Molecular Mechanisms of Macular Degeneration Associated with the Complement Factor H Y402H Mutation.

A single nucleotide polymorphism, tyrosine at position 402 to histidine (Y402H), within the gene encoding complement factor H (FH) predisposes individuals to acquiring age-related macular degeneration (AMD) after aging. This polymorphism occurs in short consensus repeat (SCR) 7 of FH and results in decreased binding affinity of SCR6-8 for heparin. As FH is responsible for regulating the complement system, decreased affinity for heparin results in decreased regulation on surfaces of self. To understand the involvement of the Y402H polymorphism in AMD, we leverage methods from bioinformatics and computational biophysics to quantify structural and dynamical differences between SCR7 isoforms that contribute to decreased pattern recognition in SCR7H402. Our data from molecular and Brownian dynamics simulations suggest a revised mechanism for decreased heparin binding. In this model, transient contacts not observed in structures for SCR7 are predicted to occur in molecular dynamics simulations between coevolved residues Y402 and I412, stabilizing SCR7Y402 in a conformation that promotes association with heparin. H402 in the risk isoform is less likely to form a contact with I412 and samples a larger conformational space than Y402. We observe energy minima for sidechains of Y402 and R404 from SCR7Y402 that are predicted to associate with heparin at a rate constant faster than energy minima for sidechains of H402 and R404 from SCR7H402. As both carbohydrate density and degree of sulfation decrease with age in Bruch's membrane of the macula, the decreased heparin recognition of SCR7H402 may contribute to the pathogenesis of AMD.

Crosslinked flagella as a stabilized vaccine adjuvant scaffold.

BACKGROUND: Engineered vaccine proteins incorporating both antigen and adjuvant components are constructed with the aim of combining functions to induce effective protective immunity. Bacterial flagellin is a strong candidate for an engineered vaccine scaffold as it is known to provide adjuvant activity through its TLR5 and inflammasome activation. Moreover, polymerized flagellin filaments can elicit a more robust immunoglobulin response than monomeric flagellin, and the multimeric antigen form can also promote T cell-independent antibody responses. Here, we aim to produce and test a covalently stabilized polymerized flagellar filament, providing additional immune efficacy through stabilization of its polymeric filament structure, as well as stabilization for long-term storage. RESULTS: Computational modeling of monomer packing in flagellin filaments helped identify amino acids with proximity to neighboring flagella protofilaments. Paired cysteine substitutions were made at amino acids predicted to form inter-monomer disulfide cross-links, and these substitutions were capable of forming flagella when transfected into a flagellin-negative strain of Salmonella enterica subspecies Typhimurium. Interestingly, each paired substitution stabilized different helical conformational polymorphisms; the stabilized filaments lost the ability to transition between conformations, reducing bacterial motility. More importantly, the paired substitutions enabled extensive disulfide cross links and intra-filament multimer formation, and in one of the three variants, permitted filament stability in high acidic and temperature conditions where wild-type filaments would normally rapidly depolymerize. In addition, with regard to potential adjuvant activity, all crosslinked flagella filaments were able to induce wild-type levels of epithelial NF-κB in a cell reporter system. Finally, bacterial virulence was unimpaired in epithelial adherence and invasion, and the cysteine substitutions also appeared to increase bacterial resistance to oxidizing and reducing conditions. CONCLUSIONS: We identified amino acid pairs, with cysteine substitutions, were able to form intermolecular disulfide bonds that stabilized the resulting flagellar filaments in detergent, hydrochloric acid, and high temperatures while retaining its immunostimulatory function. Flagellar filaments with disulfide-stabilized protofilaments introduce new possibilities for the application of flagella as a vaccine adjuvant. Specifically, increased stability and heat tolerance permits long-term storage in a range of temperature environments, as well as delivery under a range of clinical conditions.

Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis.

The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. This study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease.

AESOP: A Python Library for Investigating Electrostatics in Protein Interactions.

Electric fields often play a role in guiding the association of protein complexes. Such interactions can be further engineered to accelerate complex association, resulting in protein systems with increased productivity. This is especially true for enzymes where reaction rates are typically diffusion limited. To facilitate quantitative comparisons of electrostatics in protein families and to describe electrostatic contributions of individual amino acids, we previously developed a computational framework called AESOP. We now implement this computational tool in Python with increased usability and the capability of performing calculations in parallel. AESOP utilizes PDB2PQR and Adaptive Poisson-Boltzmann Solver to generate grid-based electrostatic potential files for protein structures provided by the end user. There are methods within AESOP for quantitatively comparing sets of grid-based electrostatic potentials in terms of similarity or generating ensembles of electrostatic potential files for a library of mutants to quantify the effects of perturbations in protein structure and protein-protein association.

Discovery of functionally selective C5aR2 ligands: novel modulators of C5a signalling.

The complement cascade is comprised of a highly sophisticated network of innate immune proteins that are activated in response to invading pathogens or tissue injury. The complement activation peptide, C5a, binds two seven transmembrane receptors, namely the C5a receptor 1 (C5aR1) and C5a receptor 2 (C5aR2, or C5L2). C5aR2 is a non-G-protein-signalling receptor whose biological role remains controversial. Some of this controversy arises owing to the lack of selective ligands for C5aR2. In this study, a library of 61 peptides based on the C-terminus of C5a was assayed for the ability to selectively modulate C5aR2 function. Two ligands (P32 and P59) were identified as functionally selective C5aR2 ligands, exhibiting selective recruitment of ß-arrestin 2 via C5aR2, partial inhibition of C5a-induced ERK1/2 activation and lipopolysaccharide-stimulated interleukin-6 release from human monocyte-derived macrophages. Importantly, neither ligand could induce ERK1/2 activation or inhibit C5a-induced ERK1/2 activation via C5aR1 directly. Finally, P32 inhibited C5a-mediated neutrophil mobilisation in wild-type, but not C5aR2(-/-) mice. These functionally selective ligands for C5aR2 are novel tools that can selectively modulate C5a activity in vitro and in vivo, and thus will be valuable tools to interrogate C5aR2 function.

Peptide redesign for inhibition of the complement system: Targeting age-related macular degeneration.

PURPOSE: To redesign a complement-inhibiting peptide with the potential to become a therapeutic for dry and wet age-related macular degeneration (AMD). METHODS: We present a new potent peptide (Peptide 2) of the compstatin family. The peptide is developed by rational design, based on a mechanistic binding hypothesis, and structural and physicochemical properties derived from molecular dynamics (MD) simulation. The inhibitory activity, efficacy, and solubility of Peptide 2 are evaluated using a hemolytic assay, a human RPE cell-based assay, and ultraviolet (UV) absorption properties, respectively, and compared to the respective properties of its parent peptide (Peptide 1). RESULTS: The sequence of Peptide 2 contains an arginine-serine N-terminal extension (a characteristic of parent Peptide 1) and a novel 8-polyethylene glycol (PEG) block C-terminal extension. Peptide 2 has significantly improved aqueous solubility compared to Peptide 1 and comparable complement inhibitory activity. In addition, Peptide 2 is more efficacious in inhibiting complement activation in a cell-based model that mimics the pathobiology of dry AMD. CONCLUSIONS: We have designed a new peptide analog of compstatin that combines N-terminal polar amino acid extensions and C-terminal PEGylation extensions. This peptide demonstrates significantly improved aqueous solubility and complement inhibitory efficacy, compared to the parent peptide. The new peptide overcomes the aggregation limitation for clinical translation of previous compstatin analogs and is a candidate to become a therapeutic for the treatment of AMD.

Molecular Mechanism of Biased Ligand Conformational Changes in CC Chemokine Receptor 7.

Biased ligand binding to G protein-coupled receptors enables functional selectivity of intracellular effectors to mediate cellular function. Despite the significant advances made in characterizing the conformational states (transmembrane helical arrangements) capable of discriminating between G protein and arrestin binding, the role of the ligand in stabilizing such conformations remains unclear. To address this issue, we simulate microsecond dynamics of CC chemokine receptor 7 (CCR7) bound to its native biased ligands, CCL19 and CCL21, and detect a series of molecular switches that are mediated by various ligand-induced allosteric events. These molecular switches involve three tyrosine residues (Y112(3.32), Y255(6.51), and Y288(7.39)), three phenylalanine residues (F116(3.36), F208(5.47), and F248(6.44)), and a polar interaction between Q252(6.48) and R294(7.45) in the transmembrane domain of CCR7. Conformational changes within these switches, particularly hydrogen bond formation between Y112(3.32) and Y255(6.51), lead to global helical movements in the receptor's transmembrane helices and contribute to the transitioning of the receptor to distinct states. Ligand-induced helical movements in the receptor highlight the ability of biased ligands to stabilize the receptor in different states through a dynamic network of allosteric events.

Hybrid flagellin as a T cell independent vaccine scaffold.

BACKGROUND: To extend the potency of vaccines against infectious diseases, vaccines should be able to exploit multiple arms of the immune system. One component of the immune system that is under-used in vaccine design is the subset of B cells known to be capable of responding to repetitive antigenic epitopes and differentiate into plasma cells even in the absence of T cell help (T-independent, TI). RESULTS: To target vaccine responses from T-independent B cells, we reengineered a bacterial Flagellin (FliC) by replacing its exposed D3 domain with a viral envelope protein from Dengue virus (DENV2). The resulting hybrid FliC protein (hFliC) was able to form stable filaments decorated with conformationally intact DENV2 envelope domains. These filaments were not only capable of inducing a T cell-dependent (TD) humoral antibody response, but also significant IgM and IgG3 antibody response in a helper T cell repertoire-restricted transgenic mouse model. CONCLUSIONS: Our results provide proof-of-principle demonstration that a reengineered hybrid FliC could be used as a platform for polymeric subunit vaccines, enhancing T cell-dependent and possibly inducing T-independent antibody responses from B-1 B cells as well.

Molecular analysis of the interaction between staphylococcal virulence factor Sbi-IV and complement C3d.

Staphylococcus aureus expresses numerous virulence factors that aid in immune evasion. The four-domain staphylococcal immunoglobulin binding (Sbi) protein interacts with complement component 3 (C3) and its thioester domain (C3d)-containing fragments. Recent structural data suggested two possible modes of binding of Sbi domain IV (Sbi-IV) to C3d, but the physiological binding mode remains unclear. We used a computational approach to provide insight into the C3d-Sbi-IV interaction. Molecular dynamics (MD) simulations showed that the first binding mode (PDB code 2WY8) is more robust than the second (PDB code 2WY7), with more persistent polar and nonpolar interactions, as well as conserved interfacial solvent accessible surface area. Brownian dynamics and steered MD simulations revealed that the first binding mode has faster association kinetics and maintains more stable intermolecular interactions compared to the second binding mode. In light of available experimental and structural data, our data confirm that the first binding mode represents Sbi-IV interaction with C3d (and C3) in a physiological context. Although the second binding mode is inherently less stable, we suggest a possible physiological role. Both binding sites may serve as a template for structure-based design of novel complement therapeutics.

Novel compstatin family peptides inhibit complement activation by drusen-like deposits in human retinal pigmented epithelial cell cultures.

We have used a novel human retinal pigmented epithelial (RPE) cell-based model that mimics drusen biogenesis and the pathobiology of age-related macular degeneration to evaluate the efficacy of newly designed peptide inhibitors of the complement system. The peptides belong to the compstatin family and, compared to existing compstatin analogs, have been optimized to promote binding to their target, complement protein C3, and to enhance solubility by improving their polarity/hydrophobicity ratios. Based on analysis of molecular dynamics simulation data of peptide-C3 complexes, novel binding features were designed by introducing intermolecular salt bridge-forming arginines at the N-terminus and at position -1 of N-terminal dipeptide extensions. Our study demonstrates that the RPE cell assay has discriminatory capability for measuring the efficacy and potency of inhibitory peptides in a macular disease environment.

The two sides of complement C3d: evolution of electrostatics in a link between innate and adaptive immunity.

The interaction between complement fragment C3d and complement receptor 2 (CR2) is a key aspect of complement immune system activation, and is a component in a link between innate and adaptive immunities. The complement immune system is an ancient mechanism for defense, and can be found in species that have been on Earth for the last 600 million years. However, the link between the complement system and adaptive immunity, which is formed through the association of the B-cell co-receptor complex, including the C3d-CR2 interaction, is a much more recent adaptation. Human C3d and CR2 have net charges of -1 and +7 respectively, and are believed to have evolved favoring the role of electrostatics in their functions. To investigate the role of electrostatics in the function and evolution of human C3d and CR2, we have applied electrostatic similarity methods to identify regions of evolutionarily conserved electrostatic potential based on 24 homologues of complement C3d and 4 homologues of CR2. We also examine the effects of structural perturbation, as introduced through molecular dynamics and mutations, on spatial distributions of electrostatic potential to identify perturbation resistant regions, generated by so-called electrostatic "hot-spots". Distributions of electrostatic similarity based on families of perturbed structures illustrate the presence of electrostatic "hot-spots" at the two functional sites of C3d, while the surface of CR2 lacks electrostatic "hot-spots" despite its excessively positive nature. We propose that the electrostatic "hot-spots" of C3d have evolved to optimize its dual-functionality (covalently attaching to pathogen surfaces and interaction with CR2), which are both necessary for the formation B-cell co-receptor complexes. Comparison of the perturbation resistance of the electrostatic character of the homologues of C3d suggests that there was an emergence of a new role of electrostatics, and a transition in the function of C3d, after the divergence of jawless fish.

A gain-of-function mutation of Arabidopsis lipid transfer protein 5 disturbs pollen tube tip growth and fertilization.

During compatible pollination of the angiosperms, pollen tubes grow in the pistil transmitting tract (TT) and are guided to the ovule for fertilization. Lily (Lilium longiflorum) stigma/style Cys-rich adhesin (SCA), a plant lipid transfer protein (LTP), is a small, secreted peptide involved in pollen tube adhesion-mediated guidance. Here, we used a reverse genetic approach to study biological roles of Arabidopsis thaliana LTP5, a SCA-like LTP. The T-DNA insertional gain-of-function mutant plant for LTP5 (ltp5-1) exhibited ballooned pollen tubes, delayed pollen tube growth, and decreased numbers of fertilized eggs. Our reciprocal cross-pollination study revealed that ltp5-1 results in both male and female partial sterility. RT-PCR and beta-glucuronidase analyses showed that LTP5 is present in pollen and the pistil TT in low levels. Pollen-targeted overexpression of either ltp5-1 or wild-type LTP5 resulted in defects in polar tip growth of pollen tubes and thereby decreased seed set, suggesting that mutant ltp5-1 acts as a dominant-active form of wild-type LTP5 in pollen tube growth. The ltp5-1 protein has additional hydrophobic C-terminal sequences, compared with LTP5. In our structural homology/molecular dynamics modeling, Tyr-91 in ltp5-1, replacing Val-91 in LTP5, was predicted to interact with Arg-45 and Tyr-81, which are known to interact with a lipid ligand in maize (Zea mays) LTP. Thus, Arabidopsis LTP5 plays a significant role in reproduction.

Influence of electrostatics on the complement regulatory functions of Kaposica, the complement inhibitor of Kaposi's sarcoma-associated herpesvirus.

Kaposica, the complement regulator of Kaposi's sarcoma-associated herpesvirus, inhibits complement by supporting factor I-mediated inactivation of the proteolytically activated form of C3 (C3b) and C4 (C4b) (cofactor activity [CFA]) and by accelerating the decay of classical and alternative pathway C3-convertases (decay-accelerating activity [DAA]). Previous data suggested that electrostatic interactions play a critical role in the binding of viral complement regulators to their targets, C3b and C4b. We therefore investigated how electrostatic potential on Kaposica influences its activities. We built a homology structure of Kaposica and calculated the electrostatic potential of the molecule, using the Poisson-Boltzmann equation. Mutants were then designed to alter the overall positive potential of the molecule or of each of its domains and linkers by mutating Lys/Arg to Glu/Gln, and the functional activities of the expressed mutants were analyzed. Our data indicate that 1) positive potential at specific sites and not the overall positive potential on the molecule guides the CFAs and classical pathway DAA; 2) positive potential around the linkers between complement control protein domains (CCPs) 1-2 and 2-3 is more important for DAAs than for CFAs; 3) positive potential in CCP1 is crucial for binding to C3b and C4b, and thereby its functional activities; 4) conversion to negative or enhancement of negative potential for CCPs 2-4 has a marked effect on C3b-linked activities as opposed to C4b-linked activities; and 5) reversal of the electrostatic potential of CCP4 to negative has a differential effect on classical and alternative pathway DAAs. Together, our data provide functional relevance to conservation of positive potential in CCPs 1 and 4 and the linkers of viral complement regulators.

Design of a modified mouse protein with ligand binding properties of its human analog by molecular dynamics simulations: the case of C3 inhibition by compstatin.

The peptide compstatin and its derivatives inhibit the complement-component protein C3 in primate mammals and are potential therapeutic agents against the unregulated activation of complement in humans, but are inactive against C3 from lower mammals. Recent molecular dynamics (MD) simulations showed that the most potent compstatin analog comprised entirely of natural amino acids (W4A9) had a smaller affinity for rat C3, due to reproducible changes in the rat protein structure with respect to the human protein, which eliminated or weakened specific protein-ligand interactions seen in the human C3:W4A9 complex. Here, we study by MD simulations three W4A9 complexes with the mouse C3 protein, and two "transgenic" mouse derivatives, containing a small number (6-9) of human C3 substitutions. The mouse complex experiences the conformational changes and affinity reduction of the rat complex. In the "transgenic" complexes, the conformation remains closer to that of the human complex, the protein-ligand interactions are improved, and the affinity for compstatin becomes "human-like." The present work creates new avenues for a compstatin-sensitive animal model. A similar strategy, involving the comparison of a series of complexes by MD simulations, could be used to design "transgenic" sequences in other systems.

Contribution of specific amino acid changes in penicillin binding protein 1 to amoxicillin resistance in clinical Helicobacter pylori isolates.

Amoxicillin is commonly used to treat Helicobacter pylori, a major cause of peptic ulcers, stomach cancer, and B-cell mucosa-associated lymphoid tissue lymphoma. Amoxicillin resistance in H. pylori is increasing steadily, especially in developing countries, leading to treatment failures. In this study, we characterize the mechanism of amoxicillin resistance in the U.S. clinical isolate B258. Transformation of amoxicillin-susceptible strain 26695 with the penicillin binding protein 1 gene (pbp1) from B258 increased the amoxicillin resistance of 26695 to equal that of B258, while studies using biotinylated amoxicillin showed a decrease in the binding of amoxicillin to the PBP1 of B258. Transformation with 4 pbp1 fragments, each encompassing several amino acid substitutions, combined with site-directed mutagenesis studies, identified 3 amino acid substitutions in PBP1 of B258 which affected amoxicillin susceptibility (Val 469 Met, Phe 473 Leu, and Ser 543 Arg). Homology modeling showed the spatial orientation of these specific amino acid changes in PBP1 from 26695 and B258. The results of these studies demonstrate that amoxicillin resistance in the clinical U.S. isolate B258 is due solely to an altered PBP1 protein with a lower binding affinity for amoxicillin. Homology modeling analyses using previously identified amino acid substitutions of amoxicillin-resistant PBP1s demonstrate the importance of specific amino acid substitutions in PBP1 that affect the binding of amoxicillin in the putative binding cleft, defining those substitutions deemed most important in amoxicillin resistance.

Systems Biology Modeling of the Complement System Under Immune Susceptible Pathogens.

The complement system is assembled from a network of proteins that function to bring about the first line of defense of the body against invading pathogens. However, complement deficiencies or invasive pathogens can hijack complement to subsequently increase susceptibility of the body to infections. Moreover, invasive pathogens are increasingly becoming resistant to the currently available therapies. Hence, it is important to gain insights into the highly dynamic interaction between complement and invading microbes in the frontlines of immunity. Here, we developed a mathematical model of the complement system composed of 670 ordinary differential equations with 328 kinetic parameters, which describes all three complement pathways (alternative, classical, and lectin) and includes description of mannose-binding lectin, collectins, ficolins, factor H-related proteins, immunoglobulin M, and pentraxins. Additionally, we incorporate two pathogens: (type 1) complement susceptible pathogen and (type 2) Neisseria meningitidis located in either nasopharynx or bloodstream. In both cases, we generate time profiles of the pathogen surface occupied by complement components and the membrane attack complex (MAC). Our model shows both pathogen types in bloodstream are saturated by complement proteins, whereas MACs occupy <<1.0% of the pathogen surface. Conversely, the MAC production in nasopharynx occupies about 1.5-10% of the total N. meningitidis surface, thus making nasal MAC levels at least about eight orders of magnitude higher. Altogether, we predict complement-imbalance, favoring overactivation, is associated with nasopharynx homeostasis. Conversely, orientating toward complement-balance may cause disruption to the nasopharynx homeostasis. Thus, for sporadic meningococcal disease, our model predicts rising nasal levels of complement regulators as early infection biomarkers.

Role of Electrostatic Hotspots in the Selectivity of Complement Control Proteins Toward Human and Bovine Complement Inhibition.

Poxviruses are dangerous pathogens, which can cause fatal infection in unvaccinated individuals. The causative agent of smallpox in humans, variola virus, is closely related to the bovine vaccinia virus, yet the molecular basis of their selectivity is currently incompletely understood. Here, we examine the role of the electrostatics in the selectivity of the smallpox protein SPICE and vaccinia protein VCP toward the human and bovine complement protein C3b, a key component of the complement immune response. Electrostatic calculations, in-silico alanine-scan and electrostatic hotspot analysis, as introduced by Kieslich and Morikis (PLoS Comput. Biol. 2012), are used to assess the electrostatic complementarity and to identify sites resistant to local perturbation where the electrostatic potential is likely to be evolutionary conserved. The calculations suggest that the bovine C3b is electrostatically prone to selectively bind its VCP ligand. On the other hand, the human isoform of C3b exhibits a lower electrostatic complementarity toward its SPICE ligand. Yet, the human C3b displays a highly preserved electrostatic core, which suggests that this isoform could be less selective in binding different ligands like SPICE and the human Factor H. This is supported by experimental cofactor activity assays revealing that the human C3b is prone to bind both SPICE and Factor H, which exhibit diverse electrostatic properties. Additional investigations considering mutants of SPICE and VCP that revert their selectivity reveal an "electrostatic switch" into the central modules of the ligands, supporting the critical role of the electrostatics in the selectivity. Taken together, these evidences provide insights into the selectivity mechanism of the complement regulator proteins encoded by the variola and vaccinia viruses to circumvent the complement immunity and exert their pathogenic action. These fundamental aspects are valuable for the development of novel vaccines and therapeutic strategies.

SPARC coordinates extracellular matrix remodeling and efficient recruitment to and migration of antigen-specific T cells in the brain following infection.

Central nervous system (CNS) injury and infection can result in profound tissue remodeling in the brain, the mechanism and purpose of which is poorly understood. Infection with the protozoan parasite Toxoplasma gondii causes chronic infection and inflammation in the brain parenchyma. Control of parasite replication requires the continuous presence of IFNγ-producing T cells to keep T. gondii in its slowly replicating cyst form. During infection, a network of extracellular matrix fibers, revealed using multiphoton microscopy, forms in the brain. The origin and composition of these structures are unknown but the fibers have been observed to act as a substrate for migrating T cells. In this study, we show a critical regulator of extracellular matrix (ECM) remodeling, Secreted Protein, Acidic, Rich in Cysteine (SPARC), is upregulated in the brain during the early phases of infection in the frontal cortex. In the absence of SPARC, a reduced and disordered fibrous network, increased parasite burden, and reduced antigen-specific T cell entry into the brain points to a role for SPARC in T cell recruitment to and migration within the brain. We also report SPARC can directly bind to CCR7 ligands CCL19 and CCL21 but not CXCL10, and enhance migration toward a chemokine gradient. Measurement of T cell behavior points to tissue remodeling being important for access of immune cells to the brain and facilitating cellular locomotion. Together, these data identify SPARC as an important regulatory component of immune cell trafficking and access to the inflamed CNS.

Species specificity of the complement inhibitor compstatin investigated by all-atom molecular dynamics simulations.

The development of compounds to regulate the activation of the complement system in non-primate species is of profound interest because it can provide models for human diseases. The peptide compstatin inhibits protein C3 in primate mammals and is a potential therapeutic agent against unregulated activation of complement in humans but is inactive against nonprimate species. Here, we elucidate this species specificity of compstatin by molecular dynamics simulations of complexes between the most potent natural compstatin analog and human or rat C3. The results are compared against an experimental conformation of the human complex, determined recently by X-ray diffraction at 2.4-A resolution. The human complex simulations provide information on the relative contributions to stability of specific C3 and compstatin residues. In the rat simulations, the protein undergoes reproducible conformational changes, which eliminate or weaken specific interactions and reduce the complex stability. The simulation insights can be used to design improved compstatin-based inhibitors for human C3 and active inhibitors against lower mammals.

A multifaceted study of stigma/style cysteine-rich adhesin (SCA)-like Arabidopsis lipid transfer proteins (LTPs) suggests diversified roles for these LTPs in plant growth and reproduction.

Lily stigma/style cysteine-rich adhesin (SCA), a plant lipid transfer protein (LTP) which is secreted into the extracellular matrix, functions in pollen tube guidance in fertilization. A gain-of-function mutant (ltp5-1) for Arabidopsis LTP5, an SCA-like molecule, was recently shown to display defects in sexual reproduction. In the current study, it is reported that ltp5-1 plants have dwarfed primary shoots, delayed hypocotyl elongation, various abnormal tissue fusions, and display multibranching. These mutant phenotypes in vegetative growth are recessive. No abnormality was found in ltp5-1/+ plants. In a phylogenetic analysis of plant LTPs, SCA-like Arabidopsis LTPs were classified with conventional plant LTPs. Homology modelling-based electrostatic similarity index (ESI) clustering was used to show diversity in spatial distributions of electrostatic potentials of SCA-like LTPs, suggestive of their various roles in interaction in the extracellular matrix space. ß-Glucuronidase (GUS) analysis showed that SCA-like Arabidopsis LTP genes are diversely present in various tissues. LTP4 was found specifically in the guard cells and LTP6 in trichomes as well as in other tissues. LTP1 levels were specifically abundant in the stigma, and both LTP3 and LTP6 in the ovules. LTP2 and LTP4 gene levels were up-regulated in whole seedlings with 20% polyethylene glycol (PEG) and 300 mM NaCl treatments, respectively. LTP5 was up-regulated in the hypocotyl with 3 d dark growth conditions. LTP6 was specifically expressed in the tip of the cotyledon under drought stress conditions. The results suggest that SCA-like Arabidopsis LTPs are multifunctional, with diversified roles in plant growth and reproduction.