HCV NS3 helicase is a member of a superfamily of helicases, termed superfamily II. Residues of NS3 helicase which are conserved among superfamily II helicases line an interdomain cleft between the first two domains. The oligonucleotide binds in an orthogonal binding site and contacts relatively few conserved residues. There are no strong sequence-specific interactions with the oligonucleotide bases.

The X-ray structure of the ternary complex of a calcineurin A fragment, calcineurin B, FKBP12, and the immunosuppressant drug FK506 (also known as tacrolimus) has been determined at 2.5 A resolution, providing a description of how FK506 functions at the atomic level. In the structure, the FKBP12-FK506 binary complex does not contact the phosphatase active site on calcineurin A that is more than 10 A removed. Instead, FKBP12-FK506 is so positioned that it can inhibit the dephosphorylation of its macromolecular substrates by physically hindering their approach to the active site. The ternary complex described here represents the three-dimensional structure of a Ser/Thr protein phosphatase and provides a structural basis for understanding calcineurin inhibition by FKBP12-FK506.

Interleukin-1 beta converting enzyme (ICE) processes an inactive precursor to the proinflammatory cytokine, interleukin-1 beta, and may regulate programmed cell death in neuronal cells. The high-resolution structure of human ICE in complex with an inhibitor has been determined by X-ray diffraction. The structure confirms the relationship between human ICE and cell-death proteins in other organisms. The active site spans both the 10 and 20K subunits, which associate to form a tetramer, suggesting a mechanism for ICE autoactivation.

An estimated 1% of the global human population is infected by hepatitis C viruses (HCVs), and there are no broadly effective treatments for the debilitating progression of chronic hepatitis C. A serine protease located within the HCV NS3 protein processes the viral polyprotein at four specific sites and is considered essential for replication. Thus, it emerges as an attractive target for drug design. We report here the 2.5 angstrom resolution X-ray crystal structure of the NS3 protease domain complexed with a synthetic NS4A activator peptide. The protease has a chymotrypsin-like fold and features a tetrahedrally coordinated metal ion distal to the active site. The NS4A peptide intercalates within a beta sheet of the enzyme core.

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We have investigated the phase diagram of aqueous solutions of the bovine lens protein y1l-crystallin. For temperatures T < Tc = 278.5 K, we find that these solutions exhibit a reversible coexistence between two isotropic liquid phases differing in protein concentration. The dilute and concentrated branches of the coexistence curve were characterized, consistently, both by measurements of the two coexisting concentrations, c(T), and by measuring the cloud temperatures for various initial concentrations. The importance of studies of phase transitions in protein/ water solutions derives also from their physiological relevance to the supramolecular organization of normal tissues and to certain pathological states. For example, such phase transitions play an important role in the deformation of the erythrocyte in sickle-cell disease (4) and in the cryoprecipitation of immunoglobulins in cryoglobulinemia and rheumatoid arthritis (5, 6). Perhaps the most striking example is the involvement of liquid-liquid phase separation in the opacification of the ocular lens fiber cell cytoplasm in certain forms of mammalian cataracts (7-11). Our work has indicated (12, 13) that the phase separation of one class of lens proteins, the y-crystallins, may be the dominant mechanism responsible for the loss of transparency in such cataracts.Liquid-liquid phase separation in protein solutions has been studied by a number of investigators. Bungenberg de Jong (14) studied liquid-liquid phase separation extensively in a variety of colloidal systems, including aqueous protein solutions. He induced phase separation by the addition of simple electrolytes, polyelectrolytes, and organic solvents miscible with water. He regarded such phase separations as a subset of a broader phenomenon, which he termed coacervation. Dervichian (15) also studied liquid-liquid phase separation in aqueous protein solutions and constructed phase diagrams with which to represent his findings. Similar investigations continue to be reported. Most of these studies have been largely exploratory in character, with an emphasis on application to protein separation methods (16,17) and to microencapsulation (18,19) and on the qualitative classification of the effects of additives on the phase separations (14, 15). Relatively little work has been devoted to the study of phase separation in protein water solutions as a model system to investigate the basic statistical mechanics of critical phenomena and to examine the fundamental interaction energies acting between the constituent protein molecules. One notable exception has been the work of Ishimoto and Tanaka (20) and Tanaka, Nishio, and Sun (21), who studied phase separation in aqueous lysozyme solutions using light scattering methods.In this paper, we report our development of an experimental system that we believe will prove quite useful for the study of liquid-liquid phase separation and crystallization in protein solutions. The system comprises aqueous solutions of the bovine lens protein yl1-crystallin. The y...

FK506 (tacrolimus) is a natural product now approved in the US and Japan for organ transplantation. FK506, in complex with its 12 kDa cytosolic receptor (FKBP12), is a potent agonist of immunosuppression through the inhibition of the phosphatase activity of calcineurin. Rapamycin (sirolimus), which is itself an immunosuppressant by a different mechanism, completes with FK506 for binding to FKBP12 and thereby acts as an antagonist of calcineurin inhibition. We have solved the X-ray structure of unliganded FKBP12 and of FKBP12 in complex with FK506 and with rapamycin; these structures show localized differences in conformation and mobility in those regions of the protein that are known, by site-directed mutagenesis, to be involved in calcineurin inhibition. A comparison of 16 additional X-ray structures of FKBP12 in complex with FKBP12-binding ligands, where those structures were determined from different crystal forms with distinct packing arrangements, lends significance to the observed structural variability and suggests that it represents an intrinsic functional characteristic of the protein. Similar differences have been observed for FKBP12 before, but were considered artifacts of crystal-packing interactions. We suggest that immunosuppressive ligands express their differential effects in part by modulating the conformation of FKBP12, in agreement with mutagenesis experiments on the protein, and not simply through differences in the ligand structures themselves.

A virus-encoded serine proteinase mediates four site-specific cleavages in the hepatitis C virus polyprotein. In addition to the catalytic domain, which is located in the N-terminal one-third of nonstructural protein NS3, the 54-residue NS4A protein is required for cleavage at some but not all sites. Here, we provide evidence for a non-ionic detergent-stable interaction between NS4A and the NS3 serine proteinase domain and demonstrate that the central region of NS4A plays a key role in NS4A-dependent processing. Hydrophobic residues, in particular Ile-29, were shown to be important for NS4A activity, and a synthetic peptide, spanning NS4A residues 22 to 34, could substitute for intact NS4A in a cell-free trans cleavage assay. Furthermore, NS4A mutations, which abolished or inhibited processing, correlated with destabilization of the NS3-NS4A complex. These results suggest that a stable interaction exists between the central region of NS4A and the NS3 catalytic domain which is required for NS4A-dependent processing. Since NS4A is required for processing at certain serine proteinase-dependent cleavage sites, this interaction may represent a new target for development of antiviral compounds.