No abstract

Bisphosphonate drugs (e.g., Fosamax and Zometa) are thought to act primarily by inhibiting farnesyl diphosphate synthase (FPPS), resulting in decreased prenylation of small GTPases. Here, we show that some bisphosphonates can also inhibit geranylgeranyl diphosphate synthase (GGPPS), as well as undecaprenyl diphosphate synthase (UPPS), a cis-prenyltransferase of interest as a target for antibacterial therapy. Our results on GGPPS (10 structures) show that there are three bisphosphonate-binding sites, consisting of FPP or isopentenyl diphosphate substrate-binding sites together with a GGPP product-or inhibitor-binding site. In UPPS, there are a total of four binding sites (in five structures). These results are of general interest because they provide the first structures of GGPPSand UPPS-inhibitor complexes, potentially important drug targets, in addition to revealing a remarkably broad spectrum of binding modes not seen in FPPS inhibition.cell wall ͉ geranylgeranyl diphosphate synthase ͉ undecaprenyl diphosphate synthase ͉ x-ray structure I soprenoid biosynthesis involves the condensation of C 5 -diphosphates to form a very broad range of compounds used in cell membrane (cholesterol, ergosterol), cell wall (lipid I, II, peptidoglycan) and terpene biosynthesis, electron transfer (quinone, heme a, carotenoid, chlorophyll), and in many eukaryotes, cell signaling pathways (Ras, Rho, Rap, Rac). There has, therefore, been considerable interest in developing specific inhibitors of some of these pathways to modify cell function. For example, the bisphosphonate drugs used to treat bone resorption diseases such as osteoporosis (1) have been thought to function by targeting farnesyl diphosphate synthase (FPPS, EC 2.5.1.10) in osteoclasts, leading to dysregulation of cell-signaling pathways involving small GTPases, and in some parasitic protozoa, leading to inhibition of ergosterol biosynthesis (2). However, in recent work Goffinet et al. (3) proposed that the main biological activity of the most potent bisphosphonate zoledronate (Zometa) in humans cells is directed against protein geranylgeranylation. This opens up the intriguing possibility that it might be possible to enhance potency by developing drugs that work by inhibiting geranylgeranyl diphosphate synthase (GGPPS, EC 2.5.1.30), the enzyme that produces the geranylgeranyl diphosphate (GGPP) used to geranylgeranylate e.g., Rac, Rap, and Rho. Based on the recent observation of a previously uncharacterized (GGPP) inhibitor site in GGPPS (4), we reasoned that larger, more hydrophobic species than those in current use might bind to this site and exhibit enhanced activity, because of increased hydrophobic stabilization and, in cells, enhanced lipophilicity. Here, we thus report structures of a series of five bisphosphonates bound to GGPPS together with, for comparative purposes, the structures of five isoprenoid diphosphate-GGPPS complexes. We find three quite different binding modes, corresponding to FPP/GPP (substrate), IPP (substrate), and GGPP [product/ inhibitor (4)...

We have analyzed the functions of several pre-mRNA processing (PRP) proteins in yeast spliceosome formation. Here, we show that PRP5 (a DEAD box helicase-like protein}, PRP9, and PRPll are each required for the U2 snRNP to bind to the pre-spliceosome during spliceosome assembly in vitro. Genetic analyses of their functions suggest that they and another protein, PRP21, act concertedly and/or interact physically with each other and with the stem-loop IIa of U2 snRNA to bind U2 snRNP to the pre-mRNA. Biochemical complementation experiments also indicate that the PRP9 and PRPll proteins interact. The PRP9 and PRPll proteins may be functioning similarly in yeast and mammalian cells. The requirement for ATP and the helicase-like PRP5 protein suggests that these factors might promote a conformational change {involving either the U1 or U2 snRNP) that is required for the association of U2 snRNP with the pre-mRNA.[Key Words: Splicing; pre-mRNA; spliceosome; U2 snRNP; PRP proteins] Received June 28, 1993; revised version accepted August 9, 1993.A key to understanding the mechanism and regulation of nuclear precursor messenger RNA (pre-mRNA) splicing lies in discovering the functions of numerous trans-acting factors. These factors can be grouped into two classes: the small nuclear ribonucleoprotein particles (snRNPs)--U1, U2, U4/U6, and U5--and a multitude of non-snRNP factors (for review, see Green 1991;Guthrie 1991;Ruby and Abelson 1991;Brown et al. 1992;Rymond and Rosbash 1992;Moore et al. 1993). The snRNPs and some non-snRNP factors assemble on the premRNA to form the spliceosome on which splicing occurs. U1 snRNP binds first to the pre-mRNA, followed by the U2 snRNP and, finally, by the tri-snRNP U4/US/ U6 particle. Some non-snRNP factors may become integral components of the spliceosome, whereas others may only loosely or transiently associate with the snRNPs and/or the spliceosome. The functions of these non-sn-RNP factors are particularly intriguing as their elucidation may lead to our understanding of why the premRNA splicing apparatus is so complex and requires ATP.Pre-mRNA splicing occurs in two transesterification reactions, which are mechanistically the same as those of the group II self-splicing introns (for discussion, see 4Corresponding author.Weiner 1993). As the group II selfsplicing introns require no nucleotide or protein cofactors in vitro (for review, see ]acquier 19901, it is thought that pre-mRNA splicing is likely to be catalyzed by RNA as well. The spliceosomal small nuclear RNAs (snRNAs) may have this function. Thus, it is a quandary as to the functions of the numerous snRNP and non-snRNP proteins in premRNA splicing. Some non-snRNP factors such as the mammalian ASF/SF2 (Ge et al. 1991;Krainer et al. 1991) and SC35 (Fu and Maniatis 1992), and the Drosophila transformer (Tra}, Tra2 (Tian and Maniatis 1992), and sex-lethal (Sxl) [Baker 1989) proteins function in the recognition and selection of introns and splice sites. One non-snRNP protein has been proposed to regulate the fidelity of splicing [Bu...