Structural studies of kinesin-nucleotide intermediates.

We have investigated the structural changes that occur in the molecular motor kinesin during its ATPase cycle, utilizing two bacterially expressed constructs. The structure of both constructs has been examined as a function of the nature of the nucleotide intermediate occupying the active site by means of sedimentation velocity, sedimentation equilibrium, fluorescence solute quenching, fluorescence anisotropy decay, and limited proteolysis. While the molecular weight of monomeric and dimeric human kinesin constructs, as measured by sedimentation velocity and sedimentation equilibrium, and the tryptic cleavage pattern are unaffected by the nucleotide intermediate occupying the active site, significant changes in the rotational correlation time of fluorescently labeled kinesin-nucleotide intermediates can be detected. These results suggest that kinesin contains an internal "hinge" whose flexibility varies through the course of the ATPase cycle. In prehydrolytic, "strong" binding states, this hinge is relatively rigid, while in posthydrolytic, "weak" binding states, it is more flexible. Our results, in conjunction with anisotropy decay studies of myosin, suggest that these two molecular motors may share a common structural feature; viz. weak binding states are characterized by segmental flexibility, which is lost upon assumption of a strong binding conformation.

Equilibrium studies of kinesin-nucleotide intermediates.

We have examined the energetics of the interactions of two kinesin constructs with nucleotide and microtubules to develop a structural model of kinesin-dependent motility. Dimerization of the constructs was found to reduce the maximum rate of the microtubule-activated kinesin ATPase 5-fold. Beryllium fluoride and aluminum fluoride also reduce this rate, and they increase the affinity of kinesin for microtubules. By contrast, inorganic phosphate reduces the affinity of a dimeric kinesin construct for microtubules. These findings are consistent with a model in which the kinesin head can assume one of two conformations, "strong" or "weak" binding, determined by the nature of the nucleotide that occupies the active site. Data for dimeric kinesin are consistent with a model in which kinesin.ATP binds to the microtubule in a strong state with positive cooperativity; hydrolysis of ATP to ADP+P(i) leads to dissociation of one of the attached heads and converts the second, attached head to a weak state; and dissociation of phosphate allows the second head to reattach. These results also argue that a large free energy change is associated with formation of kinesin.ADP.P(i) and that this step is the major pathway for dissociation of kinesin from the microtubule.

Structural and kinetic studies of the 10 S<==>6 S transition in smooth muscle myosin.

The conformational transitions that smooth muscle myosin undergoes after nucleotide binding have been examined using fluorescently labeled nucleotides and regulatory light chain. The 10 S conformation of smooth muscle myosin could be induced by addition of 1-N6-ethenoadenosine or mant ADP plus beryllium fluoride, as well as by mant adenosine 5'-(beta,gamma-iminotriphosphate) (AMPPNP). Fluorescence lifetime studies using 1-N6-ethenoadenosine plus beryllium fluoride reveal two components for both (10 S)- and (6 S)-myosins, with little difference in the values of these lifetimes, their fractional amplitudes, or solute accessibilities. Anisotropy decay studies of myosin-mant nucleotide complexes demonstrate that the rotational correlation time for (10 S)-myosin is nearly 4-fold longer than that for (6 S)-myosin. Qualitatively similar results were obtained with a 5-[[[2(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid fluorescent probe attached to the regulatory light chain. Mant AMPPNP can be trapped in the active site by (10S)-myosin. Actin accelerates this release rate by 40-50-fold. These studies reveal: 1) reduction in nucleotide release rate by converting (6S) to (10S)-myosin is not due to a reduction in solute accessibility of the nucleotide 2) the heads in (10 S)-myosin are rigidly attached to the rest of the molecule, while in (6 S)-myosin, they have segmental flexibility, 3) regulatory light chain phosphorylation mimics the effect of high salt in enhancing segmental flexibility of the myosin heads, and 4) actin can induce the unfolding of (10 S)-myosin in the absence of regulatory light chain phosphorylation.

The GPQ-rich segment of Dictyostelium myosin IB contains an actin binding site.

Myosin I has been implicated as the motor that drives protrusion of the leading edge of motile cells. This function requires a close association with the plasma membrane and the cytoskeleton. Association with the actin cytoskeleton is mediated by an ATP-dependent binding site in the motor-containing myosin head, as well as by a second, ATP-independent actin binding site. In myosin IC from Acanthamoeba, the ATP-independent actin binding site is located in the carboxy-terminal tail, in a domain composed of two segments. The first segment is basic and is referred to as the GPA-rich segment. The second is a highly conserved sequence called src homology region 3 (SH3), found in a variety of cytoskeletal-associated proteins. We have used bacterially-expressed fusion proteins containing portions of Dictyostelium myosin IB to determine if the tail of this myosin I isoform also binds to actin and to establish precisely where the actin binding site is located. We have determined that the carboxy-terminal portion of the tail of Dictyostelium myosin IB can bind to actin in an ATP-independent manner and that the actin binding site is contained within residues 922-1059, corresponding to the GPA-rich segment of Acanthamoeba myosin IC. We conclude that this region contains a specific actin binding site which may be responsible for the cytoskeletal association of this myosin I isoform.

Structure of the type-specific polysaccharide antigen of Streptococcus rattus.

The structure of the type-specific polysaccharide antigen of Streptococcus rattus was determined by methylation analysis, periodate oxidation, and by 2D-1H- and 13C-n.m.r.-spectroscopy. The polysaccharide was found to possess the trisaccharide repeating unit----3)-alpha-L-Rhap-(1----2)-[alpha-D-Galp-(1----3)]-alpha-L-+ ++Rhap- (1----.

Structure of the group G streptococcal polysaccharide.

The structure of the group-specific polysaccharide of group G Streptococcus was determined by means of methylation analysis and selective chemical degradations. The anomeric configurations and conformations of the sugar residues were studied by 1H- and 13C-n.m.r. spectroscopy. The tetrasaccharide repeating unit, ----3)-alpha-D-Galp-(1----2)-[alpha-L-Rhap-(1----3)-beta-D-GalpNAc - (1----4)]-alpha-L-Rhap-(1----, was determined.