The repressors of temperate bacteriophages such as 434 and lambda control transcription by binding to a set of DNA operator sites. The different affinity of repressor for each of these sites ensures efficient regulation. High-resolution x-ray crystallography was used to study the DNA-binding domain of phage 434 repressor in complex with a synthetic DNA operator. The structure shows recognition of the operator by direct interactions with base pairs in the major groove, combined with the sequence-dependent ability of DNA to adopt the required conformation on binding repressor. In particular, a network of three-centered bifurcated hydrogen bonds among base pairs in the operator helps explain why 434 repressor prefers certain sites over others. These bonds, which stabilize the conformation of the bound DNA, can form only with certain sequences.

The multimeric membrane-tethering complexes TRAPPI and TRAPPII share seven subunits, of which four (Bet3p, Bet5p, Trs23p, and Trs31p) are minimally needed to activate the Rab GTPase Ypt1p in an event preceding membrane fusion. Here, we present the structure of a heteropentameric TRAPPI assembly complexed with Ypt1p. We propose that TRAPPI facilitates nucleotide exchange primarily by stabilizing the nucleotide-binding pocket of Ypt1p in an open, solvent-accessible form. Bet3p, Bet5p, and Trs23p interact directly with Ypt1p to stabilize this form, while the C terminus of Bet3p invades the pocket to participate in its remodeling. The Trs31p subunit does not interact directly with the GTPase but allosterically regulates the TRAPPI interface with Ypt1p. Our findings imply that TRAPPII activates Ypt1p by an identical mechanism. This view of a multimeric membrane-tethering assembly complexed with a Rab provides a framework for understanding events preceding membrane fusion at the molecular level.

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 structure of an N-terminal fragment of CD4 has been determined to 2.4 A resolution. It has two tightly abutting domains connected by a continuous beta strand. Both have the immunoglobulin fold, but domain 2 has a truncated beta barrel and a non-standard disulphide bond. The binding sites for monoclonal antibodies, class II major histocompatibility complex molecules, and human immunodeficiency virus gp120 can be mapped on the molecular surface.

The zinc metallopeptidase neurolysin is shown by x-ray crystallography to have large structural elements erected over the active site region that allow substrate access only through a deep narrow channel. This architecture accounts for specialization of this neuropeptidase to small bioactive peptide substrates without bulky secondary and tertiary structures. In addition, modeling studies indicate that the length of a substrate N-terminal to the site of hydrolysis is restricted to approximately 10 residues by the limited size of the active site cavity. Some structural elements of neurolysin, including a five-stranded ␤-sheet and the two active site helices, are conserved with other metallopeptidases. The connecting loop regions of these elements, however, are much extended in neurolysin, and they, together with other open coil elements, line the active site cavity. These potentially flexible elements may account for the ability of the enzyme to cleave a variety of sequences.

Crystallographic studies are being extended to increasingly challenging systems: ever larger molecules, more complex viruses, membrane proteins, and assemblies. The crystals are often radiation-sensitive and scatter X-rays weakly, in many cases posing severe data collection problems. Synchrotron sources, high-performance area detectors and enhanced computational power have made it possible to tackle more difficult crystals and have greatly improved and simplified collection in general. Another tool, data collection at cryogenic temperatures, addresses more directly some of the problems associated with macromolecular crystals. Low-temperature techniques are coming into widespread use, and have become the routine method of data collection in a few laboratories. Cryogenic collection can make possible otherwise intractable projects, helping to expand the range of applications of crystallography to include some of the most exciting problems in biology. It is now clear that low-temperature techniques also offer real advantages with more conventional crystals, so that they should be considered for all macromolecular X-ray studies.

[1] We used detailed microtopographic surveys to measure fault offset along the southern trace of the Wairarapa fault, near Wellington, New Zealand, which most recently experienced a M w > 8.1 earthquake in 1855. Our measurements at 16 localities support the inference that dextral slip in 1855 reached 18.7 m and averaged $16 m over the 16 km length that we studied. Five measurements were made where a single active strand comprises the fault zone, yielding ''smallest'' dextral offsets of 13.0-18.7 m. At Pigeon Bush, sequential beheading of a stream and new 14 C dating support the interpretation that its 18.7 ± 1.0 m of offset accumulated in 1855. We also measured three ''nextsmallest'' offsets on single-strand faults of 26.3-32.7 m, evidence that dextral slip during the previous event was $14 m. Eight measurements were made where the Wairarapa fault includes two closely spaced strands, yielding smallest dextral offsets of 12.9-16.0 m. At Tauherenikau River, 14 C dating of postoffset mud yielded ages indistinguishable from A.D. 1855. Combining all single-strand and two-strand (minimum) estimates yields an average dextral slip of 15.5 ± 1.4 m in the study area. Historical observations and our data indicate that vertical slip reached $2.5 m. The large displacement and short ($145 km) strike length yield an unusually high displacement/length ratio for the rupture. As suggested by previous dislocation modeling, we propose that the rupture extended tens of kilometers downdip (W) to merge with the underlying subduction interface. Alternatively, the rupture may have been strongly segmented at depth, yielding an earthquake with an unusually large static stress drop.

Alanine scanning mutagenesis was undertaken to evaluate the structural significance of Met230-His235 of the 66 kDa subunit of p66/p51 human immunodeficiency virus reverse transcriptase (HIV-1 RT). Together with Glu224-Trp229, these residues provide the framework of the p66 "primer grip", whose proposed role is maintaining the primer terminus in an orientation appropriate for nucleophilic attack on an incoming dNTP. Of these residues, altering Leu234 results in a p66 subunit incapable of associating into heterodimer. The remaining selectively mutated enzymes were successfully reconstituted and purified to homogeneity for evaluation of RT-associated activities. We show here that alterations to any residue within the p66-Trp229-Met230-Gly231-Tyr232-quartet alter functions associated with both the DNA polymerase and ribonuclease H (RNase H) domains. Detailed analysis of mutant p66Y232A/p51 with an intact or a model "precleaved" RNA-DNA hybrid suggests an altered RNase H phenotype could result from relocation of template-primer in the nucleic acid binding cleft. As a consequence, template nucleotide-8 is positioned in the immediate vicinity of the RNase H catalytic center rather than nucleotide-17.