Type IIA topoisomerases both manage the topological state of chromosomal DNA and are the targets of a variety of clinical agents. Bisdioxopiperazines are anticancer agents that associate with ATP-bound eukaryotic topoisomerase II (topo II) and convert the enzyme into an inactive, salt-stable clamp around DNA. To better understand both topo II and bisdioxopiperazine function, we determined the structures of the adenosine 5′-[β,γ-imino]-triphosphate-bound yeast topo II ATPase region ( Sc T2-ATPase) alone and complexed with the bisdioxopiperazine ICRF-187. The drug-free form of the protein is similar in overall fold to the equivalent region of bacterial gyrase but unexpectedly displays significant conformational differences. The ternary drug-bound complex reveals that ICRF-187 acts by an unusual mechanism of inhibition in which the drug does not compete for the ATP-binding pocket, but bridges and stabilizes a transient dimer interface between two ATPase protomers. Our data explain why bisdioxopiperazines target ATP-bound topo II, provide a structural rationale for the effects of certain drug-resistance mutations, and point to regions of bisdioxopiperazines that might be modified to improve or alter drug specificity.

The family of calcium binding proteins called KChIPs associates with Kv4 family K(+) channels and modulates their biophysical properties. Here, using mutagenesis and X-ray crystallography, we explore the interaction between Kv4 subunits and KChIP1. Two regions in the Kv4.2 N terminus, residues 7-11 and 71-90, are necessary for KChIP1 modulation and interaction with Kv4.2. When inserted into the Kv1.2 N terminus, residues 71-90 of Kv4.2 are also sufficient to confer association with KChIP1. To provide a structural framework for these data, we solved the crystal structures of Kv4.3N and KChIP1 individually. Taken together with the mutagenesis data, the individual structures suggest that that the Kv4 N terminus is required for stable association with KChIP1, perhaps through a hydrophobic surface interaction, and that residues 71-90 in Kv4 subunits form a contact loop that mediates the specific association of KChIPs with Kv4 subunits.

A member of the novel protein kinase C (PKC) subfamily, PKC, is an essential component of the T cell synapse and is required for optimal T cell activation and interleukin-2 production. Selective involvement of PKC in TCR signaling makes this enzyme an attractive therapeutic target in T cell-mediated disease processes. In this report we describe the crystal structure of the catalytic domain of PKC at 2.0-Å resolution. Human recombinant PKC kinase domain was expressed in bacteria as catalytically active phosphorylated enzyme and co-crystallized with its subnanomolar, ATP site inhibitor staurosporine. The structure follows the classic bilobal kinase fold and shows the enzyme in its active conformation and phosphorylated state. Inhibitory interactions between conserved features of staurosporine and the ATP-binding cleft are accompanied by closing of the glycine-rich loop, which also maintains an inhibitory arrangement by blocking the phosphate recognition subsite. The two major phosphorylation sites, Thr-538 in the activation loop and Ser-695 in the hydrophobic motif, are both occupied in the structure, playing key roles in stabilizing active conformation of the enzyme and indicative of PKC autocatalytic phosphorylation and activation during bacterial expression. The PKC-staurosporine complex represents the first kinase domain crystal structure of any PKC isotypes to be determined and as such should provide valuable insight into PKC specificity and into rational drug design strategies for PKC selective leads. Inhibitors of PKC1 are currently being used in clinical trials for various types of cancer, and a PKC␤ inhibitor is being used in clinical trials for diabetes-related retinopathy (1).PKC and PKB/AKT kinase domains are related by sequence homology; however, there are key structural differences in the regulatory domains and second messenger cofactor requirements. PKB/AKT contains an N-terminal pleckstrin homology domain regulated by phosphoinositide second messengers, a central catalytic kinase domain, and a C-terminal regulatory region facilitating key protein-protein interactions with signal-

Aggrecanases are now believed to be the principal proteinases responsible for aggrecan degradation in osteoarthritis. Given their potential as a drug target, we solved crystal structures of the two most active human aggrecanase isoforms, ADAMTS4 and ADAMTS5, each in complex with bound inhibitor and one wherein the enzyme is in apo form. These structures show that the unliganded and inhibitor-bound enzymes exhibit two essentially different catalytic-site configurations: an autoinhibited, nonbinding, closed form and an open, binding form. On this basis, we propose that mature aggrecanases exist as an ensemble of at least two isomers, only one of which is proteolytically active.Keywords: protein structure; enzymes; metalloproteins; aggrecanases Supplemental material: see www.proteinscience.org Osteoarthritis (OA) is a progressive disease that results in degradation of articular cartilage and chronic pain. The extracellular matrix is composed of two major components, aggrecan and collagen. Aggrecan is a large multidomain proteoglycan that provides cartilage with compressibility and elasticity by swelling and hydrating the collagen network (Vertel and Ratcliffe 2000). Loss of aggrecan is considered a critical early event in OA, occurring initially at the joint surface and progressing to the deeper zones. This is followed by degradation of collagen fibrils and mechanical failure of the tissue (Nagase and Kashiwagi 2003). Aggrecanase-1 (ADAMTS4) and aggrecanase-2 (ADAMTS5), members of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) gene family, cleave aggrecan at a unique site termed the ''aggrecanase site Tortorella et al. 1999). ADAMTS4 and ADAMTS5 are expressed in human normal and OA cartilage (Yamanishi et al. 2002) and in OA synovium, and contribute to the structural damage that characterizes human OA (Powell et al. 2007;Song et al. 2007). However, there is no consensus in the literature as to which aggrecanase is the most important in human OA. In mice, ADAMTS5 (but not ADAMTS4) is responsible for disease progression in a surgically induced model of OA (Glasson et al. 2004(Glasson et al. , 2005. ADAMTS4/ADAMTS5 double knockout mice are physiologically normal (Majumdar et al. 2007) and also protected from developing OA. Given the normal phenotype of the double knockout mice, dual inhibition Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

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