Unstructured regions in elongation factors EF1A from three overkingdom of the living world

A new characteristic for classification of the Living World which based on the ability of amino acid sequences to form unstructured regions that appear as loops in their 3D structure is described. Our approach is in principle different from RNA and protein phylogenies that are based on the alignment of amino acid sequences from different organisms. Introduction of new structural-functional characteristic in itself is of undoubted interest because megataxonomy and macrophylogeny lack features that may be resolve evolutionary relation between different groups of organisms though apparent abundance of such characteristics are present. We used the program FoldUnfold to search for unstructured regions in the elongation factors EF1A. The reliability of loop prediction was checked against five factors whose structure is known from X-ray analysis. In addition to two cross-bridges between three structural domains in the elongation factors, the program predicts extra loops. Not counting the effector loop that is inherent to all factors, there are six. Three (A, B and C) of the six different loops are revealed in the first domain, one loop (D) in the second, and two loops (E and F) in the third domain of the factor, all six of which are never found in the same factor. Signatures of elongation factors for each Superkingdom of the Living World have been found for several dozen typical representatives from each Superkingdom. These signatures lead to the variation of the number of loops and their localization within the factor domains. The obtained data leads us to believe that the approach based on the prediction of unstructured protein loops--up to six--must have higher resolution than the method based on the indels (insertion + deletion), the number of which equals one for the same elongation factors. In our analysis, the specificity of sequences it is important, in addition to the existence of loops. Since the total number of loops predicted in the factors increases with the complexity of an organism, we propose the following about the role of the loops in evolution: holding to the principle of "thrifty inventiveness", Nature operates with different universal inserts (loops) adapting their number and location among the factor domains as well as their amino acid composition so that the protein will perform special functions: one in protozoa and several in higher organisms.     

Structure and function in rhodopsin. Studies of the interaction between the rhodopsin cytoplasmic domain and transducin.

Structural requirements for the activation of transducin by rhodopsin have been studied by site-specific mutagenesis of bovine rhodopsin. A variety of single amino acid replacements and amino acid insertions and deletions of varying sizes were carried out in the two cytoplasmic loops CD (amino acids 134-151) and EF (amino acids 231-252). Except for deletion mutant delta 137-150, all the mutants bound 11-cis-retinal and displayed normal spectral characteristics. Deletion mutant delta 236-239 in loop EF caused a 50% reduction of transducin activation, whereas deletion mutant delta 244-249 and the larger deletions in loop EF abolished transducin activation. An 8-amino acid deletion in the cytoplasmic loop CD as well as a replacement of 13 amino acids with an unrelated sequence showed no transducin activation. Several single amino acid substitutions also caused significant reduction in transducin activation. The conserved charged pair Glu-134/Arg-135 in the cytoplasmic loop CD was required for transducin activation; its reversal or neutralization abolished transducin activation. Three amino acid replacements in loop EF (S240A, T243V, and K248L) resulted in significant reduction in transducin activation. We conclude that 1) both the cytoplasmic loops CD and EF are required for transducin activation, and 2) effective functional interaction between rhodopsin and transducin involves relatively large peptide sequences in the cytoplasmic loops.     

A minimal transmembrane beta-barrel platform protein studied by nuclear magnetic resonance

In this study, we were concerned with the structural role of the surface-exposed extracellular loops of the N-terminal transmembrane (TM) domain of OmpA. A variant of the TM domain of outer membrane protein A (OmpA) with all four such loops shortened, which we call the beta-barrel platform (BBP), was successfully refolded. This indicates that the removed parts of the surface-exposed loops indeed do not contain amino acid sequences critical for this membrane protein's refolding in vitro. BBP has the potential to be used as a template beta-barrel membrane protein structure for the development of novel functions, although our results also highlight the potential difficulties that can arise when functionality is being engineered into the loop regions of membrane proteins. We have used solution nuclear magnetic resonance spectroscopy to determine the global fold of BBP+EF, BBP with a metal ion-binding EF-hand inserted in one of the shortened loops. BBP and BBP+EF in dihexanoylphosphatidylcholine micelles are eight-stranded antiparallel beta-barrels, and BBP represents the smallest beta-structured integral membrane protein known to date.     

Prediction of short loops in intrinsically disordered proteins

A new possibility of predicting short disordered regions (loops) at a small window size (three amino acid residues) by the FoldUnfold program is described. As demonstrated with the example of three G proteins, FoldUnfold predicted almost all existing loops at the positions fitting well the X-ray structural data. The loops predicted in the Ras p21 structure were classified into two types. The loops of the first type display high Debye-Waller factor values, characteristic of the so-called functional loops (flexible loops). The second-type loops had lower Debye-Waller factor values and, consequently, were regarded as the loops connecting secondary structure elements (rigid loops). Comparison of the results predicted by FoldUnfold with the predictions of other programs (PONDR, RONN, DisEMBL, PreLINK, IUPred, GlobPlot 2, and FoldIndex) demonstrated that the first program was much better in predicting the positions of short loops. FoldUnfold made it possible to solve the problem difficult for the other programs, that is, to determine the boundary between the ordered and disordered regions in proteins with a large fraction of disordered regions, exemplified by the ubiquitin-like domain. In particular, FoldUnfold predicted a boundary between the ordered and disordered regions at residues 30 and 31, whereas the other programs predicted the boundary in the range of 28–70 amino acid residues.     

Improvement of domain linker prediction by incorporating loop-length-dependent characteristics

Protein dissection into structural domains that can fold in isolation is an important issue in both functional and structural proteomics. Here, we analyzed inter- and intradomain loop sequences (respectively named domain linker and nonlinker loops) and computed a domain linker likelihood score, which was used for developing a domain boundary prediction protocol. The analysis confirmed our previous results indicating that the amino acid composition in terms of glycine, proline, aspartic acid, asparagine, lysine, and histidine significantly differs between linker and nonlinker loops. However, a detailed examination revealed that the amino acid composition bias actually depends on the loop length. Indeed, significant frequency deviations were observed for glycine, proline, and aspartic acid in short linker and nonlinker loops, whereas deviations were observed for aspartic acid, proline, asparagine, and lysine in long linker and nonlinker loops. Finally, we incorporated this loop-length-dependent amino acid composition bias in a simple linker prediction protocol, which predicted linkers with a 40.6% specificity and a 36.1% sensitivity. These figures are 4.4 and 2.4% higher than those obtained with our former prediction protocol that does not incorporate loop-length-dependent characteristics. This result should have practical significance for experimental protein dissection, since the probability of obtaining a stably folding structural domain by randomly dissecting a protein sequence is estimated to be 12.6%.     

The position of the disulfide bonds in human plasma alpha 2 HS-glycoprotein and the repeating double disulfide bonds in the domain structure

The positions of the inter- and intra-chain disulfide bonds of human plasma alpha 2 HS-glycoprotein were determined. alpha 2 HS-glycoprotein was digested with acid proteinase and then with thermolysin. The disulfide bonds containing peptides were separated by reversed-phase HPLC and detected by SBD-F (7-fluorobenzo-2-oxa-1,3-diasole-4-sulfonic acid ammonium salt) method. One inter-disulfide bond containing peptide and five intra-disulfide bond containing peptides (A-chain) were purified and identified as Cys-18 (B-chain)--Cys-14 (A-chain), Cys-71--Cys-82, Cys-96--Cys-114, Cys-128--Cys-131, Cys-190--Cys-201 and Cys-212--Cys-229, respectively. The location of the intra-disulfide bonds revealed that the A-chain of alpha 2 HS-glycoprotein is composed of three domains. Two domains were shown to possess intramolecular homology judging from the total chain length of the domains, size of the loops formed by the S--S bonds, the location of two disulfide loops near the C-terminal end of domains A and B, the distance between two S--S bonds of each domain, the amino acid sequence homology between these two domains (22.6%), number of amino acid residues between the second S--S loops and the end of domains A and B, and the positions of the ordered structures.     

Intrinsically disordered regions of p53 family are highly diversified in evolution

Proteins of the p53 family are expressed in vertebrates and in some invertebrate species. The main function of these proteins is to control and regulate cell cycle in response to various cellular signals, and therefore to control the organism's development. The regulatory functions of the p53 family members originate mostly from their highly-conserved and well-structured DNA-binding domains. Many human diseases (including various types of cancer) are related to the missense mutations within this domain. The ordered DNA-binding domains of the p53 family members are surrounded by functionally important intrinsically disordered regions. In this study, substitution rates and propensities in different regions of p53 were analyzed. The analyses revealed that the ordered DNA-binding domain is conserved, whereas disordered regions are characterized by high sequence diversity. This diversity was reflected both in the number of substitutions and in the types of substitutions to which each amino acid was prone. These results support the existence of a positive correlation between protein intrinsic disorder and sequence divergence during the evolutionary process. This higher sequence divergence provides strong support for the existence of disordered regions in p53 in vivo for if they were structured, they would evolve at similar rates as the rest of the protein.     

Conformational dynamics in loop swap mutants of homologous fibronectin type III domains

Fibronectin type III (FN-III) domains are autonomously folded modules found in a variety of multidomain proteins. The 10th FN-III domain from fibronectin (fnFN10) and the 3rd FN-III domain from tenascin-C (tnFN3) have 27% sequence identity and the same overall fold; however, the CC' loop has a different pattern of backbone hydrogen bonds and the FG loop is longer in fnFN10 compared to tnFN3. To examine the influence of length, sequence, and context in determining dynamical properties of loops, CC' and FG loops were swapped between fnFN10 and tnFN3 to generate four mutant proteins and backbone conformational dynamics on ps-ns and mus-ms timescales were characterized by solution (15)N-NMR spin relaxation spectroscopy. The grafted loops do not strongly perturb the properties of the protein scaffold; however, specific effects of the mutations are observed for amino acids that are proximal in space to the sites of mutation. The amino acid sequence primarily dictates conformational dynamics when the wild-type and grafted loop have the same length, but both sequence and context contribute to conformational dynamics when the loop lengths differ. The results suggest that changes in conformational dynamics of mutant proteins must be considered in both theoretical studies and protein design efforts.     

Surface loop dynamics in adeno-associated virus capsid assembly

The HI loop is a prominent domain on the adeno-associated virus (AAV) capsid surface that extends from each viral protein (VP) subunit overlapping the neighboring fivefold VP. Despite the highly conserved nature of the residues at the fivefold pore, the HI loops surrounding this critical region vary significantly in amino acid sequence between the AAV serotypes. In order to understand the role of this unique capsid domain, we ablated side chain interactions between the HI loop and the underlying EF loop in the neighboring VP subunit by generating a collection of deletion, insertion, and substitution mutants. A mutant lacking the HI loop was unable to assemble particles, while a substitution mutant (10 glycine residues) assembled particles but was unable to package viral genomes. Substitution mutants carrying corresponding regions from AAV1, AAV4, AAV5, and AAV8 yielded (i) particles with titers and infectivity identical to those of AAV2 (AAV2 HI1 and HI8), (ii) particles with a decreased virus titer (1 log) but normal infectivity (HI4), and (iii) particles that synthesized VPs but were unable to assemble into intact capsids (HI5). AAV5 HI is shorter than all other HI loops by one amino acid. Replacing the missing residue (threonine) in AAV2 HI5 resulted in a moderate particle assembly rescue. In addition, we replaced the HI loop with peptides varying in length and amino acid sequence. This region tolerated seven-amino-acid peptide substitutions unless they spanned a conserved phenylalanine at amino acid position 661. Mutation of this highly conserved phenylalanine to a glycine resulted in a modest decrease in virus titer but a substantial decrease (1 log order) in infectivity. Subsequently, confocal studies revealed that AAV2 F661G is incapable of efficiently completing a key step in the infectious pathway nuclear entry, hinting at a possible perturbation of VP1 phospholipase activity. Molecular modeling studies with the F661G mutant suggest that disruption of interactions between F661 and an underlying P373 residue in the EF loop of the neighboring subunit might adversely affect incorporation of the VP1 subunit at the fivefold axis. Western blot analysis confirmed inefficient incorporation of VP1, as well as a proteolytically processed VP1 subunit that could account for the markedly reduced infectivity. In summary, our studies show that the HI loop, while flexible in amino acid sequence, is critical for AAV capsid assembly, proper VP1 subunit incorporation, and viral genome packaging, all of which implies a potential role for this unique surface domain in viral infectivity.     

Light-driven activation of beta 2-adrenergic receptor signaling by a chimeric rhodopsin containing the beta 2-adrenergic receptor cytoplasmic loops

Structure-function studies of rhodopsin indicate that both intradiscal and transmembrane (TM) domains are required for retinal binding and subsequent light-induced structural changes in the cytoplasmic domain. Further, a hypothesis involving a common mechanism for activation of G-protein-coupled receptor (GPCR) has been proposed. To test this hypothesis, chimeric receptors were required in which the cytoplasmic domains of rhodopsin were replaced with those of the beta(2)-adrenergic receptor (beta(2)-AR). Their preparation required identification of the boundaries between the TM domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR necessary for formation of the rhodopsin chromophore and its activation by light and subsequent optimal activation of beta(2)-AR signaling. Chimeric receptors were constructed in which the cytoplasmic loops of rhodopsin were replaced one at a time and in combination. In these replacements, size of the third cytoplasmic (EF) loop critically determined the extent of chromophore formation, its stability, and subsequent signal transduction specificity. All the EF loop replacements showed significant decreases in transducin activation, while only minor effects were observed by replacements of the CD and AB loops. Light-dependent activation of beta(2)-AR leading to Galphas signaling was observed only for the EF2 chimera, and its activation was further enhanced by replacements of the other loops. The results demonstrate coupling between light-induced conformational changes occurring in the transmembrane domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR.     

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.     

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.     

Phylomat: an automated protein motif analysis tool for phylogenomics

Recent progress in genomics, proteomics, and bioinformatics enables unprecedented opportunities to examine the evolutionary history of molecular, cellular, and developmental pathways through phylogenomics. Accordingly, we have developed a motif analysis tool for phylogenomics (Phylomat, http://alg.ncsa.uiuc.edu/pmat) that scans predicted proteome sets for proteins containing highly conserved amino acid motifs or domains for in silico analysis of the evolutionary history of these motifs/domains. Phylomat enables the user to download results as full protein or extracted motif/domain sequences from each protein. Tables containing the percent distribution of a motif/domain in organisms normalized to proteome size are displayed. Phylomat can also align the set of full protein or extracted motif/domain sequences and predict a neighbor-joining tree from relative sequence similarity. Together, Phylomat serves as a user-friendly data-mining tool for the phylogenomic analysis of conserved sequence motifs/domains in annotated proteomes from the three domains of life.     

Similarities in protein amino acid composition in connection with principles of protein evolution

The amino acid composition of human alcohol dehydrogenase (ADH) was compared with alcohol dehydrogenases from different organisms and with other proteins. Similar amino acid sequences in human ADH (template protein) and in other proteins were determined by means of an original computer program. Analysis of amino acid motifs reveals that the ADHs from evolutionary more close organisms have more common amino acid sequences. The quantity measure of amino acid similarity was the number of similar motifs in analyzed protein per protein length. This value was measured for ADHs and for different proteins. For ADHs, this quotient was higher than for proteins with different functions; for vertebrates it correlated with evolutionary closeness. The similar operation of motif comparison was made with the help of program complex “MEME”. The analysis of ADHs revealed 4 motifs common to 6 of 10 tested organisms and no such motifs for proteins of different function. The conclusion is that general amino composition is more important for protein function than amino acid order and for enzymes of similar function it better correlates with evolutionary distance between 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.     

Interstrand loops CD and EF act as pH-dependent gates to regulate fatty acid ligand binding in tear lipocalin

Tear lipocalin (TL), a major component of human tears, shows pH-dependent endogenous ligand binding. The structural and conformational changes associated with ligand release in the pH range of 7.5-3.0 are monitored by circular dichroism spectroscopy and site-directed tryptophan fluorescence. In the transition from pH 7.5 to pH 5.5, the ligand affinity for 16-(9-anthroyloxy)palmitic acid (16AP) and 8-anilino-1-naphthalenesulfonic acid is reduced. At pH 4.0 these ligands no longer bind within the TL calyx. From pH 7.3 to pH 3.0, the residues on loops CD and EF, which overhang the calyx entrance, show reduced accessibility to acrylamide. In addition resonance energy transfer is enhanced between residues on the two loops; the distance between the loops narrows. These findings suggest that apposition of the loops at low pH excludes the ligand from the intracavitary binding site. The conformational changes observed in transition from pH 7.3 to pH 3.0 for loops CD and EF are quite different. The CD loop shows less population reshuffling than the EF loop with an acidic environment, probably because backbone motion is restrained by the adjacent disulfide bond. The Trp fluorescence wavelength maximum (lambda(max)) reflects internal electrostatic interactions for positions on loops CD and EF. The titration curves of lambda(max) for mutants on the EF loop fit the Hendersen-Hasselbalch equation for two apparent pK(a) values, while the CD loop positions fit satisfactorily with one pK(a) value. Midpoints of transition for the binding affinity of TL tryptophan mutants to 16AP occur at pH 5.5-6.1. Replacement of each amino acid on either loop by single tryptophan mutation does not disrupt the pH-dependent binding affinity to 16AP. Taken together the data suggest that pH-driven ligand release involves ionization changes in several titratable residues associated with CD and EF loop apposition and occlusion of the calyx.     

Crystal structure and RNA binding properties of the RNA recognition motif (RRM) and AlkB domains in human AlkB homolog 8 (ABH8), an enzyme catalyzing tRNA hypermodification

Humans express nine paralogs of the bacterial DNA repair enzyme AlkB, an iron/2-oxoglutarate-dependent dioxygenase that reverses alkylation damage to nucleobases. The biochemical and physiological roles of these paralogs remain largely uncharacterized, hampering insight into the evolutionary expansion of the AlkB family. However, AlkB homolog 8 (ABH8), which contains RNA recognition motif (RRM) and methyltransferase domains flanking its AlkB domain, recently was demonstrated to hypermodify the anticodon loops in some tRNAs. To deepen understanding of this activity, we performed physiological and biophysical studies of ABH8. Using GFP fusions, we demonstrate that expression of the Caenorhabditis elegans ABH8 ortholog is widespread in larvae but restricted to a small number of neurons in adults, suggesting that its function becomes more specialized during development. In vitro RNA binding studies on several human ABH8 constructs indicate that binding affinity is enhanced by a basic α-helix at the N terminus of the RRM domain. The 3.0-Å-resolution crystal structure of a construct comprising the RRM and AlkB domains shows disordered loops flanking the active site in the AlkB domain and a unique structural Zn(II)-binding site at its C terminus. Although the catalytic iron center is exposed to solvent, the 2-oxoglutarate co-substrate likely adopts an inactive conformation in the absence of tRNA substrate, which probably inhibits uncoupled free radical generation. A conformational change in the active site coupled to a disorder-to-order transition in the flanking protein segments likely controls ABH8 catalytic activity and tRNA binding specificity. These results provide insight into the functional and structural adaptations underlying evolutionary diversification of AlkB domains.     

Structured proteins and proteins with intrinsic disorder

Until recently, the point of view that the unique tertiary structure is necessary for protein function has prevailed. However, recent data have demonstrated that many cell proteins do not possess such structure in isolation, although displaying a distinct function under physiological conditions. These proteins were named the naturally, or intrinsically, disordered proteins. The fraction of intrinsically disordered regions in such proteins may vary from several amino acid residues to a completely unordered sequence of several tens or even several hundreds of residues. The main distinction of these proteins from structured (globular) proteins is that they have no unique tertiary structure in isolation and acquire it only upon interaction with their partners. The conformation of these proteins in a complex is determined not only by their own amino acid sequence (as is typical of structured, or globular, proteins) but also by the interacting partner. This review discusses the structure-function relationships in structured and intrinsically disordered proteins. The intricateness of this problem and the possible ways to solve it are illustrated by the example of the EF1A elongation factor family.     

Backbone-driven collapse in unfolded protein chains

Collapse of unfolded protein chains is an early event in folding. It affects structural properties of intrinsically disordered proteins, which take a considerable fraction of the human proteome. Collapse is generally believed to be driven by hydrophobic forces imposed by the presence of nonpolar amino acid side chains. Contributions from backbone hydrogen bonds to protein folding and stability, however, are controversial. To date, the experimental dissection of side-chain and backbone contributions has not yet been achieved because both types of interactions are integral parts of protein structure. Here, we realized this goal by applying mutagenesis and chemical modification on a set of disordered peptides and proteins. We measured the protein dimensions and kinetics of intra-chain diffusion of modified polypeptides at the level of individual molecules using fluorescence correlation spectroscopy, thereby avoiding artifacts commonly caused by aggregation of unfolded protein material in bulk. We found no contributions from side chains to collapse but, instead, identified backbone interactions as a source sufficient to form globules of native-like dimensions. The presence of backbone hydrogen bonds decreased polypeptide water solubility dramatically and accelerated the nanosecond kinetics of loop closure, in agreement with recent predictions from computer simulation. The presence of side chains, instead, slowed loop closure and modulated the dimensions of intrinsically disordered domains. It appeared that the transient formation of backbone interactions facilitates the diffusive search for productive conformations at the early stage of folding and within intrinsically disordered proteins.