Core Residues Research Articles

DNA–RNA Hybrid Quadruplexes Reveal Interactions that Favor RNA Parallel Topologies

A DNA G-quadruplex adopting a (3+1)-hybrid structure was substituted at its 5'-tetrad by riboguanosine (rG) analogs. Incorporation of anti-favoring rG at appropriate syn-positions of the 5'-outer tetrad induced conformational rearrangements to yield a quadruplex featuring a 5'-tetrad with reversed polarity. A high-resolution structure of a disubstituted quadruplex variant as well as direct NMR experimental evidence reveals a non-conventional C-H⋅⋅⋅O hydrogen bond in a medium groove between the 2'-OH of an rG residue adopting a C2'-endo sugar pucker and H8 of a 3'-neighboring anti-G residue. In contrast, a C3'-endo sugar conformation for another guanine ribonucleotide prevents formation of a corresponding hydrogen bond but relocates its 2'-OH substituent from the quadruplex narrow groove into a medium groove. Both the formation of favorable CHO hydrogen bridges and unfavorable interactions of the 2'-hydroxyl group in a narrow groove will promote RNA folding into a parallel topology featuring all-anti core residues and four grooves of medium size.

Fine-tuning of the stability of β-strands by Y181 in perfringolysin O directs the prepore to pore transition

Perfringolysin O (PFO) is a toxic protein that forms β-barrel transmembrane pores upon binding to cholesterol-containing membranes. The formation of lytic pores requires conformational changes in PFO that lead to the conversion of water-soluble monomers into membrane-bound oligomers. Although the general outline of stepwise pore formation has been established, the underlying mechanistic details await clarification. To extend our understanding of the molecular mechanisms that control the pore formation, we compared the hydrogen-deuterium exchange patterns of PFO with its derivatives bearing mutations in the D3 domain. In the case of two of these mutations F318A, Y181A, known from previous work to lead to a decreased lytic activity, global destabilization of all protein domains was observed in their water-soluble forms. This was accompanied by local changes in D3 β-sheet, including unexpected stabilization of functionally important β1 strand in Y181A. In case of the double mutation (F318A/Y181A) that completely abolished the lytic activity, several local changes were retained, but the global destabilization effects of single mutations were reverted and hydrogen-deuterium exchange (HDX) pattern returned to PFO level. Strong structural perturbations were not observed in case of remaining variants in which other residues of the hydrophobic core of D3 domain were substituted by alanine. Our results indicate the existence in PFO of a well-tuned H-bonding network that maintains the stability of the D3 β-strands at appropriate level at each transformation step. F318 and Y181 moieties participate in this network and their role extends beyond their direct intermolecular interaction during oligomerization that was identified previously.

Precise Binding of Tropomyosin on Actin Involves Sequence-Dependent Variance in Coiled-Coil Twisting

Often considered an archetypal dimeric coiled coil, tropomyosin nonetheless exhibits distinctive “noncanonical” core residues located at the hydrophobic interface between its component α-helices. Notably, a charged aspartate, D137, takes the place of nonpolar residues otherwise present. Much speculation has been offered to rationalize potential local coiled-coil instability stemming from D137 and its effect on regulatory transitions of tropomyosin over actin filaments. Although experimental approaches such as electron cryomicroscopy reconstruction are optimal for defining average tropomyosin positions on actin filaments, to date, these methods have not captured the dynamics of tropomyosin residues clustered around position 137 or elsewhere. In contrast, computational biochemistry, involving molecular dynamics simulation, is a compelling choice to extend the understanding of local and global tropomyosin behavior on actin filaments at high resolution. Here, we report on molecular dynamics simulation of actin-free and actin-associated tropomyosin, showing noncanonical residue D137 as a locus for tropomyosin twist variation, with marked effects on actin-tropomyosin interactions. We conclude that D137-sponsored coiled-coil twisting is likely to optimize electrostatic side-chain contacts between tropomyosin and actin on the assembled thin filament, while offsetting disparities between tropomyosin pseudorepeat and actin subunit periodicities. We find that D137 has only minor local effects on tropomyosin coiled-coil flexibility, (i.e., on its flexural mobility). Indeed, D137-associated overtwisting may actually augment tropomyosin stiffness on actin filaments. Accordingly, such twisting-induced stiffness of tropomyosin is expected to enhance cooperative regulatory translocation of the tropomyosin cable over actin.

Changes in soybean trypsin inhibitor by varying pressure and temperature of processing: A molecular modeling study

Abstract In recent years, molecular dynamic (MD) simulations have been used to understand the effects of various processing methods on the structural properties and stability of proteins. In this study, the conformational changes in soybean trypsin inhibitor molecule were evaluated with the use of high temperature and pressure. The MD simulations have been performed at various temperatures (300 K, 345 K and 373 K) and pressure (1 bar, 3 kbar, 6 kbar) combinations. The results showed that the soybean trypsin inhibitor (STI) molecule is relatively stable at high temperatures, primarily due to the presence of disulphide bonds. However, at higher pressures, significant compaction was observed. Root mean square deviations, Radius of gyration and volume of the STI molecule were evaluated. Furthermore, Ramachandran plots were used to evaluate the stability of the various simulated molecules. It was found that the compaction resulted in high steric interferences among the core residues.

Differential regulation of the Wnt/\u03b2-catenin pathway by hepatitis C virus recombinants expressing core from various genotypes

Clinical studies have suggested association of some hepatitis C virus (HCV) subtypes or isolates with progression toward hepatocellular carcinoma (HCC). HCV core protein has been reported to interfere with host Wnt/β-catenin pathway, a cell fate-determining pathway, which plays a major role in HCC. Here, we investigated the impact of HCV core genetic variability in the dysregulation of Wnt/β-catenin pathway. We used both transient expression of core proteins from clinical isolates of HCV subtypes 1a (Cambodia), 4a (Romania) and 4f (Cameroon) and infection systems based on a set of engineered intergenotypic recombinant viruses encoding core from these various clinical strains. We found that TCF transcription factor-dependent reporter activity was upregulated by core in a strain-specific manner. We documented core sequence-specific transcriptional upregulation of several β-catenin downstream target genes associated with cell proliferation and malignant transformation, fibrogenesis or fat accumulation. The extent of β-catenin nuclear translocation varied in accordance with β-catenin downstream gene upregulation in infected cells. Pairwise comparisons of subgenotypic core recombinants and mutated core variants unveiled the critical role of core residues 64 and 71 in these dysregulations. In conclusion, this work identified natural core polymorphisms involved in HCV strain-specific activation of Wnt/β-catenin pathway in relevant infection systems.

Residue-Specific Dynamics and Local Environmental Changes in Aβ40 Oligomer and Fibril Formation.

Elucidating local dynamics of protein aggregation is crucial for understanding the mechanistic details of protein amyloidogenesis. Herein, we studied the residue-specific dynamics and local environmental changes of Aβ40 along the course of aggregation by using para-cyanophenylalanine (PheCN ) as a fluorescent and vibrational probe. Our results show that the PheCN residues introduced at various positions all exhibited an immediate decay of fluorescence intensity, indicating a relatively synergistic process in early oligomer formation. The fast decreases in the fluorescence intensities of residues 19 and 20 in the central hydrophobic core region and residue 10 in the N-terminal region suggest that they play crucial roles in the formation of the oligomeric core. The PheCN 4 residue exhibits a remarkably slower decrease in fluorescence intensity, implicating its dynamic conformational characteristics in oligomer and fibril formation. Our results also suggest that the N-terminal residues in fibrils are surrounded by a relatively hydrophobic local environment, as opposed to being solvated.

Residue‐Specific Dynamics and Local Environmental Changes in Aβ40 Oligomer and Fibril Formation

AbstractElucidating local dynamics of protein aggregation is crucial for understanding the mechanistic details of protein amyloidogenesis. Herein, we studied the residue‐specific dynamics and local environmental changes of Aβ40 along the course of aggregation by using para‐cyanophenylalanine (PheCN) as a fluorescent and vibrational probe. Our results show that the PheCN residues introduced at various positions all exhibited an immediate decay of fluorescence intensity, indicating a relatively synergistic process in early oligomer formation. The fast decreases in the fluorescence intensities of residues 19 and 20 in the central hydrophobic core region and residue 10 in the N‐terminal region suggest that they play crucial roles in the formation of the oligomeric core. The PheCN4 residue exhibits a remarkably slower decrease in fluorescence intensity, implicating its dynamic conformational characteristics in oligomer and fibril formation. Our results also suggest that the N‐terminal residues in fibrils are surrounded by a relatively hydrophobic local environment, as opposed to being solvated.

Network analysis of a proposed exit pathway for protons to the P-side of cytochrome c oxidase

Cytochrome c Oxidase (CcO) reduces O2, the terminal electron acceptor, to water in the aerobic, respiratory electron transport chain. The energy released by O2 reductions is stored by removing eight protons from the high pH, N-side, of the membrane with four used for chemistry in the active site and four pumped to the low pH, P-side. The proton transfers must occur along controllable proton pathways that prevent energy dissipating movement towards the N-side. The CcO N-side has well established D- and K-channels to deliver protons to the protein interior. The P-side has a buried core of hydrogen-bonded protonatable residues designated the Proton Loading Site cluster (PLS cluster) and many protonatable residues on the P-side surface, providing no obvious unique exit. Hydrogen bond pathways were identified in Molecular Dynamics (MD) trajectories of Rb. sphaeroides CcO prepared in the PR state with the heme a3 propionate and Glu286 in different protonation states. Grand Canonical Monte Carlo sampling of water locations, polar proton positions and residue protonation states in trajectory snapshots identify a limited number of water mediated, proton paths from PLS cluster to the surface via a (P-exit) cluster of residues. Key P-exit residues include His93, Ser168, Thr100 and Asn96. The hydrogen bonds between PLS cluster and P-exit clusters are mediated by a water wire in a cavity centered near Thr100, whose hydration can be interrupted by a hydrophobic pair, Leu255B (near CuA) and Ile99. Connections between the D channel and PLS via Glu286 are controlled by a second, variably hydrated cavity. Significance statementCytochrome C oxidase plays a crucial role in cellular respiration and energy generation. It reduces O2 to water and uses the released free energy to move protons across mitochondrial and bacterial cell membranes adding to the essential electrochemical gradient. Energy storage requires that protons are taken up from the high pH, N-side and released to the low pH, P-side of the membrane. We identify a potential proton exit from a buried cluster of polar residues (the proton loading site) to the P-side of CcO via paths made up of waters and conserved residues. Two water cavities connect the proton exit pathway to the surface only when hydrated. Changing the degree of hydration may control otherwise energetically favorable proton backflow from the P-side.

Structural and functional dissection of differentially expressed tomato WRKY transcripts in host defense response against the vascular wilt pathogen (Fusarium oxysporum f. sp. lycopersici).

The WRKY transcription factors have indispensable role in plant growth, development and defense responses. The differential expression of WRKY genes following the stress conditions has been well demonstrated. We investigated the temporal and tissue-specific (root and leaf tissues) differential expression of plant defense-related WRKY genes, following the infection of Fusarium oxysporum f. sp. lycopersici (Fol) in tomato. The genome-wide computational analysis revealed that during the Fol infection in tomato, 16 different members of WRKY gene superfamily were found to be involved, of which only three WRKYs (SolyWRKY4, SolyWRKY33, and SolyWRKY37) were shown to have clear-cut differential gene expression. The quantitative real time PCR (qRT-PCR) studies revealed different gene expression profile changes in tomato root and leaf tissues. In root tissues, infected with Fol, an increased expression for SolyWRKY33 (2.76 fold) followed by SolyWRKY37 (1.93 fold) gene was found at 24 hrs which further increased at 48 hrs (5.0 fold). In contrast, the leaf tissues, the expression was more pronounced at an earlier stage of infection (24 hrs). However, in both cases, we found repression of SolyWRKY4 gene, which further decreased at an increased time interval. The biochemical defense programming against Fol pathogenesis was characterized by the highest accumulation of H2O2 (at 48 hrs) and enhanced lignification. The functional diversity across the characterized WRKYs was explored through motif scanning using MEME suite, and the WRKYs specific gene regulation was assessed through the DNA protein docking studies The functional WRKY domain modeled had β sheets like topology with coil and turns. The DNA-protein interaction results revealed the importance of core residues (Tyr, Arg, and Lys) in a feasible WRKY-W-box DNA interaction. The protein interaction network analysis revealed that the SolyWRKY33 could interact with other proteins, such as mitogen-activated protein kinase 5 (MAPK), sigma factor binding protein1 (SIB1) and with other WRKY members including WRKY70, WRKY1, and WRKY40, to respond various biotic and abiotic stresses. The STRING results were further validated through Predicted Tomato Interactome Resource (PTIR) database. The CELLO2GO web server revealed the functional gene ontology annotation and protein subcellular localization, which predicted that SolyWRKY33 is involved in amelioration of biological stress (39.3%) and other metabolic processes (39.3%). The protein (SolyWRKY33) most probably located inside the nucleus (91.3%) with having transcription factor binding activity. We conclude that the defense response following the Fol challenge was accompanied by differential expression of the SolyWRKY4(↓), SolyWRKY33(↑) and SolyWRKY37(↑) transcripts. The biochemical changes are occupied by elicitation of H2O2 generation and accumulation and enhanced lignified tissues.

Protein-solvent interfaces in human Y145Stop prion protein amyloid fibrils probed by paramagnetic solid-state NMR spectroscopy

The C-terminally truncated Y145Stop variant of prion protein (PrP23-144), which is associated with heritable PrP cerebral amyloid angiopathy in humans and also capable of triggering a transmissible prion disease in mice, serves as a useful in vitro model for investigating the molecular and structural basis of amyloid strains and cross-seeding specificities. Here, we determine the protein-solvent interfaces in human PrP23-144 amyloid fibrils generated from recombinant 13C,15N-enriched protein and incubated in aqueous solution containing paramagnetic Cu(II)-EDTA, by measuring residue-specific 15N longitudinal paramagnetic relaxation enhancements using two-dimensional magic-angle spinning solid-state NMR spectroscopy. To further probe the interactions of the amyloid core residues with solvent molecules we perform complementary measurements of amide hydrogen/deuterium exchange detected by solid-state NMR and solution NMR methods. The solvent accessibility data are evaluated in the context of the structural model for human PrP23-144 amyloid.

Structural Characterization and Directed Evolution of a Novel Acetyl Xylan Esterase Reveals Thermostability Determinants of the Carbohydrate Esterase 7 Family.

A hot desert hypolith metagenomic DNA sequence data set was screened in silico for genes annotated as acetyl xylan esterases (AcXEs). One of the genes identified encoded an ∼36-kDa protein (Axe1NaM1). The synthesized gene was cloned and expressed, and the resulting protein was purified. NaM1 was optimally active at pH 8.5 and 30°C and functionally stable at salt concentrations of up to 5 M. The specific activity and catalytic efficiency were 488.9 U mg-1 and 3.26 × 106 M-1 s-1, respectively. The crystal structure of wild-type NaM1 was solved at a resolution of 2.03 Å, and a comparison with the structures and models of more thermostable carbohydrate esterase 7 (CE7) family enzymes and variants of NaM1 from a directed evolution experiment suggests that reduced side-chain volume of protein core residues is relevant to the thermal stability of NaM1. Surprisingly, a single point mutation (N96S) not only resulted in a simultaneous improvement in thermal stability and catalytic efficiency but also increased the acyl moiety substrate range of NaM1.IMPORTANCE AcXEs belong to nine carbohydrate esterase families (CE1 to CE7, CE12, and CE16), of which CE7 enzymes possess a unique and narrow specificity for acetylated substrates. All structurally characterized members of this family are moderately to highly thermostable. The crystal structure of a novel, mesophilic CE7 AcXE (Axe1NaM1), from a soil metagenome, provides a basis for comparisons with thermostable CE7 enzymes. Using error-prone PCR and site-directed mutagenesis, we enhanced both the stability and activity of the mesophilic AcXE. With comparative structural analyses, we have also identified possible thermal stability determinants. These are valuable for understanding the thermal stability of enzymes within this family and as a guide for future protein engineering of CE7 and other α/β hydrolase enzymes.

Site-Mutation of Hydrophobic Core Residues Synchronically Poise Super Interleukin 2 for Signaling: Identifying Distant Structural Effects through Affordable Computations.

A superkine variant of interleukin-2 with six site mutations away from the binding interface developed from the yeast display technique has been previously characterized as undergoing a distal structure alteration which is responsible for its super-potency and provides an elegant case study with which to get insight about how to utilize allosteric effect to achieve desirable protein functions. By examining the dynamic network and the allosteric pathways related to those mutated residues using various computational approaches, we found that nanosecond time scale all-atom molecular dynamics simulations can identify the dynamic network as efficient as an ensemble algorithm. The differentiated pathways for the six core residues form a dynamic network that outlines the area of structure alteration. The results offer potentials of using affordable computing power to predict allosteric structure of mutants in knowledge-based mutagenesis.

Identification of evolutionarily conserved residues required for the bioactivity of a pedal peptide/orcokinin-type neuropeptide

Pedal peptides and orcokinins are structurally related neuropeptides that were first discovered in protostomian invertebrates – mollusks and arthropods, respectively. Recently, pedal peptide/ocokinin (PP/OK)-type neuropeptides were discovered in a deuterostomian phylum, the echinoderms, indicating that the evolutionary origin of this neuropeptide family can be traced back to the common ancestor of bilaterian animals. Sequences comparison of PP/OK-type neuropeptides reveals several conserved residues, including N- and C-terminally located hydrophobic residues that are important for the bioactivity of orcokinin. Here we report the first comprehensive analysis of the structure-activity relationships of a PP/OK-type neuropeptide – starfish myorelaxant peptide (SMP; FGKGGAYDPLSAGFTD) from the starfish Patiria pectinifera (Phylum Echinodermata). Comparison of the bioactivity of SMP with N-terminally and/or C-terminally truncated and alanine-substituted SMP analogs revealed a core peptide (GAYDPLSAGF; SMP(5-14)) that retains the muscle-relaxing activity of SMP, albeit with reduced potency and efficacy. Within this core peptide, alanine-substitution of several residues resulted in complete or partial loss of bioactivity, whilst loss or substitution of the N-terminal phenylalanine residue of SMP also caused a substantial reduction in bioactivity. Furthermore, analysis of the bioactivity of other SMP-like peptides derived from the same precursor as SMP revealed that none of these were more potent/effective than SMP as muscle relaxants. In conclusion, we have identified key residues required for the bioactivity of a PP/OK-type neuropeptide (SMP), including hydrophobic residues located in the N- and C-terminal regions that are conserved in PP/OK-type peptides from other phyla as well as core residues that are conserved in echinoderm PP/OK-type peptides.

Comparing side chain packing in soluble proteins, protein‐protein interfaces, and transmembrane proteins

We compare side chain prediction and packing of core and non-core regions of soluble proteins, protein-protein interfaces, and transmembrane proteins. We first identified or created comparable databases of high-resolution crystal structures of these 3 protein classes. We show that the solvent-inaccessible cores of the 3 classes of proteins are equally densely packed. As a result, the side chains of core residues at protein-protein interfaces and in the membrane-exposed regions of transmembrane proteins can be predicted by the hard-sphere plus stereochemical constraint model with the same high prediction accuracies (>90%) as core residues in soluble proteins. We also find that for all 3 classes of proteins, as one moves away from the solvent-inaccessible core, the packing fraction decreases as the solvent accessibility increases. However, the side chain predictability remains high (80% within 30°) up to a relative solvent accessibility, rSASA≲0.3, for all 3 protein classes. Our results show that ≈40% of the interface regions in protein complexes are "core", that is, densely packed with side chain conformations that can be accurately predicted using the hard-sphere model. We propose packing fraction as a metric that can be used to distinguish real protein-protein interactions from designed, non-binding, decoys. Our results also show that cores of membrane proteins are the same as cores of soluble proteins. Thus, the computational methods we are developing for the analysis of the effect of hydrophobic core mutations in soluble proteins will be equally applicable to analyses of mutations in membrane proteins.

Comprehensive Analysis of Constraint on the Spatial Distribution of Missense Variants in Human Protein Structures

The spatial distribution of genetic variation within proteins is shaped by evolutionary constraint and provides insight into the functional importance of protein regions and the potential pathogenicity of protein alterations. Here, we comprehensively evaluate the 3D spatial patterns of human germline and somatic variation in 6,604 experimentally derived protein structures and 33,144 computationally derived homology models covering 77% of all human proteins. Using a systematic approach, we quantify differences in the spatial distributions of neutral germline variants, disease-causing germline variants, and recurrent somatic variants. Neutral missense variants exhibit a general trend toward spatial dispersion, which is driven by constraint on core residues. In contrast, germline disease-causing variants are generally clustered in protein structures and form clusters more frequently than recurrent somatic variants identified from tumor sequencing. In total, we identify 215 proteins with significant spatial constraints on the distribution of disease-causing missense variants in experimentally derived protein structures, only 65 (30%) of which have been previously reported. This analysis identifies many clusters not detectable from sequence information alone; only 12% of proteins with significant clustering in 3D were identified from similar analyses of linear protein sequence. Furthermore, spatial analyses of mutations in homology-based structural models are highly correlated with those from experimentally derived structures, supporting the use of computationally derived models. Our approach highlights significant differences in the spatial constraints on different classes of mutations in protein structure and identifies regions of potential function within individual proteins.

Rare Dissipative Transitions Punctuate the Initiation of Chemical Denaturation in Proteins

Protein unfolding dynamics are bound by their degree of entropy production, a quantity that relates the amount of heat dissipated by a nonequilibrium process to a system’s forward and time-reversed trajectories. We here explore the statistics of heat dissipation that emerge in protein molecules subjected to a chemical denaturant. Coupling large molecular dynamics datasets and Markov state models with the theory of entropy production, we demonstrate that dissipative processes can be rigorously characterized over the course of the urea-induced unfolding of the protein chymotrypsin inhibitor 2. By enumerating full entropy production probability distributions as a function of time, we first illustrate that distinct passive and dissipative regimes are present in the denaturation dynamics. Within the dissipative dynamical region, we next find that chymotrypsin inhibitor 2 is strongly driven into unfolded states in which the protein’s hydrophobic core has been penetrated by urea molecules and disintegrated. Detailed analyses reveal that urea’s interruption of key hydrophobic contacts between core residues causes many of the protein’s native structural features to dissolve.

Elucidating the Unfolding Step in the Mechanism of Protein Translocation using Anthrax Toxin

Protein translocation across membranes is a critical step in many cellular processes, including bacterial pathogenesis. However, its molecular mechanism is poorly understood. The three-protein virulence factor, anthrax toxin that intoxicates cells via transmembrane protein translocation, is a well-defined model for studying this process. Upon endocytosis of the anthrax toxin, the acidic endosomal pH triggers the protective antigen (PA) to form a transmembrane channel, through which the two active enzyme components, the lethal factor (LF) and the edema factor (EF) are translocated to the host cell cytosol. Protein translocation across the PA channel is catalyzed by two key non-specific polypeptide binding sites – the α-clamp and Φ-clamp. The Φ-clamp being a very narrow opening, requires proteins to unfold in order for them to translocate. We hypothesize that small heterologous proteins such as Im7 can be translocated through the PA channel and that they unfold through a rate-limiting transition state during translocation. To test our hypothesis, we expressed and purified small proteins composed of the first 30 amino acids of LF followed by Im7 wild type (LF1-30.Im7 WT) and its mutants. Single channel translocation assays of the WT and its mutants were carried out using planar lipid bilayer electrophysiology. Results showed that the mutants translocated faster than the wild type, indicating that mutating the core residues caused protein destabilization, which facilitated the unfolding process and thus required less driving force for their translocation. Furthermore, denaturation studies using guanidine HCl will help determine the transition states, from which, the critical step in translocation can be determined. Understanding this mechanism can potentially facilitate in developing a new model for targeted drug delivery.

Short linear motif core and flanking regions modulate retinoblastoma protein binding affinity and specificity.

Pocket proteins retinoblastoma (pRb), p107 and p130 are negative regulators of cellular proliferation and multifunctional proteins regulating development, differentiation and chromatin structure. The retinoblastoma protein is a potent tumor suppressor mutated in a wide range of human cancers, and oncogenic viruses often interfere with cell cycle regulation by inactivating pRb. The LxCxE and pRb AB groove short linear motifs (SLiMs) are key to many pocket protein mediated interactions including host and viral partners. A review of available experimental evidence reveals that several core residues composing each motif instance are determinants for binding. In the LxCxE motif, a fourth hydrophobic position that might allow variable spacing is required for binding. In both motifs, flanking regions including charged stretches and phosphorylation sites can fine-tune the binding affinity and specificity of pocket protein SLiM-mediated interactions. Flanking regions can modulate pocket protein binding specificity, or tune the high affinity interactions of viral proteins that hijack the pRb network. The location of SLiMs within intrinsically disordered regions allows faster evolutionary rates that enable viruses to acquire a functional variant of the core motif by convergent evolution, and subsequently test numerous combinations of flanking regions towards maximizing interaction specificity and affinity. This knowledge can guide future efforts directed at the design of peptide-based compounds that can target pocket proteins to regulate the G1/S cell cycle checkpoint or impair viral mediated pRb inactivation.

Functional role of the core gap in the middle part of tropomyosin.

Tropomyosin (Tpm) is an α-helical coiled-coil actin-binding protein playing an essential role in the regulation of muscle contraction. The middle part of the Tpm molecule has some specific features, such as the presence of noncanonical residues as well as a substantial gap at the interhelical interface, which are believed to destabilize a coiled-coil and impart structural flexibility to this part of the molecule. To study how the gap affects structural and functional properties of α-striated Tpm (the Tpm1.1 isoform that is expressed in cardiac and skeletal muscles) we replaced large conserved apolar core residues located at both sides of the gap with smaller ones by mutations M127A/I130A and M141A/Q144A. We found that in contrast with the stabilizing substitutions D137L and G126R studied earlier, these substitutions have no appreciable influence on thermal unfolding and domain structure of the Tpm molecule. They also do not affect actin-binding properties of Tpm. However, they strongly increase sliding velocity of regulated actin filaments in an invitro motility assay and cause an oversensitivity of the velocity to Ca2+ similar to the stabilizing substitutions D137L and G126R. Molecular dynamics shows that the substitutions studied here increase bending stiffness of the coiled-coil structure of Tpm, like that of G126R/D137L, probably due to closure of the interhelical gap in the area of the substitutions. Our results clearly indicate that the conserved middle part of Tpm is important for the fine tuning of the Ca2+ regulation of actin-myosin interaction in muscle.

Globular domain structure and function of restriction-like-endonuclease LINEs: similarities to eukaryotic splicing factor Prp8

BackgroundR2 elements are a clade of early branching Long Interspersed Elements (LINEs). LINEs are retrotransposable elements whose replication can have profound effects on the genomes in which they reside. No crystal or EM structures exist for the reverse transcriptase (RT) and linker regions of LINEs.ResultsUsing limited proteolysis as a probe for globular domain structure, we show that the protein encoded by the Bombyx mori R2 element has two major globular domains: (1) a small globular domain consisting of the N-terminal zinc finger and Myb motifs, and (2) a large globular domain consisting of the RT, linker, and type II restriction-like endonuclease (RLE). Further digestion of the large globular domain occurred within the RT. Mapping these RT cleavages onto an updated model of the R2Bm RT indicated that the thumb of the RT was largely protected from proteolytic cleavage. The crystal structure of the large globular domain of Prp8, a eukaryotic splicing factor, was a major template used in building the R2Bm RT model, particularly the thumb region. The large fragment of Prp8 consists not only of a RT similar to R2Bm, but also an RLE and a linker connecting the two regions. The linker sequences adjacent to the RLE in LINEs and Prp8 share a set of two important α-helices and a (presumptive) knuckle/ββα structural motif that are closely associated with the thumb. The RLEs of LINEs and Prp8 share a unique catalytic core residue spacing as well as other key residues.ConclusionsThe protein encoded by RLE LINEs consists of two major globular domains. The larger of the two globular domain contains the RT, linker, and RLE and is similar to the large fragment of the spliceosomal protein Prp8. The similarities are suggestive of possible common ancestry.