Conservation and Diversity in Allosteric Fingerprints of Proteins for Evolutionary-inspired Engineering and Design

Hand-in-hand work of physics and evolution delivered protein universe with diversity of forms, sizes, and functions. Pervasiveness and advantageous traits of allostery made it an important component of the protein function regulation, calling for thorough investigation of its structural determinants and evolution. Learning directly from nature, we explored here allosteric communication in several major folds and repeat proteins, including α/β and β-barrels, β-propellers, Ig-like fold, ankyrin and α/β leucine-rich repeat proteins, which provide structural platforms for many different enzymatic and signalling functions. We obtained a picture of conserved allosteric communication characteristic in different fold types, modifications of the structure-driven signalling patterns via sequence-determined divergence to specific functions, as well as emergence and potential diversification of allosteric regulation in multi-domain proteins and oligomeric assemblies. Our observations will be instrumental in facilitating the engineering and de novo design of proteins with allosterically regulated functions, including development of therapeutic biologics. In particular, results described here may guide the identification of the optimal structural platforms (e.g. fold type, size, and oligomerization states) and the types of diversifications/perturbations, such as mutations, effector binding, and order–disorder transition. The tunable allosteric linkage across distant regions can be used as a pivotal component in the design/engineering of modular biological systems beyond the traditional scaffolding function.     

Method for comparing the structures of protein ligand-binding sites and application for predicting protein-drug interactions

Many drugs, even ones that are designed to act selectively on a target protein, bind unintended proteins. These unintended bindings can explain side effects or indicate additional mechanisms for a drug's medicinal properties. Structural similarity between binding sites is one of the reasons for binding to multiple targets. We developed a method for the structural alignment of atoms in the solvent-accessible surface of proteins that uses similarities in the local atomic environment, and carried out all-against-all structural comparisons for 48,347 potential ligand-binding regions from a nonredundant protein structure subset (nrPDB, provided by NCBI). The relationships between the similarity of ligand-binding regions and the similarity of the global structures of the proteins containing the binding regions were examined. We found 10,403 known ligand-binding region pairs whose structures were similar despite having different global folds. Of these, we detected 281 region pairs that had similar ligands with similar binding modes. These proteins are good examples of convergent evolution. In addition, we found a significant correlation between Z-score of structural similarity and true positive rate of "active" entries in the PubChem BioAssay database. Moreover, we confirmed the interaction between ibuprofen and a new target, porcine pancreatic elastase, by NMR experiment. Finally, we used this method to predict new drug-target protein interactions. We obtained 540 predictions for 105 drugs (e.g., captopril, lovastatin, flurbiprofen, metyrapone, and salicylic acid), and calculated the binding affinities using AutoDock simulation. The results of these structural comparisons are available at http://www.tsurumi.yokohama-cu.ac.jp/fold/database.html.     

Analysis of protein homology by assessing the (dis)similarity in protein loop regions

Two proteins are considered to have a similar fold if sufficiently many of their secondary structure elements are positioned similarly in space and are connected in the same order. Such a common structural scaffold may arise due to either divergent or convergent evolution. The intervening unaligned regions ("loops") between the superimposable helices and strands can exhibit a wide range of similarity and may offer clues to the structural evolution of folds. One might argue that more closely related proteins differ less in their nonconserved loop regions than distantly related proteins and, at the same time, the degree of variability in the loop regions in structurally similar but unrelated proteins is higher than in homologs. Here we introduce a new measure for structural (dis)similarity in loop regions that is based on the concept of the Hausdorff metric. This measure is used to gauge protein relatedness and is tested on a benchmark of homologous and analogous protein structures. It has been shown that the new measure can distinguish homologous from analogous proteins with the same or higher accuracy than the conventional measures that are based on comparing proteins in structurally aligned regions. We argue that this result can be attributed to the higher sensitivity of the Hausdorff (dis)similarity measure in detecting particularly evident dissimilarities in structures and draw some conclusions about evolutionary relatedness of proteins in the most populated protein folds.     

Structural Analysis of Metal Sites in Proteins: Non-heme Iron Sites as a Case Study

In metalloproteins, the protein environment modulates metal properties to achieve the required goal, which can be protein stabilization or function. The analysis of metal sites at the atomic level of detail provided by protein structures can thus be of benefit in functional and evolutionary studies of proteins. In this work, we propose a structural bioinformatics approach to the study of metalloproteins based on structural templates of metal sites that include the PDB coordinates of protein residues forming the first and the second coordination sphere of the metal. We have applied this approach to non-heme iron sites, which have been analyzed at various levels. Templates of sites located in different protein domains have been compared, showing that similar sites can be found in unrelated proteins as the result of convergent evolution. Templates of sites located in proteins of a large superfamily have been compared, showing possible mechanisms of divergent evolution of proteins to achieve different functions. Furthermore, template comparisons have been used to predict the function of uncharacterized proteins, showing that similarity searches focused on metal sites can be advantageously combined with typical whole-domain comparisons. Structural templates of metal sites, finally, may constitute the basis for a systematic classification of metalloproteins in databases.     

In vitro selection of GTP-binding proteins by block shuffling of estrogen-receptor fragments

To what extent has alternative splicing contributed to the evolution of protein-function diversity? We previously constructed a pool of block-deletion mutants of the human estrogen receptor α ligand binding domain by random multi-recombinant PCR. Here we performed iterative in vitro selection of GTP-binding proteins by using the library of mRNA-displayed proteins and GTP-affinity chromatography combined with quantitative real-time PCR. We obtained a novel GTP-binding protein with moderate affinity and substrate-specificity. The results of our in vitro simulation imply that alternative splicing may have contributed substantially to the diversification of protein function during evolution.     

Identification of Atg3 as an intrinsically disordered polypeptide yields insights into the molecular dynamics of autophagy-related proteins in yeast

The mechanism of autophagy relies on complex cell signaling and regulatory processes. Each cell contains many proteins that lack a rigid 3-dimensional structure under physiological conditions. These dynamic proteins, called intrinsically disordered proteins (IDPs) and protein regions (IDPRs), are predominantly involved in cell signaling and regulation. Yet, very little is known about their presence among proteins of the core autophagy machinery. In this work, we characterized the autophagy protein Atg3 from yeast and human along with 2 variants to show that Atg3 is an IDPRs-containing protein and that disorder/order predicted for these proteins from their amino acid sequence corresponds to their experimental characteristics. Based on this consensus, we applied the same prediction methods to all known Atg proteins from Saccharomyces cerevisiae. The data presented here provide an insight into the structural dynamics of each Atg protein. They also show that intrinsic disorder at various levels has to be taken into consideration for about half of the Atg proteins. This work should become a useful tool that will facilitate and encourage exploration of protein intrinsic disorder in autophagy.     

Structural trees for protein superfamilies

Structural trees for large protein superfamilies, such as β proteins with the aligned β sheet packing, β proteins with the orthogonal packing of α helices, two-layer and three-layer α/β proteins, have been constructed. The structural motifs having unique overall folds and a unique handedness are taken as root structures of the trees. The larger protein structures of each superfamily are obtained by a stepwise addition of α helices and/or β strands to the corresponding root motif, taking into account a restricted set of rules inferred from known principles of the protein structure. Among these rules, prohibition of crossing connections, attention to handedness and compactness, and a requirement for α helices to be packed in α-helical layers and β strands in β layers are the most important. Proteins and domains whose structures can be obtained by stepwise addition of α helices and/or β strands to the same root motif can be grouped into one structural class or a superfamily. Proteins and domains found within branches of a structural tree can be grouped into subclasses or subfamilies. Levels of structural similarity between different proteins can easily be observed by visual inspection. Within one branch, protein structures having a higher position in the tree include the structures located lower. Proteins and domains of different branches have the structure located in the branching point as the common fold. Proteins 28:241–260, 1997. © 1997 Wiley-Liss Inc.     

Structural classification of small, disulfide-rich protein domains

Disulfide-rich domains are small protein domains whose global folds are stabilized primarily by the formation of disulfide bonds and, to a much lesser extent, by secondary structure and hydrophobic interactions. Disulfide-rich domains perform a wide variety of roles functioning as growth factors, toxins, enzyme inhibitors, hormones, pheromones, allergens, etc. These domains are commonly found both as independent (single-domain) proteins and as domains within larger polypeptides. Here, we present a comprehensive structural classification of approximately 3000 small, disulfide-rich protein domains. We find that these domains can be arranged into 41 fold groups on the basis of structural similarity. Our fold groups, which describe broader structural relationships than existing groupings of these domains, bring together representatives with previously unacknowledged similarities; 18 of the 41 fold groups include domains from several SCOP folds. Within the fold groups, the domains are assembled into families of homologs. We define 98 families of disulfide-rich domains, some of which include newly detected homologs, particularly among knottin-like domains. On the basis of this classification, we have examined cases of convergent and divergent evolution of functions performed by disulfide-rich proteins. Disulfide bonding patterns in these domains are also evaluated. Reducible disulfide bonding patterns are much less frequent, while symmetric disulfide bonding patterns are more common than expected from random considerations. Examples of variations in disulfide bonding patterns found within families and fold groups are discussed.     

Sequence and structure classification of kinases

Kinases are a ubiquitous group of enzymes that catalyze the phosphoryl transfer reaction from a phosphate donor (usually ATP) to a receptor substrate. Although all kinases catalyze essentially the same phosphoryl transfer reaction, they display remarkable diversity in their substrate specificity, structure, and the pathways in which they participate. In order to learn the relationship between structural fold and functional specificities in kinases, we have done a comprehensive survey of all available kinase sequences (>17,000) and classified them into 30 distinct families based on sequence similarities. Of these families, 19, covering nearly 98% of all sequences, fall into seven general structural folds for which three-dimensional structures are known. These fold groups include some of the most widespread protein folds, such as Rossmann fold, ferredoxin fold, ribonuclease H fold, and TIM beta/alpha-barrel. On the basis of this classification system, we examined the shared substrate binding and catalytic mechanisms as well as variations of these mechanisms in the same fold groups. Cases of convergent evolution of identical kinase activities occurring in different folds are discussed.     

A novel member of the YchN-like fold: solution structure of the hypothetical protein Tm0979 from Thermotoga maritima

We report herein the NMR structure of Tm0979, a structural proteomics target from Thermotoga maritima. The Tm0979 fold consists of four beta/alpha units, which form a central parallel beta-sheet with strand order 1234. The first three helices pack toward one face of the sheet and the fourth helix packs against the other face. The protein forms a dimer by adjacent parallel packing of the fourth helices sandwiched between the two beta-sheets. This fold is very interesting from several points of view. First, it represents the first structure determination for the DsrH family of conserved hypothetical proteins, which are involved in oxidation of intracellular sulfur but have no defined molecular function. Based on structure and sequence analysis, possible functions are discussed. Second, the fold of Tm0979 most closely resembles YchN-like folds; however the proteins that adopt these folds differ in secondary structural elements and quaternary structure. Comparison of these proteins provides insight into possible mechanisms of evolution of quaternary structure through a simple mechanism of hydrophobicity-changing mutations of one or two residues. Third, the Tm0979 fold is found to be similar to flavodoxin-like folds and beta/alpha barrel proteins, and may provide a link between these very abundant folds and putative ancestral half-barrel proteins.     

Exploring Potential Signals of Selection for Disordered Residues in Prokaryotic and Eukaryotic Proteins

Intrinsically disordered proteins (IDPs) are an important class of proteins in all domains of life for their functional importance. However, how nature has shaped the disorder potential of prokaryotic and eukaryotic proteins is still not clearly known. Randomly generated sequences are free of any selective constraints, thus these sequences are commonly used as null models. Considering different types of random protein models, here we seek to understand how the disorder potential of natural eukaryotic and prokaryotic proteins differs from random sequences. Comparing proteome-wide disorder content between real and random sequences of 12 model organisms, we noticed that eukaryotic proteins are enriched in disordered regions compared to random sequences, but in prokaryotes such regions are depleted. By analyzing the position-wise disorder profile, we show that there is a generally higher disorder near the N- and C-terminal regions of eukaryotic proteins as compared to the random models; however, either no or a weak such trend was found in prokaryotic proteins. Moreover, here we show that this preference is not caused by the amino acid or nucleotide composition at the respective sites. Instead, these regions were found to be endowed with a higher fraction of protein–protein binding sites, suggesting their functional importance. We discuss several possible explanations for this pattern, such as improving the efficiency of protein–protein interaction, ribosome movement during translation, and post-translational modification. However, further studies are needed to clearly understand the biophysical mechanisms causing the trend.     

Universal Sharing Patterns in Proteomes and Evolution of Protein Fold Architecture and Life

Protein evolution is imprinted in both the sequence and the structure of evolutionary building blocks known as protein domains. These domains share a common ancestry and can be unified into a comparatively small set of folding architectures, the protein folds. We have traced the distribution of protein folds between and within proteomes belonging to Eukarya, Archaea, and Bacteria along the branches of a universal phylogeny of protein architecture. This tree was reconstructed from global fold-usage statistics derived from a structural census of proteomes. We found that folds shared by the three organismal domains were placed almost exclusively at the base of the rooted tree and that there were marked heterogeneities in fold distribution and clear evolutionary patterns related to protein architecture and organismal diversification. These include a relative timing for the emergence of prokaryotes, congruent episodes of architectural loss and diversification in Archaea and Bacteria, and a late and quite massive rise of architectural novelties in Eukarya perhaps linked to multicellularity.Reviewing Editor : Dr. David Pollock     

Nodal modulator (NOMO) is required to sustain endoplasmic reticulum morphology

The endoplasmic reticulum (ER) is a membrane-bound organelle responsible for protein folding, lipid synthesis, and calcium homeostasis. Maintenance of ER structural integrity is crucial for proper function, but much remains to be learned about the molecular players involved. To identify proteins that support the structure of the ER, we performed a proteomic screen and identified nodal modulator (NOMO), a widely conserved type I transmembrane protein of unknown function, with three nearly identical orthologs specified in the human genome. We found that overexpression of NOMO1 imposes a sheet morphology on the ER, whereas depletion of NOMO1 and its orthologs causes a collapse of ER morphology concomitant with the formation of membrane-delineated holes in the ER network positive for the lysosomal marker lysosomal-associated protein 1. In addition, the levels of key players of autophagy including microtubule-associated protein light chain 3 and autophagy cargo receptor p62/sequestosome 1 strongly increase upon NOMO depletion. In vitro reconstitution of NOMO1 revealed a “beads on a string” structure likely representing consecutive immunoglobulin-like domains. Extending NOMO1 by insertion of additional immunoglobulin folds results in a correlative increase in the ER intermembrane distance. Based on these observations and a genetic epistasis analysis including the known ER-shaping proteins Atlastin2 and Climp63, we propose a role for NOMO1 in the functional network of ER-shaping proteins.     

MAMMOTH (matching molecular models obtained from theory): an automated method for model comparison

Advances in structural genomics and protein structure prediction require the design of automatic, fast, objective, and well benchmarked methods capable of comparing and assessing the similarity of low-resolution three-dimensional structures, via experimental or theoretical approaches. Here, a new method for sequence-independent structural alignment is presented that allows comparison of an experimental protein structure with an arbitrary low-resolution protein tertiary model. The heuristic algorithm is given and then used to show that it can describe random structural alignments of proteins with different folds with good accuracy by an extreme value distribution. From this observation, a structural similarity score between two proteins or two different conformations of the same protein is derived from the likelihood of obtaining a given structural alignment by chance. The performance of the derived score is then compared with well established, consensus manual-based scores and data sets. We found that the new approach correlates better than other tools with the gold standard provided by a human evaluator. Timings indicate that the algorithm is fast enough for routine use with large databases of protein models. Overall, our results indicate that the new program (MAMMOTH) will be a good tool for protein structure comparisons in structural genomics applications. MAMMOTH is available from our web site at http://physbio.mssm.edu/~ortizg/.     

Intra-chain 3D segment swapping spawns the evolution of new multidomain protein architectures

Multidomain proteins form in evolution through the concatenation of domains, but structural domains may comprise multiple segments of the chain. In this work, we demonstrate that new multidomain architectures can evolve by an apparent three-dimensional swap of segments between structurally similar domains within a single-chain monomer. By a comprehensive structural search of the current Protein Data Bank (PDB), we identified 32 well-defined segment-swapped proteins (SSPs) belonging to 18 structural families. Nearly 13% of all multidomain proteins in the PDB may have a segment-swapped evolutionary precursor as estimated by more permissive searching criteria. The formation of SSPs can be explained by two principal evolutionary mechanisms: (i) domain swapping and fusion (DSF) and (ii) circular permutation (CP). By large-scale comparative analyses using structural alignment and hidden Markov model methods, it was found that the majority of SSPs have evolved via the DSF mechanism, and a much smaller fraction, via CP. Functional analyses further revealed that segment swapping, which results in two linkers connecting the domains, may impart directed flexibility to multidomain proteins and contributes to the development of new functions. Thus, inter-domain segment swapping represents a novel general mechanism by which new protein folds and multidomain architectures arise in evolution, and SSPs have structural and functional properties that make them worth defining as a separate group.     

Structural anatomy of telomere OB proteins

Telomere DNA-binding proteins protect the ends of chromosomes in eukaryotes. A subset of these proteins are constructed with one or more OB folds and bind with G+T-rich single-stranded DNA found at the extreme termini. The resulting DNA-OB protein complex interacts with other telomere components to coordinate critical telomere functions of DNA protection and DNA synthesis. While the first crystal and NMR structures readily explained protection of telomere ends, the picture of how single-stranded DNA becomes available to serve as primer and template for synthesis of new telomere DNA is only recently coming into focus. New structures of telomere OB fold proteins alongside insights from genetic and biochemical experiments have made significant contributions towards understanding how protein-binding OB proteins collaborate with DNA-binding OB proteins to recruit telomerase and DNA polymerase for telomere homeostasis. This review surveys telomere OB protein structures alongside highly comparable structures derived from replication protein A (RPA) components, with the goal of providing a molecular context for understanding telomere OB protein evolution and mechanism of action in protection and synthesis of telomere DNA.     

Beta turn propensity and a model polymer scaling exponent identify intrinsically disordered phase-separating proteins

The complex cellular milieu can spontaneously demix, or phase separate, in a process controlled in part by intrinsically disordered (ID) proteins. A protein''s propensity to phase separate is thought to be driven by a preference for protein–protein over protein–solvent interactions. The hydrodynamic size of monomeric proteins, as quantified by the polymer scaling exponent (v), is driven by a similar balance. We hypothesized that mean v, as predicted by protein sequence, would be smaller for proteins with a strong propensity to phase separate. To test this hypothesis, we analyzed protein databases containing subsets of proteins that are folded, disordered, or disordered and known to spontaneously phase separate. We find that the phase-separating disordered proteins, on average, had lower calculated values of v compared with their non-phase-separating counterparts. Moreover, these proteins had a higher sequence-predicted propensity for β-turns. Using a simple, surface area-based model, we propose a physical mechanism for this difference: transient β-turn structures reduce the desolvation penalty of forming a protein-rich phase and increase exposure of atoms involved in π/sp² valence electron interactions. By this mechanism, β-turns could act as energetically favored nucleation points, which may explain the increased propensity for turns in ID regions (IDRs) utilized biologically for phase separation. Phase-separating IDRs, non-phase-separating IDRs, and folded regions could be distinguished by combining v and β-turn propensity. Finally, we propose a new algorithm, ParSe (partition sequence), for predicting phase-separating protein regions, and which is able to accurately identify folded, disordered, and phase-separating protein regions based on the primary sequence.     

Progressive combinatorial algorithm for multiple structural alignments: application to distantly related proteins

MALECON is a progressive combinatorial procedure for multiple alignments of protein structures. It searches a library of pairwise alignments for all three-protein alignments in which a specified number of residues is consistently aligned. These alignments are progressively expanded to include additional proteins and more spatially equivalent residues, subject to certain criteria. This action involves superimposing the aligned proteins by their hitherto equivalent residues and searching for additional Calpha atoms that lie close in space. The performance of MALECON is illustrated and compared with several extant multiple structure alignment methods by using as test the globin homologous superfamily, the OB and the Jellyrolls folds. MALECON gives better definitions of the common structural features in the structurally more diverse proteins of the OB and Jellyrolls folds, but it yields comparable results for the more similar globins. When no consistent multiple alignments can be derived for all members of a protein group, our procedure is still capable of automatically generating consistent alignments and common core definitions for subgroups of the members. This finding is illustrated for proteins of the OB fold and SH3 domains, believed to share common structural features, and should be very instrumental in homology modeling and investigations of protein evolution.