Eckhard Hofmann

Structural Basis of Light Harvesting by Carotenoids: Peridinin-Chlorophyll-Protein from Amphidinium carterae

Peridinin-chlorophyll-protein, a water-soluble light-harvesting complex that has a blue-green absorbing carotenoid as its main pigment, is present in most photosynthetic dinoflagellates. Its high-resolution (2.0 angstrom) x-ray structure reveals a noncrystallographic trimer in which each polypeptide contains an unusual jellyroll fold of the α-helical amino- and carboxyl-terminal domains. These domains constitute a scaffold with pseudo-twofold symmetry surrounding a hydrophobic cavity filled by two lipid, eight peridinin, and two chlorophyll a molecules. The structural basis for efficient excitonic energy transfer from peridinin to chlorophyll is found in the clustering of peridinins around the chlorophylls at van der Waals distances.

A conserved structural motif for lipopolysaccharide recognition by procaryotic and eucaryotic proteins

BACKGROUND Lipopolysaccharide (LPS), a lipoglycan from the outer membrane of Gram-negative bacteria, is an immunomodulatory molecule that stimulates the innate immune response. High levels of LPS cause excessive release of inflammatory mediators and are responsible for the septic shock syndrome. The interaction of LPS with its cognate binding proteins has not, as yet, been structurally elucidated. RESULTS The X-ray crystallographic structure of LPS in complex with the integral outer membrane protein FhuA from Escherichia coli K-12 is reported. It is in accord with data obtained using mass spectroscopy and nuclear magnetic resonance. Most of the important hydrogen-bonding or electrostatic interactions with LPS are provided by eight positively charged residues of FhuA. Residues in a similar three-dimensional arrangement were searched for in all structurally known proteins using a fast template-matching algorithm, and a subset of four residues was identified that is common to known LPS-binding proteins. CONCLUSIONS These four residues, three of which form specific interactions with lipid A, appear to provide the structural basis of pattern recognition in the innate immune response. Their arrangement can serve to identify LPS-binding sites on proteins known to interact with LPS, and could serve as a template for molecular modeling of a LPS scavenger designed to reduce the septic shock syndrome.

The structure of the periplasmic ligand-binding domain of the sensor kinase CitA reveals the first extracellular PAS domain

The integral membrane sensor kinase CitA of Klebsiella pneumoniae is part of a two-component signal transduction system that regulates the transport and metabolism of citrate in response to its environmental concentration. Two-component systems are widely used by bacteria for such adaptive processes, but the stereochemistry of periplasmic ligand binding and the mechanism of signal transduction across the membrane remain poorly understood. The crystal structure of the CitAP periplasmic sensor domain in complex with citrate reveals a PAS fold, a versatile ligand-binding structural motif that has not previously been observed outside the cytoplasm or implicated in the transduction of conformational signals across the membrane. Citrate is bound in a pocket that is shared among many PAS domains but that shows structural variation according to the nature of the bound ligand. In CitAP, some of the citrate contact residues are located in the final strand of the central β-sheet, which is connected to the C-terminal transmembrane helix. These secondary structure elements thus provide a potential conformational link between the periplasmic ligand binding site and the cytoplasmic signaling domains of the receptor.

The ABC maltose transporter

Bacterial ATP‐binding cassette (ABC) transporters and their homologues in eukaryotic cells form one of the largest superfamilies known today. They function as primary pumps that couple substrate translocation across the cytoplasmic membrane to ATP hydrolysis. Although ABC transporters have been studied for more than three decades, the structure of these multicomponent systems is unknown, and the mechanism of transport is not understood. This article reviews one of the most widely studied ABC systems, the maltose transporter of Escherichia coli. A first structural model of the transport channel allows discussion of possible mechanisms of transport. In addition, recent experimental evidence suggests that regulation of gene expression and transport activity is far more complex than expected.

Förster Excitation Energy Transfer in Peridinin-Chlorophyll-a-Protein

Time-resolved fluorescence anisotropy spectroscopy has been used to study the chlorophyll a (Chl a) to Chl a excitation energy transfer in the water-soluble peridinin-chlorophyll a-protein (PCP) of the dinoflagellate Amphidinium carterae. Monomeric PCP binds eight peridinins and two Chl a. The trimeric structure of PCP, resolved at 2 A (, Science. 272:1788-1791), allows accurate calculations of energy transfer times by use of the Förster equation. The anisotropy decay time constants of 6.8 +/- 0.8 ps (tau(1)) and 350 +/- 15 ps (tau(2)) are respectively assigned to intra- and intermonomeric excitation equilibration times. Using the ratio tau(1)/tau(2) and the amplitude of the anisotropy, the best fit of the experimental data is achieved when the Q(y) transition dipole moment is rotated by 2-7 degrees with respect to the y axis in the plane of the Chl a molecule. In contrast to the conclusion of, Biochemistry. 23:1564-1571) that the refractive index (n) in the Förster equation should be equal to that of the solvent, n can be estimated to be 1.6 +/- 0.1, which is larger than that of the solvent (water). Based on our observations we predict that the relatively slow intermonomeric energy transfer in vivo is overruled by faster energy transfer from a PCP monomer to, e.g., the light-harvesting a/c complex.

The Crystal Structure of Arabidopsis thaliana Allene Oxide Cyclase: Insights into the Oxylipin Cyclization Reaction

We describe the crystallization and structure elucidation of Arabidopsis thaliana allene oxide cyclase 2 (AOC2), a key enzyme in the biosynthesis of jasmonates. In a coupled reaction with allene oxide synthase, AOC2 releases the first cyclic and biologically active metabolite, 12-oxo-phytodienoic acid (OPDA). AOC2 (AT3G25770) folds into an eight-stranded antiparallel β-barrel with a C-terminal partial helical extension. The protein forms a hydrophobic binding cavity with two distinct polar patches. AOC2 is trimeric in crystals, in vitro and in planta. Based on the observed folding pattern, we assigned AOC2 as a low molecular weight member of the lipocalin family with enzymatic activity in plants. We determined the binding position of the competitive inhibitor vernolic acid (a substrate analog) in the binding pocket. Based on models for bound substrate 12,13-epoxy-9,11,15-octadecatrienoic acid and product OPDA, we propose a reaction scheme that explains the influence of the C15 double bond on reactivity. Reaction is promoted by anchimeric assistance through a conserved Glu residue. The transition state with a pentadienyl carbocation and an oxyanion is stabilized by a strongly bound water molecule and favorable π–π interactions with aromatic residues in the cavity. Stereoselectivity results from steric restrictions to the necessary substrate isomerizations imposed by the protein.

Identification of a single peridinin sensing Chl-a excitation in reconstituted PCP by crystallography and spectroscopy

The peridinin-chlorophyll a-protein (PCP) of dinoflagellates is unique among the large variety of natural photosynthetic light-harvesting systems. In contrast to other chlorophyll protein complexes, the soluble PCP is located in the thylakoid lumen, and the carotenoid pigments outnumber the chlorophylls. The structure of the PCP complex consists of two symmetric domains, each with a central chlorophyll a (Chl-a) surrounded by four peridinin molecules. The protein provides distinctive surroundings for the pigment molecules, and in PCP, the specific environment around each peridinin results in overlapping spectral line shapes, suggestive of different functions within the protein. One particular Per, Per-614, is hypothesized to show the strongest electronic interaction with the central Chl-a. We have performed an in vitro reconstitution of pigments into recombinant PCP apo-protein (RFPCP) and into a mutated protein with an altered environment near Per-614. Steady-state and transient optical spectroscopic experiments comparing the RFPCP complex with the reconstituted mutant protein identify specific amino acid-induced spectral shifts. The spectroscopic assignments are reinforced by a determination of the structures of both RFPCP and the mutant by x-ray crystallography to a resolution better than 1.5 Å. RFPCP and mutated RFPCP are unique in representing crystal structures of in vitro reconstituted light-harvesting pigment-protein complexes.

Directional Proton Transfer in Membrane Proteins Achieved through Protonated Protein-Bound Water Molecules: A Proton Diode†

The key function of energy-transducing membrane proteins is the creation of a proton gradient by directional proton transfer. The role of protein-bound water molecules herein is not fully understood, as X-ray diffraction analysis has resolved the positions of oxygen, but not of hydrogen atoms in such protein–water complexes. Here we show, now timeresolved at atomic resolution, how a membrane protein achieves directional proton transfer via protein-bound water molecules in contrast to random proton transfer in liquid water. A combination of X-ray structure analysis, timeresolved FTIR spectroscopy, and molecular dynamics (MD) simulations elucidates how directionality is achieved. Using the proton-pump bacteriorhodopsin as the paradigm, we show how controlled conformational changes of few amino acid residues rearrange preordered water molecules and induce directional proton transfer. This mechanism is analogous to an electronic diode: a “proton diode”. According to the chemiosmotic theory, the creation of a proton gradient in photosynthesis and oxidative phosphorylation by means of directional proton transfer is the key step for energy transduction in living cells. ATPases use this proton gradient to produce ATP, the fuel for life. In contrast to this directional mechanism in proteins, proton transfer in liquid water is random. Bacteriorhodopsin (bR), a protein that belongs to the microbial rhodopsin family, achieves this directional proton transfer by a light-driven protonpumping mechanism. Like other microbial rhodopsins, bR exhibits a structural motif of seven transmembrane a-helices and a retinal chromophore covalently bound to a lysine through a protonated Schiff base. The light-induced retinal isomerization from all-trans in the ground state (BR) to the 13-cis conformer drives bR through a photocycle with intermediates named J, K, L, M, N, and O in order of their appearance. During the L to M transition, the protonated Schiff base (C=NH), the central proton-binding site, deprotonates and protonates its counterion Asp85 (step 1 in Figure 1a). Protonation of Asp85 breaks its salt bridge to Arg82, which then moves towards Glu194/Glu204 (step 2). The orientation of Arg82 depends on the protonation state of Asp85. The arginine movement destabilizes a protonated water cluster between Arg82, Glu194, and Glu204 (step 3 in Figure 1a), and a proton is released to the bulk. However, the detailed nature of the proton-release group is still under debate. QM/MM simulations of the protonrelease group propose a shared proton between Glu194 and Glu204, a Zundel cation with two water molecules (H5O2 ), or an asymmetric Eigen cation of four water molecules (H9O4 ). From time-resolved FTIR experiments with site-directed mutations around the protonated water cluster and H/D-exchange experiments we have concluded that the proton-release group forms a protonated water cluster, most likely an asymmetric Eigen ion as shown in Figure 1a in purple. Glu194 and Glu204 are clearly deprotonated in the bR ground state. This experimental result was recently confirmed by L renz-Fonfr a et al. Nevertheless, the exact nature of the protonated water cluster and the release mechanism has still to be determined. Here, we used X-ray structure analysis to determine the positions of the water oxygen atoms and FTIR difference spectroscopy to determine the dynamics of the corresponding water hydrogen atoms. The proton release to the bulk in the L [*] Dr. S. Wolf, E. Freier, Dr. M. Potschies, Prof. Dr. E. Hofmann, Prof. Dr. K. Gerwert Lehrstuhl f r Biophysik, Ruhr-University Bochum Universit tsstrasse 150, 44780 Bochum (Germany) Fax: (+49)234-321-4238 E-mail: gerwert@bph.rub.de Homepage: http://www.bph.rub.de Dr. S. Wolf, Prof. Dr. K. Gerwert Department of Biophysics CAS–Max-Planck Partner Institute for Computational Biology Shanghai Institutes for Biological Sciences 320 Yue Yang Road, 200031 Shanghai (P.R. China) [] Current address: Lehrstuhl f r Biophysik, Universit t Konstanz (Germany) [] These authors contributed equally to this work.

Phycoerythrobilin Synthase (Pebs) of a Marine Virus: Crystal Structures of the Biliverdin Complex and the Substrate-Free Form.

The reddish purple open chain tetrapyrrole pigment phycoerythrobilin (PEB; Aλmax ∼ 550 nm) is an essential chromophore of the light-harvesting phycobiliproteins of most cyanobacteria, red algae, and cryptomonads. The enzyme phycoerythrobilin synthase (PebS), recently discovered in a marine virus infecting oceanic cyanobacteria of the genus Prochlorococcus (cyanophage PSSM-2), is a new member of the ferredoxin-dependent bilin reductase (FDBR) family. In a formal four-electron reduction, the substrate biliverdin IXα is reduced to yield 3Z-PEB, a reaction that commonly requires the action of two individual FDBRs. The first reaction catalyzed by PebS is the reduction of the 15,16-methine bridge of the biliverdin IXα tetrapyrrole system. This reaction is exclusive to PEB biosynthetic enzymes. The second reduction site is the A-ring 2,3,31,32-diene system, the most common target of FDBRs. Here, we present the first crystal structures of a PEB biosynthetic enzyme. Structures of the substrate complex were solved at 1.8- and 2.1-Å resolution and of the substrate-free form at 1.55-Å resolution. The overall folding revealed an α/β/α-sandwich with similarity to the structure of phycocyanobilin:ferredoxin oxidoreductase (PcyA). The substrate-binding site is located between the central β-sheet and C-terminal α-helices. Eight refined molecules with bound substrate, from two different crystal forms, revealed a high flexibility of the substrate-binding pocket. The substrate was found to be either in a planar porphyrin-like conformation or in a helical conformation and is coordinated by a conserved aspartate/asparagine pair from the β-sheet side. From the α-helix side, a conserved highly flexible aspartate/proline pair is involved in substrate binding and presumably catalysis.

Peridinin-chlorophyll-protein reconstituted with chlorophyll mixtures: preparation, bulk and single molecule spectroscopy.

Reconstitution of the 16 kDa N‐terminal domain of the peridinin–chlorophyll–protein, N‐PCP, with mixtures of chlorophyll a (Chl a) and Chl b, resulted in 32 kDa complexes containing two pigment clusters, each bound to one N‐PCP. Besides homo‐chlorophyllous complexes, hetero‐chlorophyllous ones were obtained that contain Chl a in one pigment cluster, and Chl b in the other. Binding of Chl b is stronger than that of the native pigment, Chl a. Energy transfer from Chl b to Chl a is efficient, but there are only weak interactions between the two pigments. Individual homo‐ and hetero‐chlorophyllous complexes were investigated by single molecule spectroscopy using excitation into the peridinin absorption band and scanning of the Chl fluorescence, the latter show frequently well resolved emissions of the two pigments.