Neil Bate

The structure of an integrin/talin complex reveals the basis of inside‐out signal transduction

Fundamental to cell adhesion and migration, integrins are large heterodimeric membrane proteins that uniquely mediate inside‐out signal transduction, whereby adhesion to the extracellular matrix is activated from within the cell by direct binding of talin to the cytoplasmic tail of the β integrin subunit. Here, we report the first structure of talin bound to an authentic full‐length β integrin tail. Using biophysical and whole cell measurements, we show that a specific ionic interaction between the talin F3 domain and the membrane–proximal helix of the β tail disrupts an integrin α/β salt bridge that helps maintain the integrin inactive state. Second, we identify a positively charged surface on the talin F2 domain that precisely orients talin to disrupt the heterodimeric integrin transmembrane (TM) complex. These results show key structural features that explain the ability of talin to mediate inside‐out TM signalling.

Functional architecture of a late pollen promoter: pollen-specific transcription is developmentally regulated by multiple stage-specific and co-dependent activator elements

The tomato lat52 gene encodes an essential cysteine-rich protein preferentially transcribed in the vegetative cell during pollen maturation. Detailed analyses of the identity, organization and role of cis-regulatory elements in controlling the precise developmental and tissue-specific expression of lat52 during pollen development were performed. Analysis of a series of 5′ promoter deletion mutants stably introduced into tobacco demonstrated differential developmental activation of deletion mutants during pollen development. All major cis-regulatory elements required for pollen-specific transcription were located within the upstream region −492 to −52. This region was shown to comprise three independent activator domains A, B and C, each sufficient to activate the minimal CaMV 35S promoter in a pollen-specific manner. 5′ deletion and gain of function approaches were used to show that domain A and the previously defined motif PBII (sub-domain B1) were largely redundant in the presence of downstream sequences in mature pollen. Within domain B two novel pollen-specific sub-domains B2 and B3 were identified. Within domain C, the activity of the PBI motif (sub-domain C1) was shown to be strictly dependent upon a downstream 20 bp pollen-specific activator unit −72 to −52 (sub-domain C2), containing two novel co-dependent regulatory elements AGAAA and TCCACCATA. These results demonstrate that transcriptional activation of lat52 is controlled by a complex of pollen-specific cis-regulatory elements which cooperate to achieve maximum levels of gene expression throughout pollen maturation. Alternative models of the interaction of identified cis-regulatory elements with putative trans-acting factors within the lat52 promoter and their developmental utilization are presented.

Activation of a vinculin-binding site in the talin rod involves rearrangement of a five-helix bundle.

The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin‐mediated cell adhesion and migration. We have determined the crystal structures of two domains from the talin rod spanning residues 482–789. Talin 482–655, which contains a vinculin‐binding site (VBS), folds into a five‐helix bundle whereas talin 656–789 is a four‐helix bundle. We show that the VBS is composed of a hydrophobic surface spanning five turns of helix 4. All the key side chains from the VBS are buried and contribute to the hydrophobic core of the talin 482–655 fold. We demonstrate that the talin 482–655 five‐helix bundle represents an inactive conformation, and mutations that disrupt the hydrophobic core or deletion of helix 5 are required to induce an active conformation in which the VBS is exposed. We also report the crystal structure of the N‐terminal vinculin head domain in complex with an activated form of talin. Activation of the VBS in talin and the recruitment of vinculin may support the maturation of small integrin/talin complexes into more stable adhesions.

Mechanotransduction in vivo by repeated talin stretch-relaxation events depends upon vinculin.

The focal adhesion protein talin undergoes cycles of stretching and relaxation in living cells, suggesting a role in the transduction of mechanical into biochemical signals.

Impact of thioesterase activity on tylosin biosynthesis in Streptomyces fradiae.

BACKGROUND The polyketide lactone, tylactone, is produced in Streptomyces fradiae by the TylG complex of five multifunctional proteins. As with other type I polyketide synthases, the enzyme catalysing the final elongation step (TylGV) possesses an integral thioesterase domain that is believed to be responsible for chain termination and ring closure to form tylactone, which is then glycosylated to yield tylosin. In common with other macrolide producers, S. fradiae also possesses an additional thioesterase gene (orf5) located within the cluster of antibiotic biosynthetic genes. The function of the Orf5 protein is addressed here. RESULTS Disruption of orf5 reduced antibiotic accumulation in S. fradiae by at least 85%. Under such circumstances, the strain accumulated desmycosin (demycarosyl-tylosin) due to a downstream polar effect on the expression of orf6, which encodes a mycarose biosynthetic enzyme. High levels of desmycosin production were restored in the disrupted strain by complementation with intact orf5, or with the corresponding thioesterase gene, nbmB, from S. narbonensis, but not with DNA encoding the integral thioesterase domain of TylGV. CONCLUSIONS Polyketide metabolism in S. fradiae is strongly dependent on the thioesterase activity encoded by orf5 (tylO). It is proposed that the TylG complex might operate with a significant error frequency and be prone to blockage with aberrant polyketides. A putative editing activity associated with TylO might be essential to unblock the polyketide synthase complex and thereby promote antibiotic accumulation.

The structure of the C-terminal actin-binding domain of talin

Talin is a large dimeric protein that couples integrins to cytoskeletal actin. Here, we report the structure of the C‐terminal actin‐binding domain of talin, the core of which is a five‐helix bundle linked to a C‐terminal helix responsible for dimerisation. The NMR structure of the bundle reveals a conserved surface‐exposed hydrophobic patch surrounded by positively charged groups. We have mapped the actin‐binding site to this surface and shown that helix 1 on the opposite side of the bundle negatively regulates actin binding. The crystal structure of the dimerisation helix reveals an antiparallel coiled‐coil with conserved residues clustered on the solvent‐exposed face. Mutagenesis shows that dimerisation is essential for filamentous actin (F‐actin) binding and indicates that the dimerisation helix itself contributes to binding. We have used these structures together with small angle X‐ray scattering to derive a model of the entire domain. Electron microscopy provides direct evidence for binding of the dimer to F‐actin and indicates that it binds to three monomers along the long‐pitch helix of the actin filament.

The Structure of the Talin Head Reveals a Novel Extended Conformation of the FERM Domain

Summary FERM domains are found in a diverse superfamily of signaling and adaptor proteins at membrane interfaces. They typically consist of three separately folded domains (F1, F2, F3) in a compact cloverleaf structure. The crystal structure of the N-terminal head of the integrin-associated cytoskeletal protein talin reported here reveals a novel FERM domain with a linear domain arrangement, plus an additional domain F0 packed against F1. While F3 binds β-integrin tails, basic residues in F1 and F2 are required for membrane association and for integrin activation. We show that these same residues are also required for cell spreading and focal adhesion assembly in cells. We suggest that the extended conformation of the talin head allows simultaneous binding to integrins via F3 and to PtdIns(4,5)P2-enriched microdomains via basic residues distributed along one surface of the talin head, and that these multiple interactions are required to stabilize integrins in the activated state.

Structure of a double ubiquitin-like domain in the talin head: a role in integrin activation

Talin is a 270‐kDa protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N‐terminal FERM domain comprised of F1, F2 and F3 domains, but it is atypical in that F1 contains a large insert and is preceded by an extra domain F0. Although F3 contains the binding site for β‐integrin tails, F0 and F1 are also required for activation of β1‐integrins. Here, we report the solution structures of F0, F1 and of the F0F1 double domain. Both F0 and F1 have ubiquitin‐like folds joined in a novel fixed orientation by an extensive charged interface. The F1 insert forms a loop with helical propensity, and basic residues predicted to reside on one surface of the helix are required for binding to acidic phospholipids and for talin‐mediated activation of β1‐integrins. This and the fact that basic residues on F2 and F3 are also essential for integrin activation suggest that extensive interactions between the talin FERM domain and acidic membrane phospholipids are required to orientate the FERM domain such that it can activate integrins.

The structure of an interdomain complex that regulates talin activity.

Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. It exists in both globular and extended conformations, and an intramolecular interaction between the N-terminal F3 FERM subdomain and the C-terminal part of the talin rod contributes to an autoinhibited form of the molecule. Here, we report the solution structure of the primary F3 binding domain within the C-terminal region of the talin rod and use intermolecular nuclear Overhauser effects to determine the structure of the complex. The rod domain (residues 1655–1822) is an amphipathic five-helix bundle; Tyr-377 of F3 docks into a hydrophobic pocket at one end of the bundle, whereas a basic loop in F3 (residues 316–326) interacts with a cluster of acidic residues in the middle of helix 4. Mutation of Glu-1770 abolishes binding. The rod domain competes with β3-integrin tails for binding to F3, and the structure of the complex suggests that the rod is also likely to sterically inhibit binding of the FERM domain to the membrane.

Multiple regulatory genes in the tylosin biosynthetic cluster of Streptomyces fradiae

BACKGROUND The macrolide antibiotic tylosin is composed of a polyketide lactone substituted with three deoxyhexose sugars. In order to produce tylosin efficiently, Streptomyces fradiae presumably requires control mechanisms that balance the yields of the constituent metabolic pathways together with switches that allow for temporal regulation of antibiotic production. In addition to possible metabolic feedback and/or other signalling devices, such control probably involves interplay between specific regulatory proteins. Prior to the present work, however, no candidate regulatory gene(s) had been identified in S. fradiae. RESULTS DNA sequencing has shown that the tylosin biosynthetic gene cluster, within which four open reading frames utilise the rare TTA codon, contains at least five candidate regulatory genes, one of which (tylP) encodes a gamma-butyrolactone signal receptor for which tylQ is a probable target. Two other genes (tylS and tylT) encode pathway-specific regulatory proteins of the Streptomyces antibiotic regulatory protein (SARP) family and a fifth, tylR, has been shown by mutational analysis to control various aspects of tylosin production. CONCLUSIONS The tyl genes of S. fradiae include the richest collection of regulators yet encountered in a single antibiotic biosynthetic gene cluster. Control of tylosin biosynthesis is now amenable to detailed study, and manipulation of these various regulatory genes is likely to influence yields in tylosin-production fermentations.