A method for the analysis of domain movements in large biomolecular complexes.
ABSTRACT A new method for the analysis of domain movements in large, multichain, biomolecular complexes is presented. The method is applicable to any molecule for which two atomic structures are available that represent a conformational change indicating a possible domain movement. The method is blind to atomic bonding and atom type and can, therefore, be applied to biomolecular complexes containing different constituent molecules such as protein, RNA, or DNA. At the heart of the method is the use of blocks located at grid points spanning the whole molecule. The rotation vector for the rotation of atoms from each block between the two conformations is calculated. Treating components of these vectors as coordinates means that each block is associated with a point in a "rotation space" and that blocks with atoms that rotate together, perhaps as part of the same rigid domain, will have colocated points. Thus a domain can be identified from the clustering of points from blocks that span it. Domain pairs are accepted for analysis of their relative movements in terms of screw axes based upon a set of reasonable criteria. Here, we report on the application of the method to biomolecules covering a considerable size range: hemoglobin, liver alcohol dehydrogenase, S-Adenosylhomocysteine hydrolase, aspartate transcarbamylase, and the 70S ribosome. The results provide a depiction of the conformational change within each molecule that is easily understood, giving a perspective that is expected to lead to new insights. Of particular interest is the allosteric mechanism in some of these molecules. Results indicate that common boundaries between subunits and domains are good regions to focus on as movement in one subunit can be transmitted to another subunit through such interfaces.
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ABSTRACT: A simple algorithm is described for the identification of spatially contiguous regions in crystallographic envelopes. In a single pass through the grid points of the envelope map, the occupied points are assigned to a series of locally contiguous sets based on consideration of the connections within single voxels. A spatially contiguous region is identified as the union of all of the locally contiguous sets that share an element in common. Therefore, chains of spatial connectivity are traced implicitly by performing simple set operations. This algorithm has been implemented in the program CNCTDENV as part of the DEMON/ANGEL suite of density-modification programs.Acta Crystallographica Section D Biological Crystallography 08/1997; 53(Pt 4):434-7. · 14.10 Impact Factor
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ABSTRACT: The ribosome is a macromolecular assembly that is responsible for protein biosynthesis following genetic instructions in all organisms. It is composed of two unequal subunits: the smaller subunit binds messenger RNA and the anticodon end of transfer RNAs, and helps to decode the mRNA; and the larger subunit interacts with the amino-acid-carrying end of tRNAs and catalyses the formation of the peptide bonds. After peptide-bond formation, elongation factor G (EF-G) binds to the ribosome, triggering the translocation of peptidyl-tRNA from its aminoacyl site to the peptidyl site, and movement of mRNA by one codon. Here we analyse three-dimensional cryo-electron microscopy maps of the Escherichia coli 70S ribosome in various functional states, and show that both EF-G binding and subsequent GTP hydrolysis lead to ratchet-like rotations of the small 30S subunit relative to the large 50S subunit. Furthermore, our finding indicates a two-step mechanism of translocation: first, relative rotation of the subunits and opening of the mRNA channel following binding of GTP to EF-G; and second, advance of the mRNA/(tRNA)2 complex in the direction of the rotation of the 30S subunit, following GTP hydrolysis.Nature 08/2000; 406(6793):318-22. · 38.60 Impact Factor
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ABSTRACT: In an X-ray diffraction study using the method of multiple isomorphous replacement, the structure of aspartate Carbamoyltransferase (EC 220.127.116.11) complexed with the bisubstrate analog N-(phosphonacetyl)-l-aspartate (PALA)‡ has been solved to 2.5 Å. Ten rounds of model building and 123 cycles of restrained reciprocal space refinement have resulted in a model containing 94.4% of the theoretical atoms of the protein-inhibitor complex with an R-factor of 0.231. The fit of the model to the density is excellent, except for occasional side-chains and two sections of the regulatory chains that may be disordered.The electron density for the PALA molecule is readily identifiable for both catalytic (c) chains of the asymmetric unit and bonding interactions with several important residues including Ser52, Arg54, Thr55, Ser80, Lys84, Arg105, His134, Arg165, Arg229 and Gln231 are apparent. The carboxylate groups of the PALA molecule are in a nearly cis conformation.Gross quaternary changes between the T and R forms are noted and in agreement with earlier work from this laboratory. Namely, in the new structure the catalytic trimers move apart by 12 Å along the 3-fold axis of the enzyme and relocate by 10 ° relative to each other, adopting a more eclipsed position. The regulatory (r) chains in the new structure reorient about their 2-fold axis by 15 °.Large tertiary changes that include domain migration and rearrangement are also present between these two forms. In the R form both domains of the catalytic chain relocate closer to each other in order to bind to the inhibitor. The polar domain seems to bind primarily to the carbamoyl phosphate moiety of PALA, and the equatorial domain binds primarily to the l-aspartate moiety. Other changes in tertiary structure bring the 80s loop (from an adjacent catalytic chain) and the 240s loop into a position to interact with the PALA molecule.Changes have been searched for in all interface regions of the enzyme. While the C1–C4§ and C1–R4 regions have been completely altered, most of the other interchain interfaces are similar in the T and R forms. The intrachain interfaces, between domains of the same catalytic chains, have undergone some reorganization as these domains move closer to each other when the inhibitor is bound.This new structure allows a reinterpretation of genetic and chemical modification studies done to date. As such, the roles of Ser52, Lys84 and His134 are supported. Other residues implicated in similar studies such as Cys47, Tyr165, Lys232 and Tyr240 are too far from the inhibitor to have a direct interaction.Finally, on the basis of the above results, a preliminary model for the homotropic transition is offered. In it, the change to the R-form is triggered by the binding of l-aspartate to the equatorial domain of a catalytic chain in which carbamoyl phosphate is bound to its site in the polar domain. Each C1–C4 group within the enzyme appears to be able to adopt its more active form somewhat independently, but the reorientation of the regulatory dimers that accompanies the homotropic transition serves to facilitate a T to R state transition in the other C1–C4 units. During the change from the T to R states most contact areas between pairs and between pairs are preserved. This is consistent with the suggestion that c-r-r-c is a functionally important unit. In addition, direct communication between C1–C4 is also consistent with the new structure.Journal of Molecular Biology 03/1987; · 3.91 Impact Factor