Crystal structures of the MJ1267 ATP binding cassette reveal an induced-fit effect at the ATPase active site of an ABC transporter.

BACKGROUND: ATP binding cassette (ABC) transporters are ubiquitously distributed transmembrane solute pumps that play a causative role in numerous diseases. Previous structures have defined the fold of the ABC and established the flexibility of its alpha-helical subdomain. But the nature of the mechanical changes that occur at each step of the chemical ATPase cycle have not been defined. RESULTS: Crystal structures were determined of the MJ1267 ABC from Methanococcus jannaschii in Mg-ADP-bound and nucleotide-free forms. Comparison of these structures reveals an induced-fit effect at the active site likely to be a consequence of nucleotide binding. In the Mg-ADP-bound structure, the loop following the Walker B moves toward the Walker A (P-loop) coupled to backbone conformational changes in the intervening "H-loop", which contains an invariant histidine. These changes affect the region believed to mediate intercassette interaction in the ABC transporter complex. Comparison of the Mg-ADP-bound structure of MJ1267 to the ATP-bound structure of HisP suggests that an outward rotation of the alpha-helical subdomain is coupled to the loss of a molecular contact between the gamma-phosphate of ATP and an invariant glutamine in a segment connecting this subdomain to the core of the cassette. CONCLUSIONS: The induced-fit effect and rotation of the alpha-helical subdomain may play a role in controlling the nucleotide-dependent change in cassette-cassette interaction affinity believed to represent the power-stroke of ABC transporters. Outward rotation of the alpha-helical subdomain also likely facilitates Mg-ADP release after hydrolysis. The MJ1267 structures therefore define features of the nucleotide-dependent conformational changes that drive transmembrane transport in ABC transporters.

Structural analysis and molecular modeling of two anti-trichosanthin IgE clones from phage antibody library.

Recently we constructed a murine IgE phage surface display library and screened out two IgE (Fab) clones with specific binding activity to Trichosanthin (TCS). In this work, the V epsilon and V kappa genes of the two clones were sequenced and their putative germline gene usages were studied. On the basis of the known 3D structure of Trichosanthin and antibody, molecular modeling was carried out to study the antigen-antibody interaction. The possible antigenic determinant sites on the surface of TCS recognized by both the clones were analyzed, and the reaction forces between TCS and two Fab fragments were also analyzed respectively.

A possible hydrolysis mechanism of beta-naphthyl acetate catalyzed by antibodies.

The mechanism of ester hydrolysis has been extensively studied; however, the precise function of active-site residues in promoting catalysis is unclear. We describe here the structural models for the complex of a catalytic antibody Fv fragment with a phosphonate transition-state analogue, constructed by using gene cloning, sequencing and molecular modeling, mainly based on a known X-ray structure of a catalytic antibody. Hydrophobic and electrostatic analyses of the Fv/analog and Fv/substrate interaction suggest the hydrolysis mechanism: Tyr L91 and Tyr H97 play important roles to stabilize the beta-naphthyl group of hapten through pi-stack; His H35 donates a pair of free electrons at the atom NE2 to an active water and let it to be a partial hydroxide, which attacks the carbon atom of the carbonyl group of the substrate. Both His H35 and Arg L96 can form hydrogen bonds and stabilize the anionic tetrahedral intermediate formed during turnover. This mechanism emphasizes that an active water bridge may be formed during hydrolysis process.

Structural biology of RNA silencing and its functional implications.

We outline structure-function contributions from our laboratories on protein-RNA recognition events that monitor siRNA length, 5 -phosphate and 2-nucleotide 3 overhangs, as well as the architecture of Argonaute, its externally bound siRNA complex, and Argonaute-based models involving guide-strand-mediated mRNA binding, cleavage, and release.

An improved rapid method for selecting monoclonal antibodies with high catalytic activities.

Monoclonal antibodies were raised against a beta-naphthyl phosphonate hapten (1) to elicit antibodies capable of catalyzing the hydrolysis of beta-naphthyl acetate (3). After cell fusion, potential catalytic antibody-producing hybridomas were selected, by use of a competitive inhibition assay on the basis of the binding activity for a short transition-state analogue (inhibitor 5), followed by use of high-performance liquid chromatography analysis for the hybridoma supernatants to screen the antibodies processing catalytic activities. It was shown that supernatants of 12 wells had high binding activity with inhibitor and of them, 7 had catalytic activities. After cloning by limiting dilution, we got two hybridoma clones producing monoclonal antibodies which catalyzed the hydrolysis of beta-naphthyl acetate. This combination of competitive inhibition assay with high-performance liquid chromatography analysis represents an improved rapid approach for the screening of potential catalytic antibodies and significantly increases the possibility of obtaining efficient catalytic monoclonal antibodies. Further study of the catalytic antibodies revealed significant rate enhancement (Kcat/K(uncat) approximately 10(6) and specificity.

Three-dimensional structure of beta-momorcharin at 2.55 A resolution.

Beta-Momorcharin (Mr approximately 29 kDa) is a single-chained ribosome-inactivating protein (RIP) with a branched hexasaccharide bound to Asn51. The crystal structure of beta-momorcharin has been determined using the molecular-replacement method and refined to 2. 55 A resolution. The final structural model gave an R factor of 17. 2% and root-mean-square deviations of 0.016 A and 1.76 degrees from ideal bond lengths and bond angles, respectively. beta-Momorcharin contains nine alpha-helices, two 310 helices and three beta-sheets, and its overall structure is similar to those of other single-chained RIPs. Residues Tyr70, Tyr109, Glu158 and Arg161 are expected to define the active site of beta-momorcharin as an rRNA N-glycosidase. The oligosaccharide is linked to the protein through an N-glycosidic bond, beta-GlcNAc-(1-N)-Asn51, and stretches from the surface of the N-terminal domain far from the active site, which suggests that it should not play a role in enzymatic function. The oligosaccharide of each beta-momorcharin molecule interacts with the protein through hydrogen bonds, although in the crystals most of these are intermolecular interactions with the protein atoms in an adjacent unit cell. This is the first example of an RIP structure which provides information about the three-dimensional structure and binding site of the oligosaccharide in the active chains of RIPs.

The crystal structure of the MJ0796 ATP-binding cassette. Implications for the structural consequences of ATP hydrolysis in the active site of an ABC transporter.

The crystal structure of the MJ0796 ATP-binding cassette, a member of the o228/LolD transporter family, has been determined at 2.7-A resolution with MgADP bound at its active site. Comparing this structure with that of the ATP-bound form of the HisP ATP-binding cassette (Hung, L. W., Wang, I. X., Nikaido, K., Liu, P. Q., Ames, G. F., and Kim, S. H. (1998) Nature 396, 703-707) shows a 5-A withdrawal of a phylogenetically invariant glutamine residue from contact with the gamma-phosphate of ATP in the active site. This glutamine is located in a protein segment that links the rigid F(1)-type ATP-binding core of the enzyme to an ABC transporter-specific alpha-helical subdomain that moves substantially away from the active site in the MgADP-bound structure of MJ0796 compared with the ATP-bound structure of HisP. A similar conformational effect is observed in the MgADP-bound structure of MJ1267 (Karpowich, N., et al. (2001) Structure, in press), establishing the withdrawal of the glutamine and the coupled outward rotation of the alpha-helical subdomain as consistent consequences of gamma-phosphate release from the active site of the transporter. Considering this subdomain movement in the context of a leading model for the physiological dimer of cassettes present in ABC transporters indicates that it produces a modest mechanical change that is likely to play a role in facilitating nucleotide exchange out of the ATPase active site. Finally, it is noteworthy that one of the intersubunit packing interactions in the MJ0796 crystal involves antiparallel beta-type hydrogen bonding interactions between the outermost beta-strands in the two core beta-sheets, leading to their fusion into a single extended beta-sheet, a type of structural interaction that has been proposed to play a role in mediating the aggregation of beta-sheet-containing proteins.

["Effort index"--estimating abilities of respiratory muscles].

This paper analyzes the actual values of FEV1 and MBC in 240 normal subjects, 147 patients with COPD and 85 cases of cancer tumors, and shows that the actual values of MBC in both COPD patients and cancerous cases are much lower than the calculated values from FEV1. The authors believe that the ratio between the actual values of MBC and its calculated values may be used to reflect the strengths or abilities of the respiratory muscles efforted, called "Effort Index". The formula of MBC calculated from FEV1 is based on the linear regression equation formulated from the actual values of FEV1 and MBC in 240 normal subjects (aged 16-80 yrs). The "Effort Index" is equal to the actual value of MBC/the calculated value of MBC. The "Effort Index" in 240 normal subjects is 1.00 +/- 0.14 (M +/- SD), in mild, moderate and severe cases of COPD being 0.907, 0.807 and 0.626 respectively, and in markedly wasted cancer cases, 0.861 +/- 0.130. The results show that the formula of MBC calculated from FEV1 is useful only for the subjects with normal respiratory muscular abilities; while in COPD patients and markedly emaciated cancerous cases, owing to the chronically tired-out or exhausted respiratory muscular strength, the actual values of MBC are actually lower than those calculated from FEV1, hence the decreased "Effort Index".