Summary Clamp loaders load sliding clamps onto primer-template DNA. The structure of the E. coli clamp loader bound to DNA reveals the formation of an ATP-dependent spiral of ATPase domains that tracks only the template strand, allowing recognition of both RNA and DNA primers. Unlike hexameric helicases, in which DNA translocation requires distinct conformations of the ATPase domains, the clamp loader spiral is symmetric and is set up to trigger release upon DNA recognition. Specificity for primed DNA arises from blockage of the end of the primer and accommodation of the emerging template along a surface groove. A related structure reveals how the ψ protein, essential for coupling the clamp loader to single-stranded DNA binding protein (SSB), binds to the clamp loader. By stabilizing a conformation of the clamp loader that is consistent with the ATPase spiral observed upon DNA binding, ψ binding promotes the clamp loading activity of the complex.

ions are seen to bind to the enzyme-active site. Residues Asp-10, Glu-48, and Asp-70 make direct (inner sphere) coordination contacts to the first (activating) metal, whereas residues Asp-10 and Asp-134 make direct contacts to the second (attenuating) metal. This structure is consistent with biochemical evidence suggesting that two metal ions may bind RNase H but liganding a second ion inhibits RNase H activity.The RNase H class of enzymes cleaves the RNA moiety of RNA⅐DNA hybrids in a divalent cation-dependent manner leaving 5Ј-phosphate and 3Ј-hydroxyl products. RNase H proteins are found in a wide variety of organisms ranging from bacteria to vertebrates (for review see Refs. 1 and 2). From a medical perspective, the most significant function of RNase H is its critical action in the life cycle of retroviruses such as the human immunodeficiency virus (HIV).1 This activity arises from the C-terminal region of reverse transcriptase. The RNase H domain from HIV is homologous to other members of this class including RNase HI from Escherichia coli. Because inhibiting RNase H activity prohibits production of infectious virions (3), understanding the function and mechanism of RNase H is an important avenue toward the development of anti-retroviral compounds.RNase H requires divalent cations (either Mg 2ϩ or Mn 2ϩ ) for catalysis. Five conserved residues form the metal-binding active site of RNase H (Fig. 1a). Two RNases H with metal ions bound have been studied by x-ray crystallography. E. coli RNase HI binds a single Mg 2ϩ ion via three carboxylates (Asp-10, Glu-48, and Asp-70) that form Site 1 (4, 5), whereas the HIV RNase H domain shows two Mn 2ϩ ions, one in a position similar to Site 1 and another (Site 2) liganded by the equivalents of Asp-10 and Asp-134 (6). Mutations in RNases H, which eliminate Mg 2ϩ -dependent activity, often allow retention of Mn 2ϩ -dependent activity (7-11). This, together with the different stoichiometry observed via crystallography, raises the possibility of alternate binding modes and/or catalytic requirements for these metals. In E. coli RNase HI, conservative mutation of the residues comprising Site 1 (Asp-10, Glu-48, and Asp-70) eliminates activity (12), whereas mutations of the conserved histidine (His-124) (13) and aspartate (Asp-134) of Site 2 (14) have smaller effects on catalysis. Despite structural efforts to identify additional weaker binding sites (5), no metal binding to Site 2 in E. coli RNase HI has been reported.Our laboratory has recently proposed a model for the metal dependence of E. coli RNase HI termed an "activation/attenuation mechanism" (15). In this model, a single metal bound at Site 1 is required for catalysis, and the binding of a second metal at Site 2 reduces catalysis ϳ100-fold. In this report, we present a high resolution crystal structure of E. coli RNase HI in complex with Mn 2ϩ ions. These data demonstrate that the active site of E. coli RNase HI can bind two Mn 2ϩ ions in the sites predicted by the activation/attenuation hypothesis. Furthermore, we s...

BackgroundSliding clamps, such as Proliferating Cell Nuclear Antigen (PCNA) in eukaryotes, are ring-shaped protein complexes that encircle DNA and enable highly processive DNA replication by serving as docking sites for DNA polymerases. In an ATP-dependent reaction, clamp loader complexes, such as the Replication Factor-C (RFC) complex in eukaryotes, open the clamp and load it around primer-template DNA.ResultsWe built a model of RFC bound to PCNA and DNA based on existing crystal structures of clamp loaders. This model suggests that DNA would enter the clamp at an angle during clamp loading, thereby interacting with positively charged residues in the center of PCNA. We show that simultaneous mutation of Lys 20, Lys 77, Arg 80, and Arg 149, which interact with DNA in the RFC-PCNA-DNA model, compromises the ability of yeast PCNA to stimulate the DNA-dependent ATPase activity of RFC when the DNA is long enough to extend through the clamp. Fluorescence anisotropy binding experiments show that the inability of the mutant clamp proteins to stimulate RFC ATPase activity is likely caused by reduction in the affinity of the RFC-PCNA complex for DNA. We obtained several crystal forms of yeast PCNA-DNA complexes, measuring X-ray diffraction data to 3.0 Å resolution for one such complex. The resulting electron density maps show that DNA is bound in a tilted orientation relative to PCNA, but makes different contacts than those implicated in clamp loading. Because of apparent partial disorder in the DNA, we restricted refinement of the DNA to a rigid body model. This result contrasts with previous analysis of a bacterial clamp bound to DNA, where the DNA was well resolved.ConclusionMutational analysis of PCNA suggests that positively charged residues in the center of the clamp create a binding surface that makes contact with DNA. Disruption of this positive surface, which had not previously been implicated in clamp loading function, reduces RFC ATPase activity in the presence of DNA, most likely by reducing the affinity of RFC and PCNA for DNA. The interaction of DNA is not, however, restricted to one orientation, as indicated by analysis of the PCNA-DNA co-crystals.

Ribonucleases H (RNases H) comprise a family of metaldependent enzymes that catalyze the hydrolysis of the 3-OOP bond of RNA in RNA⅐DNA hybrids. The mechanism by which RNases H use active-site metal(s) for catalysis is unclear. Based upon the seemingly contradictory structural observations of one divalent metal bound to Escherichia coli RNase HI and two divalent metals bound to the HIV RNase H domain, two models explaining RNase H metal dependence have been proposed: a one-metal mechanism and a two-metal mechanism. In this paper, we show that the Mn 2؉ -dependent activity of E. coli RNase HI is not consistent with either of these mechanisms. RNase H activity in the presence of Mn 2؉ is complex, with activation and inhibition of the enzyme at low and high Mn 2؉ concentrations, respectively. Mutations at Asp-134 result in a partial loss of this inhibition, with little effect on activation. Neutralization of His-124 by mutation to Ala results in an enzyme with a significantly decreased specific activity and an absolute loss of Mn 2؉ inhibition. Inhibition by high Mn 2؉ concentrations is shown to be due to a reduction in k cat ; this attenuation has a critical dependence on the presence of His-124. Based upon these results, we propose an "activation/attenuation" model explaining the metal dependence of RNase H activity where one metal is required for enzyme activation and binding of a second metal is inhibitory. The ribonuclease H (RNase H)1 family consists of ubiquitous, metal-dependent enzymes that catalyze the hydrolysis of RNA in RNA⅐DNA hybrids (for review, see Ref. 1). RNases H are unusual among well studied ribonucleases in that they require active-site divalent metals for activity, a property commonly observed in deoxyribonucleases. RNases H are also unusual in their specificity for leaving free 3Ј-hydroxyl products after phosphodiester hydrolysis. By contrast, the more familiar RNase A or RNase T1 leaves 3Ј-phosphate products, using the RNA 2Ј-hydroxyl as a nucleophile. RNases H are thought to employ an hydroxyl ion (activated water) as the attacking nucleophile, but the manner in which RNases H use active-site metal(s) to catalyze this specific ribonuclease reaction remains unclear.RNases H and related enzymes have emerged as important therapeutic targets because RNase H activity is absolutely required for proliferation of HIV and other retroviruses. Mutations in the RNase H domain of HIV reverse transcriptase that reduce activity result in a loss of virulence (2), making RNase H an attractive target for anti-HIV therapies. Understanding the cofactor requirements for RNase H should therefore aid in the development of such drugs. Moreover, the three-dimensional structures of a number of proteins with structural homology to RNase H have recently been solved by x-ray analysis, all with metal-dependent nucleic acid-modifying functions (reviewed in Refs. 3-5). This superfamily of proteins, termed "polynucleotide transferases," includes RNase H (6 -9), resolvase (10), integrase (11, 12), transposase (13), and ex...

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38α (involved in the formation of TNFα and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional (1)H/(13)C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38α both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in similar fashion to Jnk-1 siRNA and to rosiglitazone treatment. Together, the data suggest that these new ligand series bind to a novel, allosteric, and physiologically relevant site and therefore represent a unique approach to identify kinase inhibitors.

Background: Janus kinase 3 (Jak3) inhibitors hold promise for treatment of autoimmunity, but developing selective inhibitors is challenging. Results: We designed Jak3 inhibitors that avoid inhibition of the other JAKs. Conclusion: Our inhibitors possess high selectivity against other kinases and can potently inhibit Jak3 activity in cell-based assays. Significance: This class of irreversible inhibitors may be useful as selective agents of Jak3 inhibition.

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Proteins often require cofactors to perform their biological functions and must fold in the presence of their cognate ligands. Using circular dichroism spectroscopy, we investigated the effects of divalent metal binding upon the folding pathway of Escherichia coli RNase HI. This enzyme binds divalent metal in its active site, which is proximal to the folding core of RNase HI as defined by hydrogen0deuterium exchange studies. Metal binding increases the apparent stability of native RNase HI chiefly by reducing the unfolding rate. As with the apo-form of the protein, refolding from high denaturant concentrations in the presence of Mg 2ϩ follows three-state kinetics: formation of a rapid burst phase followed by measurable single exponential kinetics. Therefore, the overall folding pathway of RNase HI is minimally perturbed by the presence of metal ions. Our results indicate that the metal cofactor enters the active site pocket only after the enzyme reaches its native fold, and therefore, divalent metal binding stabilizes the protein by decreasing its unfolding rate. Furthermore, the binding of the cofactor is dependent upon a carboxylate critical for activity~Asp10!. A mutation in this residue~D10A! alters the folding kinetics in the absence of metal ions such that they are similar to those observed for the unaltered enzyme in the presence of metal.Keywords: cofactors; folding kinetics; metal ions; protein folding; ribonuclease H A major goal of modern biophysics is to understand quantitatively where, when, and why structure accumulates during the folding of a protein and how critical tertiary interactions are maintained within living cells. Many proteins require the binding of cofactors to perform their biochemical activity, and these molecules fold in a cellular environment where their cognate cofactors are present. Previous studies with dihydrofolate reductase~Iwakura et Jennings et al., 1993! and a-lactalbumin~Kuwajima et al., 1989;Forge et al., 1999; Troullier et al., 2000! have shown that the presence of cofactors can perturb the folding behavior of these proteins; however, protein folding studies are frequently conducted in the absence of potentially complicating ligands to simplify biophysical experiments. Hence, despite the functional importance of cofactors, the manner in which they affect the folding pathway of proteins remains poorly understood.To address these questions in a well-studied model system, we examined the consequences of divalent-metal ion binding to the stability and folding pathway of Escherichia coli ribonuclease HI. RNase H catalyzes the hydrolysis of RNA within RNA0DNA hybrids~Crouch, 1990; Hostomsky et al., 1993!, and E. coli RNase HI binds a single Mg 2ϩ ion cofactor in the active site of the enzyme~Katayanagi et al ., 1990, 1993bBlack & Cowan, 1994!. The stability and folding pathway of RNase HI in the absence of Mg 2ϩ ions have been extensively characterized by circular dichroism~CD! and by hydrogen0deuterium exchange~Dabora & Marqusee, 1994;Yamasaki et al., 1995;Chamberl...