Directed movement is a characteristic of many living organisms and occurs as a result of the transformation of chemical energy into mechanical energy. Myosin is one of three families of molecular motors that are responsible for cellular motility. The three-dimensional structure of the head portion of myosin, or subfragment-1, which contains both the actin and nucleotide binding sites, is described. This structure of a molecular motor was determined by single crystal x-ray diffraction. The data provide a structural framework for understanding the molecular basis of motility.

Muscle contraction consists of a cyclical interaction between myosin and actin driven by the concomitant hydrolysis of adenosine triphosphate (ATP). A model for the rigor complex of F actin and the myosin head was obtained by combining the molecular structures of the individual proteins with the low-resolution electron density maps of the complex derived by cryo-electron microscopy and image analysis. The spatial relation between the ATP binding pocket on myosin and the major contact area on actin suggests a working hypothesis for the crossbridge cycle that is consistent with previous independent structural and biochemical studies.

The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II complexed with beryllium and aluminum fluoride and magnesium ADP are reported at 2.0 and 2.6 A resolution, respectively. Crystals of the beryllium fluoride-MgADP complex belong to space group P2(1)2(1)2 with unit cell parameters of a = 105.3 A, b = 182.6 A, and c = 54.7 A, whereas the crystals of the aluminum fluoride complex belong to the orthorhombic space group C222(1) with unit cell dimensions of a = 87.9 A, b = 149.0 A, and c = 153.8 A. Chemical modification was not necessary to obtain these crystals. These structures reveal the location of the nucleotide complexes and define the amino acid residues that form the active site. The tertiary structure of the protein complexed with MgADP.BeFx is essentially identical to that observed previously in the three-dimensional model of chicken skeletal muscle myosin subfragment-1 in which no nucleotide was present. By contrast, the complex with MgADP.AlF4- exhibits significant domain movements. The structures suggest that the MgADP.BeFx complex mimics the ATP bound state and the MgADP.AlF4- complex is an analog of the transition state for hydrolysis. The domain movements observed in the MgADP.AlF4- complex indicate that myosin undergoes a conformational change during hydrolysis that is not associated with the nucleotide binding pocket but rather occurs in the COOH-terminal segment of the myosin motor domain.

The application of human embryonic stem cells (HESCs) to provide differentiated cells for regenerative medicine will require the continuous maintenance of the undifferentiated stem cells for long periods in culture. However, chromosomal stability during extended passaging cannot be guaranteed, as recent cytogenetic studies of HESCs have shown karyotypic aberrations. The observed karyotypic aberrations probably reflect the progressive adaptation of self-renewing cells to their culture conditions. Genetic change that increases the capacity of cells to proliferate has obvious parallels with malignant transformation, and we propose that the changes observed in HESCs in culture reflect tumorigenic events that occur in vivo, particularly in testicular germ cell tumors. Further supporting a link between culture adaptation and malignancy, we have observed the formation of a chromosomal homogeneous staining region in one HESC line, a genetic feature almost a hallmark of cancer cells. Identifying the genes critical for culture adaptation may thus reveal key players for both stem cell maintenance in vitro and germ cell tumorigenesis in vivo.

Carbamoyl phosphate synthetase catalyzes the production of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of MgATP. As isolated from Escherichia coli, the enzyme has a total molecular weight of approximately 160K and consists of two polypeptide chains referred to as the large and small subunits. Here we describe the X-ray crystal structure of this enzyme determined to 2.8 A resolution in the presence of ADP, Mn2+, phosphate, and ornithine. The small subunit is distinctly bilobal with the active site residues located in the interface formed by the NH2- and COOH-terminal domains. Interestingly, the structure of the COOH-terminal half is similar to that observed in the trpG-type amidotransferase family. The large subunit can be envisioned as two halves referred to as the carboxyphosphate and carbamoyl phosphate synthetic components. Each component contains four distinct domains. Strikingly, the two halves of the large subunit are related by a nearly exact 2-fold rotational axis, thus suggesting that this polypeptide chain evolved from a homodimeric precursor. The molecular motifs of the first three domains observed in each synthetic component are similar to those observed in biotin carboxylase. A linear distance of approximately 80 A separates the binding sites for the hydrolysis of glutamine in the small subunit and the ATP-dependent phosphorylations of bicarbonate and carbamate in the large subunit. The reactive and unstable enzyme intermediates must therefore be sequentially channeled from one active site to the next through the interior of the protein.