Vaccine potentials of an intrinsically unstructured fragment derived from the blood stage-associated Plasmodium falciparum protein PFF0165c.

We have identified new malaria vaccine candidates through the combination of bioinformatics prediction of stable protein domains in the Plasmodium falciparum genome, chemical synthesis of polypeptides, in vitro biological functional assays, and association of an antigen-specific antibody response with protection against clinical malaria. Within the predicted open reading frame of P. falciparum hypothetical protein PFF0165c, several segments with low hydrophobic amino acid content, which are likely to be intrinsically unstructured, were identified. The synthetic peptide corresponding to one such segment (P27A) was well recognized by sera and peripheral blood mononuclear cells of adults living in different regions where malaria is endemic. High antibody titers were induced in different strains of mice and in rabbits immunized with the polypeptide formulated with different adjuvants. These antibodies recognized native epitopes in P. falciparum-infected erythrocytes, formed distinct bands in Western blots, and were inhibitory in an in vitro antibody-dependent cellular inhibition parasite-growth assay. The immunological properties of P27A, together with its low polymorphism and association with clinical protection from malaria in humans, warrant its further development as a malaria vaccine candidate.

"Fluorescent timer": protein that changes color with time.

We generated a mutant of the red fluorescent protein drFP583. The mutant (E5) changes its fluorescence from green to red over time. The rate of color conversion is independent of protein concentration and therefore can be used to trace time-dependent expression. We used in vivo labeling with E5 to measure expression from the heat shock-dependent promoter in Caenorhabditis elegans and from the Otx-2 promoter in developing Xenopus embryos. Thus, E5 is a "fluorescent timer" that can be used to monitor both activation and down-regulation of target promoters on the whole-organism scale.

When protein folding is simplified to protein coiling: the continuum of solenoid protein structures.

Solenoid proteins contain repeating structural units that form a continuous superhelix. This category of proteins conveys the least complicated relationship between a sequence and the corresponding three-dimensional structure. Although solenoid proteins are divided into different classes according to commonly used classification schemes, they share many structural and functional properties.

The leucine-rich repeat as a protein recognition motif.

Leucine-rich repeats (LRRs) are 20-29-residue sequence motifs present in a number of proteins with diverse functions. The primary function of these motifs appears to be to provide a versatile structural framework for the formation of protein-protein interactions. The past two years have seen an explosion of new structural information on proteins with LRRs. The new structures represent different LRR subfamilies and proteins with diverse functions, including GTPase-activating protein rna1p from the ribonuclease-inhibitor-like subfamily; spliceosomal protein U2A', Rab geranylgeranyltransferase, internalin B, dynein light chain 1 and nuclear export protein TAP from the SDS22-like subfamily; Skp2 from the cysteine-containing subfamily; and YopM from the bacterial subfamily. The new structural information has increased our understanding of the structural determinants of LRR proteins and our ability to model such proteins with unknown structures, and has shed new light on how these proteins participate in protein-protein interactions.

Modeling of the three-dimensional structure of proteins with the typical leucine-rich repeats.

BACKGROUND: Leucine-rich repeats (LRRs) are present in proteins with diverse functions. The horseshoe-shaped structure of a ribonuclease inhibitor (RI), with a parallel beta sheet lining the inner circumference of the horseshoe and alpha helices flanking its outer circumference, is the only X-ray structure containing these repeats to be determined. Despite the fact that the lengths and sequences of the RI repeats differ from those of the most commonly occurring LRRs, it was deemed worthwhile to derive a three-dimensional structural framework of these more typical LRR proteins, using the RI structure as a template. RESULTS: Sequence alignments of 569 LRRs from 68 proteins were obtained by a profile search and used in a comparative sequence analysis to distinguish between residues with a probable structural role and those which seemed essential for function. This knowledge, along with the known atomic structure of RI, was used to model the three-dimensional structure of the most common LRR units. These modeled units were then used to build the three-dimensional structure of the extracellular domain of the thyrotropin receptor (TSHR)--a 'typical' LRR protein. CONCLUSIONS: The modeled TSHR structure adopts a non-globular arrangement, similar to that in RI. The beta regions of this typical LRR protein are the same as in the RI structure, whereas the alpha helices are shorter and the conformations of the alpha beta and beta alpha connections are different. As a result of these differences it was not possible to pack together typical LRR units using repeats such as those found in RI. This mutually exclusive relationship is supported by sequence analysis. The predicted structure of the typical LRRs obtained here can be used to build models for any of the known LRR proteins and the approach used for the prediction could be applied to other proteins containing internal repeats.

Use of transcription reporters with novel p53 binding sites to target tumour cells expressing endogenous or virally transduced p53 mutants with altered sequence-specificity.

p53 triple mutants (120N/121G/277H, 120H/121G/ 277H, 120S/121G/277H and 120H/121G/277Y) have altered sequence specificity in bandshift assays in vitro and transcription assays in vivo. These mutants activate transcription from the site TTT CATG AAA but not from wild type sites. The triple mutants activate more strongly than p53 with a single 277Y mutation. The TTT site matches the wild type p53 consensus at only 4/10 positions and is not recognised by wild type p53. 277Y mutations have been described in human tumours, and Ewing tumour cells expressing this mutant from the endogenous p53 locus selectively activate transcription from transfected luciferase reporters regulated by TTT-mutant p53 binding sites. p53 mutants with altered sequence specificity have potential advantages for cancer gene therapy: if used to activate transcription of conditionally toxic genes they would allow tumour-targeting by p53, which acts as a sensor for the malignant state, but place control over cell killing in the hands of the clinician. Rare tumours expressing such mutants from the endogenous p53 locus could be targeted directly with p53-regulated suicide vectors, but for most tumours both the p53 mutant and the reporter would need to be encoded by the virus.

Molecular packing in type I collagen fibrils. A model with neighbouring collagen molecules aligned in axial register.

A detailed stereochemical analysis of intermolecular interactions of collagens made with molecular models and summarized experimental data resulted in a new three-dimensional structural model for collagen fibrils. In this model collagen molecules aligned in axial register form a bunch. The bunches are aligned head to tail and penetrate by 300 A into each other, forming microfibrils; these in turn assemble into fibrils. The new model differs from all the others in that its characteristic axial regularity, with a period of 670 A, results from staggering of the adjacent microfibrils formed by unstaggered molecules rather than from the axial staggering of neighbouring collagen molecules.

Structural diversity of leucine-rich repeat proteins.

The superfamily of leucine-rich repeat proteins can be subdivided into at least six subfamilies, characterised by different lengths and consensus sequences of the repeats. It was proposed that the repeats from different subfamilies retain a similar superhelical fold, but differ in the three-dimensional structures of individual repeats. The sequence-structure relationship of three new subfamilies was examined by molecular modelling. I provide structural models for the repeats of all subfamilies. The models enable me to explain residue conservations within each subfamily. Furthermore, the difference in the packing explains why the repeats from different subfamilies never occur simultaneously in the same protein. Finally, these studies suggest different evolutionary origins for the different subfamilies. The approach used for the prediction of the leucine-rich repeat protein structures can be applied to other proteins containing internal repeats of about 20 to 30 residue in length.

A model of the three-dimensional structure of ice nucleation proteins.

Bacterial ice-nucleation proteins are among the most active natural ice nucleants and can reduce the supercooling point of water in plants, thereby reducing the ability of sensitive plants to avoid damaging ice formation. We describe a structural model for bacterial ice-nucleation proteins based on molecular modelling. This model predicts a largely planar extended molecule, with one side serving as a template for orienting water into an ice lattice and the other side interacting with the membrane. The model also predicts that single molecules can form aggregates of unlimited size by interdigitation.

A model of Cdc25 phosphatase catalytic domain and Cdk-interaction surface based on the presence of a rhodanese homology domain.

Mammalian Cdc25 phosphatase is responsible for the dephosphorylation of Cdc2 and other cyclin-dependent kinases at Thr14 and Tyr15, thus activating the kinase and allowing cell cycle progression. The catalytic domain of this dual-specificity phosphatase has recently been mapped to the 180 most C-terminal amino acids. Apart from a CX3R motif, which is present at the active site of all known tyrosine phosphatases, Cdc25 does not share any obvious sequence similarity with any of those enzymes. Until very recently, the Cdc25 family was the only subfamily of tyrosine phosphates for which no three-dimensional structural data were available. Using the generalized profile technique, a sensitive method for sequence database searches, we found an extended and highly significant sequence similarity between the Cdc25 catalytic domain and similarly sized regions in other proteins: the non-catalytic domain of two distinct families of MAP-kinase phosphates, the non-catalytic domain of several ubiquitin protein hydrolases, the N and C-terminal domain of rhodanese, and a large and heterogeneous groups of stress-response proteins from all phyla. The relationship of Cdc25 to the structurally well-characterized rhodanese spans the entire catalytic domain and served as template for a structural model for human Cdc25a, which is fundamentally different from previously suggested models for Cdc25 catalytic domain organization. The surface positioning of subfamily-specific conserved residues allows us to predict the sites of interaction with Cdk2, a physiological target of Cdc25a. Based on the results of this analysis, we also predict that the budding yeast arsenate resistance protein Acr2 and the ORF Ygr203w encode protein phosphatases with catalytic properties similar to that of the Cdc25 family. Recent determination of the crystal structure of the Cdc25a catalytic domain supports the validity of the model and demonstrates the power of the generalized sequence profile technique in homology-based modeling of the three-dimensional structure of a protein having a weak but significant sequence similarity with a structurally characterized protein.

Processing of Escherichia coli alkaline phosphatase: role of the primary structure of the signal peptide cleavage region.

A wide range (69) of mutant Escherichia coli alkaline phosphatases with single amino acid substitutions at positions from -5 to +1 of the signal peptide were obtained for studying protein processing as a function of the primary structure of the cleavage region. Amber suppressor mutagenesis, used to create mutant proteins, included: (i) introduction of amber mutations into respective positions of the phoA gene; and (ii) expression of each mutant phoA allele in E. coli strains producing amber suppressor tRNAs specific to Ala, Cys, Gln, Glu, Gly, His, Leu, Lys, Phe, Pro, Ser and Tyr. Most amino acid substitutions at positions -3 and -1 resulted in a complete block of protein processing. These data give new experimental support for the "-3, -1 rule". Only Ala, Gly and Ser at position -1 allowed protein processing, and Ala provided the highest rate of processing. The results revealed the more conservative nature of the amino acids at the -1 position of signal peptides of Gram-negative bacteria as compared with those of eukaryotic organisms. Position -3 was less regular, since not only Ala, Ser and Gly, but also Leu and Cys at this position, allowed the processing. Mutations at position -4 had an insignificant effect on the processing. Surprisingly, efficient processing was provided mainly by large amino acid residues at position -2 and by middle-sized residues at position -5, indicating that the processing rate is affected by the size of amino acid residues not only at positions -1 and -3. Conformation analysis of the cleavage site taken together with the mutation and statistical data suggests an extended beta-conformation of the -5 to -1 region in the signal peptidase binding pocket.

Thermodynamic studies of the collagen-like region of human subcomponent C1q. A water-containing structural model.

Thermal transitions of Clq were investigated by methods of differential scanning calorimetry, circular dichroism and fluorescence. The melting curves of Clq display two pronounced heat absorption peaks with enables determination of the thermodynamic parameters characterizing each transition. The low temperature peak was assigned to melting of the Clq collagenous part. Analysis of the data has revealed unusual, as compared with the monomeric collagen molecules, thermodynamic features of the Clq collagenous part: (1) higher thermal stability strongly dependent on pH; (2) less linear co-operative regions; and (3) a noticeable change in the partial specific heat capacity (delta Cp) in contrast to both the monomeric collagen and the collagen fibrils. This unusually large delta Cp value suggested a conclusion that the fibril-like endpiece of Clq may have a cavity filled with ice-like ordered water molecules.

De novo design of fibrils made of short alpha-helical coiled coil peptides.

BACKGROUND: The alpha-helical coiled coil structures formed by 25-50 residues long peptides are recognized as one of Nature's favorite ways of creating an oligomerization motif. Known de novo designed and natural coiled coils use the lateral dimension for oligomerization but not the axial one. Previous attempts to design alpha-helical peptides with a potential for axial growth led to fibrous aggregates which have an unexpectedly big and irregular thickness. These facts encouraged us to design a coiled coil peptide which self-assembles into soluble oligomers with a fixed lateral dimension and whose alpha-helices associate in a staggered manner and trigger axial growth of the coiled coil. Designing the coiled coil with a large number of subunits, we also pursue the practical goal of obtaining a valuable scaffold for the construction of multivalent fusion proteins. RESULTS: The designed 34-residue peptide self-assembles into long fibrils at slightly acid pH and into spherical aggregates at neutral pH. The fibrillogenesis is completely reversible upon pH change. The fibrils were characterized using circular dichroism spectroscopy, sedimentation diffusion, electron microscopy, differential scanning calorimetry and X-ray fiber diffraction. The peptide was deliberately engineered to adopt the structure of a five-stranded coiled coil rope with adjacent alpha-helices, staggered along the fibril axis. As shown experimentally, the most likely structure matches the predicted five-stranded arrangement. CONCLUSIONS: The fact that the peptide assembles in an expected fibril arrangement demonstrates the credibility of our conception of design. The discovery of a short peptide with fibril-forming ability and stimulus-sensitive behavior opens new opportunities for a number of applications.

Heart-specific splice-variant of a human mitochondrial ribosomal protein (mRNA processing; tissue specific splicing).

It has been proposed that splice-variants of proteins involved in mitochondrial RNA processing and translation may be involved in the tissue specificity of mitochondrial DNA disease mutations (Fischel-Ghodsian, 1998. Mol. Genet. Metab. 65, 97-104). To identify and characterize the structural components of mitochondrial RNA processing and translation, the Mammalian Mitochondrial Ribosomal Consortium has been formed. The 338 amino acid (aa) residues long MRP-L5 was identified (O'Brien et al., 1999. J. Biol. Chem. 274, 36043-36051), and its transcript was screened for tissue specific splice-variants. Screening of the EST databases revealed a single putative splice-variant, due to the insertion of an exon consisting of 89 nucleotides prior to the last exon. Screening of multiple cDNA libraries revealed this inserted exon to be present only in heart tissue, in addition to the predominant MRP-L5 transcript. Sequencing of this region confirmed the EST sequence, and showed in the splice-variant a termination triplet at the beginning of the last exon. Thus the inserted exon replaces the coding sequence of the regular last exon, and creates a new 353 aa long protein (MRP-L5V1). Sequence analysis and 3D modeling reveal similarity between MRP-L5 and threonyl-t-RNA synthetases, and a likely RNA binding site within MRP-L5, with the C-terminus in proximity to the RNA binding site. Sequence analysis of MRP-L5V1 also suggests a likely transmembrane domain at the C-terminus. Thus it is possible that the MRP-L5V1 C-terminus could interfere with RNA binding and may have gained a transmembrane domain. Further studies will be required to elucidate the functional significance of MRP-L5V1.

alpha-Helical solenoid model for the human involucrin.

Involucrin is a key component of the cross-linked envelope of terminally differentiated keratinocytes. The human molecule largely consists of 10 residue repeats and forms a thin 460 A long rod. Summarized experimental data and a detailed stereochemical analysis made with computer modeling resulted in a structural model for the involucrin molecule. The suggested structure is a left-handed alpha-helical solenoid built of a tandem array of helix-turn-helix folds. The structure enables us to explain the whole set of experimental data and residue conservations within the repeats. It is ideally suited to serve as a scaffold for cell envelope assembly and proposes a possible mode of the intermolecular interactions of involucrin during cell cornification.

Left-handed topology of super-secondary structure formed by aligned alpha-helix and beta-hairpin.

A novel super-secondary structure common for many non-homological proteins is considered. This folding pattern, consisting of adjacent along the chain alpha-helix and beta-hairpin, has an aligned packing. It is found that one of the two possible 'mirror-symmetrical' topologies is observed in proteins. The alpha-helix + beta-hairpin structures have a similar pattern of hydrophobic residues in their amino acid sequences. The remaining part of a molecule or a domain is almost always located on the same side of the considered folding pattern. These results can be used in the prediction of three-dimensional protein structure and protein design.

Positively charged lysine at the N-terminus of the signal peptide of the Escherichia coli alkaline phosphatase provides the secretion efficiency and is involved in the interaction with anionic phospholipids.

Positively charged amino acid residues at the N-terminus of the signal peptide (SP) have been proposed to play a significant role in the initial step of protein secretion in bacteria. To test this hypothesis, Lys(-20) of the Escherichia coli alkaline phosphatase SP was replaced by other amino acid residues, and the effect of these substitutions on protein maturation was studied. The introduction of negatively charged and hydrophobic amino acids resulted in a decrease in secretion efficiency and impaired the SP-APL interaction, whereas His and Tyr had no significant effect. A structural analysis of the SP-APL interaction suggests that the positively charged Lys(-20) determines the stability of the complex.

Stereochemical analysis of interaction of signal peptide with phospholipids at the initiation of protein translocation across the membrane.

Stereochemical analysis of signal peptide interaction with E. coli membrane phospholipids revealed the structural complementarity of N-terminus of signal peptide alpha-helix and acid phospholipids. The formation of their complex leads to neutralization of charges and decrease in hydrophilicity of both components, and promotes insertion of peptide and phospholipid into the membrane, not separately but as a complex. Interaction of acid phospholipids with the E. coli alkaline phosphatase (AP) signal peptide was thoroughly analyzed, and it was shown that in this case a complex of signal peptide alpha-helix with phosphatidylglycerol is inserted into the membrane with the lowest energy expense. On the basis of the results of stereochemical analysis and the available experimental data, a molecular mechanism of protein translocation initiation across the membrane has been proposed, in which the key events are the formation of the complex "signal peptide alpha-helix-acid phospholipid", the coupled insertion of hydrophobic peptide-lipid complex into a nonpolar membrane interior and translocation across the membranes.

Interactions of Bax and tBid with lipid monolayers.

The release of cytochrome c from mitochondria to the cytosol is a crucial step of apoptosis that involves interactions of Bax and tBid proteins with the mitochondrial membrane. We investigated Bax and tBid interactions with (i) phosphatidylcholine (PC) monolayer as the main component of the outer leaflet of the outer membrane, (ii) with phosphatidylethanolamine (PE) and phosphatidylserine (PS) that are present in the inner leaflet and (iii) with a mixed PC/PE/Cardiolipin (CL) monolayer of the contact sites between the outer and inner membranes. These interactions were studied by measuring the increase of the lipidic monolayer surface pressure induced by the proteins. Our measurements suggest that tBid interacts strongly with the POPC/DOPE/CL, whereas Bax interaction with this monolayer is about 12 times weaker. Both tBid and Bax interact moderately half as strongly with negatively charged DOPS and non-lamellar DOPE monolayers. TBid also slightly interacts with DOPC. Our results suggest that tBid but not Bax interacts with the PC-containing outer membrane. Subsequent insertion of these proteins may occur at the PC/PE/CL sites of contact between the outer and inner membranes. It was also shown that Bax and tBid being mixed in solution inhibit their insertion into POPC/DOPE/CL monolayer. The known 3-D structures of Bax and Bid allowed us to propose a structural interpretation of these experimental results.

Modeling of a five-stranded coiled coil structure for the assembly domain of the cartilage oligomeric matrix protein.

The three-dimensional structure of the assembly domain of the cartilage oligomeric matrix protein (COMP) has been modeled. The model demonstrates a parallel five-stranded coiled coil and fits well with a large amount of experimental data that describe the oligomerization state, the alpha-helical conformation, the helix directionality and the properties of the (abcdefg)n repeat sequence containing apolar residues at (a) and (d) positions. Comparison of the pentamer model with the known dimer, trimer, and tetramer coiled coils revealed interactions that could mediate the switch to the formation of the pentamer coiled coil. The most distinctive feature of the pentamer model involves ion pair interactions. Charged side chains of the pentamer can form (f-g), (f-b'), and (e-c') interhelical ion pairs, which are neither experimentally observed nor modeled in the di-, tri-, and tetramers. A polar glutamine residue could be adopted at an interior (d) position of the modeled structure due to the formation of a symmetrical network of buried hydrogen bonds between five such glutamines. The pentamer model contains an axial cavity that can accept water molecules. Conformational analysis was carried out in an attempt to determine the three-dimensional structure of the disulfide bonded C-terminal region of the pentamer. Recent data on crystallization of the COMP assembly domain (Efimov et al., Proteins 24:259-262, 1996) indicate that the prediction can be tested experimentally in the near future.