The crystal structure of the reverse transcriptase (RT) from the type 1 human immunodeficiency virus has been determined at 3.2-A resolution. Comparison with complexes between RT and the polymerase inhibitor Nevirapine [Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A. & Steitz, T. A. (1992) Science 256, 1783Science 256, -1790 and between RT and an oligonucleotide [Jacobo-Molina, A., Ding, J., Nanni, R., Clark, A. D., Lu, X., Tantillo, C., Williams, R. L., Kamer a heterodimer (p66/p5i), with domains labeled "fingers," "thumb," "palm," and "connection" in both subunits, and a ribonuclease H domain in the larger subunit only. The most striking difference between RT and both complex structures is the change in orientation of the p66 thumb (-33°rotation). Smaller shifts relative to the core of the molecule were also found in other domains, including the p66 fingers and palm, which contain the polymerase active site. Within the polymerase catalytic region itself, there are no rearrangements between RT and the RT/DNA complex. In RT/Nevirapine, the drug binds in the p66 palm near the polymerase active site, a region that is well-packed hydrophobic core in the unliganded enzyme. Room for the drug is provided by movement of a small ,B-sheet within the palm domain of the Nevirapine complex. The rearrangement within the palm and thumb, as well as domain shifts relative to the enzyme core, may prevent correct placement of the oligonucleotide substrate when the drug is bound.The reverse transcriptase (RT) from the type 1 human immunodeficiency virus type 1 (HIV-1) is a heterodimer composed of a 66-kDa subunit (p66) and a 51-kDa subunit (pSi) derived from p66 by proteolytic removal of the C-terminal domain. RT possesses both DNA polymerase activity, which ultimately produces double-stranded DNA from the viral genomic RNA, and a ribonuclease H (RNase H) activity, which cleaves the viral genome after it is copied. A crystal structure of RT complexed with the drug Nevirapine (RT/ Nevirapine), a non-nucleoside-analog polymerase inhibitor, has been reported (1, 2), as well as the structure (3) of a complex with an 18/19-mer oligonucleotide (RT/DNA). We report here the structure of the unliganded enzyme at 3.2-A resolution." By comparing it with RT/Nevirapine and RT/ DNA, we can begin to examine the mechanisms of drug and nucleic acid binding. These processes are found to involve changes in domain arrangement within the enzyme but no major repositioning of the polymerase catalytic residues. Differences between the unliganded enzyme and RT/Nevirapine suggest a possible mechanism for the action of nonnucleoside inhibitors. MATERIALS AND METHODSCrystallization. Expression and purification of HIV-1 (BH10 strain) RT Data Collection. RT crystals used for data collection were dialyzed against buffer containing 50% ammonium sulfate, 60 mM sodium phosphate (pH 6.8), and 20% (vol/vol) glycerol. Crystals were mounted in loops (6) made from nylon fibers and flash-cooled in the gaseous nitrogen stream from a modified c...

The crystal structure of human endostatin reveals a zinc-binding site. Atomic absorption spectroscopy indicates that zinc is a constituent of both human and murine endostatin in solution. The human endostatin zinc site is formed by three histidines at the N terminus, residues 1, 3, and, 11, and an aspartic acid at residue 76. The N-terminal loop ordered around the zinc makes a dimeric contact in human endostatin crystals. The location of the zinc site at the amino terminus, immediately adjacent to the precursor cleavage site, suggests the possibility that the zinc may be involved in activation of the antiangiogenic activity following cleavage from the inactive collagen XVIII precursor or in the cleavage process itself.

Regulators of G protein signaling (RGS) proteins that contain DEP (disheveled, EGL-10, pleckstrin) and GGL (G protein ␥ subunit-like) domains form a subfamily that includes the mammalian RGS proteins RGS6, RGS7, RGS9, and RGS11. We describe the cloning of RGS6 cDNA, the specificity of interaction of RGS6 and RGS7 with G protein ␤ subunits, and certain biochemical properties of RGS6/␤5 and RGS7/␤5 complexes. After expression in Sf9 cells, complexes of both RGS6 and RGS7 with the G␤5 subunit (but not G␤s 1-4) are found in the cytosol. When purified, these complexes are similar to RGS11/␤5 in that they act as GTPase-activating proteins specifically toward G␣ o . Unlike conventional G ␤␥ complexes, RGS6/␤5 and RGS7/␤5 do not form heterotrimeric complexes with either G␣ o -GDP or G␣ q -GDP. Neither RGS6/␤5 nor RGS7/␤5 altered the activity of adenylyl cyclases types I, II, or V, nor were they able to activate either phospholipase C-␤1 or -␤2. However, the RGS/␤5 complexes inhibited ␤ 1 ␥ 2 -mediated activation of phospholipase C-␤2. RGS/␤5 complexes may contribute to the selectivity of signal transduction initiated by receptors coupled to G i and G o by binding to phospholipase C and stimulating the GTPase activity of G␣ o .Initial studies of proteins that belong to the regulators of G protein signaling (RGS) 1 family demonstrated that they are GTPase-activating proteins (GAPs) that accelerate hydrolysis of GTP bound to the ␣ subunits of certain heterotrimeric G proteins (1-3). As such, RGS proteins can function as negative regulators of G protein-mediated signal transduction by speeding deactivation of the active form of G ␣ subunits, thereby promoting formation of inactive G protein heterotrimers (G␣ GDP ␤␥). It is now clear that some proteins that contain an RGS domain have more complex functions. For example, p115 RhoGEF, which contains an RGS domain near its amino terminus, is an effector for G protein action, catalyzing guanine nucleotide exchange on the monomeric G protein Rho more efficiently when stimulated by G␣ 13 -GTP. The capacity of G␣ 13 to activate p115 RhoGEF is dependent on the RGS domain of p115, which also accelerates hydrolysis of GTP by G␣ 13 (4, 5). p115 RhoGEF thus resembles phospholipase C␤, a well characterized effector for G protein action that also deactivates its regulator (G␣ q ) by acting as a GAP (6 -8).A subfamily of RGS proteins has been identified in which each member possesses so-called DEP (disheveled, EGL-10, pleckstrin) and GGL (G protein ␥ subunit-like) domains in addition to an RGS domain (9, 10). Members of this group include mammalian RGS proteins (RGS6, RGS7, RGS9, and RGS11), a Drosophila RGS protein (dRGS7), and EGL-10, an RGS protein found in Caenorhabditis elegans (10 -12) Functionally, the GGL domain was shown to specify binding of RGS11 or a fragment of RGS7 to the G protein ␤5 subunit (10). It has also been shown that both RGS7 and RGS9 can be isolated from brain and retina as a complex with G␤5 (13-15) 2 and that the distribution of mRNA for the GGL-containing RG...

A complementary DNA clone encoding the alpha subunit of the adenylate cyclase stimulatory G protein (Gs) was isolated and identified. A bovine brain complementary DNA library was screened with an oligonucleotide probe derived from amino acid sequence common to known G proteins. The only clone that was obtained with this probe has a complementary DNA insert of approximately 1670 base pairs. An antibody to a peptide synthesized according to deduced amino acid sequence reacts specifically with the alpha subunit of Gs. In addition, RNA that hybridizes with probes made from the clone is detected in wild-type S49 cells; however, cyc- S49 cells, which are deficient in Gs alpha activity, are devoid of this messenger RNA.