Acetylcholinesterases--the structural similarities and differences

Acetylcholinesterase (AChE) is a widely spread enzyme playing a very important role in nerve signal transmission. As AChE controls key processes, its inhibition leads to the very fast death of an organism, including humans. However, when this feature is to be used for killing of unwanted organisms (i.e. mosquitoes), one is faced with the question - how much do AChEs differ between species and what are the differences? Here, a theoretical point of view was utilized to identify the structural basis for such differences. The various primary and tertiary alignments show that AChEs are very evolutionary conserved enzymes and this fact could lead to difficulties, for example, in the search for inhibitors specific for a particular species.     

Assessment of structural and functional similarities and differences between caeca of the bluegill

Bluegill Lepomis macrochirus possess six to eight gastrointestinal caeca, >70% having seven. The terminal caeca toward abdominal edge (C6, C7) were always larger than the terminal caeca toward the vertebral column (C1, C2) and middle caeca (C3, C4, C5). The caeca varied in relative thickness of the histological layers and lumen space. The terminal caeca (C1, C2, C6, C7) have anatomical positional advantage over, and possess more contents, than the middle ones (C3, C4, C5) in both field and laboratory conditions. Hence, all caeca in bluegill may have the same function but their functional potentials are not the same. Two factors, size of caeca and anatomical position of caeca, apparently work synergistically to cause variation in their functional efficiency.     

Biophysical and structural studies of the human calcium- and integrin-binding protein family: understanding their functional similarities and differences

The human calcium- and integrin-binding protein 1 (CIB1) plays important roles in various cellular functions. In this study, three other members of this protein family (CIB2-4: CIB2, CIB3, and CIB4) were purified and subsequently characterized using biophysical and structural approaches. As expected from sequence alignments, CIB2-4 were shown to bind calcium (Ca(2+)) and magnesium (Mg(2+)) ions. Binding of Ca(2+) or Mg(2+) ions changes the secondary structure of CIB2-4 and the exposure of hydrophobic surface area. Ca(2+) and Mg(2+) ions also stabilize the tertiary structures for CIB2 and CIB3. Through in vitro binding experiments, we show that CIB2 can interact with the integrin αIIb cytoplasmic domain and the integrin α7b membrane-proximal fragment. Fluorescence experiments using a 7-azatryptophan labeled peptide demonstrate that CIB2, CIB3, and CIB4 are binding partners for the integrin αIIb subunit, which suggests that they are potentially involved in regulating integrin αIIb subunit activation. The distinct responses of αIIb to the different CIB3 and CIB4 metal (Ca(2+) and Mg(2+)) binding states imply a potential connection between the calcium and integrin signaling pathways.     

Nek1 shares structural and functional similarities with NIMA kinase

The Aspergillus NIMA serine/threonine kinase plays a pivotal role in controlling entrance into mitosis. A major function attributed to NIMA is the induction of chromatin condensation. We show here that the founder murine NIMA-related kinase, Nek1, is larger than previously reported, and that the full-length protein conserves the structural hallmarks of NIMA. Even though Nek1 bears two classical nuclear localization signals (NLS), the endogenous protein localizes to the cytoplasm. Ectopic overexpression of various Nek1 constructs suggests that the C-terminus of Nek1 bears cytoplasmic localization signal(s). Overexpression of nuclear constructs of Nek1 resulted in abnormal chromatin condensation, with the DNA mainly confined to the periphery of the nucleus. Advanced condensation phenotype was associated with nuclear pore complex dispersal. The condensation was not accompanied by up-regulation of mitotic or apoptotic markers. A similar phenotype has been described following NIMA overexpression, strengthening the notion that the mammalian Nek1 kinase has functional similarity to NIMA.     

Protein structural alignments and functional genomics

Structural genomics-the systematic solution of structures of the proteins of an organism-will increasingly often produce molecules of unknown function with no close relative of known function. Prediction of protein function from structure has thereby become a challenging problem of computational molecular biology. The strong conservation of active site conformations in homologous proteins suggests a method for identifying them. This depends on the relationship between size and goodness-of-fit of aligned substructures in homologous proteins. For all pairs of proteins studied, the root-mean-square deviation (RMSD) as a function of the number of residues aligned varies exponentially for large common substructures and linearly for small common substructures. The exponent of the dependence at large common substructures is well correlated with the RMSD of the core as originally calculated by Chothia and Lesk (EMBO J 1986;5:823-826), affording the possibility of reconciling different structural alignment procedures. In the region of small common substructures, reduced aligned subsets define active sites and can be used to suggest the locations of active sites in homologous proteins.     

Alpha-synuclein has structural and functional similarities to small heat shock proteins

The aggregation and fibrillization of α-synuclein, a major component of Lewy bodies, is a key event in Parkinson’s disease. Although the mechanisms of fibrils formation are largely investigated, physiological function of α-synuclein is not yet clearly elucidated. Here, we showed that C-terminal region of α-synuclein is similar to α-crystalline domain of small heat shock proteins. In our experiments, α-synuclein, like small heat shock proteins, protected cellular proteins from denaturation, and confer Escherichia coli cellular tolerances against thermal- and oxidative-stresses.     

Protein structural similarities predicted by a sequence-structure compatibility method.

A method for protein structure prediction has been developed, which evaluates the compatibility of an amino acid sequence with known 3-dimensional structures and identifies the most likely structure. The method was applied to a large number of sequences in a database, and the structures of the following proteins were predicted: (1) shikimate kinase (SKase), (2) the hydrophilic subunit of mannose permease (IIABMan), (3) rat tyrosine aminotransferase (Tyr AT), and (4) threonine dehydratase (TDH). The functional and evolutionary implications of the predictions are discussed. (1) The structural similarity between SKase and adenylate kinase was predicted. Alignment of their sequences reveals that the ATP-binding type A sequence motif and 2 ATP-binding arginine residues are conserved. The prediction suggests a similarity in their functional mechanisms as well as an evolutionary relationship. (2) The structural similarity between IIABMan and galactose/glucose-binding protein (GGBP) was predicted. The IIA and IIB domains are aligned with the N- and C-terminal domains of GGBP, respectively. The 2 phosphorylated residues, His 10 and His 175, of IIABMan are threaded onto loops located in the substrate-binding cleft of GGBP. The prediction accounts for the phosphoryl transfer from His 10 to His 175, and to the sugar substrate. (3) The structural similarity between rat Tyr AT and Escherichia coli aspartate AT was predicted, as well as (4) the structural similarity between TDH and the tryptophan synthase beta subunit. Predictions (3) and (4) support the previous predictions based on observations of the functional similarities between the proteins.     

Centrosomal localization of cyclins E and A: Structural similarities and functional differences

Recent identification of the modular CLS motifs responsible for cyclins A and E localization on centrosomes has revealed a tight linkage between the nuclear and centrosomal cycles. These G₁/S cyclins must localize on the centrosome in order for DNA replication to occur in the nucleus, whereas essential DNA replication factors also function on the centrosome to prevent centrosome overduplication. Both events are dependent on the presence of an intact CLS within each cyclin. Here we compare the cyclins A and E CLSs at the structural and functional levels and identify a new cyclin A CLS mutant that disrupts all CLS functions and reduces the affinity of cyclin A for Cdk2. Analysis of interactions of the CLS motif within the cyclin molecules highlights the importance of the cyclin CBOX1 region for Cdk2 binding.Key words: cyclin A, cyclin E, Cdk2, centrosome, CLS, PSTAIRE, DNA synthesis     

AMPA and NMDA receptors: similarities and differences in their synaptic distribution

Recent technical developments, including antigen-retrieval and electron microscopic immunogold methods, are making it possible to determine some of the basic principles governing the subcellular distribution of ionotropic glutamate receptors. Distinct AMPA and NMDA receptor subtypes are selectively targeted to functionally different synapses of a single cell, resulting in an input-selective fine-tuning and regulation of the postsynaptic responses. The amount, density and variability of AMPA receptors at a given glutamatergic synapse is governed by both pre- and postsynaptic factors, resulting in functionally distinct glutamatergic connections that display characteristic patterns of receptor expression.     

Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes.

Many viruses express their genome, or part of their genome, initially as a polyprotein precursor that undergoes proteolytic processing. Molecular genetic analyses of viral gene expression have revealed that many of these processing events are mediated by virus-encoded proteinases. Biochemical activity studies and structural analyses of these viral enzymes reveal that they have remarkable similarities to cellular proteinases. However, the viral proteinases have evolved unique features that permit them to function in a cellular environment. In this article, the current status of plant and animal virus proteinases is described along with their role in the viral replication cycle. The reactions catalyzed by viral proteinases are not simple enzyme-substrate interactions; rather, the processing steps are highly regulated, are coordinated with other viral processes, and frequently involve the participation of other factors.     

Characterization of spindle checkpoint kinase Mps1 reveals domain with functional and structural similarities to tetratricopeptide repeat motifs of Bub1 and BubR1 checkpoint kinases

Kinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region.     

Protein structure similarities.

Comparison of protein structures can reveal distant evolutionary relationships that would not be detected by sequence information alone. This helps to infer functional properties. In recent years, many methods for pairwise protein structure alignment have been proposed and are now available on the World Wide Web. Although these methods have made it possible to compare all available protein structures, they also highlight the remaining difficulties in defining a reliable score for protein structure similarities.     

Protein semi-synthesis: new proteins for functional and structural studies

Our ability to alter and control the structure and function of biomolecules, and of proteins in particular, will be of utmost importance in order to understand their respective biological roles in complex systems such as living organisms. This challenge has prompted the development of powerful modern techniques in the fields of molecular biology, physical biochemistry and chemical biology. These fields complement each other and their successful combination has provided unique insights into protein structure and function at the level of isolated molecules, cells and organisms. Chemistry is without doubt most suited for introducing subtle changes into biomolecules down to the atomic level, but often struggles when it comes to large targets, such as proteins. In this review, we attempt to give an overview of modern and broadly applicable techniques that permit chemical synthesis to be applied to complex protein targets in order to gain control over their structure and function. As will be demonstrated, these approaches offer unique possibilities in our efforts to understand the molecular basis of protein functioning in vitro and in vivo. We will discuss modern synthetic reactions that can be applied to proteins and give examples of recent highlights. Another focus of this review will be the application of inteins as versatile protein engineering tools.     

Three human interferon-alpha 2 subvariants disclose structural and functional differences.

The human interferon-alpha 2 subvariants 2a, 2b and 2c differ by only one or two amino acids at positions 23 and/or 34 of the mature protein. In this study, the coding regions of the three interferon-alpha 2 subvariants were derived from the cDNA of interferon-alpha 2c by site-directed in vitro mutagenesis. The interferon-alpha subvariants were synthesized using the same Escherichia coli strain for production and were subsequently purified. Comparative studies revealed that they differ significantly in their biological and antigenic properties. Therefore, amino acid positions 23 and 34 seem to be crucial for structure/function of human interferon-alpha. Furthermore, the study points to the importance of defining, whether such minor structural variants of naturally occurring polypeptides represent functional variants.     

Yeast calmodulin: structural and functional differences compared with vertebrate calmodulin

Calmodulin of the baker's yeast (Saccharomyces cerevisiae) showed a similar affinity for Ca2+ to that of vertebrate calmodulin. The maximum binding number of Ca2+ to yeast calmodulin was, however, 3 mol/mol, which is lower than that of vertebrate calmodulin (4 mol/mol). The same maximum activity of porcine brain phosphodiesterase was attained when 100 times higher concentration of yeast calmodulin than that of vertebrate calmodulin was added. On the other hand, the maximum activation of chicken gizzard myosin light chain kinase was attained with 1,000 times higher concentration of yeast calmodulin than that of vertebrate calmodulin, and the maximum activity with yeast calmodulin was less than 1/5 of that with vertebrate calmodulin. Several amino acid substitutions observed in the yeast calmodulin, particularly at the alpha-helical rod connecting the two globular domains, may affect the interaction mode of various target enzymes with this calmodulin.     

Arteriogenesis versus angiogenesis: similarities and differences

Cardiovascular diseases account for more than half of total mortality before the age of 75 in industrialized countries. To develop therapies promoting the compensatory growth of blood vessels could be superior to palliative surgical interventions. Therefore, much effort has been put into investigating underlying mechanisms. Depending on the initial trigger, growth of blood vessels in adult organisms proceeds via two major processes, angiogenesis and arteriogenesis. While angiogenesis is induced by hypoxia and results in new capillaries, arteriogenesis is induced by physical forces, most importantly fluid shear stress. Consequently, chronically elevated fluid shear stress was found to be the strongest trigger under experimental conditions. Arteriogenesis describes the remodelling of pre-existing arterio-arteriolar anastomoses to completely developed and functional arteries. In both growth processes, enlargement of vascular wall structures was proposed to be covered by proliferation of existing wall cells. Recently, increasing evidence emerges, implicating a pivotal role for circulating cells, above all blood monocytes, in vascular growth processes. Since it has been shown that monocytes/ macrophage release a cocktail of chemokines, growth factors and proteases involved in vascular growth, their contribution seems to be of a paracrine fashion. A similar role is currently discussed for various populations of bone-marrow derived stem cells and endothelial progenitors. In contrast, the initial hypothesis that these cells -after undergoing a (trans-)differentiation- contribute by a structural integration into the growing vessel wall, is increasingly challenged.     

Conservation of structural fluctuations in homologous protein kinases and its implications on functional sites

Our aim is to explore the similarities in structural fluctuations of homologous kinases. Gaussian Network Model based Normal Mode Analysis was performed on 73 active conformation structures in Ser/Thr/Tyr kinase superfamily. Categories of kinases with progressive evolutionary divergence, viz. (i) Same kinase with many crystal structures, (ii) Within‐Subfamily, (iii) Within‐Family, (iv) Within‐Group, and (v) Across‐Group, were analyzed. We identified a flexibility signature conserved in all kinases involving residues in and around the catalytic loop with consistent low‐magnitude fluctuations. However, the overall structural fluctuation profiles are conserved better in closely related kinases (Within‐Subfamily and Within‐family) than in distant ones (Within‐Group and Across‐Group). A substantial 65.4% of variation in flexibility was not accounted by variation in sequences or structures. Interestingly, we identified substructural residue‐wise fluctuation patterns characteristic of kinases of different categories. Specifically, we recognized statistically significant fluctuations unique to families of protein kinase A, cyclin‐dependent kinases, and nonreceptor tyrosine kinases. These fluctuation signatures localized to sites known to participate in protein‐protein interactions typical of these kinase families. We report for the first time that residues characterized by fluctuations unique to the group/family are involved in interactions specific to the group/family. As highlighted for Src family, local regions with differential fluctuations are proposed as attractive targets for drug design. Overall, our study underscores the importance of consideration of fluctuations, over and above sequence and structural features, in understanding the roles of sites characteristic of kinases. Proteins 2016; 84:957–978. © 2016 Wiley Periodicals, Inc.