Microbial sensory rhodopsins are a family of membrane-embedded photoreceptors in prokaryotic and eukaryotic organisms. Structures of archaeal rhodopsins, which function as light-driven ion pumps or photosensors, have been reported. We present the structure of a eubacterial rhodopsin, which differs from those of previously characterized archaeal rhodopsins in its chromophore and cytoplasmic-side portions. Anabaena sensory rhodopsin exhibits light-induced interconversion between stable 13-cis and all-trans states of the retinylidene protein. The ratio of its cis and trans chromophore forms depends on the wavelength of illumination, thus providing a mechanism for a single protein to signal the color of light, for example, to regulate color-sensitive processes such as chromatic adaptation in photosynthesis. Its cytoplasmic half channel, highly hydrophobic in the archaeal rhodopsins, contains numerous hydrophilic residues networked by water molecules, providing a connection from the photoactive site to the cytoplasmic surface believed to interact with the receptor's soluble 14-kilodalton transducer.Over the past 4 years, microbial genomics has revealed a large family of photoactive, seventransmembrane-helix retinylidene proteins called microbial rhodopsins in phylogenetically diverse species, including haloarchaea, proteobacteria, cyanobacteria, fungi, and algae (1-4). *To whom correspondence should be addressed. hudel@uci.edu (H.L.) or john.l.spudich@uth.tmc.edu (J.L.S. Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThe first members of this family were discovered in halophilic archaea: the light-driven ion pumps bacteriorhodopsin and halorhodopsin and the phototaxis receptors sensory rhodopsins I and II. These four related haloarchaeal pigments are among the bestcharacterized membrane proteins in terms of structure and function, and nearly all of our knowledge of the properties of microbial rhodopsins, such as isomeric configuration and conformation of their chromophore, photochemical reactions, light-induced conformational changes in the protein, and function, derives from the study of these four, including atomic resolution structures that have been obtained for three of them (5-9). Studies of nonhaloarchaeal rhodopsins, of which >800 are known to exist (10, 11), are needed to examine the diversity of properties of this widespread family (12). Anabaena sensory rhodopsin, a recently discovered sensory representative outside of archaea (2), is well suited for exploration. It is the only bacterial sensory rhodopsin so far expressed in a photoactive form. Unlike the haloarchaeal sensory rhodopsins, which transmit signals to other integral membrane proteins, its function appears to involve modulation of a soluble cytoplasmic transducer, analogous to animal visual pigments (2).In this study, we report the structure of the retinal-complexed protein at 2.0 Å resolution, obtained by X-ray diffraction of crystals grown in a cubic lipid phase (table S1). The overall membrane-embedded seven-he...

Diacylglycerol kinase (DgkA) catalyzes the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid for use in shuttling water-soluble components to membrane derived oligosaccharide and lipopolysaccharide in the cell envelope of Gram-negative bacteria1. For half a century, this 121-residue kinase has served as a paradigm for investigating membrane protein enzymology1,3-7, folding8,9, assembly10-13, and stability1,14. Here, we present crystal structures for three functional forms of this unique and paradigmatic kinase, one of which is wild type (WT). These reveal a homo-trimeric enzyme with three transmembrane helices and an N-terminal amphiphilic helix per monomer. Bound lipid substrate and docked ATP identify the putative active site which is of the composite, shared site type. The crystal structures rationalize extensive biochemical and biophysical data on the enzyme. They are however at variance with a published solution NMR model2 in that domain swapping, a key feature of the solution form, is not observed in the crystal structures.

With functions that range from cell envelope structure to signal transduction and transport, lipoproteins constitute 2 to 3% of bacterial genomes and play critical roles in bacterial physiology, pathogenicity, and antibiotic resistance. Lipoproteins are synthesized with a signal peptide securing them to the cytoplasmic membrane with the lipoprotein domain in the periplasm or outside the cell. Posttranslational processing requires a signal peptidase II (LspA) that removes the signal peptide. Here, we report the crystal structure of LspA from Pseudomonas aeruginosa complexed with the antimicrobial globomycin at 2.8 angstrom resolution. Mutagenesis studies identify LspA as an aspartyl peptidase. In an example of molecular mimicry, globomycin appears to inhibit by acting as a noncleavable peptide that sterically blocks the active site. This structure should inform rational antibiotic drug discovery.

Aurodox is a member of the family of kirromycin antibiotics, which inhibit protein biosynthesis by binding to elongation factor Tu (EF-Tu). We have determined the crystal structure of the 1:1:1 complex of Thermus thermophilus EF-Tu with GDP and aurodox to 2.0-Å resolution. During its catalytic cycle, EF-Tu adopts two strikingly different conformations depending on the nucleotide bound: the GDP form and the GTP form. In the present structure, a GTP complex-like conformation of EF-Tu is observed, although GDP is bound to the nucleotide-binding site. This is consistent with previous proposals that aurodox fixes EF-Tu on the ribosome by locking it in its GTP form. Binding of EF-Tu⅐GDP to aminoacyl-tRNA and mutually exclusive binding of kirromycin and elongation factor Ts to EF-Tu can be explained on the basis of the structure. For many previously observed mutations that provide resistance to kirromycin, it can now be understood how they prevent interaction with the antibiotic. An unexpected feature of the structure is the reorientation of the His-85 side chain toward the nucleotide-binding site. We propose that this residue stabilizes the transition state of GTP hydrolysis, explaining the acceleration of the reaction by kirromycin-type antibiotics.

SignificanceThe uptake of nutrients from the environment is an essential process that is achieved in most cells through the use of secondary active transporters. The POT family of proton-coupled peptide transporters are one of the most diverse nutrient uptake systems, recognizing amino acids, peptides, nitrate, and seed-defense compounds. A long-standing question is how this family achieves such ligand diversity. A high-resolution crystal structure combined with multiscale molecular dynamics simulations demonstrate water molecules are able to shuttle protons using a Grotthuss-type mechanism, suggesting a separation of ligand recognition from proton movement. This would have clear advantages for a transporter family that must accommodate chemically diverse ligands while retaining the ability to couple transport to the proton electrochemical gradient.