Targeting of proteins to bacterial microcompartments (BMCs) is mediated by an 18-amino-acid peptide sequence. Herein, we report the solution structure of the N-terminal targeting peptide (P18) of PduP, the aldehyde dehydrogenase associated with the 1,2-propanediol utilization metabolosome from Citrobacter freundii. The solution structure reveals the peptide to have a well-defined helical conformation along its whole length. Saturation transfer difference and transferred NOE NMR has highlighted the observed interaction surface on the peptide with its main interacting shell protein, PduK. By tagging both a pyruvate decarboxylase and an alcohol dehydrogenase with targeting peptides, it has been possible to direct these enzymes to empty BMCs in vivo and to generate an ethanol bioreactor. Not only are the purified, redesigned BMCs able to transform pyruvate into ethanol efficiently, but the strains containing the modified BMCs produce elevated levels of alcohol.

Data relating to the structural basis of ligand recognition by integrins are limited. Here we describe the physical requirements for high affinity binding of ligands to ␣v␤6. By combining a series of structural analyses with functional testing, we show that 20-mer peptide ligands, derived from high affinity ligands of ␣v␤6 (foot-and-mouth-disease virus, latency associated peptide), have a common structure comprising an Arg-Gly-Asp motif at the tip of a hairpin turn followed immediately by a C-terminal helix. This arrangement allows two conserved Leu/ Ile residues at Asp ؉1 and Asp ؉4 to be presented on the outside face of the helix enabling a potential hydrophobic interaction with the ␣v␤6 integrin, in addition to the Arg-Gly-Asp interaction. The extent of the helix determines peptide affinity for ␣v␤6 and potency as an ␣v␤6 antagonist. A major role of this C-terminal helix is likely to be the correct positioning of the Asp ؉1 and Asp ؉4 residues. These data suggest an explanation for several biological functions of ␣v␤6 and provide a structural platform for design of ␣v␤6 antagonists.Understanding of the molecular basis of the binding interface of integrins with their ligands still is relatively poor. The most detailed information available comes from x-ray crystallography of RGD 4 peptide binding to ␣v␤3, where the RGD motif bridged the ␣ and ␤ subunits, the arginine associating with the ␣v subunit and the aspartate coordinating with the bivalent metal ion on the ␤3 subunit (1). However, the RGD motif occurs in many extracellular matrix ligands so specificity is modified by other residues, often flanking the RGD site (2), although distant residues also can affect ligand binding (3). Detailed comparison of the different ligands of one integrin could illuminate the essential elements that determine specificity and affinity, improve biological understanding of integrinligand interactions, and allow rational design of targeting peptides and peptidomimetics (4 -6); we have made just such a comparison for ␣v␤6.The integrin ␣v␤6 is an epithelial specific integrin that is expressed at low or undetectable levels in adult tissues but can be up-regulated during tissue remodeling. Thus increased ␣v␤6 expression occurs during wound healing, development and inflammation (7), and in more severe pathologies, including chronic skin wounds (8) and cancer (9). The ability of ␣v␤6 to promote migration and invasion, in part through protease upregulation (10 -12), may explain why ␣v␤6 expression is an independent indicator of colon cancer aggressiveness (13). These data suggest that reagents designed to specifically antagonize ␣v␤6 could have clinical utility in colon cancer and possibly other diseases.␣v␤6 binds to the arginine-glycine-aspartate (RGD) motif in its ligands, which include fibronectin, tenascin, the latency associated peptides (LAP) of TGF␤1 (14) and TGF␤3 (15), and the VP1 coat protein of foot-and-mouth disease virus (FMDV) (16). In addition, the motif DLXXL was identified by phage display of 7-and 12-residue peptid...

Modified tetrapyrroles such as chlorophyll, heme, siroheme, vitamin B 12 , coenzyme F 430 , and heme d 1 underpin a wide range of essential biological functions in all domains of life, and it is therefore surprising that the syntheses of many of these life pigments remain poorly understood. It is known that the construction of the central molecular framework of modified tetrapyrroles is mediated via a common, core pathway. Herein a further branch of the modified tetrapyrrole biosynthesis pathway is described in denitrifying and sulfate-reducing bacteria as well as the Archaea. This process entails the hijacking of siroheme, the prosthetic group of sulfite and nitrite reductase, and its processing into heme and d 1 heme. The initial step in these transformations involves the decarboxylation of siroheme to give didecarboxysiroheme. For d 1 heme synthesis this intermediate has to undergo the replacement of two propionate side chains with oxygen functionalities and the introduction of a double bond into a further peripheral side chain. For heme synthesis didecarboxysiroheme is converted into Fe-coproporphyrin by oxidative loss of two acetic acid side chains. Fe-coproporphyrin is then transformed into heme by the oxidative decarboxylation of two propionate side chains. The mechanisms of these reactions are discussed and the evolutionary significance of another role for siroheme is examined.

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PDI (protein disulfide-isomerase) catalyses the formation of native disulfide bonds of secretory proteins in the endoplasmic reticulum. PDI consists of four thioredoxin-like domains, of which two contain redox-active catalytic sites (a and a'), and two do not (b and b'). The b' domain is primarily responsible for substrate binding, although the nature and specificity of the substrate-binding site is still poorly understood. In the present study, we show that the b' domain of human PDI is in conformational exchange, but that its structure is stabilized by the addition of peptide ligands or by binding the x-linker region. The location of the ligand-binding site in b' was mapped by NMR chemical shift perturbation and found to consist primarily of residues from the core beta-sheet and alpha-helices 1 and 3. This site is where the x-linker region binds in the X-ray structure of b'x and we show that peptide ligands can compete with x binding at this site. The finding that x binds in the principal ligand-binding site of b' further supports the hypothesis that x functions to gate access to this site and so modulates PDI activity.

Membrane scission is a crucial step in all budding processes, from endocytosis to viral budding. Many proteins have been associated with scission, though the underlying molecular details of how scission is accomplished often remain unknown. Here, we investigate the process of M2-mediated membrane scission during the budding of influenza viruses. Residues 50–61 of the viral M2 protein bind membrane and form an amphipathic α-helix (AH). Membrane binding requires hydrophobic interactions with the lipid tails but not charged interactions with the lipid headgroups. Upon binding, the M2AH induces membrane curvature and lipid ordering, constricting and destabilizing the membrane neck, causing scission. We further show that AHs in the cellular proteins Arf1 and Epsin1 behave in a similar manner. Together, they represent a class of membrane-induced AH domains that alter membrane curvature and fluidity, mediating the scission of constricted membrane necks in multiple biological pathways.

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