The rickettsia surface cell antigen 4 applies mimicry to bind to and activate vinculin

Pathogenic Rickettsia species cause high morbidity and mortality, especially R. prowazekii, the causative agent of typhus. Like many intracellular pathogens, Rickettsia exploit the cytoskeleton to enter and spread within the host cell. Here we report that the cell surface antigen sca4 of Rickettsia co-localizes with vinculin in cells at sites of focal adhesions in sca4-transfected cells and that sca4 binds to and activates vinculin through two vinculin binding sites (VBSs) that are conserved across all Rickettsia. Remarkably, this occurs through molecular mimicry of the vinculin-talin interaction that is also seen with the IpaA invasin of the intracellular pathogen Shigella, where binding of these VBSs to the vinculin seven-helix bundle head domain (Vh1) displaces intramolecular interactions with the vinculin tail domain that normally clamp vinculin in an inactive state. Finally, the vinculin·sca4-VBS crystal structures reveal that vinculin adopts a new conformation when bound to the C-terminal VBS of sca4. Collectively, our data define the mechanism by which sca4 activates vinculin and interacts with the actin cytoskeleton, and they suggest important roles for vinculin in Rickettsia pathogenesis.     

Crystal structure of vinculin in complex with vinculin binding site 50 (VBS50), the integrin binding site 2 (IBS2) of talin

The cytoskeletal protein talin activates integrin receptors by binding of its FERM domain to the cytoplasmic tail of β‐integrin. Talin also couples integrins to the actin cytoskeleton, largely by binding to and activating the cytoskeletal protein vinculin, which binds to F‐actin through the agency of its five‐helix bundle tail (Vt) domain. Talin activates vinculin by means of buried amphipathic α‐helices coined vinculin binding sites (VBSs) that reside within numerous four‐ and five‐helix bundle domains that comprise the central talin rod, which are released from their buried locales by means of mechanical tension on the integrin:talin complex. In turn, these VBSs bind to the N‐terminal seven‐helix bundle (Vh1) domain of vinculin, creating an entirely new helix bundle that severs its head‐tail interactions. Interestingly, talin harbors a second integrin binding site coined IBS2 that consists of two five‐helix bundle domains that also contain a VBS (VBS50). Here we report the crystal structure of VBS50 in complex with vinculin at 2.3 Å resolution and show that intramolecular interactions of VBS50 within IBS2 are much more extensive versus its interactions with vinculin. Indeed, the IBS2‐vinculin interaction only occurs at physiological temperature and the affinity of VBS50 for vinculin is about 30 times less than other VBSs. The data support a model where integrin binding destabilizes IBS2 to allow it to bind to vinculin.     

Arabidopsis thaliana FLA4 functions as a glycan‐stabilized soluble factor via its carboxy‐proximal Fasciclin 1 domain

Fasciclin‐like arabinogalactan proteins (FLAs) are involved in numerous important functions in plants but the relevance of their complex structure to physiological function and cellular fate is unresolved. Using a fully functional fluorescent version of Arabidopsis thaliana FLA4 we show that this protein is localized at the plasma membrane as well as in endosomes and soluble in the apoplast. FLA4 is likely to be GPI‐anchored, is highly N‐glycosylated and carries two O‐glycan epitopes previously associated with arabinogalactan proteins. The activity of FLA4 was resistant against deletion of the amino‐proximal fasciclin 1 domain and was unaffected by removal of the GPI‐modification signal, a highly conserved N‐glycan or the deletion of predicted O‐glycosylation sites. Nonetheless these structural changes dramatically decreased endoplasmic reticulum (ER)‐exit and plasma membrane localization of FLA4, with N‐glycosylation acting at the level of ER‐exit and O‐glycosylation influencing post‐secretory fate. We show that FLA4 acts predominantly by molecular interactions involving its carboxy‐proximal fasciclin 1 domain and that its amino‐proximal fasciclin 1 domain is required for stabilization of plasma membrane localization. FLA4 functions as a soluble glycoprotein via its carboxy‐proximal Fas1 domain and its normal cellular trafficking depends on N‐ and O‐glycosylation.     

Intermolecular versus intramolecular interactions of the vinculin binding site 33 of talin

The cytoskeletal proteins talin and vinculin are localized at cell‐matrix junctions and are key regulators of cell signaling, adhesion, and migration. Talin couples integrins via its FERM domain to F‐actin and is an important regulator of integrin activation and clustering. The 220 kDa talin rod domain comprises several four‐ and five‐helix bundles that harbor amphipathic α‐helical vinculin binding sites (VBSs). In its inactive state, the hydrophobic VBS residues involved in binding to vinculin are buried within these helix bundles, and the mechanical force emanating from bound integrin receptors is thought necessary for their release and binding to vinculin. The crystal structure of a four‐helix bundle of talin that harbors one of these VBSs, coined VBS33, was recently determined. Here we report the crystal structure of VBS33 in complex with vinculin at 2 Å resolution. Notably, comparison of the apo and vinculin bound structures shows that intermolecular interactions of the VBS33 α‐helix with vinculin are more extensive than the intramolecular interactions of the VBS33 within the talin four‐helix bundle.     

Crystal structure of the variable domain of the Streptococcus gordonii surface protein SspB

The Antigen I/II (AgI/II) family of proteins are cell wall anchored adhesins expressed on the surface of oral streptococci. The AgI/II proteins interact with molecules on other bacteria, on the surface of host cells, and with salivary proteins. Streptococcus gordonii is a commensal bacterium, and one of the primary colonizers that initiate the formation of the oral biofilm. S. gordonii expresses two AgI/II proteins, SspA and SspB that are closely related. One of the domains of SspB, called the variable (V‐) domain, is significantly different from corresponding domains in SspA and all other AgI/II proteins. As a first step to elucidate the differences among these proteins, we have determined the crystal structure of the V‐domain from S. gordonii SspB at 2.3 Å resolution. The domain comprises a β‐supersandwich with a putative binding cleft stabilized by a metal ion. The overall structure of the SspB V‐domain is similar to the previously reported V‐domain of the Streptococcus mutans protein SpaP, despite their low sequence similarity. In spite of the conserved architecture of the binding cleft, the cavity is significantly smaller in SspB, which may provide clues about the difference in ligand specificity. We also verified that the metal in the binding cleft is a calcium ion, in concurrence with previous biological data. It was previously suggested that AgI/II V‐domains are carbohydrate binding. However, we tested that hypothesis by screening the SspB V‐domain for binding to over 400 glycoconjucates and found that the domain does not interact with any of the carbohydrates.     

Structural analysis of ligand‐bound states of the Salmonella type III secretion system ATPase InvC

Translocation of virulence effector proteins through the type III secretion system (T3SS) is essential for the virulence of many medically relevant Gram‐negative bacteria. The T3SS ATPases are conserved components that specifically recognize chaperone–effector complexes and energize effector secretion through the system. It is thought that functional T3SS ATPases assemble into a cylindrical structure maintained by their N‐terminal domains. Using size‐exclusion chromatography coupled to multi‐angle light scattering and native mass spectrometry, we show that in the absence of the N‐terminal oligomerization domain the Salmonella T3SS ATPase InvC can form monomers and dimers in solution. We also present for the first time a 2.05 å resolution crystal structure of InvC lacking the oligomerization domain (InvCΔ79) and map the amino acids suggested for ATPase intersubunit interaction, binding to other T3SS proteins and chaperone–effector recognition. Furthermore, we validate the InvC ATP‐binding site by co‐crystallization of InvCΔ79 with ATPγS (2.65 å) and ADP (2.80 å). Upon ATP‐analogue recognition, these structures reveal remodeling of the ATP‐binding site and conformational changes of two loops located outside of the catalytic site. Both loops face the central pore of the predicted InvC cylinder and are essential for the function of the T3SS ATPase. Our results present a fine functional and structural correlation of InvC and provide further details of the homo‐oligomerization process and ATP‐dependent conformational changes underlying the T3SS ATPase activity.     

Structural mapping of the coiled‐coil domain of a bacterial condensin and comparative analyses across all domains of life suggest conserved features of SMC proteins

The structural maintenance of chromosomes (SMC) proteins form the cores of multisubunit complexes that are required for the segregation and global organization of chromosomes in all domains of life. These proteins share a common domain structure in which N‐ and C‐ terminal regions pack against one another to form a globular ATPase domain. This “head” domain is connected to a central, globular, “hinge” or dimerization domain by a long, antiparallel coiled coil. To date, most efforts for structural characterization of SMC proteins have focused on the globular domains. Recently, however, we developed a method to map interstrand interactions in the 50‐nm coiled‐coil domain of MukB, the divergent SMC protein found in γ‐proteobacteria. Here, we apply that technique to map the structure of the Bacillus subtilis SMC (BsSMC) coiled‐coil domain. We find that, in contrast to the relatively complicated coiled‐coil domain of MukB, the BsSMC domain is nearly continuous, with only two detectable coiled‐coil interruptions. Near the middle of the domain is a break in coiled‐coil structure in which there are three more residues on the C‐terminal strand than on the N‐terminal strand. Close to the head domain, there is a second break with a significantly longer insertion on the same strand. These results provide an experience base that allows an informed interpretation of the output of coiled‐coil prediction algorithms for this family of proteins. A comparison of such predictions suggests that these coiled‐coil deviations are highly conserved across SMC types in a wide variety of organisms, including humans. Proteins 2015; 83:1027–1045. © 2015 Wiley Periodicals, Inc.     

Distinct modes of recruitment of the CCR4–NOT complex by Drosophila and vertebrate Nanos

Nanos proteins repress the expression of target mRNAs by recruiting effector complexes through non‐conserved N‐terminal regions. In vertebrates, Nanos proteins interact with the NOT1 subunit of the CCR4–NOT effector complex through a NOT1 interacting motif (NIM), which is absent in Nanos orthologs from several invertebrate species. Therefore, it has remained unclear whether the Nanos repressive mechanism is conserved and whether it also involves direct interactions with the CCR4–NOT deadenylase complex in invertebrates. Here, we identify an effector domain (NED) that is necessary for the Drosophila melanogaster (Dm) Nanos to repress mRNA targets. The NED recruits the CCR4–NOT complex through multiple and redundant binding sites, including a central region that interacts with the NOT module, which comprises the C‐terminal domains of NOT1–3. The crystal structure of the NED central region bound to the NOT module reveals an unanticipated bipartite binding interface that contacts NOT1 and NOT3 and is distinct from the NIM of vertebrate Nanos. Thus, despite the absence of sequence conservation, the N‐terminal regions of Nanos proteins recruit CCR4–NOT to assemble analogous repressive complexes.     

Structure and oligomerization of the PilC type IV pilus biogenesis protein from Thermus thermophilus

Type IV pili are expressed from a wide variety of Gram‐negative bacteria and play a major role in host cell adhesion and bacterial motility. PilC is one of at least a dozen different proteins that are implicated in Type IV pilus assembly in Thermus thermophilus and a member of a conserved family of integral inner membrane proteins which are components of the Type II secretion system (GspF) and the archeal flagellum. PilC/GspF family members contain repeats of a conserved helix‐rich domain of around 100 residues in length. Here, we describe the crystal structure of one of these domains, derived from the N‐terminal domain of Thermus thermophilus PilC. The N‐domain forms a dimer, adopting a six helix bundle structure with an up‐down‐up‐down‐up‐down topology. The monomers are related by a rotation of 170°, followed by a translation along the axis of the final α‐helix of approximately one helical turn. This means that the regions of contact on helices 5 and 6 in each monomer are overlapping, but different. Contact between the two monomers is mediated by a network of hydrophobic residues which are highly conserved in PilC homologs from other Gram‐negative bacteria. Site‐directed mutagenesis of residues at the dimer interface resulted in a change in oligomeric state of PilC from tetramers to dimers, providing evidence that this interface is also found in the intact membrane protein and suggesting that it is important to its function. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The Caenorhabditis elegans spe‐49 gene is required for fertilization and encodes a sperm‐specific transmembrane protein homologous to SPE‐42

Fertilization, the fusion of sperm and oocyte to form a zygote, is the first and arguably the most important cell–cell interaction event in an organism’s life. Forward and reverse genetic approaches in the nematode Caenorhabditis elegans have identified many genes that are required for gametogenesis and fertilization and thus are beginning to elucidate the molecular pathways that underlie these processes. We identified an allele of the spe‐49 gene in a second filial generation (F₂) mutagenesis screen for spermatogenesis‐defective (spe) mutants. Mutant worms for spe‐49 produce sperm that have normal morphology, activate to form ameboid spermatozoa, and can migrate to and maintain their position in the hermaphrodite reproductive tract but fail to fertilize oocytes. This phenotype puts spe‐49 in the spe‐9 class of late‐acting genes that function in sperm at the time of fertilization. We cloned the spe‐49 gene through a combination of deficiency mapping, transgenic rescue, and genomic sequencing. spe‐49 messenger RNA (mRNA) is enriched in male germ cells, and the complementary DNA (cDNA) encodes a predicted 772‐amino‐acid six‐pass transmembrane protein that is homologous to SPE‐42. Indeed, SPE‐49 and SPE‐42 have identical predicted membrane topology and domain structure, including a large extracellular domain with six conserved cysteine residues, a DC‐STAMP domain, and a C‐terminal cytoplasmic domain containing a C4–C4 RING finger motif. The presence of two SPE‐42 homologs in animal genomes from worms to humans suggests that these proteins are highly conserved components of the molecular apparatus required for the sperm–oocyte recognition, binding, and fusion.     

Palmitoylation of stathmin family proteins domain A controls Golgi versus mitochondrial subcellular targeting

Background information. Precise localization of proteins to specialized subcellular domains is fundamental for proper neuronal development and function. The neural microtubule‐regulatory phosphoproteins of the stathmin family are such proteins whose specific functions are controlled by subcellular localization. Whereas stathmin is cytosolic, SCG10, SCLIP and RB3/RB3′/RB3″ are localized to the Golgi and vesicle‐like structures along neurites and at growth cones. We examined the molecular determinants involved in the regulation of this specific subcellular localization in hippocampal neurons in culture. Results. We show that their conserved N‐terminal domain A carrying two palmitoylation sites is dominant over the others for Golgi and vesicle‐like localization. Using palmitoylation‐deficient GFP (green fluorescent protein) fusion mutants, we demonstrate that domains A of stathmin proteins have the particular ability to control protein targeting to either Golgi or mitochondria, depending on their palmitoylation. This regulation involves the co‐operation of two subdomains within domain A, and seems also to be under the control of its SLD (stathmin‐like domain) extension. Conclusions. Our results unravel that, in specific biological conditions, palmitoylation of stathmin proteins might be able to control their targeting to express their functional activities at appropriate subcellular sites. They, more generally, open new perspectives regarding the role of palmitoylation as a signalling mechanism orienting proteins to their functional subcellular compartments.     

Different Vinculin Binding Sites Use the Same Mechanism to Regulate Directional Force Transduction

Vinculin is a universal adaptor protein that transiently reinforces the mechanical stability of adhesion complexes. It stabilizes mechanical connections that cells establish between the actomyosin cytoskeleton and the extracellular matrix via integrins or to neighboring cells via cadherins, yet little is known regarding its mechanical design. Vinculin binding sites (VBSs) from different nonhomologous actin-binding proteins use conserved helical motifs to associate with the vinculin head domain. We studied the mechanical stability of such complexes by pulling VBS peptides derived from talin, α-actinin, and Shigella IpaA out of the vinculin head domain. Experimental data from atomic force microscopy single-molecule force spectroscopy and steered molecular dynamics (SMD) simulations both revealed greater mechanical stability of the complex for shear-like than for zipper-like pulling configurations. This suggests that reinforcement occurs along preferential force directions, thus stabilizing those cytoskeletal filament architectures that result in shear-like pulling geometries. Large force-induced conformational changes in the vinculin head domain, as well as protein-specific fine-tuning of the VBS sequence, including sequence inversion, allow for an even more nuanced force response.     

The crystal structure of Arabidopsis BON1 provides insights into the copine protein family

The Arabidopsis thaliana BON1 gene product is a member of the evolutionary conserved eukaryotic calcium‐dependent membrane‐binding protein family. The copine protein is composed of two C2 domains (C2A and C2B) followed by a vWA domain. The BON1 protein is localized on the plasma membrane, and is known to suppress the expression of immune receptor genes and to positively regulate stomatal closure. The first structure of this protein family has been determined to 2.5‐Å resolution and shows the structural features of the three conserved domains C2A, C2B and vWA. The structure reveals the third Ca²⁺‐binding region in C2A domain is longer than classical C2 domains and a novel Ca²⁺ binding site in the vWA domain. The structure of BON1 bound to Mn²⁺ is also presented. The binding of the C2 domains to phospholipid (PSF) has been modeled and provides an insight into the lipid‐binding mechanism of the copine proteins. Furthermore, the selectivity of the separate C2A and C2B domains and intact BON1 to bind to different phospholipids has been investigated, and we demonstrated that BON1 could mediate aggregation of liposomes in response to Ca²⁺. These studies have formed the basis of further investigations into the important role that the copine proteins play in vivo.     

Novel proteins for homocysteine biosynthesis in anaerobic microorganisms

The metabolic network for sulfide assimilation and trafficking in methanogens is largely unknown. To discover novel proteins required for these processes, we used bioinformatics to identify genes co‐occurring with the protein biosynthesis enzyme SepCysS, which converts phosphoseryl‐tRNA^Cys to cysteinyl‐tRNA^Cys in nearly all methanogens. Exhaustive analysis revealed three conserved protein families, each containing molecular signatures predicting function in sulfur metabolism. One of these families, classified within clusters of orthologous groups (COG) 1900, possesses two conserved cysteine residues and is often found in genomic contexts together with known sulfur metabolic genes. A second protein family is predicted to bind two 4Fe‐4S clusters. All three genes were also identified in more than 50 strictly anaerobic bacterial genera from nine distinct phyla. Gene‐deletion and growth experiments in Methanosarcina acetivorans, using sulfide as the sole sulfur source, demonstrate that two of the proteins (MA1821 and MA1822) are essential to homocysteine biosynthesis in a background lacking an additional gene for sulfur insertion into homocysteine. Mutational analysis confirms the importance of several structural elements, including a conserved cysteine residue and the predicted 4Fe‐4S cluster‐binding domain.     

Structure and interactions of the C‐terminal metal binding domain of Archaeoglobus fulgidus CopA

The Cu⁺‐ATPase CopA from Archaeoglobus fulgidus belongs to the P_1B family of the P‐type ATPases. These integral membrane proteins couple the energy of ATP hydrolysis to heavy metal ion translocation across membranes. A defining feature of P_1B‐1‐type ATPases is the presence of soluble metal binding domains at the N‐terminus (N‐MBDs). The N‐MBDs exhibit a conserved ferredoxin‐like fold, similar to that of soluble copper chaperones, and bind metal ions via a conserved CXXC motif. The N‐MBDs enable Cu⁺ regulation of turnover rates apparently through Cu‐sensitive interactions with catalytic domains. A. fulgidus CopA is unusual in that it contains both an N‐terminal MBD and a C‐terminal MBD (C‐MBD). The functional role of the unique C‐MBD has not been established. Here, we report the crystal structure of the apo, oxidized C‐MBD to 2.0 Å resolution. In the structure, two C‐MBD monomers form a domain‐swapped dimer, which has not been observed previously for similar domains. In addition, the interaction of the C‐MBD with the other cytoplasmic domains of CopA, the ATP binding domain (ATPBD) and actuator domain (A‐domain), has been investigated. Interestingly, the C‐MBD interacts specifically with both of these domains, independent of the presence of Cu⁺ or nucleotides. These data reinforce the uniqueness of the C‐MBD and suggest a distinct structural role for the C‐MBD in CopA transport. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Apo raver1 structure reveals distinct RRM domain orientations

Raver1 is a multifunctional protein that modulates both alternative splicing and focal adhesion assembly by binding to the nucleoplasmic splicing repressor polypyrimidine tract protein (PTB) or to the cytoskeletal proteins vinculin and α‐actinin. The amino‐terminal region of raver1 has three RNA recognition motif (RRM1, RRM2, and RRM3) domains, and RRM1 interacts with the vinculin tail (Vt) domain and vinculin mRNA. We previously determined the crystal structure of the raver1 RRM1–3 domains in complex with Vt at 2.75 Å resolution. Here, we report crystal structure of the unbound raver1 RRM1–3 domains at 2 Å resolution. The apo structure reveals that a bound sulfate ion disrupts an electrostatic interaction between the RRM1 and RRM2 domains, triggering a large relative domain movement of over 30°. Superposition with other RNA‐bound RRM structures places the sulfate ion near the superposed RNA phosphate group suggesting that this is the raver1 RNA binding site. While several single and some tandem RRM domain structures have been described, to the best of our knowledge, this is the second report of a three‐tandem RRM domain structure.     

Assigning DYW-type PPR proteins to RNA editing sites in the funariid mosses Physcomitrella patens and Funaria hygrometrica

The plant‐specific pentatricopeptide repeat (PPR) proteins with variable PPR repeat lengths (PLS‐type) and protein extensions up to the carboxyterminal DYW domain have received attention as specific recognition factors for the C‐to‐U type of RNA editing events in plant organelles. Here, we report a DYW‐protein knockout in the model plant Physcomitrella patens specifically affecting mitochondrial RNA editing positions cox1eU755SL and rps14eU137SL. Assignment of DYW proteins and RNA editing sites might best be corroborated by data from a taxon with a slightly different, yet similarly manageable low number of editing sites and DYW proteins. To this end we investigated the mitochondrial editing status of the related funariid moss Funaria hygrometrica. We find that: (i) Funaria lacks three mitochondrial RNA editing positions present in Physcomitrella, (ii) that F. hygrometrica cDNA sequence data identify nine DYW proteins as clear orthologues of their P. patens counterparts, and (iii) that the ‘missing’ 10th DYW protein in F. hygrometrica is responsible for two mitochondrial editing sites in P. patens lacking in F. hygrometrica (nad3eU230SL, nad4eU272SL). Interestingly, the third site of RNA editing missing in F. hygrometrica (rps14eU137SL) is addressed by the DYW protein characterized here and the presence of its orthologue in F. hygrometrica is explained through its simultaneous action on site cox1eU755SL conserved in both mosses.     

Crystal structure of BinB: A receptor binding component of the binary toxin from Lysinibacillus sphaericus

The binary toxin (Bin), produced by Lysinibacillus sphaericus, is composed of BinA (42 kDa) and BinB (51 kDa) proteins, which are both required for full toxicity against Culex and Anopheles mosquito larvae. Specificity of Bin toxin is determined by the binding of BinB component to a receptor present on the midgut epithelial membranes, while BinA is proposed to be a toxic component. Here, we determined the first crystal structure of the active form of BinB at a resolution of 1.75 Å. BinB possesses two distinct structural domains in its N‐ and C‐termini. The globular N‐terminal domain has a β‐trefoil scaffold which is a highly conserved architecture of some sugar binding proteins or lectins, suggesting a role of this domain in receptor‐binding. The BinB β‐rich C‐terminal domain shares similar three‐dimensional folding with aerolysin type β‐pore forming toxins, despite a low sequence identity. The BinB structure, therefore, is a new member of the aerolysin‐like toxin family, with probably similarities in the cytolytic mechanism that takes place via pore formation. Proteins 2014; 82:2703–2712. © 2014 Wiley Periodicals, Inc.     

Crystal structure of the trimeric N‐terminal domain of ciliate Euplotes octocarinatus centrin binding with calcium ions

Centrin is a member of the EF‐hand superfamily of calcium‐binding proteins, a highly conserved eukaryotic protein that binds to Ca²⁺. Its self‐assembly plays a causative role in the fiber contraction that is associated with the cell division cycle and ciliogenesis. In this study, the crystal structure of N‐terminal domain of ciliate Euplotes octocarinatus centrin (N‐EoCen) was determined by using the selenomethionine single‐wavelength anomalous dispersion method. The protein molecules formed homotrimers. Every protomer had two putative Ca²⁺ ion‐binding sites I and II, protomer A, and C bound one Ca²⁺ ion, while protomer B bound two Ca²⁺ ions. A novel binding site III was observed and the Ca²⁺ ion was located at the center of the homotrimer. Several hydrogen bonds, electrostatic, and hydrophobic interactions between the protomers contributed to the formation of the oligomer. Structural studies provided insight into the foundation for centrin aggregation and the roles of calcium ions.