Short peptides that contain the basic region of the HIV-1 Tat protein bind specifically to a bulged region in TAR RNA. A peptide that contained nine arginines (R9) also bound specifically to TAR, and a mutant Tat protein that contained R9 was fully active for transactivation. In contrast, a peptide that contained nine lysines (K9) bound TAR poorly and the corresponding protein gave only marginal activity. By starting with the K9 mutant and replacing lysine residues with arginines, a single arginine was identified that is required for specific binding and transactivation. Ethylation interference experiments suggest that this arginine contacts two adjacent phosphates at the RNA bulge. Model building suggests that the arginine eta nitrogens and the epsilon nitrogen can form specific networks of hydrogen bonds with adjacent pairs of phosphates and that these arrangements are likely to occur near RNA loops and bulges and not within double-stranded A-form RNA. Thus, arginine side chains may be commonly used to recognize specific RNA structures.

Arginine-rich sequences are found in many RNA-binding proteins and have been proposed to mediate specific RNA recognition. Fragments of the HIV-1 Tat protein that contain the arginine-rich region of Tat bind specifically to a 3-nucleotide bulge in TAR RNA. To determine the amino acid requirements for specific RNA recognition, we synthesized a series of mutant Tat peptides spanning this domain (YGRKKRRQRRRP) and measured their affinity and specificity for TAR RNA. Several corresponding mutations were introduced into the full-length Tat protein, and trans-activation activity was measured. Systematic substitution of arginine residues with alanines or lysines suggested that overall charge density is important but did not point to any specific residues as being essential for binding. A glutamine-to-alanine substitution had no effect on binding. Remarkably, peptides with scrambled or reversed sequences showed the same affinity and specificity for TAR RNA as the wild-type peptide. Trans-activation activity of the mutant Tat proteins correlated with RNA binding. Arginine-rich peptides from SIV Tat and from HIV-1 Rev, which can functionally substitute for the basic region of HIV-1 Tat, also bound specifically to TAR. Circular dichroism spectra suggest that the arginine-rich region of Tat is unstructured in the absence of RNA, becomes partially or fully structured upon binding, and induces a conformational change in the RNA. These results suggest that arginine-rich RNA-binding domains have considerable sequence flexibility, reminiscent of acidic domains found in transcriptional activators, and that RNA structure may provide much of the specificity for the interaction.

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There has been intense interest in the development of factor Xa inhibitors for the treatment of thrombotic diseases. Our laboratory has developed a series of novel non-amidine inhibitors of factor Xa. This paper presents two crystal structures of compounds from this series bound to factor Xa. The first structure is derived from the complex formed between factor Xa and compound 1. Compound 1 was the first non-amidine factor Xa inhibitor from our lab that had measurable potency in an in vitro assay of anticoagulant activity. The second compound, 2, has a molar affinity for factor Xa (K(iapp)) of 7 pM and good bioavailability. The two inhibitors bind in an L-shaped conformation with a chloroaromatic ring buried deeply in the S1 pocket. The opposite end of these compounds contains a basic substituent that extends into the S4 binding site. A chlorinated phenyl ring bridges the substituents in the S1 and S4 pockets via amide linkers. The overall conformation is similar to the previously published structures for amidine-based inhibitors complexed with factor Xa. However, there are significant differences in the interactions between the inhibitor and the protein at the atomic level. Most notably, there is no group that forms a salt bridge with the carboxylic acid at the base of the S1 pocket (Asp189). Each inhibitor forms only one well-defined hydrogen bond to the protein. There are no direct charge-charge interactions. The results indicate that electrostatic interactions play a secondary role in the binding of these potent inhibitors.

To determine which of the 86 amino acids in the Tat protein of human inmunodeficiency virus type 1 (HIV-1) are important for transactivation, peptides from Tat were synthesized and their activity was measured in cells containing a chloramphenicol acetyltransferase reporter gene under control of the HIV long terminal repeat promoter.Although the Tat sequence contains arginine-and cysteine-rich stretches that are difficult to synthesize, it was possible to prepare pure peptides in good yield by using fluoren-9-ylmethoxycarbonyl (Fmoc) chemistry. A peptide containing residues 1-58 had 5-10% the activity of full-length Tat. Deleting 4 amino acids from the N terminus of this peptide further reduced activity, while peptides with more extensive N-terminal deletions and peptides missing the basic region at the C terminus had no detectable activity. A peptide previously reported to transactivate, Tat-(37-62), was completely inactive in our assays. Inactive peptides were also tested as possible inhibitors of transactivation. Tat-(21-38), which contains the cysteine-rich region and can form heterodimers with intact Tat in vitro, showed inhibition at high peptide concentrations. However, this effect was not specific for Tat or for the HIV promoter, since the peptide also inhibited expression from the simian virus 40 early promoter.The Tat protein from human immunodeficiency virus (HIV) is a potent viral transactivator (1) that is essential for viral replication (2,3). Although the precise mechanism of transactivation remains unclear, Tat may cause accumulation of mRNA by increasing the rate of transcription from the long terminal repeat (LTR) promoter (4, 5), by acting as a transcriptional antiterminator (6), or by stabilizing viral mRNAs (7-10). Tat may also increase translational efficiency (7)(8)(9)(10)(11)(12). A region of about 40 base pairs just 3' to the start of transcription [the transactivation response (TAR) site] is necessary for transactivation by .The Tat protein is 86 amino acids long and contains a highly basic region (with 2 lysines and 6 arginines in 9 residues) and a cysteine-rich region (with 7 cysteines in 16 residues) (16,17). The basic region is important for nuclear localization (18,19). The cysteine-rich region mediates the formation of metal-linked dimers in vitro (20), although it is not known whether the metal-linked dimer is important for transactivation. An 18-amino acid peptide containing just the cysteinerich region of Tat can also form metal-linked dimers and can form heterodimers with intact Tat in vitro (21). It was suggested that, ifdimerization is essential for transactivation, this peptide might inhibit Tat activity by forming inactive heterodimers (20,21).It is now possible to directly measure the activity of Tat peptides and to test whether peptides can inhibit transactivation. Assays have recently been developed that use a procedure known as "scrape-loading" to introduce Tat into tissue culture cells that contain the HIV-1 promoter linked to a reporter gene (22,23). It is bel...

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