Simian virus 40 large tumor antigen (LT) is both a potent oncogenic protein and an efficient hexameric nanomachine that harnesses the energy from ATP binding/hydrolysis to melt origin DNA and unwind replication forks. However, how the six subunits of the helicase motor coordinate during ATP hydrolysis and DNA unwinding/translocation is unresolved. Here we investigated the subunit coordination mechanisms “binomial distribution mutant doping” experiments in the presence of various DNA substrates. For ATP hydrolysis, we observed multiple coordination modes, ranging from random and semi-random, and semi-coordinated modes, depending on which type of DNA is present. For DNA unwinding, however, the results indicated a fully-coordinated mode for the natural origin-containing duplex DNA, but a semi-coordinated mode for a pre-existing fork-DNA, providing direct evidence for LT to use potentially different mechanisms to unwind the two types of substrates. The results of this study provide insights into DNA translocation and unwinding mechanisms for LT hexameric biomotor.From the Clinical EditorThe study describes the subunit coordination of simian virus 40 large tumor antigen (LT) showing that multiple mechanisms exist that handle the specific needs of different stages of DNA replication.The fully-coordinated model for origin DNA unwinding by large T hexameric helicase motor, in which six subunits work together in a concerted fashion, with all sites binding to ATP, followed by hydrolysis to ADP, and then emptying all the sites for the next round of reloading of ATP.

The lymphoid tyrosine phosphatase (LYP), encoded by the PTPN22 gene, recently emerged as an important risk factor and drug target for human autoimmunity. Here we solved the structure of the catalytic domain of LYP, which revealed noticeable differences with previously published structures. The active center with a semi-closed conformation binds a phosphate ion, which may represent an intermediate conformation after dephosphorylation of the substrate but before release of the phosphate product. The structure also revealed an unusual disulfide bond formed between the catalytic Cys and one of the two Cys residues nearby, which is not observed in previously determined structures. Our structural and mutagenesis data suggest that the disulfide bond may play a role in protecting the enzyme from irreversible oxidation. Surprisingly, we found that the two noncatalytic Cys around the active center exert an opposite yin-yang regulation on the catalytic Cys activity. These detailed structural and functional characterizations have provided new insights into autoregulatory mechanisms of LYP function.

In recent years, tremendous progress has been made in the elucidation of the biological roles and molecular mechanisms of the apolioprotein B mRNA-editing enzyme catalytic polypeptide (APOBEC) family of enzymes. The APOBEC family of cytidine deaminases has important functional roles within the adaptive and innate immune system. Activation induced cytidine deaminase (AID) plays a central role in the biochemical steps of somatic hypermutation and class switch recombination during antibody maturation, and the APOBEC 3 enzymes are able to inhibit the mobility of retroelements and the replication of retroviruses and DNA viruses, such as the human immunodeficiency virus type-1 and hepatitis B virus. Recent advances in structural and functional studies of the APOBEC enzymes provide new biochemical insights for how these enzymes carry out their biological roles. In this review, we provide an overview of these recent advances in the APOBEC field with a special emphasis on AID and APOBEC3G.

Methanothermobacter thermautotrophicus minichromosomal maintenance protein (mtMCM) is a 75 kDa protein that self-assembles into a double hexamer structure. The double hexamer formed by the N-terminal region of mtMCM has a highly charged (overwhelmingly net positive) inner channel. Here we investigate the effects of point mutations of some of these charged residues on the biological activities of mtMCM. Although all of the mutants were similar to the wild type in protein folding and complex assembly, we found that mutations impaired helicase activity. The study of the DNA binding and ATPase activities of these mutants revealed that the impairment of the helicase activity was highly correlated with a decrease in DNA binding, providing evidence consistent with the role of these charged residues of the inner channel in interactions with DNA.

The minichromosome maintenance protein (MCM) complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. Whereas the eukaryotic complex consists of 6 homologous proteins (MCM2–7), the archaeon Sulfolobus solfataricus has only 1 MCM protein (ssoMCM), 6 subunits of which form a homohexamer. Here, we report a 4.35-Å crystal structure of the near-full-length ssoMCM. The structure shows an elongated fold, with 5 subdomains that are organized into 2 large N- and C-terminal domains. A near-full-length ssoMCM hexamer generated based on the 6-fold symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM) hexamer shows intersubunit distances suitable for bonding contacts, including the interface around the ATP pocket. Four unusual β-hairpins of each subunit are located inside the central channel or around the side channels in the hexamer. Additionally, the hexamer fits well into the double-hexamer EM map of mtMCM. Our mutational analysis of residues at the intersubunit interfaces and around the side channels demonstrates their critical roles for hexamerization and helicase function. These structural and biochemical results provide a basis for future study of the helicase mechanisms of the archaeal and eukaryotic MCM complexes in DNA replication.

The APOBEC family members are involved in diverse biological functions. APOBEC3G restricts the replication of human immunodeficiency virus (HIV), hepatitis B virus and retroelements by cytidine deamination on single-stranded DNA or by RNA binding1, 2, 3, 4. Here we report the high-resolution crystal structure of the carboxy-terminal deaminase domain of APOBEC3G (APOBEC3G-CD2) purified from Escherichia coli. The APOBEC3G-CD2 structure has a five-stranded β-sheet core that is common to all known deaminase structures and closely resembles the structure of another APOBEC protein, APOBEC2 (ref. 5). A comparison of APOBEC3G-CD2 with other deaminase structures shows a structural conservation of the active-site loops that are directly involved in substrate binding. In the X-ray structure, these APOBEC3G active-site loops form a continuous ‘substrate groove’ around the active centre. The orientation of this putative substrate groove differs markedly (by 90 degrees) from the groove predicted by the NMR structure6. We have introduced mutations around the groove, and have identified residues involved in substrate specificity, single-stranded DNA binding and deaminase activity. These results provide a basis for understanding the underlying mechanisms of substrate specificity for the APOBEC family.

APOBEC-2 (APO2) belongs to the family of apolipoprotein B messenger RNA-editing enzyme catalytic (APOBEC) polypeptides, which deaminates mRNA and single-stranded DNA1, 2. Different APOBEC members use the same deamination activity to achieve diverse human biological functions. Deamination by an APOBEC protein called activation-induced cytidine deaminase (AID) is critical for generating high-affinity antibodies3, and deamination by APOBEC-3 proteins can inhibit retrotransposons and the replication of retroviruses such as human immunodeficiency virus and hepatitis B virus4, 5, 6, 7. Here we report the crystal structure of APO2. APO2 forms a rod-shaped tetramer that differs markedly from the square-shaped tetramer of the free nucleotide cytidine deaminase, with which APOBEC proteins share considerable sequence homology. In APO2, two long α-helices of a monomer structure prevent the formation of a square-shaped tetramer and facilitate formation of the rod-shaped tetramer via head-to-head interactions of two APO2 dimers. Extensive sequence homology among APOBEC family members allows us to test APO2 structure-based predictions using AID. We show that AID deamination activity is impaired by mutations predicted to interfere with oligomerization and substrate access. The structure suggests how mutations in patients with hyper-IgM-2 syndrome inactivate AID, resulting in defective antibody maturation.

The GINS complex, which contains the four subunits Sld5, Psf1, Psf2, and Psf3, is essential for both the initiation and progression of DNA replication in eukaryotes. GINS associates with the MCM2-7 complex and Cdc45 to activate the eukaryotic minichromosome maintenance helicase. It also appears to interact with and stimulate the polymerase activities of DNA polymerase ε and the DNA polymerase α-primase complex. To further understand the functional role of GINS, we determined the crystal structure of the full-length human GINS heterotetramer. Each of the four subunits has a major domain composed of an α-helical bundle-like structure. With the exception of Psf1, each of the other subunits has a small domain containing a three-stranded β-sheet core. Each full-length protein in the crystal has unstructured regions that are all located on the surface of GINS and are probably involved in its interaction with other replication factors. The four subunits contact each other mainly through α-helices to form a ring-like tetramer with a central pore. This pore is partially plugged by a 16-residue peptide from the Psf3 N terminus, which is unique to some eukaryotic Psf3 proteins and is not required for tetramer formation. Removal of these N-terminal 16 residues of Psf3 from the GINS tetramer increases the opening of the pore by 80%, suggesting a mechanism by which accessibility to the pore may be regulated. The structural data presented here indicate that the GINS tetramer is a highly stable complex with multiple flexible surface regions.

Simian virus 40 large tumor antigen is required for DNA unwinding during viral replication. The helicase-active form of large tumor antigen is a ring-shaped hexamer/double hexamer, which has a positively charged hexameric channel for interacting with DNA. On the hexameric channel surface are six β-hairpin structures and loops, emanating from each of the six subunits. At the tips of the β-hairpin and the loop structures are two ring-shaped residues, H513 and F459, respectively. Additionally, two positively charged residues, K512 and K516, are near the tip of the β-hairpin. The positions of these ring-shaped and positively charged residues suggest that they may play a role in binding DNA for helicase function. To understand the roles of these residues in helicase function, we obtained a set of mutants and examined various activities, including oligomerization, ATPase, DNA binding, and helicase activities. We found that substitution of these residues by Ala abolished helicase activity. Extensive mutagenesis showed that substitutions by ring-shaped residues (W and Y) at position F459 and by residues with hydrophobic or long aliphatic side chains (W, Y, F, L, M, and R) at position H513 supported helicase activity. Our study demonstrated that the four residues (F459, H513, K512, and K516) play a critical role in interacting with DNA for helicase function. The results suggest a possible mechanism to explain how these residues, as well as the β-hairpin and the loop structures on which the residues reside, participate in binding and translocating DNA for origin melting and unwinding.

•We report a high-resolution filament structure of the N-terminal domain of an archaeal MCM.•The filament structure with six subunits per turn suggests a lockwasher conformation for a hexameric MCM.•Combined with other structures of MCM, the result indicates conformational dynamics of MCM.•An N-terminal hairpin may play a role in regulating MCM oligomerization.Mini-chromosome maintenance (MCM) proteins are the replicative helicase necessary for DNA replication in both eukarya and archaea. Most of archaea only have one MCM gene. Here, we report a 1.8-Å crystal structure of the N-terminal MCM from the archaeon Thermoplasma acidophilum (tapMCM). In the structure, the MCM N-terminus forms a right-handed filament that contains six subunits in each turn, with a diameter of 25 Å of the central channel opening. The inner surface is highly positively charged, indicating DNA binding. This filament structure with six subunits per turn may also suggests a potential role for an open-ring structure for hexameric MCM and dynamic conformational changes in initiation and elongation stages of DNA replication.Download high-res image (388KB)Download full-size image

•We report a high-resolution filament structure of the N-terminal domain of an archaeal MCM.•The filament structure with six subunits per turn suggests a lockwasher conformation for a hexameric MCM.•Combined with other structures of MCM, the result indicates conformational dynamics of MCM.•An N-terminal hairpin may play a role in regulating MCM oligomerization.Mini-chromosome maintenance (MCM) proteins are the replicative helicase necessary for DNA replication in both eukarya and archaea. Most of archaea only have one MCM gene. Here, we report a 1.8-Å crystal structure of the N-terminal MCM from the archaeon Thermoplasma acidophilum (tapMCM). In the structure, the MCM N-terminus forms a right-handed filament that contains six subunits in each turn, with a diameter of 25 Å of the central channel opening. The inner surface is highly positively charged, indicating DNA binding. This filament structure with six subunits per turn may also suggests a potential role for an open-ring structure for hexameric MCM and dynamic conformational changes in initiation and elongation stages of DNA replication.Download high-res image (388KB)Download full-size image