Tilman Schirmer

Structural basis for sugar translocation through maltoporin channels at 3.1 A resolution

Trimeric maltoporin (LamB protein) facilitates the diffusion of maltodextrins across the outer membrane of Gram-negative bacteria. The crystal structure of maltoporin from Escherichia coli, determined to a resolution of 3.1 angstroms, reveals an 18-stranded, antiparallel beta-barrel that forms the framework of the channel. Three inwardly folded loops contribute to a constriction about halfway through the channel. Six contingent aromatic residues line the channel and form a path from the vestibule to the periplasmic outlet. Soaking of a crystal with maltotriose revealed binding of the sugar to this hydrophobic track across the constriction, which suggests that maltose and linear oligosaccharides may be translocated across the membrane by guided diffusion along this path.

Structural and mechanistic determinants of c-di-GMP signalling

Bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is a ubiquitous second messenger that regulates cell surface-associated traits in bacteria. Components of this regulatory network include GGDEF and EAL domain-containing proteins that determine the cellular concentrations of c-di-GMP by mediating its synthesis and degradation, respectively. Crystal structure analyses in combination with functional studies have revealed the catalytic mechanisms and regulatory principles involved. Downstream, c-di-GMP is recognized by PilZ domain-containing receptors that can undergo large-scale domain rearrangements on ligand binding. Here, we review recent data on the structure and functional properties of the protein families that are involved in c-di-GMP signalling and discuss the mechanistic implications.

X-ray crystallographic structure of the light-harvesting biliprotein C-phycocyanin from the thermophilic cyanobacterium Mastigocladus laminosus and its resemblance to globin structures

The structure of the biliprotein C-phycocyanin from the thermophilic cyanobacterium Mastigocladus laminosus has been determined at 3 A resolution by X-ray diffraction methods. Phases have been obtained by the multiple isomorphous replacement method. The electron density map could be improved by solvent flattening and has been interpreted in terms of the amino acid sequence. The protein consists of three identical (alpha-beta)-units which are arranged around a threefold symmetry axis to form a disc of approximate dimensions 110 A X 30 A with a central channel of 35 A in diameter. This aggregation form is supposed to be the same as that found in the rods of native phycobilisomes. Both subunits, alpha and beta, exhibit a similar structure and are related by a local twofold rotational axis. Each subunit is folded into eight helices and irregular loops. Six helices are arranged to form a globular part, whereas two helices stick out and mediate extensive contact between the subunits. The arrangement of the helices of the globular part resembles the globin fold: 59 equivalent C alpha-atoms have a root-mean-square deviation of 2 X 9 A. The chromophores attached to cystein 84 of the alpha- and beta-subunits are topologically equivalent to the haem. All three chromophores of C-phycocyanin, open-chain tetrapyrroles, are in an extended conformation. alpha 84 and beta 84 are attached to helix E (globin nomenclature), beta 155 is linked to the G--H loop. The shortest centre-to-centre distance between chromophores in trimer is 22 A.

Crystal structure of hyaluronidase, a major allergen of bee venom

BACKGROUND Hyaluronic acid (HA) is the most abundant glycosaminoglycan of vertebrate extracellular spaces and is specifically degraded by a beta-1,4 glycosidase. Bee venom hyaluronidase (Hya) shares 30% sequence identity with human hyaluronidases, which are involved in fertilization and the turnover of HA. On the basis of sequence similarity, mammalian enzymes and Hya are assigned to glycosidase family 56 for which no structure has been reported yet. RESULTS The crystal structure of recombinant (Baculovirus) Hya was determined at 1.6 A resolution. The overall topology resembles a classical (beta/alpha)(8) TIM barrel except that the barrel is composed of only seven strands. A long substrate binding groove extends across the C-terminal end of the barrel. Cocrystallization with a substrate analog revealed the presence of a HA tetramer bound to subsites -4 to -1 and distortion of the -1 sugar. CONCLUSIONS The structure of the complex strongly suggest an acid-base catalytic mechanism, in which Glu113 acts as the proton donor and the N-acetyl group of the substrate is the nucleophile. The location of the catalytic residues shows striking similarity to bacterial chitinase which also operates via a substrate-assisted mechanism.

Structural and Functional Characterization of OmpF Porin Mutants Selected for Larger Pore Size II. FUNCTIONAL CHARACTERIZATION

The effects on the channel characteristics of four single amino acid substitutions in OmpF porin and of a deletion mutant in the constriction loop L3 have been studied. These mutations are all located in the narrow section of the channel of the protein that forms pores across the outer membrane of Escherichia coli. The single channel conductance of the deletion mutant (Δ109-114) is decreased by one third, whereas the point mutations do not exhibit significant deviations from that of the wild-type protein. The mutants exhibit drastic changes in ion selectivities. In the wild-type protein, the critical threshold potential (Vc), above which channels close reversibly, exhibits a strong pH dependence, with a titration point of ∼ pH 7.7, which is abolished in all mutants studied here. Diffusion of six monosaccharides is little affected in the point mutants, while four disaccharides are taken up at highly increased rates by the deletion mutant. The functional results, presented here, are correlated to the x-ray structures of the mutants (Lou, K.-L., Saint, N., Prilipov, A., Rummel, G., Benson, S.A., Rosenbusch, J.P., and Schirmer, T. (1996) J. Biol. Chem. 271, 20669-20675). In most, but not all, cases, the structural changes explain the functional alterations observed.

The structure of OmpF porin in a tetragonal crystal form.

BACKGROUND OmpF porin is a trimeric integral membrane protein responsible for the passive transport of small hydrophilic molecules, such as nutrients and waste products, across the outer membrane of Escherichia coli. Very few membrane proteins have been crystallized in three dimensions, yet this stable protein can be obtained in several crystal forms. Comparison of the structures of the same membrane protein in two different packing environments is of major interest, because it allows us to explore the integrity of the structure outside the natural membrane environment. RESULTS The structure of OmpF porin in a tetragonal crystal form with two trimers per asymmetric unit has been determined at 3.2 A resolution and compared with that obtained previously in a trigonal crystal form. The lattice contacts involve only polar atoms, whereas extensive hydrophobic protein-protein interactions were found in the trigonal lattice. The trimer structure is virtually identical in both. CONCLUSIONS Our comparison reveals that the overall structure of OmpF is not influenced by crystal lattice constraints and, thus, presumably bears close resemblance to the in vivo structure. The tetragonal crystal structure has provided the starting model for the phasing of neutron diffraction data obtained from this crystal form, as described in an accompanying article.

Second messenger signalling governs Escherichia coli biofilm induction upon ribosomal stress.

Biofilms are communities of surface‐attached, matrix‐embedded microbial cells that can resist antimicrobial chemotherapy and contribute to persistent infections. Using an Escherichia coli biofilm model we found that exposure of bacteria to subinhibitory concentrations of ribosome‐targeting antibiotics leads to strong biofilm induction. We present evidence that this effect is elicited by the ribosome in response to translational stress. Biofilm induction involves upregulation of the polysaccharide adhesin poly‐β‐1,6‐N‐acetyl‐glucosamine (poly‐GlcNAc) and two components of the poly‐GlcNAc biosynthesis machinery, PgaA and PgaD. Poly‐GlcNAc control depends on the bacterial signalling molecules guanosine‐bis 3′, 5′(diphosphate) (ppGpp) and bis‐(3′‐5′)‐cyclic di‐GMP (c‐di‐GMP). Treatment with translation inhibitors causes a ppGpp hydrolase (SpoT)‐mediated reduction of ppGpp levels, resulting in specific derepression of PgaA. Maximal induction of PgaD and poly‐GlcNAc synthesis requires the production of c‐di‐GMP by the dedicated diguanylate cyclase YdeH. Our results identify a novel regulatory mechanism that relies on ppGpp signalling to relay information about ribosomal performance to the Pga machinery, thereby inducing adhesin production and biofilm formation. Based on the important synergistic roles of ppGpp and c‐di‐GMP in this process, we suggest that interference with bacterial second messenger signalling might represent an effective means for biofilm control during chronic infections.

Crystal structure and functional characterization of OmpK36, the osmoporin of Klebsiella pneumoniae

BACKGROUND Porins are channel-forming membrane proteins that confer solute permeability to the outer membrane of Gram-negative bacteria. In Escherichia coli, major nonspecific porins are matrix porin (OmpF) and osmoporin (OmpC), which show high sequence homology. In response to high osmolarity of the medium, OmpC is expressed at the expense of OmpF porin. Here, we study osmoporin of the pathogenic Klebsiella pneumoniae (OmpK36), which shares 87% sequence identity with E. coliOmpC in an attempt to establish why osmoporin is best suited to function at high osmotic pressure. RESULTS The crystal structure of OmpK36 has been determined to a resolution of 3.2 A by molecular replacement with the model of OmpF. The structure of OmpK36 closely resembles that of the search model. The homotrimeric structure is composed of three hollow 16-stranded antiparallel beta barrels, each delimiting a separate pore. Most insertions and deletions with respect to OmpF are found in the loops that protrude towards the cell exterior. A characteristic ten-residue insertion in loop 4 contributes to the subunit interface. At the pore constriction, the replacement of an alanine by a tyrosine residue does not alter the pore profile of OmpK36 in comparison with OmpF because of the different course of the mainchain. Functionally, as characterized in lipid bilayers and liposomes, OmpK36 resembles OmpC with decreased conductance and increased cation selectivity in comparison with OmpF. CONCLUSIONS The osmoporin structure suggests that not an altered pore size but an increase in charge density is the basis for the distinct physico-chemical properties of this porin that are relevant for its preferential expression at high osmotic strength.

Regulatory cohesion of cell cycle and cell differentiation through interlinked phosphorylation and second messenger networks

In Caulobacter crescentus, phosphorylation of key regulators is coordinated with the second messenger cyclic di-GMP to drive cell-cycle progression and differentiation. The diguanylate cyclase PleD directs pole morphogenesis, while the c-di-GMP effector PopA initiates degradation of the replication inhibitor CtrA by the AAA+ protease ClpXP to license S phase entry. Here, we establish a direct link between PleD and PopA reliant on the phosphodiesterase PdeA and the diguanylate cyclase DgcB. PdeA antagonizes DgcB activity until the G1-S transition, when PdeA is degraded by the ClpXP protease. The unopposed DgcB activity, together with PleD activation, upshifts c-di-GMP to drive PopA-dependent CtrA degradation and S phase entry. PdeA degradation requires CpdR, a response regulator that delivers PdeA to the ClpXP protease in a phosphorylation-dependent manner. Thus, CpdR serves as a crucial link between phosphorylation pathways and c-di-GMP metabolism to mediate protein degradation events that irreversibly and coordinately drive bacterial cell-cycle progression and development.

Artificial Transfer Hydrogenases for the Enantioselective Reduction of Cyclic Imines

Man-made activity: Introduction of a biotinylated iridium piano stool complex within streptavidin affords an artificial imine reductase (see scheme). Saturation mutagenesis allowed optimization of the activity and the enantioselectivity of this metalloenzyme, and its X-ray structure suggests that a nearby lysine residue acts as a proton source during the transfer hydrogenation.