HIV-1 protease is a dimeric aspartic protease that plays an essential role in viral replication. To further understand the catalytic mechanism and inhibitor recognition of HIV-1 protease, we need to determine the locations of key hydrogen atoms in the catalytic aspartates Asp-25 and Asp-125. The structure of HIV-1 protease in complex with transition-state analog KNI-272 was determined by combined neutron crystallography at 1.9-Å resolution and X-ray crystallography at 1.4-Å resolution. The resulting structural data show that the catalytic residue Asp-25 is protonated and that Asp-125 (the catalytic residue from the corresponding diad-related molecule) is deprotonated. The proton on Asp-25 makes a hydrogen bond with the carbonyl group of the allophenylnorstatine (Apns) group in KNI-272. The deprotonated Asp-125 bonds to the hydroxyl proton of Apns. The results provide direct experimental evidence for proposed aspects of the catalytic mechanism of HIV-1 protease and can therefore contribute substantially to the development of specific inhibitors for therapeutic application.drug target ͉ neutron diffraction ͉ reaction mechanism ͉ transition-state analog

A crystal structure of the signaling complex between human granulocyte colony-stimulating factor (GCSF) and a ligand binding region of GCSF receptor (GCSF-R), has been determined to 2.8 Å resolution. The GCSF:GCSF-R complex formed a 2:2 stoichiometry by means of a cross-over interaction between the Ig-like domains of GCSF-R and GCSF. The conformation of the complex is quite different from that between human GCSF and the cytokine receptor homologous domain of mouse GCSF-R, but similar to that of the IL-6͞gp130 signaling complex. The Ig-like domain cross-over structure necessary for GCSF-R activation is consistent with previously reported thermodynamic and mutational analyses.ligand-receptor interaction ͉ x-ray crystallography ͉ IL-6 ͉ gp130

UDP-glucose: anthocyanidin 3-O-glucosyltransferase (UGT78K6) from Clitoria ternatea catalyzes the transfer of glucose from UDP-glucose to anthocyanidins such as delphinidin. After the acylation of the 3-O-glucosyl residue, the 3 0 -and 5 0 -hydroxyl groups of the product are further glucosylated by a glucosyltransferase in the biosynthesis of ternatins, which are anthocyanin pigments. To understand the acceptor-recognition scheme of UGT78K6, the crystal structure of UGT78K6 and its complex forms with anthocyanidin delphinidin and petunidin, and flavonol kaempferol were determined to resolutions of 1.85 Å , 2.55 Å , 2.70 Å , and 1.75 Å , respectively. The enzyme recognition of unstable anthocyanidin aglycones was initially observed in this structural determination. The anthocyanidin-and flavonol-acceptor binding details are almost identical in each complex structure, although the glucosylation activities against each acceptor were significantly different. The 3-hydroxyl groups of the acceptor substrates were located at hydrogen-bonding distances to the Ne2 atom of the His17 catalytic residue, supporting a role for glucosyl transfer to the 3-hydroxyl groups of anthocyanidins and flavonols. However, the molecular orientations of these three acceptors are different from those of the known flavonoid glycosyltransferases, VvGT1 and UGT78G1. The acceptor substrates in UGT78K6 are reversely bound to its binding site by a 180 rotation about the O1-O3 axis of the flavonoid backbones observed in VvGT1 and UGT78G1; consequently, the 5-and 7-hydroxyl groups are protected from glucosylation. These substrate recognition schemes are useful to understand the unique reaction mechanism of UGT78K6 for the ternatin biosynthesis, and suggest the potential for controlled synthesis of natural pigments.

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A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild-type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X-ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L-and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L-RNase 1) was also evaluated. N4L-RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.

The ultimate goal of catalytic antibody research is to develop new patient therapies that use the advantages offered by human catalytic antibodies. The establishment of a high-throughput method for obtaining valuable candidate catalytic antibodies must be accelerated to achieve this objective. In this study, based on our concept that we can find antibody light chains with a high probability of success if they include a serine protease-like catalytic triad composed of Ser, His, and Asp on a variable region of the antibody structure, we amplified and cloned DNAs encoding human antibody light chains from germline genes of subgroup II by seminested PCR using two primer sets designed for this purpose. Seven DNA fragments encoding light chains in 17 clones were derived from germline gene A18b, 6 DNA fragments from A3/A19, 2 DNA fragments from A17, and a clone DNA fragment from A5 and O11/O1. All light chains expressed in Escherichia coli and highly purified under nondenaturing conditions exhibited amidolytic activity against synthetic peptides. Some of the light chains exhibited unique features that suppressed the infectious activity of the rabies virus. Furthermore, the survival rate of mice in which a lethal level of the rabies virus was coinoculated directly into the brain with light chain 18 was significantly improved. In the case of humans, these results demonstrate that high-throughput selection of light chains possessing catalytic functions and specificity for a target molecule can be attained from a light-chain DNA library amplified from germline genes belonging to subgroup II.

Flowers of the butterfly pea (Clitoria ternatea) accumulate a group of polyacylated anthocyanins, named ternatins, in their petals. The first step in ternatin biosynthesis is the transfer of glucose from UDP‐glucose to anthocyanidins such as delphinidin, a reaction catalyzed in C. ternatea by UDP‐glucose:anthocyanidin 3‐O‐glucosyltransferase (Ct3GT‐A; AB185904). To elucidate the structure–function relationship of Ct3GT‐A, recombinant Ct3GT‐A was expressed in Escherichia coli and its tertiary structure was determined to 1.85 Å resolution by using X‐ray crystallography. The structure of Ct3GT‐A shows a common folding topology, the GT‐B fold, comprised of two Rossmann‐like β/α/β domains and a cleft located between the N‐ and C‐domains containing two cavities that are used as binding sites for the donor (UDP‐Glc) and acceptor substrates. By comparing the structure of Ct3GT‐A with that of the flavonoid glycosyltransferase VvGT1 from red grape (Vitis vinifera) in complex with UDP‐2‐deoxy‐2‐fluoro glucose and kaempferol, locations of the catalytic His‐Asp dyad and the residues involved in recognizing UDP‐2‐deoxy‐2‐fluoro glucose were essentially identical in Ct3GT‐A, but certain residues of VvGT1 involved in binding kaempferol were found to be substituted in Ct3GT‐A. These findings are important for understanding the differentiation of acceptor‐substrate recognition in these two enzymes.

Pokeweed antiviral proteins (PAPs) are single-chain ribosome-inactivating proteins (RIPs) isolated from several organs of Phytolacca americana (Pokeweed) that are characterized by their ability to depurinate not only ribosomes but also various nucleic acids. PAP-S is one of the isoforms found in seeds. In this study, we obtained three different genomic clones encoding two forms of PAP-S (here designated as PAP-S1 and PAP-S2) and alpha-PAP after PCR using a pair of degenerated primers based on the known N- and C-terminal amino acid sequences of PAP-S. The nucleotide sequences of the genomic clones contained no introns. The deduced amino acid sequences of PAP-S1 and PAP-S2, which showed 83% identity to each other, were found to correspond to sequences reported independently for PAP-S protein and cDNA, respectively, demonstrating that at least two forms of PAP-S actually exist in seeds of the same plant. The recombinant PAP-S1, PAP-S2, alpha-PAP, and PAP I (a form appearing in spring leaves) exhibit the same level of depurinating activity on rat ribosomes, while their efficiencies on Escherichia coli ribosomes and salmon sperm DNA differ substantially from one another in the order of PAP I > alpha-PAP > PAP-S1 > PAP-S2 and alpha-PAP > PAP I > PAP-S1 > PAP-S2. Structural comparisons suggest that the large difference in ribosome recognition between PAP-S1 (or S2) and PAP I is caused by the alteration of residues adjacent to the adenine-binding site.