Ho S. Cho

Crystal structure of an activated response regulator bound to its target

The chemotactic regulator CheY controls the direction of flagellar rotation in Escherichia coli. We have determined the crystal structure of BeF3−-activated CheY from E. coli in complex with an N-terminal peptide derived from its target, FliM. The structure reveals that the first seven residues of the peptide pack against the β4-H4 loop and helix H4 of CheY in an extended conformation, whereas residues 8–15 form two turns of helix and pack against the H4-β5-H5 face. The peptide binds the only region of CheY that undergoes noticeable conformational change upon activation and would most likely be sandwiched between activated CheY and the remainder of FliM to reverse the direction of flagellar rotation.

A glimpse of a possible amyloidogenic intermediate of transthyretin.

Studies have indicated that partially unfolded states occur under conditions that favor amyloid formation by transthyretin (TTR), as well as other amyloidogenic proteins. In this study, we used hydrogen exchange measurements to show that there is selective destabilization of one half of the β-sandwich structure of TTR under such conditions. This provides more direct information about conformational fluctuations than previously available, and will facilitate design of future experiments to probe the intermediates critical to amyloid formation.

Phosphoaspartates in bacterial signal transduction

Bacteria use a strategy referred to as two-component signal transduction to sense a variety of stimuli and initiate an appropriate response. Signal processing begins with proteins referred to as histidine kinases. In most cases, these are membrane-bound receptors that respond to environmental cues. Histidine kinases use ATP as a phosphodonor to phosphorylate a conserved histidine residue. Subsequent transfer of the phosphoryl group to a conserved aspartyl residue in the cognate response regulator results in an appropriate output. Recent structural studies of activated (phosphorylated) response regulators and their aspartate-bearing regulatory domains have provided insight into the links between the chemistry and biology of these ubiquitous regulatory proteins. Chemical aspects of their function appear to generalize broadly to enzymes that adopt a phosphoaspartate intermediate.

The NMR Solution Structure of BeF3−-Activated Spo0F Reveals the Conformational Switch in a Phosphorelay System

Two-component systems, which are comprised of a single histidine-aspartate phosphotransfer module, are the dominant signaling pathways in bacteria and have recently been identified in several eukaryotic organisms as well. A tandem connection of two or more histidine-aspartate motifs forms complex phosphorelays. While response regulators from simple two-component systems have been characterized structurally in their inactive and active forms, we address here the question of whether a response regulator from a phosphorelay has a distinct structural basis of activation. We report the NMR solution structure of BeF(3)(-)-activated Spo0F, the first structure of a response regulator from a phosphorelay in its activated state. Conformational changes were found in regions previously identified to change in simple two-component systems. In addition, a downward shift by half a helical turn in helix 1, located on the opposite side of the common activation surface, was observed as a consequence of BeF(3)(-) activation. Conformational changes in helix 1 can be rationalized by the distinct function of phosphoryl transfer to the second histidine kinase, Spo0B, because helix 1 is known to interact directly with Spo0B and the phosphatase RapB. The identification of structural rearrangements in Spo0F supports the hypothesis of a pre-existing equilibrium between the inactive and active state prior to phosphorylation that was suggested on the basis of previous NMR dynamics studies on Spo0F. A shift of a pre-existing equilibrium is likely a general feature of response regulators.

Structural characterization of the complex of the Rev response element RNA with a selected peptide.

INTRODUCTION The RSG-1.2 peptide was selected for specific binding to the Rev response element RNA, as the natural Rev peptide does. The RSG-1.2 sequence has features incompatible with the helical structure of the bound Rev peptide, indicating that it must bind in a different conformation. RESULTS The binding of the RSG-1.2 peptide to the Rev response element RNA was characterized using multinuclear, multidimensional NMR. The RSG-1.2 peptide is shown to bind with the N-terminal segment of the peptide along the major groove in an extended conformation and turn preceding a C-terminal helical segment, which crosses the RNA groove in the region widened by the presence of purine-purine base pairs. These features make the details of the bound state rather different than that of the Rev peptide which targets the same RNA sequence binding as a single helix along the groove axis. CONCLUSIONS These studies further demonstrate the versatility of arginine-rich peptides in recognition of specific RNA elements and the lack of conserved structural features in the bound state.

Solution nuclear magnetic resonance structure of a protein disulfide oxidoreductase from Methanococcus jannaschii

The solution structure of the protein disulfide oxidoreductase Mj0307 in the reduced form has been solved by nuclear magnetic resonance. The secondary and tertiary structure of this protein from the archaebacterium Methanococcus jannaschii is similar to the structures that have been solved for the glutaredoxin proteins from Escherichia coli, although Mj0307 also shows features that are characteristic of thioredoxin proteins. Some aspects of Mj0307s unique behavior can be explained by comparing structure‐based sequence alignments with mesophilic bacterial and eukaryotic glutaredoxin and thioredoxin proteins. It is proposed that Mj0307, and similar archaebacterial proteins, may be most closely related to the mesophilic bacterial NrdH proteins. Together these proteins may form a unique subgroup within the family of protein disulfide oxidoreductases.

Isotope Labeling of Proteins for NMR Spectroscopy Using Cell-Free Methods

NMR spectroscopy is uniquely capable of providing information on the structure, function, and dynamics of proteins and other biomolecules in solution. Typically, milligram quantities of proteins are required, although recent technological improvements, including cryoprobes (see, for example, Serber et al. 2000) and high-field magnets (900 MHz), have improved the sensitivity of the method considerably. Recent experimental advances have also increased the molecular weight range assessable for study. With advanced techniques it is now possible to obtain limited information on complexes as large as 800 kDa (Riek et al. 2002). Isotopic labeling of the protein with 15N, 13C, 2H, or a combination of these isotopes is often required. In vitro protein synthesis is well suited to producing these types of samples. Herein we discuss the advantages of using in vitro methods to produce isotopically labeled proteins for NMR studies and show examples of the specialized information obtainable from such experiments.

In Vitro Protein Production for Structure Determination with the RTS System

The goal of the Berkeley Structural Genomics Center is to determine the structures of all proteins encoded in the genomes of Mycoplasma pneumoniae and Mycoplasma genatalium or structural homologs from other organisms. To achieve this goal, we are working to develop high-throughput methods for protein expression for use in X-ray and NMR structure determination. In collaboration with Roche Molecular Biochemicals (Penzburg, Germany), we are investigating the usefulness of the Rapid Translation System (RTS) of in vitro protein production to generate target proteins in quantities suitable for structure determination. Very encouraging results have been obtained with the test protein phosphoserine phosphatase (PSP) from Methanococcus jannaschii (Mj).