5′-monophosphate or 2′-deoxyriboadenosine-5′-monophosphate disclose the novel binding mode of deoxyribonucleotides in the active site. Residue R18 stabilizes the phosphate on the Co²⁺, and residue D77 forms a strong hydrogen bond critical for binding the ribose. The indole side chain of W19 is located close to the 2′-carbon atom of the deoxyribose moiety and is proposed to act as the selectivity switch for deoxyribonucleotide, which is supported by comparison to YfdR, another 5′-nucleotidase in E. coli. The nucleotide bases of both deoxyriboadenosine-5′-monophosphate and thymidine-5′-monophosphate make no specific hydrogen bonds with the protein, explaining the lack of nucleotide base selectivity. The YfbR E72A substrate complex structures also suggest a plausible single-step nucleophilic substitution mechanism. This is the first proposed molecular mechanism for an HD-domain phosphohydrolase based directly on substrate-bound crystal structures.     

2′,5′-Oligoadenylate synthetase-like gene highly induced by hepatitis C virus infection in human liver is inhibitory to viral replication in vitro

We found the 2′,5′-oligoadenylate synthetase-like (OASL) gene to be significantly elevated by high virus loads in human liver infected with hepatitis C virus (HCV). Here, we determined whether OASL inhibited HCV replication using an in vitro system. We constructed three expression vectors of OASL to produce isoform a (OASLa), isoform b (OASLb), and the C-terminal ubiquitin-like domain of isoform a (Ub). When Huh7 JFH-1 HCV replicon cells were separately transfected with these three vectors, colony formation of HCV-replicating cells was inhibited by 95%, 94%, and 65%, respectively. Both OASLa and OASLb were also inhibitory for cells as well as the virus because colony formation of OASL-producing cells was reduced to 41% and 8%, respectively. Stable Huh7 clones producing each of the three OASLs were established and assessed for their inhibition of HCV replication using luciferase reporter gene-containing JFH-1 replicon RNA. HCV replication was inhibited by 50-90% in several stable OASL clones. Association analysis in six Ub clones expressing different levels of Ub mRNA showed that the degree of inhibition of HCV replication was significantly associated with the amount of Ub present. In conclusion, OASL possesses two domains with HCV inhibitory activity. The N-terminal OAS-homology domain without OAS activity is inhibitory for cell growth as well as HCV replication, whereas C-terminal Ub is inhibitory only for HCV replication. Therefore, OASLa, a major isoform of this molecule induced in human liver, may mediate anti-HCV activity through two different domains.     

The crystal structure of an ADP complex of Bacillus subtilis pyridoxal kinase provides evidence for the parallel emergence of enzyme activity during evolution

Pyridoxal kinase catalyses the phosphorylation of pyridoxal, pyridoxine and pyridoxamine to their 5' phosphates and plays an important role in the pyridoxal 5' phosphate salvage pathway. The crystal structure of a dimeric pyridoxal kinase from Bacillus subtilis has been solved in complex with ADP to 2.8 A resolution. Analysis of the structure suggests that binding of the nucleotide induces the ordering of two loops, which operate independently to close a flap on the active site. Comparisons with other ribokinase superfamily members reveal that B. subtilis pyridoxal kinase is more closely related in both sequence and structure to the family of HMPP kinases than to other pyridoxal kinases, suggesting that this structure represents the first for a novel family of "HMPP kinase-like" pyridoxal kinases. Moreover this further suggests that this enzyme activity has evolved independently on multiple occasions from within the ribokinase superfamily.     

Crystal structures of Streptococcus mutans 2'-deoxycytidylate deaminase and its complex with substrate analog and allosteric regulator dCTP x Mg2+

2′-Deoxycytidylate deaminase [or deoxycytidine-5′-monophosphate (dCMP) deaminase, dCD] catalyzes the deamination of dCMP to deoxyuridine-5′-monophosphate to provide the main nucleotide substrate for thymidylate synthase, which is important in DNA synthesis. The activity of this homohexameric enzyme is allosterically regulated by deoxycytidine-5′-triphosphate (dCTP) as an activator and by deoxythymidine-5′-triphosphate as an inhibitor. In this article, we report the crystal structures of dCMP deaminase from Streptococcus mutans and its complex with dCTP and an intermediate analog at resolutions of 3.0 and 1.66 Å. The protein forms a hexamer composed of subunits adopting a three-layer α/β/α sandwich fold. The positive allosteric regulator dCTP mainly binds at the interface between two monomers in a molar ratio of 1:1 and rearranges the neighboring interaction networks. Structural comparisons and sequence alignments revealed that dCMP deaminase from Streptococcus mutans belongs to the cytidine deaminase superfamily, wherein the proteins exhibit a similar catalytic mechanism. In addition to the two conserved motifs involved in the binding of Zn^2 +, a new conserved motif, (G⁴³YNG⁴⁶), related to the binding of dCTP was also identified. N-terminal Arg4, a key residue located between two monomers, binds strongly to the γ phosphate group of dCTP. The regulation signal was transmitted by Arg4 from the allosteric site to the active site via modifications in the interactions at the interface where the substrate-binding pocket was involved and the relocations of Arg26, His65, Tyr120, and Arg121 to envelope the active site in order to stabilize substrate binding in the complex. Based on the enzyme-regulator complex structure observed in this study, we propose an allosteric mechanism for dCD regulation.     

A crystallographic study of the role of sequence context in thymine glycol bypass by a replicative DNA polymerase serendipitously sheds light on the exonuclease complex

Thymine glycol (Tg) is the most common oxidation product of thymine and is known to be a strong block to replicative DNA polymerases. A previously solved structure of the bacteriophage RB69 DNA polymerase (RB69 gp43) in complex with Tg in the sequence context 5′-G-Tg-G shed light on how Tg blocks primer elongation: The protruding methyl group of the oxidized thymine displaces the adjacent 5′-G, which can no longer serve as a template for primer elongation [Aller, P., Rould, M. A., Hogg, M, Wallace, S. S. & Doublié S. (2007). A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative thymine lesion, thymine glycol. Proc. Natl. Acad. Sci. USA, 104, 814-818.].Several studies showed that in the sequence context 5′-C-Tg-purine, Tg is more likely to be bypassed by Klenow fragment, an A-family DNA polymerase. We set out to investigate the role of sequence context in Tg bypass in a B-family polymerase and to solve the crystal structures of the bacteriophage RB69 DNA polymerase in complex with Tg-containing DNA in the three remaining sequence contexts: 5′-A-Tg-G, 5′-T-Tg-G, and 5′-C-Tg-G. A combination of several factors—including the associated exonuclease activity, the nature of the 3′ and 5′ bases surrounding Tg, and the cis-trans interconversion of Tg—influences Tg bypass. We also visualized for the first time the structure of a well-ordered exonuclease complex, allowing us to identify and confirm the role of key residues (Phe123, Met256, and Tyr257) in strand separation and in the stabilization of the primer strand in the exonuclease site.     

Binding of LARP6 to the Conserved 5′ Stem-Loop Regulates Translation of mRNAs Encoding Type I Collagen

Type I collagen is the most abundant protein in the human body, produced by folding of two α1(I) polypeptides and one α2(I) polypeptide into the triple helix. A conserved stem-loop structure is found in the 5′ untranslated region of collagen mRNAs, encompassing the translation start codon. We cloned La ribonucleoprotein domain family member 6 (LARP6) as the protein that binds the collagen 5′ stem-loop in a sequence-specific manner. LARP6 has a distinctive bipartite RNA binding domain not found in other members of the La superfamily. LARP6 interacts with the two single-stranded regions of the 5′ stem-loop. The K_d for binding of LARP6 to the 5′ stem-loop is 1.4 nM. LARP6 binds the 5′ stem-loop in both the nucleus and the cytoplasm. In the cytoplasm, LARP6 does not associate with polysomes; however, overexpression of LARP6 blocks ribosomal loading on collagen mRNAs. Knocking down LARP6 by small interfering RNA also decreased polysomal loading of collagen mRNAs, suggesting that it regulates translation. Collagen protein is synthesized at discrete regions of the endoplasmic reticulum. Using collagen-GFP (green fluorescent protein) reporter protein, we could reproduce this focal pattern of synthesis, but only when the reporter was encoded by mRNA with the 5′ stem-loop and in the presence of LARP6. When the reporter was encoded by mRNA without the 5′ stem-loop, or in the absence of LARP6, it accumulated diffusely throughout the endoplasmic reticulum. This indicates that LARP6 activity is needed for focal synthesis of collagen polypeptides. We postulate that the LARP6-dependent mechanism increases local concentration of collagen polypeptides for more efficient folding of the collagen heterotrimer.