Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Fatty Acids Modulate Toll-like Receptor 4 Activation through Regulation of Receptor Dimerization and Recruitment into Lipid Rafts in a Reactive Oxygen Species-dependent Manner

The saturated fatty acids acylated on Lipid A of lipopolysaccharide (LPS) or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for Toll-like Receptor 4 (TLR4) and TLR2. The results from our previous studies demonstrated that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4. However, the underlying mechanism has not been understood. Here, we report for the first time that the saturated fatty acid lauric acid induced dimerization and recruitment of TLR4 into lipid rafts, however, dimerization was not observed in non-lipid raft fractions. Similarly, LPS and lauric acid enhanced the association of TLR4 with MD-2 and downstream adaptor molecules, TRIF and MyD88, into lipid rafts leading to the activation of downstream signaling pathways and target gene expression. However, docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, inhibited LPS- or lauric acid-induced dimerization and recruitment of TLR4 into lipid raft fractions. Together, these results demonstrate that lauric acid and DHA reciprocally modulate TLR4 activation by regulation of the dimerization and recruitment of TLR4 into lipid rafts. In addition, we showed that TLR4 recruitment to lipid rafts and dimerization were coupled events mediated at least in part by NADPH oxidase-dependent reactive oxygen species generation. These results provide a new insight in understanding the mechanism by which fatty acids differentially modulate TLR4-mediated signaling pathway and consequent inflammatory responses which are implicated in the development and progression of many chronic diseases.Toll-like receptors (TLRs)³ are one of the key pattern recognition receptor families that play a critical role in inducing innate and adaptive immune responses in mammals by recognizing conserved pathogen-associated molecular pattern of invading microbes. So far, at least thirteen TLRs have been identified in mammalian species (1, 2).Lipopolysaccharide (LPS) from Gram-negative bacteria is the ligand for the TLR4 complex (3), whereas, TLR2 can recognize lipoproteins/lipopeptides of Gram-positive bacteria and mycoplasma (1, 2). LPS forms a complex with LPS-binding protein in serum leading to the conversion of oligomeric micelles of LPS to monomers, which are delivered to CD14. Monomeric LPS is known to bind TLR4/MD-2/CD14 complex (4). Lipid A, which possesses most of the biological activities of LPS, is acylated with hydroxy saturated fatty acids. The 3-hydroxyl groups of these saturated fatty acids are further 3-Ο-acylated by saturated fatty acids. Removal of these Ο-acylated saturated fatty acids from Lipid A not only results in complete loss of endotoxic activity, but also makes Lipid A act as an antagonist against the native Lipid A (5, 6). One or more Lipid As containing unsaturated fatty acids are known to be non-toxic and act as an antagonist against endotoxin (7, 8). In addition, deacylated lipoproteins are unable to activate TLR2 and to induce cytokine expression in monocytes (9). Together, these results suggest that saturated fatty acids acylated on Lipid A or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for TLR4 and TLR2. Indeed, it is suggested that the rapid interaction of bacterial lipopeptides with plasma membrane of macrophages occurs via insertion of their acylated saturated fatty acids as determined by electron energy loss spectroscopy and freeze-fracture techniques (10, 11). TLR2 can form a heterodimer with TLR1 or TLR6, which can discriminate the molecular structure of triacyl or diacyl lipopeptides (12–14). So far there is no evidence that microbial ligands for other TLRs are acylated by saturated fatty acids.Results from our previous studies demonstrated that saturated fatty acids activate TLR4 and polyunsaturated fatty acids (PUFA) inhibit both saturated fatty acid- and LPS-induced activation of TLR4 (15, 16). In addition, the saturated fatty acid lauric acid potentiates, but the n-3 PUFA docosahexaenoic acid (DHA) inhibits lipopeptide (TLR2 agonist)-induced TLR2 activation (17). Together, these results suggest that both TLR2 and TLR4 signaling pathways and target gene expression are reciprocally modulated by saturated and polyunsaturated fatty acids. However, the mechanism for this modulation by fatty acids is not understood.TLR4 is recruited to lipid raft factions after cells are treated with LPS and subsequently induces tumor necrosis factor-α expression in RAW264.7 cells (18). This process occurs in an ROS-dependent manner because inhibition of NADPH oxidase suppresses TLR4 recruitment to lipid rafts (19). Methyl-β-dextrin, a lipid raft inhibitor, significantly inhibits the LPS-induced expression of cytokine (19), suggesting that lipid rafts are essential for TLR4-mediated signal transduction and target gene expression. Lipid rafts are a collection of lipid membrane microdomains characterized by insolubility in non-ionic detergents. Lipid rafts serve as a platform where receptor-mediated signal transduction is initiated (20). Lipid rafts have a special lipid composition that is rich in cholesterol, sphingomyelin, and glycolipids (21). The polar lipids in detergent-resistant membrane contain predominantly saturated fatty acyl residues with underrepresented PUFAs (22–24), suggesting that saturated fatty acyl chains favor lipid raft association. On the other hand, n-3 PUFAs displace signaling proteins from lipid rafts by altering lipid composition, and the displacement leads to the suppression of T-cell receptor-mediated signaling (25). It is now well documented that TLRs form homo- or hetero-oligomers (1, 2). TLR4 homodimerization is the initial step of the receptor activation. Results from our previous studies suggest that the molecular target by which saturated fatty acids and n-3 PUFAs reciprocally modulate TLR4 activation is the receptor complex itself or the event leading to the receptor activation instead of the downstream signaling components (15, 16). Therefore, we determined whether the reciprocal modulation of TLR4 activation is mediated by regulation of the dimerization and recruitment of TLR4 into lipid rafts, and if these processes occur in an ROS-dependent manner.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Aldo-keto Reductase Family 1 Member B10 Promotes Cell Survival by Regulating Lipid Synthesis and Eliminating Carbonyls

Aldo-keto reductase family 1 member B10 (AKR1B10) is primarily expressed in the normal human colon and small intestine but overexpressed in liver and lung cancer. Our previous studies have shown that AKR1B10 mediates the ubiquitin-dependent degradation of acetyl-CoA carboxylase-α. In this study, we demonstrate that AKR1B10 is critical to cell survival. In human colon carcinoma cells (HCT-8) and lung carcinoma cells (NCI-H460), small-interfering RNA-induced AKR1B10 silencing resulted in caspase-3-mediated apoptosis. In these cells, the total and subspecies of cellular lipids, particularly of phospholipids, were decreased by more than 50%, concomitant with 2–3-fold increase in reactive oxygen species, mitochondrial cytochrome c efflux, and caspase-3 cleavage. AKR1B10 silencing also increased the levels of α,β-unsaturated carbonyls, leading to the 2–3-fold increase of cellular lipid peroxides. Supplementing the HCT-8 cells with palmitic acid (80 μm), the end product of fatty acid synthesis, partially rescued the apoptosis induced by AKR1B10 silencing, whereas exposing the HCT-8 cells to epalrestat, an AKR1B10 inhibitor, led to more than 2-fold elevation of the intracellular lipid peroxides, resulting in apoptosis. These data suggest that AKR1B10 affects cell survival through modulating lipid synthesis, mitochondrial function, and oxidative status, as well as carbonyl levels, being an important cell survival protein.Aldo-keto reductase family 1 member B10 (AKR1B10,² also designated aldose reductase-like-1, ARL-1) is primarily expressed in the human colon, small intestine, and adrenal gland, with a low level in the liver (1–3). However, this protein is overexpressed in hepatocellular carcinoma, cervical cancer, lung squamous cell carcinoma, and lung adenocarcinoma in smokers, being a potential diagnostic and/or prognostic marker (1, 2, 4–6).The biological function of AKR1B10 in the intestine and adrenal gland, as well as its role in tumor development and progression, remains unclear. AKR1B10 is a monomeric enzyme that efficiently catalyzes the reduction to corresponding alcohols of a range of aromatic and aliphatic aldehydes and ketones, including highly electrophilic α,β-unsaturated carbonyls and antitumor drugs containing carbonyl groups, with NADPH as a co-enzyme (1, 7–12). The electrophilic carbonyls are constantly produced by lipid peroxidation, particularly in oxidative conditions, and are highly cytotoxic; through interaction with proteins, peptides, and DNA, the carbonyls cause protein dysfunction and DNA damage (breaks and mutations), resulting in mutagenesis, carcinogenesis, or apoptosis (10, 13–19). AKR1B10 also shows strong enzymatic activity toward all-trans-retinal, 9-cis-retinal, and 13-cis-retinal, reducing them to the corresponding retinols, which may regulate the intracellular retinoic acid, a signaling molecule modulating cell proliferation and differentiation (6, 20–23). In lung cancer, AKR1B10 expression is correlated with the patient smoking history and activates procarcinogens in cigarette smoke, such as polycyclic aromatic hydrocarbons, thus involved in lung tumorigenesis (24–26).Recent studies have shown that in breast cancer cells, AKR1B10 associates with acetyl-CoA carboxylase-α (ACCA) and blocks its ubiquitination and proteasome degradation (27). ACCA is a rate-limiting enzyme of de novo synthesis of long chain fatty acids, catalyzing the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA (28). Long chain fatty acids are the building blocks of biomembranes and the precursor of lipid second messengers, playing a critical role in cell growth and proliferation (29, 30). Therefore, ACCA activity is tightly regulated by both metabolite-mediated allosteric mechanisms and phosphorylation-dependent mechanisms; the latter are controlled by multiple hormones, such as insulin, glucagon, and growth factors (31–33). ACCA activity is also regulated through physical protein-protein interaction. For instance, breast cancer 1 (BRCA1) protein associates with the ACCA and blocks its Ser⁷⁹ residue from dephosphorylation (34, 35). The AKR1B10-mediated regulation on ACCA stability represents a novel regulatory mechanism, and this current study elucidated the biological significance of this regulation. The results show that AKR1B10 promotes cell survival via modulating lipid synthesis, mitochondrial function and oxidative stress, and carbonyl levels.     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

Mineralization by Inhibitor Exclusion: THE CALCIFICATION OF COLLAGEN WITH FETUIN

One of our goals is to understand the mechanisms that deposit mineral within collagen fibrils, and as a first step we recently determined the size exclusion characteristics of the fibril. This study revealed that apatite crystals up to 12 unit cells in size can access the water within the fibril, whereas molecules larger than a 40-kDa protein are excluded. Based on these observations, we proposed a novel mechanism for fibril mineralization: that macromolecular inhibitors of apatite growth favor fibril mineralization by selectively inhibiting crystal growth in the solution outside of the fibril. To test this mechanism, we developed a system in which crystal formation is driven by homogeneous nucleation at high calcium phosphate concentration and the only macromolecule in solution is fetuin, a 48-kDa inhibitor of apatite growth. Our experiments with this system demonstrated that fetuin determines the location of mineral growth; in the presence of fetuin mineral grows exclusively within the fibril, whereas in its absence mineral grows in solution outside the fibril. Additional experiments showed that fetuin is also able to localize calcification to the interior of synthetic matrices that have size exclusion characteristics similar to those of collagen and that it does so by selectively inhibiting mineral growth outside of these matrices. We termed this new calcification mechanism “mineralization by inhibitor exclusion,” the selective mineralization of a matrix using a macromolecular inhibitor of mineral growth that is excluded from that matrix. Future studies will be needed to evaluate the possible role of this mechanism in bone mineralization.The type I collagen fibril plays several critical roles in bone mineralization. The mineral in bone is located primarily within the fibril (1–6), and during mineralization the fibril is formed first and then water within the fibril is replaced with mineral (7, 8). The collagen fibril therefore provides the aqueous compartment in which mineral grows. We have recently shown that the physical structure of the collagen fibril plays an important additional role in mineralization, that of a gatekeeper allowing molecules smaller than a 6-kDa protein to freely access the water within the fibril while preventing molecules larger than a 40-kDa protein from entering the fibril (9).Molecules too large to enter the collagen fibril can have important effects on mineralization within the fibril. We have suggested that large inhibitors of apatite growth can paradoxically favor mineralization within the fibril by selectively preventing apatite growth in the solution outside of the fibril (9). We have also proposed that large nucleators of apatite formation may generate small crystals outside the collagen fibril and that some of these crystals can subsequently diffuse into the fibril and grow (9). Because the size exclusion characteristics of the fibril allow rapid penetration of molecules the size of a 6-kDa protein, apatite crystals up to 12 unit cells in size should in principle be able to freely access all of the water within the fibril (9).We subsequently tested these hypotheses for the role of large molecules in fibril mineralization by determining the impact of removing fetuin on the serum-driven calcification of collagen fibrils (10). Fetuin is the most abundant serum inhibitor of apatite crystal growth (11, 12), and with a molecular weight of 48 kDa fetuin is too large to penetrate the collagen fibril (9). Fetuin is also termed fetuin-A (to distinguish it from a recently discovered homologue, fetuin-B (13)) and is sometimes called α2-HS glycoprotein in humans. Our working hypothesis was that fetuin is required for the serum-driven calcification of a collagen fibril and that its role is to favor calcification within the collagen fibril by selectively preventing apatite crystal growth in the solution outside the fibril.The results of this study demonstrate that removing fetuin from serum eliminates the ability of serum to induce the calcification of a type I collagen matrix and that adding purified fetuin to fetuin-depleted serum restores this activity (14). This study further shows that a massive mineral precipitate forms during the incubation of fetuin-depleted serum but not during the incubation of serum containing fetuin (14). These observations are consistent with the hypothesis that a large serum nucleator generates apatite crystals in the solution outside of the collagen fibril, some of which penetrate into the aqueous interior of the fibril (14). Because fetuin can trap only those nuclei that it can access, the crystal nuclei that penetrate the fibril grow far more rapidly than those nuclei trapped by fetuin outside of the fibril, and the collagen fibril therefore selectively calcifies.The goal of the present experiments was to further understand the role of fetuin in the calcification of type I collagen fibrils. To accomplish this goal, we developed a system in which crystal formation is driven by homogeneous nucleation at high calcium phosphate concentrations and the only macromolecule in the solution is fetuin. This system allowed us to probe the impact of fetuin and only fetuin on the location and extent of collagen calcification. Because fetuin is the subject of this study, it is useful to review briefly its occurrence and calcification-inhibitory activity. Fetuin is a 48-kDa glycoprotein that is synthesized in the liver and is found at high concentrations in mammalian serum (15, 16) and bone (17–22). The serum fetuin concentration in adult mammals ranges from 0.5 to 1.5 mg/ml, whereas the serum fetuin concentration in the fetus and neonate is typically far higher (16). Fetuin is also one of the most abundant noncollagenous proteins found in bone (17–22), with a concentration of about 1 mg fetuin/g bone in rat (21), bovine (17), and human (19, 23) bone. Despite the abundance of fetuin in bone, however, it has not been possible to demonstrate the synthesis of fetuin in calcified tissues, and it is therefore presently thought that the fetuin found in bone arises from hepatic synthesis via serum (20, 22). This view is supported by the observation that fetuin binds strongly to apatite, the mineral phase of bone, and is selectively concentrated from serum onto apatite (18).In vitro studies have demonstrated that fetuin is an important inhibitor of apatite growth and precipitation in serum containing increased levels of calcium and phosphate (12) and that targeted deletion of the fetuin gene reduces the ability of serum to arrest apatite formation by over 70% (11). More recent studies have shown that a fetuin-mineral complex is formed in the course of the fetuin-mediated inhibition of apatite growth and precipitation in serum containing increased calcium and phosphate (24, 25). Purified fetuin also potently inhibits the growth of apatite crystals from supersaturated solutions of calcium phosphate (12, 24). In solutions in which a decline in calcium occurs within minutes because of the spontaneous formation of apatite crystals, the presence of added fetuin sustains elevated calcium levels for at least 24 h (24).     

Calcium Is Essential for the Major Pseudopilin in the Type 2 Secretion System

The pseudopilus is a key feature of the type 2 secretion system (T2SS) and is made up of multiple pseudopilins that are similar in fold to the type 4 pilins. However, pilins have disulfide bridges, whereas the major pseudopilins of T2SS do not. A key question is therefore how the pseudopilins, and in particular, the most abundant major pseudopilin, GspG, obtain sufficient stability to perform their function. Crystal structures of Vibrio cholerae, Vibrio vulnificus, and enterohemorrhagic Escherichia coli (EHEC) GspG were elucidated, and all show a calcium ion bound at the same site. Conservation of the calcium ligands fully supports the suggestion that calcium ion binding by the major pseudopilin is essential for the T2SS. Functional studies of GspG with mutated calcium ion-coordinating ligands were performed to investigate this hypothesis and show that in vivo protease secretion by the T2SS is severely impaired. Taking all evidence together, this allows the conclusion that, in complete contrast to the situation in the type 4 pili system homologs, in the T2SS, the major protein component of the central pseudopilus is dependent on calcium ions for activity.In Gram-negative bacteria, the type 2 secretion system (T2SS)² is used for the secretion of several important proteins across the outer membrane (1). The T2SS is also called the terminal branch of the general secretory pathway (Gsp) (2) and, in Vibrio species, the extracellular protein secretion (Eps) apparatus (3). This sophisticated multiprotein machinery spans both the inner and the outer membrane of Gram-negative bacteria and contains 11–15 different proteins. The T2SS consists of three major subassemblies (4–9): (i) the outer membrane complex comprising mainly the crucial multisubunit secretin GspD; (ii) the pseudopilus, which consists of one major and several minor pseudopilins; and (iii) an inner membrane platform, containing the cytoplasmic secretion ATPase GspE and the membrane proteins GspL, GspM, GspC, and GspF.The pseudopilus is a key element of the T2SS that forms a helical fiber spanning the periplasm. The fiber is assembled from multiple subunits of the major pseudopilin GspG (4, 5, 10–14). The pseudopilus is thought to form a plug of the secretin pore in the outer membrane and/or to function as a piston during protein secretion. In recent years, studies of the T2SS pseudopilins led to structure determinations of all individual pseudopilins (13, 15–17). The recent structure of the helical ternary complex of GspK-GspI-GspJ suggested that these three minor pseudopilins form the tip of the pseudopilus (17). A crystal structure of GspG from Klebsiella oxytoca was in a previous study combined with electron microscopy data to arrive at a helical arrangement, with no evidence for special features, such as disulfide bridges, other covalent links, or metal-binding sites, for stabilizing this major pseudopilin or the pseudopilus (13).The pseudopilins of the T2SS share a common fold with the type 4 pilins (15–21). Pilins are proteins incorporated into pili, long appendages on the surface of bacteria forming thin, strong fibers with multiple functions (19, 21). Type 4 pilins and pseudopilins contain a prepilin leader sequence that is cleaved off by a prepilin peptidase, yielding mature protein (10, 11, 22). A distinct feature of the type 4 pilins is the occurrence of a disulfide bridge connecting β4 to a Cys in the so-called “D-region” near the C terminus (21). In a recent study (23) on the thin fibers of Gram-positive bacteria, isopeptide units appeared to be essential for providing these filaments sufficient cohesion and stability. A key question was therefore whether the major pseudopilin GspG also requires a special feature to obtain sufficient stability to perform its function.