The crystal structure of a ternary complex of human _nununodeficiency virus type 1 reverse transcriptase (HIV-1 RT) heterodimer (p66/p5i), a 19-base/18-base doublestranded DNA template-primer, and a monoclonal antibody Fab fragment has been determined at 3.0 A resolution. (40)(41)(42)(43)(44)(45). The most numerous nucleic acid interactions with protein occur primarily along the sugar-phosphate backbone of the DNA and involve amino acid residues of the palm, thumb, and ringers of p66. Highly conserved regions are located in the p66 palm near the polymerase active site. These structural elements, together with two a-helices of the thumb of p66, act as a clamp to position the template-primer relative to the polymerase active site. The 3'-hydroxyl of the primer terminus is close to the catalytically essential Asp-110, Asp-185, and Asp-186 residues at the active site and is in a position for nudeophilic attack on the a-phosphate of an incoming nucleoside triphosphate. The structure of the HIV-1 RT/DNA/Fab complex should aid our understanding of general mechanisms of nucleic acid polymerization. AIDS therapies may be enhanced by a fuller understanding of drug inhibition and resistance emerging from these studies. A 3.5 A resolution structure of HIV-1 RT complexed with the nonnucleoside inhibitor nevirapine has been reported (8).Although the resolution of the study was not sufficient to determine the position of every amino acid and their side chains, the overall folding of the enzyme was described.We have prepared crystals of a ternary complex (9) of the HIV-1 RT p66/pSl heterodimer, a double-stranded DNA (dsDNA) template-primer, and the antigen-binding fragment (Fab fragment) ofa noninhibiting antibody that diffract x-rays to 2.8 A resolution, and reported the structure ofthis complex at 7 A resolution (10). At this resolution it was possible to determine the location ofthe template-primer and the relative positions of the polymerase and the RNase H active sites. In addition, it was shown that when the nucleic acid substrate was bound to RT a significant portion of the protein moved out of the nucleic acid-binding site.Here we report the structure of the RT-dsDNA-Fab28 complex at 3.0 A resolution. The overall arrangement of the enzyme is similar to that previously reported (8 ITo whom reprint requests should be addressed

FKBP ligand homodimers can be used to activate signaling events inside cells and animals that have been engineered to express fusions between appropriate signaling domains and FKBP. However, use of these dimerizers in vivo is potentially limited by ligand binding to endogenous FKBP. We have designed ligands that bind specifically to a mutated FKBP over the wild-type protein by remodeling an FKBP-ligand interface to introduce a specificity binding pocket. A compound bearing an ethyl substituent in place of a carbonyl group exhibited sub-nanomolar affinity and 1,000-fold selectivity for a mutant FKBP with a compensating truncation of a phenylalanine residue. Structural and functional analysis of the new pocket showed that recognition is surprisingly relaxed, with the modified ligand only partially filling the engineered cavity. We incorporated the specificity pocket into a fusion protein containing FKBP and the intracellular domain of the Fas receptor. Cells expressing this modified chimeric protein potently underwent apoptosis in response to AP1903, a homodimer of the modified ligand, both in culture and when implanted into mice. Remodeled dimerizers such as AP1903 are ideal reagents for controlling the activities of cells that have been modified by gene therapy procedures, without interference from endogenous FKBP.

The crystal structure of the tandem SH2 domains of human ZAP-70 in complex with a peptide derived from the zeta-subunit of the T-cell receptor reveals an unanticipated interaction between the two domains. A coiled coil of alpha-helices connects the two SH2 domains, producing an interface that constitutes one of the two critical phosphotyrosine binding sites. These and other unique features provide the molecular basis for highly selective association of ZAP-70 with the T-cell receptor.

AIMTo investigate whether patients with refractory epilepsy and healthy infants differ in gut microbiota (GM), and how ketogenic diet (KD) alters GM.METHODSA total of 14 epileptic and 30 healthy infants were recruited and seizure frequencies were recorded. Stool samples were collected for 16S rDNA sequencing using the Illumina Miseq platform. The composition of GM in each sample was analyzed with MOTHUR, and inter-group comparison was conducted by R software.RESULTSAfter being on KD treatment for a week, 64% of epileptic infants showed an obvious improvement, with a 50% decrease in seizure frequency. GM structure in epileptic infants (P1 group) differed dramatically from that in healthy infants (Health group). Proteobacteria, which had accumulated significantly in the P1 group, decreased dramatically after KD treatment (P2 group). Cronobacter predominated in the P1 group and remained at a low level both in the Health and P2 groups. Bacteroides increased significantly in the P2 group, in which Prevotella and Bifidobacterium also grew in numbers and kept increasing.CONCLUSIONGM pattern in healthy infants differed dramatically from that of the epileptic group. KD could significantly modify symptoms of epilepsy and reshape the GM of epileptic infants.

The non-nucleoside inhibitor-binding pocket has a flexible structure whose mobility may be required for effective polymerization, and may be part of a hinge that permits relative movements of two subdomains of the p66 subunit denoted the 'palm' and 'thumb'. An understanding of the structure of the inhibitor-binding pocket, of the interactions between HIV-1 RT and alpha-APA, and of the locations of mutations that confer resistance to inhibitors provides a basis for structure-based design of chemotherapeutic agents for the treatment of AIDS.