Computer simulations of the dynamics of human choriogonadotropin and its alpha subunit.

Human choriogonadotropin (hCG) belongs to a family of heterodimeric glycoprotein hormones involved in reproduction. Over 75 ns of molecular dynamics simulations of this heterodimer and the free alpha subunit were performed and validated by experimental information to arrive at a qualitative dynamical description of these molecules. A number of 5-ns simulations at 400 degrees K describe a sufficiently stable heterodimer structure, whereas the free alpha subunit shows the experimentally observed partial unfolding. From the main collective fluctuations of the free alpha subunit, it can be derived that residues alpha35-55 form a domain that is highly flexible with respect to the other domain, which contains all five disulfide bonds. The apparent loss of secondary structure in the region alpha33-58 may very well be induced by this. Dynamic domains can also be determined from the hCG heterodimer simulations. The most important collective mode of motion shows that the flexibility of the alpha subunit is reduced by concerted rotation with both the long loop and the determinant loop of the beta subunit. The motion of the free alpha subunit does not differ significantly from the motion it has in the hCG heterodimer, but the amplitudes along the most important eigenvectors are larger.

The structure of the Escherichia coli phosphotransferase IIAmannitol reveals a novel fold with two conformations of the active site.

BACKGROUND: The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) catalyses the cellular uptake and subsequent phosphorylation of carbohydrates. Moreover, the PTS plays a crucial role in the global regulation of various metabolic pathways. The PTS consists of two general proteins, enzyme I and the histidine-containing protein (HPr), and the carbohydrate-specific enzyme II (EII). EIIs are usually composed of two cytoplasmic domains, IIA and IIB, and a transmembrane domain, IIC. The IIA domains catalyse the transfer of a phosphoryl group from HPr to IIB, which phosphorylates the transported carbohydrate. Knowledge of the structures of the IIA proteins may provide insight into the mechanisms by which the PTS couples phosphorylation reactions with carbohydrate specificity. RESULTS: We have determined the crystal structure of the Escherichia coli mannitol-specific IIA domain, IIAmtl (M(r) 16.3 kDa), by multiple anomalous dispersion analysis of a selenomethionine variant of IIAmtl. The structure was refined at 1.8 A resolution to an R factor of 19.0% (Rfree 24.2%). The enzyme consists of a single five-stranded mixed beta sheet, flanked by helices on both sides. The phosphorylation site (His65) is located at the end of the third beta strand, in a shallow crevice lined with hydrophobic residues. The sidechains of two conserved active-site residues, Arg49 and His111, adopt two different conformations in the four independent IIAmtl molecules. Using a solution structure of phosphorylated HPr, and a combination of molecular modelling and NMR binding experiments, structural models of the HPr-IIAmtl complex were generated. CONCLUSIONS: The fold of IIAmtl is completely different from the structures of other IIA proteins determined so far. The two conformations of Arg49 and His111 might represent different states of the active site, required for the different phosphoryl transfer reactions in which IIAmtl is involved. A comparison of the HPr-IIAmtl model with models of HPr in complex with other IIA enzymes shows that the overall interaction mode between the two proteins is similar. Differences in the stabilisation of the invariant residue Arg17 of HPr by the different IIA proteins might be part of a subtle mechanism to control the hierarchy of carbohydrate utilisation by the bacterium.