Biochemical and Functional Evidence of p53 Homology Is Inconsistent with Molecular Phylogenetics for Distant Sequences

The tumor suppressor p53 is mutated in ～50% of all human cancer cases worldwide. It is commonly assumed that the phylogenetic history of this important tumor suppressor has been thoroughly studied; however, few detailed studies of the entire extended p53 protein family have been reported, and none comprehensively and simultaneously consider functional, molecular, and phylogenetic data. Herein we examine a diverse collection of reported p53-like protein sequences, including representatives from the arthropods, nematodes, and protists, with the goal of answering several important questions. First, what evidence supports these highly divergent proteins being true homologues to the p53 family? Second, is the inferred overall family phylogeny concordant with known structures and functions? Third, does the extended p53 family possess recognizable conserved sites outside of the within-chordate, highly-conserved DNA-binding domain? Our study shows that the biochemical and functional evidence of p53 homology for nematodes, arthropods, and protists is inconsistent with their implied phylogenetic relationship within the overall family. Although these divergent sequences are always reported as functionally similar to human p53, our results confirm and extend the hypothesis that p63 is a far more appropriate protein for comparison. Within these divergent sequences, we find minimal conservation within the DNA-binding domain, and no conservation elsewhere. Taken together, our findings suggest that these sequences are not bona fide homologues of the extended p53 family and provide baseline criteria for the future identification and characterization of distant p53-family homologues.     

Bayesian shadows of molecular mechanisms cast in the light of evolution

A great many carefully designed experiments will be required to fully understand biological mechanisms in atomic detail. A complementary approach is to use powerful statistical procedures to rapidly test numerous scientific hypotheses using vast numbers of protein sequences--the cell's own blueprints for specifying biological mechanisms. Bayesian inference of the evolutionary constraints imposed on functionally divergent proteins can reveal key components of the molecular machinery and thereby suggest likely mechanisms to test experimentally. This approach is demonstrated by considering how DNA polymerase clamp-loader AAA+ ATPases couple DNA recognition to ATP hydrolysis and clamp loading.     

Computational protein design and large-scale assessment by I-TASSER structure assembly simulations

Protein design aims at designing new protein molecules of desired structure and functionality. One of the major obstacles to large-scale protein design are the extensive time and manpower requirements for experimental validation of designed sequences. Recent advances in protein structure prediction have provided potentials for an automated assessment of the designed sequences via folding simulations. We present a new protocol for protein design and validation. The sequence space is initially searched by Monte Carlo sampling guided by a public atomic potential, with candidate sequences selected by the clustering of sequence decoys. The designed sequences are then assessed by I-TASSER folding simulations, which generate full-length atomic structural models by the iterative assembly of threading fragments. The protocol is tested on 52 nonhomologous single-domain proteins, with an average sequence identity of 24% between the designed sequences and the native sequences. Despite this low sequence identity, three-dimensional models predicted for the first designed sequence have an RMSD of < 2 Å to the target structure in 62% of cases. This percentage increases to 77% if we consider the three-dimensional models from the top 10 designed sequences. Such a striking consistency between the target structure and the structural prediction from nonhomologous sequences, despite the fact that the design and folding algorithms adopt completely different force fields, indicates that the design algorithm captures the features essential to the global fold of the target. On average, the designed sequences have a free energy that is 0.39 kcal/(mol residue) lower than in the native sequences, potentially affording a greater stability to synthesized target folds.     

Distinct Molecular Strategies for Hox-Mediated Limb Suppression in Drosophila: From Cooperativity to Dispensability/Antagonism in TALE Partnership

The emergence following gene duplication of a large repertoire of Hox paralogue proteins underlies the importance taken by Hox proteins in controlling animal body plans in development and evolution. Sequence divergence of paralogous proteins accounts for functional specialization, promoting axial morphological diversification in bilaterian animals. Yet functionally specialized paralogous Hox proteins also continue performing ancient common functions. In this study, we investigate how highly divergent Hox proteins perform an identical function. This was achieved by comparing in Drosophila the mode of limb suppression by the central (Ultrabithorax and AbdominalA) and posterior class (AbdominalB) Hox proteins. Results highlight that Hox-mediated limb suppression relies on distinct modes of DNA binding and a distinct use of TALE cofactors. Control of common functions by divergent Hox proteins, at least in the case studied, relies on evolving novel molecular properties. Thus, changes in protein sequences not only provide the driving force for functional specialization of Hox paralogue proteins, but also provide means to perform common ancient functions in distinct ways.     

The primary structure of rat liver glycogen synthase deduced by cDNA cloning. Absence of phosphorylation sites 1a and 1b

The cDNA for rat liver glycogen synthase was isolated by screening a rat liver cDNA library constructed in lambda gt11. The cDNA was 2.4 kilobases in length and encoded a protein of 703 amino acid residues with a molecular mass of 80.5 kDa. Comparison of the rat liver and the human muscle sequences show that the amino- and carboxyl-terminal regions are quite divergent as compared to the internal sequences which show an 80% identity. The rat liver carboxyl-terminal region is truncated by 33 residues and has only 46% identity with the muscle sequence but retains the common feature of a low content of hydrophobic amino acids (13%). Phosphorylation sites 1a and 1b, which are the primary targets for phosphorylation by cAMP-dependent protein kinase, are absent in the liver sequence. The presence of these divergent, structurally anomalous carboxyl-terminal regions in liver and muscle glycogen synthase suggests the absence of the requirement that they possess a tertiary structure that is integral to that of the protein core. A model is proposed in which this region interacts with a catalytic core to maintain the I state, and in which phosphorylation serves to uncouple this interaction.     

Evolution acts on enhancer organization to fine-tune gradient threshold readouts

The elucidation of principles governing evolution of gene regulatory sequence is critical to the study of metazoan diversification. We are therefore exploring the structure and organizational constraints of regulatory sequences by studying functionally equivalent cis-regulatory modules (CRMs) that have been evolving in parallel across several loci. Such an independent dataset allows a multi-locus study that is not hampered by nonfunctional or constrained homology. The neurogenic ectoderm enhancers (NEEs) of Drosophila melanogaster are one such class of coordinately regulated CRMs. The NEEs share a common organization of binding sites and as a set would be useful to study the relationship between CRM organization and CRM activity across evolving lineages. We used the D. melanogaster transgenic system to screen for functional adaptations in the NEEs from divergent drosophilid species. We show that the individual NEE modules across a genome in any one lineage have independently evolved adaptations to compensate for lineage-specific developmental and/or genomic changes. Specifically, we show that both the site composition and the site organization of NEEs have been finely tuned by distinct, lineage-specific selection pressures in each of the three divergent species that we have examined: D. melanogaster, D. pseudoobscura, and D. virilis. Furthermore, by precisely altering the organization of NEEs with different morphogen gradient threshold readouts, we show that CRM organizational evolution is sufficient for explaining changes in enhancer activity. Thus, evolution can act on CRM organization to fine-tune morphogen gradient threshold readouts over a wide dynamic range. Our study demonstrates that equivalence classes of CRMs are powerful tools for detecting lineage-specific adaptations by gene regulatory sequences.     

Multiresolution analysis uncovers hidden conservation of properties in structurally and functionally similar proteins

Physicochemcial properties of amino acids are important factors in determining protein structure and function. Most approaches make use of averaged properties over entire domains or even proteins to analyze their structure or function. This level of coarseness tends to hide the richness of the variability in the different properties across functional domains. This paper studies the conservation of physicochemical properties in a functionally similar family of proteins using a novel wavelet-based technique known as multiresolution analysis. Such an analysis can help uncover characteristics that can otherwise remain hidden. We have studied the protein kinase family of sequences and our findings are as follows: (a) a number of different properties are conserved over the functional catalytic domain irrespective of the sequence identities; (b) conservation of properties can be observed at different frequency levels and they agree well with the known structural/functional properties of the subdomains for the protein kinase family; (c) structural differences between the different kinase family members are reflected in the waveforms; and (d) functionally important mutations show distortions in the waveforms of conserved properties. The potential usefulness of the above findings in identifying functionally similar sequences in the twilight and midnight zones is demonstrated through a simple prediction model for the protein kinase family which achieved a recall of 93.7% and a precision of 96.75% in cross-validation tests.     

Natural selection and mammalian BRCA1 sequences: elucidating functionally important sites relevant to breast cancer susceptibility in humans

Comparison of orthologous gene sequences is emerging as a powerful approach to elucidating functionally important positions in human disease genes. Using a diverse array of 132 mammalian BRCA1 (exon 11) sequences, we evaluated the functional significance of specific sites in the context of selection information (purifying, neutral, or diversifying) as well as the ability to extract such information from alignments that index varying degrees of mammalian diversity. Small data sets of either closely related taxa (Primates) or divergent placental taxa were unable to distinguish sites conserved due to purifying selection from sites conserved due to chance (false-positive rate = 65%-99%). Increasing the number of placental taxa to 57 greatly reduced the potential false-positive rate (0%-1.5%). Using the larger data set, we ranked the oncogenic risk of human missense mutations using a novel method that incorporates site-specific selection level and severity of the amino acid change evaluated against the amino acids present in other mammalian taxa. In addition to sites undergoing positive selection in Marsupialia, Laurasiatheria, Euarchontoglires, and Primates, we identified sites most likely to be undergoing divergent selection pressure in different lineages and six pairs of potentially interacting sites. Our results demonstrate the necessity of including large numbers of sequences to elucidate functionally important sites of a protein when using a comparative evolutionary approach.     

Mechanisms of Protein Sequence Divergence and Incompatibility

Alignments of orthologous protein sequences convey a complex picture. Some positions are utterly conserved whilst others have diverged to variable degrees. Amongst the latter, many are non-exchangeable between extant sequences. How do functionally critical and highly conserved residues diverge? Why and how did these exchanges become incompatible within contemporary sequences? Our model is phosphoglycerate kinase (PGK), where lysine 219 is an essential active-site residue completely conserved throughout Eukaryota and Bacteria, and serine is found only in archaeal PGKs. Contemporary sequences tested exhibited complete loss of function upon exchanges at 219. However, a directed evolution experiment revealed that two mutations were sufficient for human PGK to become functional with serine at position 219. These two mutations made position 219 permissive not only for serine and lysine, but also to a range of other amino acids seen in archaeal PGKs. The identified trajectories that enabled exchanges at 219 show marked sign epistasis - a relatively small loss of function with respect to one amino acid (lysine) versus a large gain with another (serine, and other amino acids). Our findings support the view that, as theoretically described, the trajectories underlining the divergence of critical positions are dominated by sign epistatic interactions. Such trajectories are an outcome of rare mutational combinations. Nonetheless, as suggested by the laboratory enabled K219S exchange, given enough time and variability in selection levels, even utterly conserved and functionally essential residues may change.     

Integrating protein structures and precomputed genealogies in the Magnum database: Examples with cellular retinoid binding proteins

Background  

When accurate models for the divergent evolution of protein sequences are integrated with complementary biological information, such as folded protein structures, analyses of the combined data often lead to new hypotheses about molecular physiology. This represents an excellent example of how bioinformatics can be used to guide experimental research. However, progress in this direction has been slowed by the lack of a publicly available resource suitable for general use.     

Diversity, function and evolution of genes coding for putative Ni-containing superoxide dismutases

We examined the phylogenetic distribution, functionality and evolution of the sodN gene family, which has been shown to code for a unique Ni-containing isoform of superoxide dismutase (Ni-SOD) in Streptomyces . Many of the putative sodN sequences retrieved from public domain genomic and metagenomic databases are quite divergent from structurally and functionally characterized Ni-SOD. Structural bioinformatics studies verified that the divergent members of the sodN protein family code for similar three-dimensional structures and identified evolutionarily conserved amino acid residues. Structural and biochemical studies of the N-terminus 'Ni-hook' motif coded for by the putative sodN sequences confirmed both Ni (II) ligating and superoxide dismutase activity. Both environmental and organismal genomes expanded the previously noted phylogenetic distribution of sodN , and the sequences form four well-separated clusters, with multiple subclusters. The phylogenetic distribution of sodN suggests that the gene has been acquired via horizontal gene transfer by numerous organisms of diverse phylogenetic background, including both Eukaryotes and Prokaryotes . The presence of sodN correlates with the genomic absence of the gene coding for Fe-SOD, a structurally and evolutionarily distinct isoform of SOD. Given the low levels of Fe found in the marine environment from where many sequences were attained, we suggest that the replacement of Fe-SOD with Ni-SOD may be an evolutionary adaptation to reduce iron requirements.     

Pseudotyping Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV): F proteins from group II NPVs are functionally analogous to AcMNPV GP64

GP64, the major envelope glycoprotein of budded virions of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), is involved in viral attachment, mediates membrane fusion during virus entry, and is required for efficient virion budding. Thus, GP64 is essential for viral propagation in cell culture and in animals. Recent genome sequences from a number of baculoviruses show that only a subset of closely related baculoviruses have gp64 genes, while other baculoviruses have a recently discovered unrelated envelope protein named F. F proteins from Lymantria dispar MNPV (LdMNPV) and Spodoptera exigua MNPV (SeMNPV) mediate membrane fusion and are therefore thought to serve roles similar to that of GP64. To determine whether F proteins are functionally analogous to GP64 proteins, we deleted the gp64 gene from an AcMNPV bacmid and inserted F protein genes from three different baculoviruses. In addition, we also inserted envelope protein genes from vesicular stomatitis virus (VSV) and Thogoto virus. Transfection of the gp64-null bacmid DNA into Sf9 cells does not generate infectious particles, but this defect was rescued by introducing either the F protein gene from LdMNPV or SeMNPV or the G protein gene from VSV. These results demonstrate that baculovirus F proteins are functionally analogous to GP64. Because baculovirus F proteins appear to be more widespread within the family and are much more divergent than GP64 proteins, gp64 may represent the acquisition of an envelope protein gene by an ancestral baculovirus. The AcMNPV pseudotyping system provides an efficient and powerful method for examining the functions and compatibilities of analogous or orthologous viral envelope proteins, and it could have important biotechnological applications.     

Differential regulation at functionally divergent release sites along a common axon

Information transfer within neuronal networks requires the precise coordination of distinct neuronal populations within a given circuit. Evidence from a variety of central pathways indicates that such coordination is mediated in part by the ability of neurons to differentially regulate release properties at functionally divergent presynaptic elements along their individual axons according to the identity of the postsynaptic cell being innervated. Recent findings have revealed the cellular mechanisms by which central afferents modify release properties at individual presynaptic sites independent of neighboring terminals. Such autonomy of presynaptic regulation enables target-cell-dependent short-term and long-term synaptic plasticity and ensures that distinct features of afferent activity are relayed to divergent target-cell populations.     

Identification of GATC- and CCGG-recognizing Type II REases and their putative specificity-determining positions using Scan2S--a novel motif scan algorithm with optional secondary structure constraints

Restriction endonucleases (REases) are DNA-cleaving enzymes that have become indispensable tools in molecular biology. Type II REases are highly divergent in sequence despite their common structural core, function and, in some cases, common specificities towards DNA sequences. This makes it difficult to identify and classify them functionally based on sequence, and has hampered the efforts of specificity-engineering. Here, we define novel REase sequence motifs, which extend beyond the PD-(D/E)XK hallmark, and incorporate secondary structure information. The automated search using these motifs is carried out with a newly developed fast regular expression matching algorithm that accommodates long patterns with optional secondary structure constraints. Using this new tool, named Scan2S, motifs derived from REases with specificity towards GATC- and CGGG-containing DNA sequences successfully identify REases of the same specificity. Notably, some of these sequences are not identified by standard sequence detection tools. The new motifs highlight potential specificity-determining positions that do not fully overlap for the GATC- and the CCGG-recognizing REases and are candidates for specificity re-engineering.     

Molecular cloning of the human B cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains.

CD20 is an antigen expressed on normal and malignant human B cells that is thought to function as a receptor during B cell activation. Here we report the isolation of a CD20-specific cDNA clone from a lambda gt11 library using a polyclonal antiserum raised against purified CD20 antigen. Additional cDNA clones were then isolated from a lambda gt10 library. Alignment of the sequences of overlapping lambda clones reveal a single consensus sequence except for a divergence that preceded the first methionine within the open reading frame. Normal B cells and B cell lines contain a prominent 2.6 kb mRNA and a lower level of a 3.3 kb mRNA. An oligonucleotide derived from one of the divergent sequences hybridized to the 3.3 kb mRNA only, indicating that the two mRNA species are derived from an alternative splicing mechanism. The predicted amino acid sequence of CD20 reveals three major hydrophobic regions of approximately 53, 25 and 20 amino acids. CD20 lacks an NH2-terminal signal peptide and contains a highly charged COOH-terminal domain. Although CD20 is immunoprecipitated as a doublet of 33 and 35 kd proteins from B cells, in vitro translation of CD20 cDNA produced a single 33 kd protein that was specifically immunoprecipitated with monoclonal CD20 antibodies. CD20 was strongly phosphorylated on resting B cells after CDw40 stimulation, suggesting that CD20 may be functionally regulated by a protein kinase(s).     

Identification of a region in G protein γ subunits conserved across species but hypervariable among subunit isoforms

The heterotrimeric GTP binding proteins, G proteins, consist of three distinct subunits: alpha, beta, and gamma. There are 12 known mammalian gamma subunit genes whose products are the smallest and most variable of the G protein subunits. Sequencing of the bovine brain gamma(10) protein by electrospray mass spectrometry revealed that it differs from the human protein by an Ala to Val substitution near the N-terminus. Comparison of gamma isoform subunit sequences indicated that they vary substantially more at the N-terminus than at other parts of the protein. Thus, species variation of this region might reflect the lack of conservation of a functionally unimportant part of the protein. Analysis of 38 gamma subunit sequences from four different species shows that the N-terminus of a given gamma subunit isoform is as conserved between different species as any other part of the protein, including highly conserved regions. These data suggest that the N-terminus of gamma is a functionally important part of the protein exhibiting substantial isoform-specific variation.     

Cloning and sequence analysis of lily and tobacco guanylate kinases

Guanylate kinase is an essential enzyme in the nucleotide biosynthetic pathway, catalyzing the reversible transfer of the terminal phospharyl group of ATP to GMP or dGMP. This enzyme has been well studied from several organisms and many structural and functional details have been characterized. Animal GMP kinases have also been implicated in signal transduction pathways. However, the corresponding role by plant derived GMP kinases remains to be elucidated. Full-length cDNA clones encoding enzymatically active guanylate kinases were isolated from cDNA libraries of lily and tobacco. Lily cDNA is predicted to encode a 392-amino acid protein with a molecular mass of 43.1 kDa and carries amino- and carboxy- terminal extensions of the guanylate kinase (GK)-like domain. But tobacco cDNA is predicted to encode a smaller protein of 297-amino acids with a molecular mass of 32.7 kDa. The amino acid residues known to participate in the catalytic activity of functionally characterized GMP kinases, are also conserved in GK domains of LGK-1 and NGK-1. The GK domains of NGK-1, LGK-1 and previously characterized AGK-1 from Arabidopsis exhibit 74–84% identity, whereas their N- and C-terminal domains are more divergent with amino acid conservation in the order of 48-55%. Phylogenetic analysis on the deduced amino acid sequences reveals that NGK-1 and LGK-1 form one distinct subgroup along with AGK-1 and AGK-2 homologues from Arabidopsis. Isolation of GMP kinases from diverse plant species like lily and tobacco adds a new dimension in understanding their role in cell signaling pathways that are associated with plant growth and development.