Richard W. Schevitz

A Transforming Mutation in the Pleckstrin Homology Domain of Akt1 in Cancer.

Although AKT1 (v-akt murine thymoma viral oncogene homologue 1) kinase is a central member of possibly the most frequently activated proliferation and survival pathway in cancer, mutation of AKT1 has not been widely reported. Here we report the identification of a somatic mutation in human breast, colorectal and ovarian cancers that results in a glutamic acid to lysine substitution at amino acid 17 (E17K) in the lipid-binding pocket of AKT1. Lys 17 alters the electrostatic interactions of the pocket and forms new hydrogen bonds with a phosphoinositide ligand. This mutation activates AKT1 by means of pathological localization to the plasma membrane, stimulates downstream signalling, transforms cells and induces leukaemia in mice. This mechanism indicates a direct role of AKT1 in human cancer, and adds to the known genetic alterations that promote oncogenesis through the phosphatidylinositol-3-OH kinase/AKT pathway. Furthermore, the E17K substitution decreases the sensitivity to an allosteric kinase inhibitor, so this mutation may have important clinical utility for AKT drug development.

Crystal structure of a eukaryotic initiator tRNA

OUR understanding of the molecular structure–function relationship in tRNA rests mainly on three types of information. First, on the common sequence patterns which have emerged from careful examination of many primary structures1–3; second, a wide variety of spectral and other physical and chemical results must be accounted for by the molecular structure4–6; and third, there is the detailed image of the yeast tRNAPhe molecule independently determined and refined from two different—albeit similar—crystal forms7–10. It is also clear, however, that the molecular model deduced from the yeast tRNAphe crystal structure cannot be easily reconciled with all structural requirements for function and is best considered a well-defined and stable canonical form of tRNA which is packed in an unusually well-ordered way in specific crystal lattices. Notwithstanding the enormous value of this canonical form in explaining the basic architectural features of tRNA, it is clearly important to image other crystalline tRNAs; particularly tRNAs that exhibit different functions (such as, initiators) or have significantly different covalent structures (for example, class III tRNAs)1 or those that crystallise in different solvent conditions. We report here the initial results of the crystal structure determination of a eukaryotic initiator tRNA crystallised from a highly polar aqueous solvent11,12. Its architecture is essentially the same as crystalline yeast tRNAphe, except for a small but significant difference in the position of the anticodon arm.

Improving and extending the phases of medium- and low-resolution macromolecular structure factors by density modification

Non-negativity of the electron density function and constancy of the solvent regions were exploited to improve 2633 phases of crystalline yeast tRNAfMet (P6422, a = 115.3, c = 137.9 A, z = 12), which had been obtained by multiple isomorphous replacement (MIR) in the resolution zone 14-4.5 A. Phases were also determined for an additional 912 reflections not previously phased by MIR from the resolution range 100 to 4 A, the very limits of the diffraction pattern. An iterative procedure was employed in which phases for each cycle were calculated from a density map modified by imposing the above constraints and were combined with the observed amplitudes to produce a new and improved map. Initially phases calculated in each cycle were merged with the original MIR phase probability curve; convergence was achieved in seven cycles. The phases were then released from the MIR analysis by using just the calculated phases until a second convergence was achieved (four cycles). The average difference between the experimental phases and phases calculated from the refined coordinates was reduced from 68° for the original MIR analysis to 43° by the use of these real-space direct methods. Phases determined solely by density modification were as accurate as the original MIR phase set. The map calculated with improved and extended phases was dramatically superior to the MIR map and even approached in quality the map produced with phases calculated from the refined molecular coordinates.

Phasing low-resolution macromolecular structure factors by matricial direct methods

Matricial direct methods were used to phase 28 strong low-resolution (100-19 A) structure factors of crystalline yeast tRNA~ et (P6422, z = 12) which had not been phased by multiple isomorphous replacement (MIR). The starting phase set was composed of 107 terms in the resolution range 32-14 A which had been phased by MIR. Extending the phase set to the strong lowresolution terms significantly improved the electrondensity map. The goal of establishing a well defined molecular boundary was clearly achieved and provided the basis of a successful structure determination to 4.0 A resolution. The phases determined by direct methods deviated from the phases subsequently calculated from the refined atomic coordinates by an unweighted average value of 73 o; 36% of these were determined with a figure of merit greater than 0.75 and showed a discrepancy of only 44 o. The accuracy of the phases determined by matricial methods compared favorably with those of the starting MIR phase set. An analysis of the resolution dependence of the intensities suggests plausible substructures as the basis of the normalization leading to the successful extension of phases to very low resolution.

New trends in macromolecular X-ray crystallography.

Advances in experimental and computational techniques have reaffirmed the role of protein X-ray crystallography as one of the primary providers of structural information both to enhance our fundamental understanding of biological systems and also to assist the design and optimization of important therapeutics. Today, the most important challenge facing macromolecular X-ray crystallography is the need to grow suitable crystals of a given protein. Once this has been accomplished, most often the question is not whether the structure will be solved but rather how fast this will be done. A dramatic example of this is the crystal structure of cytochrome c oxidase. The search for crystallization conditions took about 15 years and then the structure was solved in about one year.

trp Repressor, A Crystallographic Study of Allostery in Genetic Regulation

The crystal structure of the E. coli trp repressor has been solved (1) and refined to 2.2 A. The two subunits (107 residues each) are related by an exact crystallographic dyad. Each subunit is composed of six helices, five of which intertwine about each other in a way that may make it seemingly impossible to disengage the subunits without altering their tertiary structure. The two symmetrically related L-tryptophan binding sites are formed by this interface.

A Crystallographic Analysis of Yeast Initiator tRNA

The initiation step in the complex process of protein synthesis always begins with the incorporation of a methionine residue at the amino terminal of the growing protein chain (for review, see Grunberg-Manago and Gros 1977). A specific methionine-accepting initiator tRNA that is excluded from the elongation steps on the ribosome is the unique adaptor for the “start” signals in mRNA. Together with initiation factors and GTP, the methionylated initiator tRNA is aligned in the correct frame on the ribosome so that the first peptide bond can be formed with the first aminoacylated elongator tRNA on the A site of the ribosome. Our understanding of the molecular structure-function relationships in initiator tRNA comes from an evaluation of a diversity of experimental results. There are the characteristic sequence patterns that have emerged from a careful examination of a great many primary structures of both initiator and elongator tRNAs (Barrell and Clark 1974; Sprinzl et al. 1978). Added to this are the variety of biochemical and biophysical studies of tRNA that have described the dynamics of molecular response to environmental perturbants (for review, see Sigler 1975; Kim 1976; Rich and RajBhandary 1976). Another very important constraint on the structure-function relationships in initiator tRNA is the detailed high-resolution structure of the elongator tRNA, yeast tRNA Phe (Quigley et al. 1975; Jack et al. 1976; Stout et al. 1976; Sussman and Kim 1976; Holbrook et al. 1978), which is known to bear many structural similarities and even functional similarities outside the ribosome. Reported here is a...