The Tumbleweed: towards a synthetic proteinmotor.

Biomolecular motors have inspired the design and construction of artificial nanoscale motors and machines based on nucleic acids, small molecules, and inorganic nanostructures. However, the high degree of sophistication and efficiency of biomolecular motors, as well as their specific biological function, derives from the complexity afforded by protein building blocks. Here, we discuss a novel bottom-up approach to understanding biological motors by considering the construction of synthetic protein motors. Specifically, we present a design for a synthetic protein motor that moves along a linear track, dubbed the "Tumbleweed." This concept uses three discrete ligand-dependent DNA-binding domains to perform cyclically ligand-gated, rectified diffusion along a synthesized DNA molecule. Here we describe how de novo peptide design and molecular biology could be used to produce the Tumbleweed, and we explore the fundamental motor operation of such a design using numerical simulations. The construction of this and more sophisticated protein motors is an exciting challenge that is likely to enhance our understanding of the structure-function relationship in biological motors.

Nucleophosmin is cleaved and inactivated by the cytotoxic granule protease granzyme M during natural killer cell-mediated killing.

Natural killer (NK) cells kill virus-infected or transformed target cells by delivering cytotoxic proteases called granzymes to the target cell cytosol. One of these proteases, granzyme M, is specifically expressed in NK cells and is thought to instigate a form of cell death distinct from that mediated by granzyme A or granzyme B. However, the mechanism of granzyme M-induced cell death is unclear at present, and few substrates for this granzyme have been reported to date. Here we show that the abundant nucleolar phosphoprotein, nucleophosmin (NPM), is cleaved and inactivated by granzyme M. NPM is essential for cell viability as RNA interference-mediated ablation of NPM expression in human cells resulted in spontaneous apoptosis. Significantly, overexpression of wild-type NPM rescued cells treated with NPM small interference RNA, whereas overexpression of the granzyme M-cleaved form of NPM did not. Because NPM is essential for cell viability, these data suggest that targeting of NPM by granzyme M may contribute to tumor cell eradication by abolishing NPM function.

Yersinia pseudotuberculosis superantigens.

Yersinia pseudotuberculosis, a gastro-intestinal bacterium, produces three closely related T cell superantigens, YPMa, YPMb and YPMc, which have no significant sequence similarity to other proteins, let alone other bacterial superantigens. Y. pseudotuberculosisderived mitogen (YPM) has been shown to play a role in the pathogenesis of human and animal Y. pseudotuberculosis infection. The three-dimensional structure of YPMa, as determined by X-ray crystallography and nuclear magnetic resonance spectroscopy, exhibits a jelly roll fold, a structural motif not observed in other superantigens. YPMa is structurally most similar to virus capsid proteins and members of the tumour necrosis factor (TNF) superfamily. In the crystal structure, YPMa forms a trimer, another feature shared with virus capsid proteins and TNF superfamily proteins. However, in solution YPMa exists as a monomer, and any functional relevance of the trimer observed in the crystals is yet to be established. Structures of YPM bound to the T cell receptor and/or the major histocompatibility complex (MHC) are not yet available and mapping of existing mutagenesis data onto the three-dimensional structure of YPMa did not reveal potential T cell receptor/MHC binding sites. Knowledge of the structure will aid the design of functional studies aimed at further characterizing this superantigen.

Crystal and solution structures of a superantigen from Yersinia pseudotuberculosis reveal a jelly-roll fold.

Superantigens are a class of microbial proteins with the ability to excessively activate T cells by binding to the T cell receptor. The staphylococcal and streptococcal superantigens are closely related in structure and possess an N-terminal domain that resembles an OB fold and a C-terminal domain similar to a beta-grasp fold. Yersinia pseudotuberculosis produces superantigens, YPMa, YPMb, and YPMc, which have no significant amino acid similarity to other proteins. We have determined the crystal and solution structures of YPMa, which show that the protein has a jelly-roll fold. The closest structural neighbors to YPMa are viral capsid proteins and members of the tumor necrosis factor superfamily. In the crystal structure, YPMa packs as a trimer, another feature shared with viral capsid proteins and TNF superfamily proteins. However, in solution YPMa behaves as a monomer, and any functional relevance of the trimer observed in the crystals is yet to be established.

Crystallization and preliminary X-ray analysis of Yersinia pseudotuberculosis-derived mitogen.

Yersinia pseudotuberculosis-derived mitogen (YPM), a superantigen with no amino-acid sequence similarity to other known superantigens, has been crystallized by the sitting-drop vapour-diffusion method. The crystals belong to space group C2, with unit-cell parameters a = 138.67, b = 78.66, c = 32.91 A, beta = 91.97 degrees. A native data set has been collected to a resolution of 1.8 A using synchrotron radiation. Self-rotation function calculations suggest the presence of three molecules in the asymmetric unit, corresponding to a solvent content of 45%.