A Structural Hinge in Eukaryotic MutY Homologues Mediates Catalytic Activity and Rad9-Rad1-Hus1 Checkpoint Complex Interactions

The DNA glycosylase MutY homologue (MYH or MUTYH) removes adenines misincorporated opposite 8-oxoguanine as part of the base excision repair pathway. Importantly, defects in human MYH (hMYH) activity cause the inherited colorectal cancer syndrome MYH-associated polyposis. A key feature of MYH activity is its coordination with cell cycle checkpoint via interaction with the Rad9-Rad1-Hus1 (9-1-1) complex. The 9-1-1 complex facilitates cell cycle checkpoint activity and coordinates this activity with ongoing DNA repair. The interdomain connector (IDC, residues 295-350) between the catalytic domain and the 8-oxoguanine recognition domain of hMYH is a critical element that maintains interactions with the 9-1-1 complex. We report the first crystal structure of a eukaryotic MutY protein, a fragment of hMYH (residues 65-350) that consists of the catalytic domain and the IDC. Our structure reveals that the IDC adopts a stabilized conformation projecting away from the catalytic domain to form a docking scaffold for 9-1-1. We further examined the role of the IDC using Schizosaccharomyces pombe MYH as model system. In vitro studies of S. pombe MYH identified residues I261 and E262 of the IDC (equivalent to V315 and E316 of the hMYH IDC) as critical for maintaining the MYH/9-1-1 interaction. We determined that the eukaryotic IDC is also required for DNA damage selection and robust enzymatic activity. Our studies also provide the first evidence that disruption of the MYH/9-1-1 interaction diminishes the repair of oxidative DNA damage in vivo. Thus, preserving the MYH/9-1-1 interaction contributes significantly to minimizing the mutagenic potential of oxidative DNA damage.     

Three eukaryote-like Orc1/Cdc6 proteins functionally interact and mutually regulate their activities of binding to the replication origin in the hyperthermophilic archaeon Sulfolobus solfataricus P2

The crenarchaeon Sulfolobus solfataricus has the potential to be a powerful model system to understand the central mechanism of eukaryotic DNA replication because it contains three active origins of replication and three eukaryote-like Orc1/Cdc6 proteins. However, it is not known whether these SsoCdc6 proteins can functionally interact and collectively contribute to DNA replication initiation. In the current work, we found that SsoCdc6-1 stimulates DNA-binding activities of SsoCdc6-3. In contrast, SsoCdc6-3 inhibits those of both SsoCdc6-1 and SsoCdc6-2. These regulatory functions are differentially affected by the C-terminal domains of these SsoCdc6 proteins. These data, in conjunction with studies on physical interactions between these replication initiators by bacterial two-hybrid and pull-down/Western blot assays, lead us to propose the possibility that multiple SsoCdc6 proteins might coordinately regulate DNA replication in the archaeon species. This is the first report on the functional interaction among the archaeal multiple Cdc6 proteins to regulate DNA replication.     

Unusual Conformation of the SxN Motif in the Crystal Structure of Penicillin-Binding Protein A from Mycobacterium tuberculosis

PBPA from Mycobacterium tuberculosis is a class B-like penicillin-binding protein (PBP) that is not essential for cell growth in M. tuberculosis, but is important for proper cell division in Mycobacterium smegmatis. We have determined the crystal structure of PBPA at 2.05 Å resolution, the first published structure of a PBP from this important pathogen. Compared to other PBPs, PBPA has a relatively small N-terminal domain, and conservation of a cluster of charged residues within this domain suggests that PBPA is more related to class B PBPs than previously inferred from sequence analysis. The C-terminal domain is a typical transpeptidase fold and contains the three conserved active-site motifs characterisitic of penicillin-interacting enzymes. Whilst the arrangement of the SxxK and KTG motifs is similar to that observed in other PBPs, the SxN motif is markedly displaced away from the active site, such that its serine (Ser281) is not involved in hydrogen bonding with residues of the other two motifs. A disulfide bridge between Cys282 (the “x” of the SxN motif) and Cys266, which resides on an adjacent loop, may be responsible for this unusual conformation. Another interesting feature of the structure is a relatively long connection between β5 and α11, which restricts the space available in the active site of PBPA and suggests that conformational changes would be required to accommodate peptide substrate or β-lactam antibiotics during acylation. Finally, the structure shows that one of the two threonines postulated to be targets for phosphorylation is inaccessible (Thr362), whereas the other (Thr437) is well placed on a surface loop near the active site.     

Regulation of the functional interactions between archaeal eukaryote-like Cdc6/Orc1 proteins on the replication origin by two different mechanisms

The crenarchaeon Sulfolobus solfataricus contains three active origins of replication and three eukaryote-like Cdc6/Orc1 proteins known as SsoCdc6 proteins. It has the potential to become a powerful model system in understanding the central mechanism of the eukaryotic DNA replication. In this research, we designed a group of duplex DNA substrates containing specific origin recognition boxes (ORBs) of the archaeon and identified the DNA-binding activities of different SsoCdc6 proteins. Furthermore, we showed that the DNA-protein interaction between the DNA substrate and the SsoCdc6-1 or SsoCdc6-3 strikingly regulated their DNA-binding activities of each other on the origin. On the other hand, the protein-protein interactions between SsoCdc6-1 and SsoCdc6-2 were observed to mutually modulate the stimulating or inhibitive effects on the DNA-binding activities of each other. Thus, two different mechanisms were demonstrated to be involved in the regulations of the functions of the SsoCdc6 proteins on the replication origins. The results of this study imply that the interactions between multiple SsoCdc6 proteins and origin DNA collectively contribute to the positive or negative regulation of DNA replication initiation in the archaeon species.     

Crystal Structure of the HA3 Subcomponent of Clostridium botulinum Type C Progenitor Toxin

The Clostridium botulinum type C 16S progenitor toxin contains a neurotoxin and several nontoxic components, designated nontoxic nonhemagglutinin (HA), HA1 (HA-33), HA2 (HA-17), HA3a (HA-22-23), and HA3b (HA-53). The HA3b subcomponent seems to play an important role cooperatively with HA1 in the internalization of the toxin by gastrointestinal epithelial cells via binding of these subcomponents to specific oligosaccharides. In this study, we investigated the sugar-binding specificity of the HA3b subcomponent using recombinant protein fused to glutathione S-transferase and determined the three-dimensional structure of the HA3a-HA3b complex based on X-ray crystallography. The crystal structure was determined at a resolution of 2.6 Å. HA3b contains three domains, domains I to III, and the structure of domain I resembles HA3a. In crystal packing, three HA3a-HA3b molecules are assembled to form a three-leaved propeller-like structure. The three HA3b domain I and three HA3a alternate, forming a trimer of dimers. In a database search, no proteins with high structural homology to any of the domains (Z score > 10) were found. Especially, HA3a and HA3b domain I, mainly composed of β-sheets, reveal a unique fold. In binding assays, HA3b bound sialic acid with high affinity, but did not bind galactose, N-acetylgalactosamine, or N-acetylglucosamine. The electron density of liganded N-acetylneuraminic acid was determined by crystal soaking. In the sugar-complex structure, the N-acetylneuraminic acid-binding site was located in the cleft formed between domains II and III of HA3b. This report provides the first determination of the three-dimensional structure of the HA3a-HA3b complex and its sialic acid binding site. Our results will provide useful information for elucidating the mechanism of assembly of the C16S toxin and for understanding the interactions with oligosaccharides on epithelial cells and internalization of the botulinum toxin complex.     

Mapping the FEN1 interaction domain with hTERT

The activity of telomerase in cancer cells is tightly regulated by numerous proteins including DNA replication factors. However, it is unclear how replication proteins regulate telomerase action in higher eukaryotic cells. Previously we have demonstrated that the multifunctional DNA replication and repair protein flap endonuclease 1 (FEN1) is in complex with telomerase and may regulate telomerase activity in mammalian cells. In this study, we further analyzed the nature of this association. Our results show that FEN1 and telomerase association occurs throughout the S phase, with the maximum association in the mid S phase. We further mapped the physical domains in FEN1 required for this association and found that the C-terminus and the nuclease domain of FEN1 are involved in this interaction, whereas the PCNA binding ability of FEN1 is dispensable for the interaction. These results provide insights into the nature of possible protein–protein associations that telomerase participates in for maintaining functional telomeres.     

Characterization of a Functional DnaG-Type Primase in Archaea: Implications for a Dual-Primase System

We have biochemically characterized the bacterial-like DnaG primase contained within the hyperthermophilic crenarchaeon Sulfolobus solfataricus (Sso) and compared in vitro priming kinetics with those of the eukaryotic-type primase (PriSL) also found in Sso. SsoDnaG exhibited metal- and temperature-dependent profiles consistent with priming at high temperatures. The distribution of primer products was discrete but highly similar to the distribution of primer products produced by the homologous Escherichia coli DnaG. The predominate primer length was 13 bases, although less abundant products are present. SsoDnaG was found to bind DNA cooperatively as a dimer with a moderate dissociation constant. Mutation of the conserved glutamate in the active site severely inhibited priming activity, suggesting a functional homology with E. coli DnaG. SsoDnaG was also found to have a greater than fourfold faster rate of DNA priming over that of SsoPriSL under optimal in vitro conditions. The presence of both enzymatically functional primase families in archaea suggests that the DNA priming role may be shared on leading or lagging strands during DNA replication.     

Oligomeric State in the Crystal Structure of Modular FAD Synthetase Provides Insights into Its Sequential Catalysis in Prokaryotes

The crystal structure of the modular flavin adenine dinucleotide (FAD) synthetase from Corynebacterium ammoniagenes has been solved at 1.95 Å resolution. The structure of C. ammoniagenes FAD synthetase presents two catalytic modules—a C-terminus with ATP-riboflavin kinase activity and an N-terminus with ATP-flavin mononucleotide (FMN) adenylyltransferase activity—that are responsible for the synthesis of FAD from riboflavin in two sequential steps. In the monomeric structure, the active sites from both modules are placed 40 Å away, preventing the direct transfer of the product from the first reaction (FMN) to the second catalytic site, where it acts as substrate. Crystallographic and biophysical studies revealed a hexameric assembly formed by the interaction of two trimers. Each trimer presents a head-tail configuration, with FMN adenylyltransferase and riboflavin kinase modules from different protomers approaching the active sites and allowing the direct transfer of FMN. Experimental results provide molecular-level evidences of the mechanism of the synthesis of FMN and FAD in prokaryotes in which the oligomeric state could be involved in the regulation of the catalytic efficiency of the modular enzyme.     

The Single-Stranded DNA Binding Protein of Sulfolobus solfataricus Acts in the Presynaptic Step of Homologous Recombination

Homologous recombination is an important pathway in the repair of DNA double-strand breaks in all organisms. In mesophiles, single-stranded DNA binding proteins (SSBs) are believed to be involved in the removal of single-stranded DNA (ssDNA) secondary structure during the presynaptic step of homologous recombination, facilitating the formation of a contiguous Rad51/RecA nucleoprotein filament. Here we report a role for the thermophilic archaeal Sulfolobus solfataricus SSB (SsoSSB) in the presynaptic step of homologous recombination. We have identified multiple quaternary structural forms of this protein in vivo and examined the activity of SsoSSB with the strand-exchange protein S. solfataricus RadA (SsoRadA). Using gel-shift analysis, we found that the two major forms of SsoSSB have different DNA binding affinities and site sizes. Biochemical examination of the monomeric form of SsoSSB suggests that it has a minor role in presynapsis and may slightly inhibit the ssDNA-dependent ATPase activity of SsoRadA. The tetrameric form of SsoSSB, however, significantly inhibits SsoRadA ssDNA-dependent ATPase activity under both saturating and subsaturating conditions. Order-of-addition experiments indicate that preincubation of tetrameric SsoSSB and SsoRadA prior to reaction initiation with ssDNA relieves the inhibition observed when SsoSSB is added either before or after SsoRadA. In addition, we demonstrate a direct interaction between SsoRadA and SsoSSB using coimmunoprecipitation. Taken together, these results suggest that a direct interaction between SsoSSB and SsoRadA may occur in vivo prior to the formation of the SsoRadA nucleoprotein filament.     

Crystal structures and mutagenesis of sucrose hydrolase from Xanthomonas axonopodis pv. glycines: insight into the exclusively hydrolytic amylosucrase fold

Neisseria polysaccharea amylosucrase (NpAS), a transglucosidase of glycoside hydrolase family 13, is a hydrolase and glucosyltransferase that catalyzes the synthesis of amylose-like polymer from a sucrose substrate. Recently, an NpAS homolog from Xanthomonas axonopodis pv. glycines was identified as a member of the newly defined carbohydrate utilization locus that regulates the utilization of plant sucrose in phytopathogenic bacteria. Interestingly, this enzyme is exclusively a hydrolase and not a glucosyltransferase; it is thus known as sucrose hydrolase (SUH). Here, we elucidated the novel functional features of SUH using X-ray crystallography and site-directed mutagenesis. Four different crystal structures of SUH, including the SUH-Tris and the SUH-sucrose and SUH-glucose complexes, represent structural snapshots along the catalytic reaction coordinate. These structures show that SUH is distinctly different from NpAS in that ligand-induced conformational changes in SUH cause the formation of a pocket-shaped active site and in that SUH lacks the three arginine residues found in the NpAS active site that appear to be crucial for NpAS glucosyltransferase activity. Mutation of SUH to insert these arginines failed to confer glucosyltransferase activity, providing evidence that its enzymatic activity is limited to sucrose hydrolysis by its pocket-shaped active site and the identity of residues in the vicinity of the active site.     

Crystal Structure of the Extracellular Domain of a Bacterial Ligand-Gated Ion Channel

The crystal structure of the extracellular domain (ECD) of the pentameric ligand-gated ion-channel from Gloeobacter violaceus (GLIC) was solved at neutral pH at 2.3 Å resolution in two crystal forms, showing a surprising hexameric quaternary structure with a 6-fold axis replacing the expected 5-fold axis. While each subunit retains the usual β-sandwich immunoglobulin-like fold, small deviations from the whole GLIC structure indicate zones of differential flexibility. The changes in interface between two adjacent subunits in the pentamer and the hexamer can be described in a downward translation by one inter-strand distance and a global rotation of the second subunit, using the first one for superposition. While global characteristics of the interface, such as the buried accessible surface area, do not change very much, most of the atom-atom interactions are rearranged. It thus appears that the transmembrane domain is necessary for the proper oligomeric assembly of GLIC and that there is an intrinsic plasticity or polymorphism in possible subunit-subunit interfaces at the ECD level, the latter behaving as a monomer in solution. Possible functional implications of these novel structural data are discussed in the context of the allosteric transition of this family of proteins. In addition, we propose a novel way to quantify elastic energy stored in the interface between subunits, which indicates a tenser interface for the open form than for the closed form (rest state). The hexameric or pentameric forms of the ECD have a similar negative curvature in their subunit-subunit interface, while acetylcholine binding proteins have a smaller and positive curvature that increases from the apo to the holo form.     

Structure of the ATPase subunit CysA of the putative sulfate ATP-binding cassette (ABC) transporter from Alicyclobacillus acidocaldarius

CysA, the ATPase subunit of a putative sulfate ATP-binding cassette transport system of the gram-positive thermoacidophilic bacterium Alicyclobacillus acidocaldarius, was structurally characterized at a resolution of 2.0 Angstroms in the absence of nucleotides. In line with previous findings on ABC-ATPases the structures of the two monomers (called CysA-1 and CysA-2) in the asymmetric unit differ substantially in the arrangement of their individual (sub)domains. CysA-2 was found as a physiological dimer composed of two crystallographically related monomers that are arranged in an open state. Interestingly, while the regulatory domain of CysA-2 packs against its opposing domain that of CysA-1 undergoes a conformational change and, in the dimer, would interfere with the opposing monomer thereby preventing solute translocation. Whether this conformational state is used for regulatory purposes will be discussed.     

The lectin-like activity of human C1q and its implication in DNA and apoptotic cell recognition

C1q, the binding subunit of the C1 complex of complement, is an archetypal pattern recognition molecule known for its striking ability to recognize a wide variety of targets, ranging from pathogenic non self to altered self. DNA is one of the C1q ligands, but the precise region of C1q and the DNA motifs that support interaction have not been characterized yet. Here, we report for the first time that the peripheral globular region of the C1q molecule displays a lectin-like activity, which contributes to DNA binding through interaction with its deoxy-d-ribose moiety and may participate in apoptotic cell recognition.     

Crystal Structure of Red Chlorophyll Catabolite Reductase: Enlargement of the Ferredoxin-Dependent Bilin Reductase Family

The key steps in the degradation pathway of chlorophylls are the ring-opening reaction catalyzed by pheophorbide a oxygenase and sequential reduction by red chlorophyll catabolite reductase (RCCR). During these steps, chlorophyll catabolites lose their color and phototoxicity. RCCR catalyzes the ferredoxin-dependent reduction of the C20/C1 double bond of red chlorophyll catabolite. RCCR appears to be evolutionarily related to the ferredoxin-dependent bilin reductase (FDBR) family, which synthesizes a variety of phytobilin pigments, on the basis of sequence similarity, ferredoxin dependency, and the common tetrapyrrole skeleton of their substrates. The evidence, however, is not robust; the identity between RCCR and FDBR HY2 from Arabidopsis thaliana is only 15%, and the oligomeric states of these enzymes are different. Here, we report the crystal structure of A. thaliana RCCR at 2.4 Å resolution. RCCR forms a homodimer, in which each subunit folds in an α/β/α sandwich. The tertiary structure of RCCR is similar to those of FDBRs, strongly supporting that these enzymes evolved from a common ancestor. The two subunits are related by noncrystallographic 2-fold symmetry in which the α-helices near the edge of the β-sheet unique in RCCR participate in intersubunit interaction. The putative RCC-binding site, which was derived by superimposing RCCR onto biliverdin-bound forms of FDBRs, forms an open pocket surrounded by conserved residues among RCCRs. Glu154 and Asp291 of A. thaliana RCCR, which stand opposite each other in the pocket, likely are involved in substrate binding and/or catalysis.