Corruption of host seven-transmembrane proteins by pathogenic microbes: a common theme in animals and plants?

Human diseases like AIDS, malaria, and pneumonia are caused by pathogens that corrupt host chemokine G-protein coupled receptors for molecular docking. Comparatively, little is known about plant host factors that are required for pathogenesis and that may serve as receptors for the entry of pathogenic microbes. Here, we review potential analogies between human chemokine receptors and the plant seven-transmembrane MLO protein, a candidate serving a dual role as docking molecule and defence modulator for the phytopathogenic powdery mildew fungus.     

Microscopy basics and the study of actin–actin-binding protein interactions

Actin is a multifunctional eukaryotic protein with a globular monomer form that polymerizes into a thin, linear microfilament in cells. Through interactions with various actin-binding proteins (ABPs), actin plays an active role in many cellular processes, such as cell motility and structure. Microscopy techniques are powerful tools for determining the role and mechanism of actin–ABP interactions in these processes. In this article, we describe the basic concepts of fluorescent speckle microscopy, total internal reflection fluorescence microscopy, atomic force microscopy, and cryoelectron microscopy and review recent studies that utilize these techniques to visualize the binding of actin with ABPs.     

Stability and amino acid preferences of type VIII reverse turn: the most common turn in peptides?

Free energies of the alpha(r)beta and betabeta conformations of 14 tetrapeptides, based on the sequence SALN and protein X-ray structures, were calculated using molecular dynamics simulations and MM-PBSA calculations. The alphaalpha conformations of five of the tetrapeptides were also studied. SALN has been earlier shown by molecular dynamics simulations and NMR spectroscopy to have a tendency to form an alpha(r)beta turn. The gas-phase energy of the molecular mechanical force field (CHARMM), the electrostatic and non-polar solvation free energies and solute entropies were used to explain the free energy differences of the alphaalpha, betabeta and alpha(r)beta conformations of the peptides. The alpha(r)beta conformation of SALN and SATN was predicted to be slightly more stable than the extended conformation (betabeta), in agreement with experimental results. The SALN mutants SAIN, SAVN, SATN, SSIN and MSHV, were also predicted to be potential alpha(r)beta turn-forming peptides. We report also revised positional potentials for the type VIII turn, based on a non-homologous set of protein structures. This protein databank analysis confirms the main results of the earlier analyses and reveals several new amino acid residues with a significant positional preference. The results of this work led us to suggest that the alpha(r)beta turn may be the most common turn type in peptides. Such turns may be readily formed in aqueous solution and thereby play important roles in the protein folding process by serving as an initiation point for structure formation.     

Is there evidence for a common amino acid sequence in proteins with membrane attaching ability?

A comparison between the primary sequence of staphylococcal protein A (SpA) and a wide range of published protein sequences revealed a limited, but striking homology in approximately two-thirds of them. The region in the SpA sequence with the common homology was identified as the octapeptide repeats comprising the cell wall peptidoglycan-binding domain. Available structural information and the known location of the proteins within their host cells suggests that this common octapeptide may be important in interaction of the protein with the cell surface (either membrane or wall).     

The effect of amino acid substitution in the imperfect repeat sequences of α-synuclein on fibrillation

Human α-synuclein is the causative protein of several neurodegenerative diseases, such as Parkinson's disease (PD) and dementia with Lewy Bodies (DLB). The N-terminal half of α-synuclein contains seven imperfect repeat sequences. One of the PD/DLB-causing point mutations, E46K, has been reported in the imperfect repeat sequences of α-synuclein, and is prone to form amyloid fibrils. The presence of seven imperfect repeats in α-synuclein raises the question of whether or not mutations corresponding to E46K in the other imperfect KTKE(Q)GV repeats have similar effects on aggregation and fibrillation, as well as their propensities to form α-helices. To investigate the effect of E(Q)/K mutations in each imperfect repeat sequence, we substituted the amino acid corresponding to E46K in each of the seven repeated sequences with a Lys residue. The mutations in the imperfect KTKE(Q)GV repeat sequences of the N-terminal region were prone to decrease the lag time of fibril formation. In addition, AFM imaging suggested that the Q24K mutant formed twisted fibrils, while the other mutants formed spherical aggregates and short fibrils. These observations indicate that the effect of the mutations on the kinetics of fibril formation and morphology of fibrils varies according to their location.     

The amino acid sequences of the α subunits of the lectins from the seeds of Lathyrus hirsutus and Lathyrus tingitanus

The amino acid sequences of the α₁ and α₂ subunits of the isolectins (LhL1 and LhL2) from seeds of Lathyrus hirsutus and the α subunit of the lectin from L. tingitanus were determined by analysis of peptides derived from the proteins by separate digestions with chymotrypsin and the protease from S. aureus V8. The α₁ subunit of the L. hirsutus LhL1 isolectin differed from the α₂ form in LhL2 only in having an extra lysine inserted near the C-terminus. The L. tingitanus α subunit differed from the L. hirsutus α₁ in three positions and from L. hirsutus α₂ in four.     

Clostridium botulinum types A,B, C1, and E produce proteins with or without hemagglutinating activity: Do they share common amino acid sequences and genes?

Clostridium botulinum produce the antigenically distinct 150 kD neurotoxin serotypes (e.g., A, B, C₁, and E) and simultaneously proteins, A Hn⁺, B Hn⁺, C Hn⁺, and E Hn^?, that have high, low, and no hemagglutinating activity. A Hn⁺ and B Hn⁺ are serologically cross-reactive. A Hn⁺, B Hn⁺, and C Hn⁺ found as large aggregates (900–220 kD) can be dissociated on SDS-PAGE into multiple subunits, the smallest for A Hn⁺, B Hn⁺ is 17 kD and 27 kD for C Hn⁺. The 116 kD E Hn^? does not aggregate. We determined the sequences of 10–33 amino terminal residues of the 17, 21.5, 35, and 57 kD subunits of A Hn⁺ and B Hn⁺. Each of these subunits have unique sequences, indicating that the larger units studied are not homomers or heteromers of smaller units. The subunits of A Hn⁺ and B Hn⁺ of comparable size have striking sequence identity (e.g., 21.5 kD subunits from the two are identical and 57 kD subunits have 80% identity).In vitro proteolysis of 116 kD E Hn^? with different proteases did not impart hemagglutinating activity to the fragments. The 116 kD E Hn^? and one of its proteolytic fragments (87 kD) were partially sequenced. Sixty-two base pairs downstream from the termination codon of the cloned 33 kD subunit of C Hn⁺, there is an initiation codon followed by an open reading frame for at least 34 amino acid residues (Tsuzukiet al., 1990). The derived amino acid sequence of this open reading frame, we found, has 73–84% sequence identity with those of the 17 kD subunits of A Hn⁺ and B Hn⁺ and significant identity with the N-terminal of E Hn^?. These highly conserved sequences show existence of genetic linkage among the Hn⁺ and Hn^? proteins.     

Comparison of amino acid sequences of eleven different α-amylases

Summary A comparison was made of the amino acid sequences of 11 different -amylases. The 6 animal -amylases tested were found to be highly homologous (about 80 to 90%, depending on the species compared). Amino acid sequence of Bacillus stearothermophilus -amylase was fairly homologous (about 60%) with that of a thermostable -amylase from Bacillus amyloliquefaciens. Homology was least among the thermolabile amylases from Bacillus subtilis, Aspergillus oryzae, plants and animals. Nevertheless, four highly homologous regions were found in the amino acid sequences of all the enzymes, despite their widely different origins. It was inferred that these four homologous regions were likely to be the active and/or substrate-binding sites.     

Comprehensive analysis and characterization of the TCR α chain sequences in the common marmoset

The common marmoset (Callithrix jacchus) is useful as a nonhuman primate model of human diseases. Although the marmoset model has great potential for studying autoimmune diseases and immune responses against pathogens, little information is available regarding the genes involved in adaptive immunity. Here, we identified one TCR α constant (TRAC), 46 TRAJ (joining), and 35 TRAV (variable) segments from marmoset cDNA. Marmoset TRAC, TRAJ, and TRAV shared 80%, 68–100%, and 79–98% identity with their human counterparts at the amino acid level, respectively. The amino acid sequences were less conserved in TRAC than in TCRβ chain constant (TRBC). Comparative analysis of TRAV between marmosets and humans showed that the rates of synonymous substitutions per site (d _S ) were not significantly different between the framework regions (FRs) and complementarity determining regions (CDRs), whereas the rates of nonsynonymous substitutions per site (d _N ) were significantly lower in the FRs than in CDRs. Interestingly, the d _N values of the CDRs were greater for TRBV than TRAV. These results suggested that after the divergence of Catarrhini from Platyrrhini, amino acid substitutions were decreased in the FRs by purifying selection and occurred more frequently in CDRβ than in CDRα by positive selection, probably depending on structural and functional constraints. This study provides not only useful information facilitating the investigation of adaptive immunity using the marmoset model but also new insight into the molecular evolution of the TCR heterodimer in primate species.     

The amino acid sequences of the Fd fragments of two human γ heavy chains

The amino acid sequences of the Fd fragments of two human pathological immunoglobulins of the immunoglobulin G1 class are reported. Comparison of the two sequences shows that the heavy-chain variable regions are similar in length to those of the light chains. The existence of heavy chain variable region subgroups is also deduced, from a comparison of these two sequences with those of another gamma 1 chain, Eu, a mu chain, Ou, and the partial sequence of a fourth gamma 1 chain, Ste. Carbohydrate has been found to be linked to an aspartic acid residue in the variable region of one of the gamma 1 chains, Cor.     

Are the conserved sequences in segment 1 of gelsolin important for binding actin?

The minimal region required for actin binding in the smallest of the three domains of gelsolin (termed Segment 1 or S1) was previously defined by deletion mutagenesis as residues 37-126. Further analysis of NH2-terminal deletions here redefines the minimal functional core as residues 41-126. Amino acid substitutions within this core further elucidate the nature of the interaction of segment 1 with actin. Of 26 point mutants analyzed, 14 reduced the affinity for actin. The charged residues His 119, Arg 120, Glu 121, and Gln 123 appear to be involved in direct interaction with actin. Substitutions of Leu 108, Leu 112, and Val 117 by polar groups all affect the structural stability of segment 1 and thereby reduce binding affinity. In addition replacement of Glu 126 by aspartic acid modifies the physical properties of segment 1 and weakens binding. We have further shown that changing charged residues within the highly conserved pentapeptide sequence LDDYL (residues 108-112) has no effect on actin binding. This sequence, found in a number of different actin binding proteins, does not therefore constitute part of the interaction site. Similarly, substitution of the two acidic residues by basic ones within the DESG motif of segment 1 (residues 96-99, but also found near the COOH terminus of actin) does not impair binding. These results show the dangers of predicting functional sites on the basis of conserved sequences.     

Relationship between structure and amino acid sequence of strongly twisted and coiled β-hairpins in globular proteins

β-Hairpins are widespread in proteins, and it is possible to find them both within β-sheets and separately. In this work, a comparative analysis of amino acid sequences of β-strands within strongly twisted β-hairpins from different structural protein subclasses has been conducted. Strongly twisted and coiled β-hairpin generates in the space a right double helix out of β-strands that are connected by a loop region (connections). The frequencies of amino acid residues on the internal (concave) and external (convex) surfaces of strongly twisted β-hairpins have been determined (220 β-hairpins from nonhomologous proteins were studied). The concave surface of these β-hairpins is mainly generated by hydrophobic residues, while the convex surface by hydrophilic residues; accordingly, the alternation of hydrophobic internal and hydrophilic external residues is observed in their amino acid sequences. Amino acid residues of glycine and alanine (especially in places of the largest twisting of the strands) were anomalously frequently found in internal positions of strongly twisted and coiled β-hairpins. It was established that internal positions never contain the proline residues, while external positions in the twisting region contain them in a relatively large amount. It was demonstrated that at least one amino acid residue in α_L- or ε-conformation is required for generation of relatively short (up to 7 amino acid residues) connection. As a rule, these positions are occupied by glycines. Thus, not only the alternation of hydrophobic and hydrophilic amino acid residues, but also the presence of one or two glycine residues in the connection region and the excess of glycines and alanines in the places of the largest strand twisting on the concave surface, as well as the presence of prolines on the convex surface, are required to generate a strongly twisted and coiled β-hairpin.     

Structure of α-keratin: Structural implication of the amino acid sequences of the type I and type II chain segments

Two amino acid sequences from potentially helical fragments of low-sulphur proteins from α-keratin have been analysed computationally and periods 9.4 and 28 residues long noted in the axial disposition of charged residues. Ionic interactions between chains have also been calculated and these indicate a preference for the helical fragments to aggregate in parallel with zero shift between chains in a manner essentially identical to that found for α-tropomyosin.     

Partial amino acid sequences of κ-chains of rat immunoglobulins: Genetic and evolutionary implications

Partial amino acid sequences have been determined for several -type light chains prepared from sera or urine of inbred LOU/C/Wsl rats bearing plasma cell tumors. Comparison of these sequences with those of human, rabbit, and mouse -chains available in the literature indicates that the constant region of rat -chains shows more amino acid sequence homology to that of the mouse -chain than to human and rabbit -chains, a result expected from the phylogenetic relationship of the species compared. Examination of the N-terminal amino acid sequences indicated that the variable regions of rat -chains can also be classified into subgroups according to degree of sequence homology in a manner similar to that done for -chains of other species (e.g., human, rabbit, and mouse). However, the prototype amino acid sequences of -chain variable region subgroups of the rat were not homologous to those of other species including the closely related mouse. The implications of this observation with respect to the genetics and evolution of immunoglobulins are discussed.Supported in part by grants from the USPHS (AI-13388 and AI-12840), National Science Foundation (BMS-75-09513), American Cancer Society (IM-161), South Carolina State Appropriation for Research, and Fonds Cancerologique de la CGER, Belgium. A.-C. W. is the recipient of American Cancer Society Faculty Research Award FRA-125; H.B. is a staff member of EURATOM, Biology Division (publication No. 1212).This is paper No. 4 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, South Carolina.     

Degradation of negative regulators: a common theme in hormone and light signaling networks?

Signal transduction pathways often modulate both positively and negatively acting components to optimize the efficiency of a signal. Recent results have shown that plants make extensive use of regulated proteolysis to modulate signal transduction pathways. An emerging theme from hormone (e.g. auxin and gibberellin) and light signaling pathways is signal or stimulus-induced degradation of negative regulators to optimize plant growth and development.     

Screening of transgenic proteins expressed in transgenic food crops for the presence of short amino acid sequences identical to potential,IgE – binding linear epitopes of allergens

Background  

Transgenic proteins expressed by genetically modified food crops are evaluated for their potential allergenic properties prior to marketing, among others by identification of short identical amino acid sequences that occur both in the transgenic protein and allergenic proteins. A strategy is proposed, in which the positive outcomes of the sequence comparison with a minimal length of six amino acids are further screened for the presence of potential linear IgE-epitopes. This double track approach involves the use of literature data on IgE-epitopes and an antigenicity prediction algorithm.     

Anion–π interactions in complexes of proteins and halogen-containing amino acids

We analyzed the potential influence of anion–π interactions on the stability of complexes of proteins and halogen-containing non-natural amino acids. Anion–π interactions are distance and orientation dependent and our ab initio calculations showed that their energy can be lower than ?8 kcal mol^?1, while most of their interaction energies lie in the range from ?1 to ?4 kcal mol^?1. About 20 % of these interactions were found to be repulsive. We have observed that Tyr has the highest occurrence among the aromatic residues involved in anion–π interactions, while His made the least contribution. Furthermore, our study showed that 67 % of total interactions in the dataset are multiple anion–π interactions. Most of the amino acid residues involved in anion–π interactions tend to be buried in the solvent-excluded environment. The majority of the anion–π interacting residues are located in regions with helical secondary structure. Analysis of stabilization centers for these complexes showed that all of the six residues capable of anion–π interactions are important in locating one or more of such centers. We found that anion–π interacting residues are sometimes involved in simultaneous interactions with halogens as well. With all that in mind, we can conclude that the anion–π interactions can show significant influence on molecular organization and on the structural stability of the complexes of proteins and halogen-containing non-natural amino acids. Their influence should not be neglected in supramolecular chemistry and crystal engineering fields as well.     

A compilation of amino acid analyses of proteins—XVI. residues per molecule—13

A compilation of the amino acid analyses as residues per molecular weight of 164 proteins is presented. In addition the molecular weight, amide distribution as glutamine and asparagine residues and carbohydrate content is also provided.