Disordered regions in elongation factors EF1A in the three superkingdoms of life

A new criterion proposed for classification of the living world is based on the ability of the protein amino acid sequence to form disordered regions, appearing as loops in the 3D structure. The approach used fundamentally differs from the approaches based on comparisons of certain RNA or protein sequences of different organisms. Introduction of any new structural-functional criterion that could resolve the evolutionary relationships between the main groups of origin organisms is of interest in itself, as megasystematics and macrophylogeny lack informative criteria despite the apparent abundance of molecular characteristics. The specialized program FoldUnfold was used to search for disordered regions in the elongation factors EF1A (EFs). The reliability of loop prediction was verified against five EFs with the structures known from X-ray analysis. It was demonstrated with the example of several dozens of typical representatives of the living world that the program predicts extra loops in addition to two linkers between three structural domains in EFs. Besides the effector loop, contained in all EFs, six loops were detected at maximum. Of them, three loops (A, B, and C) are in domain I, one (D) is in domain II, and two (E and F) are in domain III. Moreover, all six loops are never present in the same EF. The EF signatures were determined for each of the superkingdoms of life. Each superkingdom displayed variations in the number of loops and their location within the EF domains. Not only the presence of a particular loop was important in the analysis, but also the specificity of its amino acid sequence. As the total number of predicted loops in EFs increases with the increasing complexity of organisms, the following evolutionary role was postulated for the loops. Following the principle of thrifty inventiveness, nature operates with different universal inserts (loops), adapting their number, location within the EF domains, and amino acid composition so that the protein performs specialized functions—single in protozoa and several in higher organisms.     

Structures of the intradiskal loops and amino terminus of the G-protein receptor, rhodopsin.

The intradiskal surface of the transmembrane protein, rhodopsin, consists of the amino terminal domain and three loops connecting six of the seven transmembrane helices. This surface corresponds to the extracellular surface of other G-protein receptors. Peptides that represent each of the extramembraneous domains on this surface (three loops and the amino terminus) were synthesized. These peptides also included residues which, based on a hydrophobic plot, could be expected to be part of the transmembrane helix. The structure of each of these peptides in solution was then determined using two-dimensional 1H nuclear magnetic resonance. All peptide domains showed ordered structures in solution. The structures of each of the peptides from intradiskal loops of rhodopsin exhibited a turn in the central region of the peptide. The ends of the peptides show an unwinding of the transmembrane helices to form this turn. The amino terminal domain peptide exhibited alpha-helical regions with breaks and bends at proline residues. This region forms a compact domain. Together, the structures for the loop and amino terminus domains indicate that the intradiskal surface of rhodopsin is ordered. These data further suggest a structural motif for short loops in transmembrane proteins. The ordered structures of these loops, in the absence of the transmembrane helices, indicate that the primary sequences of these loops are sufficient to code for the turn.     

Loops and repeats in proteins as footprints of molecular evolution

This review is devoted to substantiation of new characteristics for classification of living organisms. The novel view of a role of flexible regions in protein functioning and evolution is suggested. It is based on the newly revealed correlation between the number of loops in elongation factors and the complexity of organisms. This correlation allowed us to formulate a hypothesis of evolution of this protein family. In addition, the study of the ribosomal protein S1 family made it possible to consider the number of structural domains as a reliable indicator of a microorganism’s affiliation with a particular division and to judge about “direction” of their evolution. The findings allow us to consider the loops and repeats in these proteins as unique imprints of molecular evolution.     

Characteristics and prediction of domain linker sequences in multi-domain proteins

To facilitate swift structural characterizations, structural genomic/proteomic projects need to divide large multi-domain proteins into structural domains and to determine their structures separately. Thus, the assignment of structural domains based solely on sequence information, especially on the physico-chemical properties of the amino acid sequences, could be very helpful for such projects. In this study, we examined the characteristics of domain linker sequences, which are loop sequences connecting two structural domains. To this end, we prepared a set of 101 non-redundant multi-domain protein sequences with known structures, and performed an analysis of the linker sequences. The analysis revealed that the frequencies of five (Pro, Gly, Asp, Asn, Lys) amino acid residues differed significantly between the linker and non-linker loop sequences. Moreover, we observed a similar deviation for the residue pair frequencies between the two types of loop sequences. Finally, we describe an automated method, based on the above analysis, to detect loops that have high probabilities of being domain linkers in a protein sequence.     

Sequence–structure relationships in RNA loops: establishing the basis for loop homology modeling

The specific function of RNA molecules frequently resides in their seemingly unstructured loop regions. We performed a systematic analysis of RNA loops extracted from experimentally determined three-dimensional structures of RNA molecules. A comprehensive loop-structure data set was created and organized into distinct clusters based on structural and sequence similarity. We detected clear evidence of the hallmark of homology present in the sequence–structure relationships in loops. Loops differing by <25% in sequence identity fold into very similar structures. Thus, our results support the application of homology modeling for RNA loop model building. We established a threshold that may guide the sequence divergence-based selection of template structures for RNA loop homology modeling. Of all possible sequences that are, under the assumption of isosteric relationships, theoretically compatible with actual sequences observed in RNA structures, only a small fraction is contained in the Rfam database of RNA sequences and classes implying that the actual RNA loop space may consist of a limited number of unique loop structures and conserved sequences. The loop-structure data sets are made available via an online database, RLooM. RLooM also offers functionalities for the modeling of RNA loop structures in support of RNA engineering and design efforts.     

The primary structure and the functional domains of an elongation factor-1 alpha from Mucor racemosus

We have determined the complete nucleotide sequence for TEF-1, one of three genes coding for elongation factor (EF)-1 alpha in Mucor racemosus. The deduced EF-1 alpha protein contains 458 amino acids encoded by two exons. The presence of an intervening sequence located near the 3' end of the gene was predicted by the nucleotide sequence data and confirmed by alkaline S1 nuclease mapping. The amino acid sequence of EF-1 alpha was compared to the published amino acid sequences of EF-1 alpha proteins from Saccharomyces cerevisiae and Artemia salina. These proteins shared nearly 85% homology. A similar comparison to the functionally analogous EF-Tu from Escherichia coli revealed several regions of amino acid homology suggesting that the functional domains are conserved in elongation factors from these diverse organisms. Secondary structure predictions indicated that alpha helix and beta sheet conformations associated with the functional domains in EF-Tu are present in the same relative location in EF-1 alpha from M. racemosus. Through this comparative structural analysis we have predicted the general location of functional domains in EF-1 alpha which interact with GTP and tRNA.     

Sequence codes for extended conformation: a neighbor-dependent sequence analysis of loops in proteins

We performed an extensive sequence analysis on the loops of proteins. By dividing a loop databank derived from the Protein Data Bank into groups, we analyzed the chemical characteristics and the sequence preferences of loops of different lengths and loops connecting different secondary structures in proteins. We found that a large population of loops in our loop databank (94.4%) is either partially or completely surface-exposed. A majority of surface loops in proteins are hydrophilic, whereas the chemical characteristics of interior loops are relatively neutral according to Eisenberg's consensus hydrophobicity scale. As a first step in investigating the intrinsic sequence-structure relationship of loop sequences in proteins, we performed a neighbor-dependent sequence analysis that calculated the effect of the neighboring amino acid type on the loop propensity of residues in loops. This method enhances the statistical significance of residue propensity, thus allowing us to explore the positional preference of amino acids in loops. Our analysis yielded a series of amino acid dyads that showed high preference for loop conformation. The data presented in this study should prove useful for developing potential codes in recognizing loop sequences in proteins.     

Determining the 3D structure of human ASC2 protein involved in apoptosis and inflammation

ASC2 structure has been well defined by 1141 NOE experimental restraints. The model consists of five alpha helices. alpha-Helices are connected by short random structure loops. The sole exception is the loop connecting helices 2 and 3, which has a 20-residue length. Folding generally agrees with the folding of recently published death domain structures in which alpha-helix structures have been reported. In spite of structural similarity, amino acid sequence homology with the most similar protein (ASC1) is just 64%. DD, DED, and CASP protein structures present six helices along their sequences; ASC2 presents 5 well-defined helices due to long distance restraints. However, a helical fragment was observed between amino acids 38 and 42 (representing helix 3) in the death domains when constructing the model.     

IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content

Intrinsically unstructured/disordered proteins and domains (IUPs) lack a well-defined three-dimensional structure under native conditions. The IUPred server presents a novel algorithm for predicting such regions from amino acid sequences by estimating their total pairwise interresidue interaction energy, based on the assumption that IUP sequences do not fold due to their inability to form sufficient stabilizing interresidue interactions. Optional to the prediction are built-in parameter sets optimized for predicting short or long disordered regions and structured domains.     

Search for functional domain boundaries and localization of functional sites based on oligopeptide vocabularies]

A new method based on the analysis of oligopeptide composition of the amino acid sequences from different protein families is presented. We assume, that any protein family can be characterized by the set of oligopeptides (oligopeptides vocabulary). We demonstrate, that oligopeptides vocabulary comparison can distinguish different families from each other and from random sequences. It should be noted, that this comparison can be successfully performed on the set of only 25 dipeptides and without preliminary alignment. We demonstrate, that characteristic peptides are localized in the regions of functional significance, as shown on the example of GTP-binding domain of translation elongation factors. We suggest how to use this method to localize the boundaries of functional domains in amino sequences. On the example of few functional domains we demonstrate, that the average error of prediction does not exceed 3-4 amino acid residue.     

Sequence of the tufA gene encoding elongation factor EF-Tu from Thermus aquaticus and overproduction of the protein in Escherichia coli.

The sequence of the tufA gene from the extreme thermophilic eubacterium Thermus aquaticus EP 00276 was determined. The GC content in third positions of codons is 89.5%, with an unusual predominance of guanosine (60.7%). The derived protein sequence differs from tufA- and tufB-encoded sequences for elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, another member of the genus Thermus, in 10 of the 405 amino acid residues. Three exchanges are located in the additional loop of ten amino acids (182-191). The loop, probably involved in nucleotide binding, is absent in EF-Tu of the mesophile Escherichia coli. Since EF-Tu from E. coli is quite unstable, the protein is well-suited for analyzing molecular changes that lead to thermostabilization. Comparison of the EF-Tu domain I from E. coli and Thermus strains revealed clustered amino acid exchanges in the C-terminal part of the first helix and in adjacent residues of the second loop inferred to interact with the ribosome. Most other exchanges in the guanine nucleotide binding domain are located in loops or nearest vicinity of loops suggesting their importance for thermostability. The T. aquaticus EF-Tu was overproduced in E. coli using the tac expression system. Identity of the recombinant T. aquaticus EF-Tu was verified by Western blot analysis, N-terminal sequencing and GDP binding assays.     

Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein.     

The association of heavy and light chain variable domains in antibodies: implications for antigen specificity

The antigen-binding site of immunoglobulins is formed by six regions, three from the light and three from the heavy chain variable domains, which, on association of the two chains, form the conventional antigen-binding site of the antibody. The mode of interaction between the heavy and light chain variable domains affects the relative position of the antigen-binding loops and therefore has an effect on the overall conformation of the binding site. In this article, we analyze the structure of the interface between the heavy and light chain variable domains and show that there are essentially two different modes for their interaction that can be identified by the presence of key amino acids in specific positions of the antibody sequences. We also show that the different packing modes are related to the type of recognized antigen.     

Conformational properties of the iso-1-cytochrome C denatured state: dependence on guanidine hydrochloride concentration

Production of seven single surface histidine variants of yeast iso-1-cytochrome c allowed measurement of the apparent pK(a), pK(a)(obs), for histidine-heme loop formation for loops of nine to 83 amino acid residues under varying denaturing conditions (2 M to 6 M guanidine hydrochloride, gdnHCl). A linear correlation between pK(a)(obs) and the log of the loop size is expected for a random coil, pK(a)(obs) proportional to k log(n), where k is a scaling factor and n is the number of monomers in the loop. For small loops of nine, 16, and 22 monomers, no dependence of pK(a)(obs) on loop size was observed at any denaturant concentration indicating effects from chain stiffness. For larger loops of 37, 56, 72, and 83 monomers, the dependence of pK(a)(obs) on log(n) was linear and the slope of that dependence decreased with increasing concentration of denaturant. The scaling factor obtained at 5 M and 6 M gdnHCl for the larger loop sizes was approximately -2.0, close to the value of -2.2 expected for a random coil with excluded volume. However, scaling factors obtained under less harsh denaturing conditions (2 M to 4.5 M gdnHCl) deviated strongly from that expected for a random coil, being in the range -3 to -4. The gdnHCl dependence of pK(a)(obs) at each loop size was also evaluated to obtain denaturant m-values. Short loops where chain stiffness dominates had similar m-values of approximately 0.25 kcal/mol M. For larger loops m-values decrease with increasing loop size indicating that less hydrophobic area is sequestered when larger loops form. It is known that the earliest events in protein folding involve the formation of simple loops. The data from these studies provide direct insight into the relative probability with which loops of different sizes will form, as well as the factors which affect loop formation.     

Fgf genes in the basal chordate <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

In vertebrates, a number of fibroblast growth factors (FGFs) have been shown to play important roles in developing embryos and adult organisms. However, the molecular relationships of the vertebrate FGFs are not yet completely understood, partly due to the divergence of their amino acid sequences. To solve this problem, we have identified six FGF genes in a basal chordate, the ascidian Ciona intestinalis. A phylogenetic analysis confidently assigned two of them to vertebrate FGF8/17/18 and FGF11/12/13/14, respectively. Based on the presence of the conserved domains within or outside of the FGF domains, we speculate that three of the other genes are orthologous to vertebrate FGF3/7/10/22, FGF4/5/6 and FGF9/16/20, respectively, although we cannot assign the sixth member to any of the vertebrate FGFs. A survey of the raw whole genome shotgun sequences of C. intestinalis demonstrated the presence of no FGF genes other than the six genes in the genome. The identification of these six FGF genes in the basal chordate gave us an insight into the diversification of specific subfamilies of vertebrate FGFs.     

Carcinoembryonic antigen family: characterization of cDNAs coding for NCA and CEA and suggestion of nonrandom sequence variation in their conserved loop-domains

We have isolated cDNAs for carcinoembryonic antigen (CEA) and for a normal cross-reacting antigen (NCA) and report here their nucleotide and derived amino acid sequences. Our data show that both the CEA and NCA polypeptides are organized into extracellular domains, some with cysteine-linked loops, that share extensive sequence homology (approximately 78% overall) with each other and appear similar to immunoglobulin superfamily members. A major difference between the two apoproteins is the presence of a single loop-domain in NCA compared to three tandemly repeated loop-domains in CEA. Sequence comparisons between the extracellular domains of CEA and NCA show that the N-terminal and adjacent loop domains of each apoprotein have high homology (85-90%) to each other, while comparison of loop-domain regions reveals a possible nonrandom distribution of base changes and altered amino acids near certain cysteine residues that are inferred to be involved in forming disulfide loops. Both apoproteins show high identity in their hydrophobic C-termini that are reminiscent of the type of transmembrane tails seen in proteins that potentiate signal transduction. These findings, coupled with distinct expression profiles of CEA and NCA mRNAs, suggest that these apoproteins may function as unique cell-surface molecules mediating cell-specific interactions in normal and neoplastic cells.     

Exploring the collagen-binding site of the DDR1 tyrosine kinase receptor

Discoidin domain receptors 1 and 2 (DDR1 and DDR2) are tyrosine kinase receptors activated by triple-helical collagens. Aberrant expression and signaling of these receptors have been implicated in several human diseases linked to accelerated matrix degradation and remodeling including tumor invasion, atherosclerosis and liver fibrosis. The objective of this study is to characterize the collagen-binding sites in the discoidin domains of DDR1 and DDR2 at a molecular level. We expressed glutathione S-transferase fusion proteins containing the discoidin and extracellular domains of DDR1 and DDR2 in insect cells and subjected them to a solid-phase collagen-binding assay. We found high affinity binding of the DDR extracellular domains to immobilized type I collagen and confirmed the discoidin-collagen interaction with an enzyme-linked immunosorbent assay-based read-out. Furthermore, we created a three-dimensional model of the DDR1 discoidin domain based on the related domains of blood coagulation factors V and VIII. This model predicts the presence of four neighboring, surface-exposed loops that are topologically equivalent to a major phospholipid-binding site in factors V and VIII. To test the involvement of these loops in collagen binding, we mutated individual amino acid residues to alanine or deleted short sequence stretches within these loops. We found that several residues within loop 1 (Ser-52-Thr-57) and loop 3 (Arg-105-Lys-112) as well as Ser-175 in loop 4 are critically involved in collagen binding. Our structure-function analysis of the DDR discoidin domains provides new insights into this non-integrin-mediated collagen-signaling mechanism and may ultimately lead to the design of small molecule inhibitors that interfere with aberrant DDR function.