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.

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 hexamer͞double hexamer, which has a positively charged hexameric channel for interacting with DNA. On the hexameric channel surface are six ␤-hairpin structures and loops, emanating from each of the six subunits. At the tips of the ␤-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 ␤-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 ␤-hairpin and the loop structures on which the residues reside, participate in binding and translocating DNA for origin melting and unwinding.AAA ϩ ͉ molecular machine ͉ replication ͉ large T antigen S imian virus 40 (SV40) large tumor antigen (LTag) is an efficient molecular machine that unwinds dsDNA (1-4). It belongs to AAA ϩ protein family and the helicase superfamily III (5, 6). LTag contains 708 residues and has four functional domains: DnaJ homology (residue 1-80), origin-DNA binding (residues 131-250), helicase (residues 260-627), and a host range domains (2, 4) ( Fig. 1). The helicase domain and a longer LTag construct (residues 131-627) have helicase activity similar to that of the full-length protein (7).LTag assembles into a double hexamer (dHex) at the origin through sequence-specific interactions via origin-DNA binding (2,8). However, the non-sequence-specific interaction with DNA via the helicase domain also plays an important role in the dHex assembly and helicase activity (9, 10). Based on high-resolution structures and previous biochemical͞genetic data, a looping model for dsDNA unwinding by a dHex helicase was proposed (11)(12)(13)(14). In this model, strand separation occurs within the wide chamber of the helicase domain when DNA is pulled inside the dHex helicase, and the separated ssDNA loops out from a side channel on the wall of the helicase chamber (12,14). Even though this model reconciled many observations, it did not offer a detailed view of how dsDNA is pulled inside the hHex for strand separation.The crystal structures of LTag at various nucleotide-bo...

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 phosphate ion, which may represent an intermediate conformation after de-phosphorylation 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 non-catalytic Cys around the active center exert an opposite yin-yang regulation on the catalytic Cys activity. These detailed structure and functional characterizations have provided new insight into auto-regulatory mechanisms of LYP function.Regulating tyrosine phosphorylation level is a fundamental mechanism for numerous important aspects of eukaryote physiology, as well as human health and disease (1-3). Cellular tyrosine phosphorylation levels are regulated by the antagonistic activities of two classes of enzymes, the protein tyrosine kinases (PTKs) 1 and the protein tyrosine phosphatases (PTPs). Recent findings have led to the emerging recognition that PTPs play specific and even dominant roles in setting the levels of tyrosine phosphorylation in cells and in the regulation of many physiological processes (2-7). Disruption of the equilibrium maintained by PTPs and PTKs causes a range of human disease, including cancer, diabetes, and autoimmunity (8-16).A major class of PTPs, known as classical PTPs, include transmembrane PTPs and nonreceptor PTPs (NRPTP), which are then further sub-classified based on their sequence similarities and non-catalytic domain structural motifs (4,8). NRPTPs display various ‖ The coordinates and structure factors for the disulfide structure of PTPN22 (PDB ID: 3H2X) The importance of LYP in immune system regulation has been recently demonstrated by the finding that a human variant W620, caused by a single nucleotide polymorphism in PTPN22 at nucleotide 1858, leads to a significantly increased risk for autoimmune diseases including type-1 diabetes, rheumatoid arthritis and systemic lupus erythematosus (11,16,18,19). Since the autoimmune-predisposing LYP-W620 variant is a gain-of-function mutation and shows increased phosphatase activity (20), LYP is currently considered a promising drug target for autoimmunity. Elucidation of the structure and regulation of LYP is important in order to understand its mechanism of action in autoimmunity and to develop innovative approaches to the pharmacological inhibition of the enzyme for therapeutic purposes.One possible mechanism of regulating cysteine-based PTP activity is through oxidation of t...

The side chain of Gln143, a conserved residue in manganese superoxide dismutase (MnSOD), forms a hydrogen bond with the manganese-bound solvent and is critical in maintaining catalytic activity. The side chains of Tyr34 and Trp123 form hydrogen bonds with the carboxamide of Gln143. We have replaced Tyr34 and Trp123 with Phe in single and double mutants of human MnSOD and measured their catalytic activity by stopped-flow spectrophotometry and pulse radiolysis. The replacements of these side chains inhibited steps in the catalysis as much as 50-fold; in addition, they altered the gating between catalysis and formation of a peroxide complex to yield a more product-inhibited enzyme. The replacement of both Tyr34 and Trp123 in a double mutant showed that these two residues interact cooperatively in maintaining catalytic activity. The crystal structure of Y34F/W123F human MnSOD at 1.95 A resolution suggests that this effect is not related to a conformational change in the side chain of Gln143, which does not change orientation in Y34F/W123F, but rather to more subtle electronic effects due to the loss of hydrogen bonding to the carboxamide side chain of Gln143. Wild-type MnSOD containing Trp123 and Tyr34 has approximately the same thermal stability compared with mutants containing Phe at these positions, suggesting the hydrogen bonds formed by these residues have functional rather than structural roles.