The neck region of the myosin motor domain acts as a lever arm to generate movement.

The myosin head consists of a globular catalytic domain that binds actin and hydrolyzes ATP and a neck domain that consists of essential and regulatory light chains bound to a long alpha-helical portion of the heavy chain. The swinging neck-level model assumes that a swinging motion of the neck relative to the catalytic domain is the origin of movement. This model predicts that the step size, and consequently the sliding velocity, are linearly related to the length of the neck. We have tested this point by characterizing a series of mutant Dictyostelium myosins that have different neck lengths. The 2xELCBS mutant has an extra binding site for essential light chain. The delta RLCBS mutant myosin has an internal deletion that removes the regulatory light chain binding site. The delta BLCBS mutant lacks both light chain binding sites. Wild-type myosin and these mutant myosins were subjected to the sliding filament in vitro motility assay. As expected, mutants with shorter necks move slower than wild-type myosin in vitro. Most significantly, a mutant with a longer neck moves faster than the wild type, and the sliding velocities of these myosins are linearly related to the neck length, as predicted by the swinging neck-lever model. A simple extrapolation to zero speed predicts that the fulcrum point is in the vicinity of the SH1-SH2 region in the catalytic domain.

X-ray absorption fine structure as a monitor of zinc coordination sites during oogenesis of Xenopus laevis.

The x-ray absorption fine structure (XAFS) zinc K-edge steps for intact stages I,II and V,VI Xenopus laevis oocytes demonstrate that the zinc concentration is about 3 and 1 mM, respectively. However, the chi(k) function for the early stage oocytes differs markedly from that for the late one. Analysis of the XAFS data for stage I,II oocytes indicates that zinc is bound to 2.0 +/- 0.5 sulfur atoms at an average coordination distance of 2.29 +/- 0.02 angstroms and 2.0 +/- 0.5 nitrogen or oxygen (N/O) atoms at 2.02 +/- 0.02 angstroms. In marked contrast, in stage V,VI oocytes, zinc is bound to 4.1 +/- 0.4 N/O atoms at an average distance of 1.98 +/- 0.01 angstroms. Our previous studies demonstrated that 90% of the zinc in stage VI oocytes is sequestered within yolk platelets, associated with a single molecule, lipovitellin, the proteolytically processed product of vitellogenin. XAFS analysis of yolk platelets, lipovitellin, and vitellogenin demonstrates that zinc is bound to 4.0 +/- 0.5 N/O ligands at an average distance of 1.98 +/- 0.01 angstroms in each case, identical to that of stage V,VI oocytes. The higher shell contributions in the Fourier transforms indicate that two of the N/O zinc ligands are His in both stage V,VI and I,II oocytes. The results show that in stage I,II oocytes, there is a high concentration of a zinc protein whose zinc coordination site likely is composed of (His)2(Cys)2, such as, e.g., TFIIIA. As the oocytes develop, the predominant zinc species becomes one that exhibits the (His)2(N/0)2 zinc site found in lipovitellin. Hence, the ligands to the zinc atoms in intact oocytes and the changes that take place as a function of oogenesis and after their fertilization, during embryogenesis, now can be examined and explored.

A role for the Msx-1 homeodomain in transcriptional regulation: residues in the N-terminal arm mediate TATA binding protein interaction and transcriptional repression.

In a previous study we showed that the murine homeodomain protein Msx-1 is a potent transcriptional repressor and that this activity is independent of its DNA binding function. The implication of these findings is that repression by Msx-1 is mediated through its association with certain protein factors rather than through its interaction with DNA recognition sites, which prompted investigation of the relevant protein factors. Here we show that Msx-1 interacts directly with the TATA binding protein (TBP) but not with several other general transcription factors. This interaction is mediated by the Msx-1 homeodomain, specifically through residues in the N-terminal arm. These same N-terminal arm residues are required for repression by Msx-1, suggesting a functional relationship between TBP association and transcriptional repression. This is further supported by the observation that addition of excess TBP blocks the repressor action of Msx-1 in in vitro transcription assays. Finally, DNA binding activity is separable from both TBP interaction and repression, which further shows that these other activities of the Msx-1 homeodomain are distinct. Therefore, these findings define a role for the Msx-1 homeodomain, particularly the N-terminal arm residues in protein-protein interaction and transcriptional repression, and implicate a more complex role overall for homeodomains in transcriptional regulation.

Subunit interactions in coordination of Ni2+ in cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) channels present a unique model for studying the molecular mechanisms of channel gating. We have studied the mechanism of potentiation of expressed rod CNG channels by Ni2+ as a first step toward understanding the channel gating process. Here we report that coordination of Ni2+ between histidine residues (H420) on adjacent channel subunits occurs when the channels are open. Mutation of H420 to lysine completely eliminated the potentiation by Ni2+ but did not markedly alter the apparent cGMP affinity of the channel, indicating that the introduction of positive charge at the Ni(2+)-binding site was not sufficient to produce potentiation. Deletion or mutation of most of the other histidines present in the channel did not diminish potentiation by Ni2+. We studied the role of subunit interactions in Ni2+ potentiation by generating heteromultimeric channels using tandem dimers of the rod CNG channel sequence. Injection of single heterodimers in which one subunit contained H420 and the other did not (wt/H420Q or H420Q/wt) resulted in channels that were not potentiated by Ni2+. However, coinjection of both heterodimers into Xenopus oocytes resulted in channels that exhibited potentiation. The H420 residues probably occurred predominantly in nonadjacent subunits when each heterodimer was injected individually, but, when the two heterodimers were coinjected, the H420 residues could occur in adjacent subunits as well. These results suggest that the mechanism of Ni2+ potentiation involves intersubunit coordination of Ni2+ by H420. Based on the preferential binding of Ni2+ to open channels, we suggest that alignment of H420 residues of neighboring subunits into the Ni(2+)-coordinating position may be associated with channel opening.

Y chromosome short arm-Sxr recombination in XSxr/Y males causes deletion of Rbm and XY female sex reversal.

We earlier described three lines of sex-reversed XY female mice deleted for sequences believed close to the testes-determining gene (Sry) on the Y chromosome short arm (Yp). The original sex-reversed females appeared among the offspring of XY males that carried the Yp duplication Sxr on their X chromosome. Earlier cytogenetic observations had suggested that the deletions resulted from asymmetrical meiotic recombination between the Y and the homologous Sxr region, but no direct evidence for this hypothesis was available. We have now analyzed the offspring of XSxr/Y males carrying an evolutionarily divergent Mus musculus domesticus Y chromosome, which permits detection and characterization of such recombination events. This analysis has enabled the derivation of a recombination map of Yp and Sxr, also demonstrating the orientation of Yp with respect to the Y centromere. The mapping data have established that Rbm, the murine homologue of a gene family cloned from the human Y chromosome, lies between Sry and the centromere. Analysis of two additional XY female lines shows that asymmetrical Yp-Sxr recombination leading to XY female sex reversal results in deletion of Rbm sequences. The deletions bring Sry closer to Y centromere, consistent with the hypothesis that position-effect inactivation of Sry is the basis for the sex reversal.

Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide.

We describe a method to facilitate radioimaging with technetium-99m (99mTc) by genetic incorporation of a 99mTc chelation site in recombinant single-chain Fv (sFv) antibody proteins. This method relies on fusion of the sFv C terminus with a Gly4Cys peptide that specifically coordinates 99mTc. By using analogues of the 26-10 anti-digoxin sFv as our primary model, we find that addition of the chelate peptide, to form 26-10-1 sFv', does not alter the antigen-binding affinity of sFv. We have demonstrated nearly quantitative chelation of 0.5-50 mCi of 99mTc per mg of 26-10-1 sFv' (1 Ci = 37 GBq). These 99mTc-labeled sFv' complexes are highly stable to challenge with saline buffers, plasma, or diethylenetriaminepentaacetic acid. We find that the 99mTc-labeled 741F8-1 sFv', specific for the c-erbB-2 tumor-associated antigen, is effective in imaging human ovarian carcinoma in a scid mouse tumor xenograft model. This fusion chelate methodology should be applicable to diagnostic imaging with 99mTc and radioimmunotherapy with 186Re or 188Re, and its use could extend beyond the sFv' to other engineered antibodies, recombinant proteins, and synthetic peptides.