Crystal Structure of a Four-Layer Aggregate of Engineered TMV CP Implies the Importance of Terminal Residues for Oligomer Assembly

Background

Crystal structures of the tobacco mosaic virus (TMV) coat protein (CP) in its helical and disk conformations have previously been determined at the atomic level. For the helical structure, interactions of proteins and nucleic acids in the main chains were clearly observed; however, the conformation of residues at the C-terminus was flexible and disordered. For the four-layer aggregate disk structure, interactions of the main chain residues could only be observed through water–mediated hydrogen bonding with protein residues. In this study, the effects of the C-terminal peptides on the interactions of TMV CP were investigated by crystal structure determination.

Methodology/Principal Findings

The crystal structure of a genetically engineered TMV CP was resolved at 3.06 Å. For the genetically engineered TMV CP, a six-histidine (His) tag was introduced at the N-terminus, and the C-terminal residues 155 to 158 were truncated (N-His-TMV CP¹⁹). Overall, N-His-TMV CP¹⁹ protein self-assembled into the four-layer aggregate form. The conformations of residues Gln36, Thr59, Asp115 and Arg134 were carefully analyzed in the high radius and low radius regions of N-His-TMV CP¹⁹, which were found to be significantly different from those observed previously for the helical and four-layer aggregate forms. In addition, the aggregation of the N-His-TMV CP¹⁹ layers was found to primarily be mediated through direct hydrogen-bonding. Notably, this engineered protein also can package RNA effectively and assemble into an infectious virus particle.

Conclusion

The terminal sequence of amino acids influences the conformation and interactions of the four-layer aggregate. Direct protein–protein interactions are observed in the major overlap region when residues Gly155 to Thr158 at the C-terminus are truncated. This engineered TMV CP is reassembled by direct protein–protein interaction and maintains the normal function of the four-layer aggregate of TMV CP in the presence of RNA.     

Induction of sister-chromatid exchanges in cultured human lymphocytes by Aldicarb, a carbamate pesticide

The aim of this work was to investigate the effects of Aldicarb on human lymphocytes in vitro in the presence of an exogenous metabolic activation system. This was done by means of an analysis of SCE and mitotic delay. CP was used to compare the chromosomal effects of Aldicarb with a known genotoxic agent. Our experiments showed that Aldicarb as well as CP induced a significant increase of SCE values in the absence of S9 mix. In vitro metabolic activation of both chemicals increased the SCE values. The addition of a metabolic system slightly decreased the successive mitotic progression of cells in culture.     

On the NMR analysis of pKa values in the unfolded state of proteins by extrapolation to zero denaturant

Detailed knowledge of the pH-dependence in both folded and unfolded states of proteins is essential to understand the role of electrostatics in protein stability. The increasing number of natively disordered proteins constitutes an excellent source for the NMR analysis of pKa values in the unfolded state of proteins. However, the tendency of many natively disordered proteins to aggregate via intermolecular hydrophobic clusters limits their NMR analysis over a wide pH range. To assess whether the pKa values in natively disordered polypeptides can be extrapolated from NMR measurements in the presence of denaturants, the natively disordered backbone of the C-terminal fragment 75 to 105 of Human Thioredoxin was studied. First, assignments using triple resonance experiments were performed to confirm lack of secondary structure. Then the pH-dependence of the amides and carboxylate side chains of Glu residues (Glu88, Glu95, Glu98, and Glu103) in the pH range from 2.0 to 7.0 was monitored using 2D 1H15N HSQC and 3D C(CO)NH experiments, and the behavior of their amides and corresponding carboxyl groups was compared to confirm the absence of nonlocal interactions. Lastly, the effect of increasing dimethyl urea concentration on the pKa values of these Glu residues was monitored. The results indicate that: (i) the dispersion in the pKa of carboxyl groups and the pH midpoints of amides in Glu residues is about 0.5 pH units and 0.6 pH units, respectively; (ii) the backbone amides of the Glu residues exhibit pH midpoints which are within 0.2 pH units from those of their carboxylates; (iii) the addition of denaturant produces upshifts in the pKa values of Glu residues that are nearly independent of their position in the sequence; and (iv) these upshifts show a nonlinear behavior in denaturant concentration, complicating the extrapolation to zero denaturant. Nevertheless, the relative ordering of the pKa values of Glu residues is preserved over the whole range of denaturant concentrations indicating that measurements at high denaturant concentration (e.g. 4 M dimethyl urea) can yield a qualitatively correct ranking of the pKa of these residues in natively disordered proteins whose pH-dependence cannot be monitored directly by NMR.     

Cell cycle kinases predicted from conserved biophysical properties

Machine-learning techniques can classify functionally related proteins where homology-transfer as well as sequence and structure motifs fail. Here, we present a method that aimed at complementing homology-transfer in the identification of cell cycle control kinases from sequence alone. First, we identified functionally significant residues in cell cycle proteins through their high sequence conservation and biophysical properties. We then incorporated these residues and their features into support vector machines (SVM) to identify new kinases and more specifically to differentiate cell cycle kinases from other kinases and other proteins. As expected, the most informative residues tend to be highly conserved and tend to localize in the ATP binding regions of the kinases. Another observation confirmed that ATP binding regions are typically not found on the surface but in partially buried sites, and that this fact is correctly captured by accessibility predictions. Using these highly conserved, semi-buried residues and their biophysical properties, we could distinguish cell cycle S/T kinases from other kinase families at levels around 70-80% accuracy and 62-81% coverage. An application to the entire human proteome predicted at least 97 human proteins with limited previous annotations to be candidates for cell cycle kinases.     

Effect of circular permutations on transient partial unfolding in proteins

Under native conditions, proteins can undergo transient partial unfolding, which may cause proteins to misfold or aggregate. A change in sequence connectivity by circular permutation may affect the energetics of transient partial unfolding in proteins without altering the three‐dimensional structures. Using Escherichia coli dihydrofolate reductase (DHFR) as a model system, we investigated how circular permutation affects transient partial unfolding in proteins. We constructed three circular permutants, CP18, CP37, and CP87, with the new N‐termini at residue 18, 37, and 87, respectively, and probed transient partial unfolding by native‐state proteolysis. The new termini in CP18, CP37, and CP87 are within, near, and distal to the Met20 loop, which is known to be dynamic and also part of the region that undergoes transient unfolding in wild‐type DHFR. The stabilities of both native and partially unfolded forms of CP18 are similar to those of wild‐type DHFR, suggesting that the influence of introducing new termini in a dynamic region to the protein is minimal. CP37 has a significantly more accessible partially unfolded form than wild‐type DHFR, demonstrating that introducing new termini near a dynamic region may promote transient partial unfolding. CP87 has significantly destabilized native and partially unfolded forms, confirming that modification of the folded region in a partially unfolded form destabilizes the partially unfolded form similar to the native form. Our findings provide valuable guidelines to control transient partial unfolding in designing circular permutants in proteins.     

On the accuracy of inferring energetic coupling between distant sites in protein families from evolutionary imprints: Illustrations using lattice model

It is suspected that correlated motions among a subset of spatially separated residues drive conformational dynamics not only in multidomain but also in single domain proteins. Sequence and structure‐based methods have been proposed to determine covariation between two sites on a protein. The statistical coupling analysis (SCA) that compares the changes in probability at two sites in a multiple sequence alignment (MSA) and a subset of the MSA has been used to infer the network of residues that encodes allosteric signals in protein families. The structural perturbation method (SPM), that probes the response of a local perturbation at all other sites, has been used to probe the allostery wiring diagram in biological machines and enzymes. To assess the efficacy of the SCA, we used an exactly soluble two dimensional lattice model and performed double‐mutant cycle (DMC) calculations to predict the extent of physical coupling between two sites. The predictions of the SCA and the DMC results show that only residues that are in contact in the native state are accurately identified. In addition, covariations among strongly interacting residues are most easily identified by the SCA. These conclusions are consistent with the DMC experiments on the PDZ family. Good correlation between the SCA and the DMC is only obtained by performing multiple experiments that vary the nature of amino acids at a given site. In contrast, the energetic coupling found in experiments for the PDZ domain are recovered using the SPM. We also predict, using the SPM, several residues that are coupled energetically. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Influence of beta-myrcene on sister-chromatid exchanges induced by mutagens in V79 and HTC cells

The influence of beta-myrcene (MC) on sister-chromatid exchanges (SCE) in V79 cells induced by 4 S9 mix-activated indirect mutagens was studied. The mutagens used were cyclophosphamide (CP), benzo[a]pyrene (BP), aflatoxin B1 (AFB) and 9,10-dimethyl-1,2-benz[a]anthracene (DMBA). MC effectively inhibited SCEs induced by CP and AFB in a dose-dependent manner, but it had no effect on SCE induction by BP and DMBA. MC also reduced CP-induced SCE frequencies in a hepatic tumor cell line (HTC). These cells are metabolically competent and activate CP into its biologically active metabolites. Our results support the suggestion that MC modulates the genotoxicity of indirect-acting mutagens by inhibiting certain forms of the cytochrome P-450 enzymes required for activation of premutagens like CP and AFB.     

Evolutionary information hidden in a single protein structure

The knowledge of conserved sequences in proteins is valuable in identifying functionally or structurally important residues. Generating the conservation profile of a sequence requires aligning families of homologous sequences and having knowledge of their evolutionary relationships. Here, we report that the conservation profile at the residue level can be quantitatively derived from a single protein structure with only backbone information. We found that the reciprocal packing density profiles of protein structures closely resemble their sequence conservation profiles. For a set of 554 nonhomologous enzymes, 74% (408/554) of the proteins have a correlation coefficient > 0.5 between these two profiles. Our results indicate that the three-dimensional structure, instead of being a mere scaffold for positioning amino acid residues, exerts such strong evolutionary constraints on the residues of the protein that its profile of sequence conservation essentially reflects that of its structural characteristics.     

Cyclophosphamide-Induced IN VIVO Sister Chromatid Exchanges (Sce) in MUS MUSCULUS. III. Quantitative Genetic Analysis

In vivo cyclophosphamide (CP)-induced sister chromatid exchanges (SCEs) were evaluated in females from five genetic strains of mice (C57BL/6J, C3H/S, 129/ReJ, BALB/c and DBA/2) and their F₁ hybrids. Baseline (noninduced) SCE values differ significantly among strains, 129/ReJ having the lowest and DBA/2 having the highest mean SCE per cell values. In general, the baseline SCE of a given F₁ is within the range of its corresponding parental strains or near the lower parental value. Furthermore, there is a genotype-dependent increase in mean SCEs per cell with CP dose. Strain differences in SCE induction are noted particularly at the two higher CP doses (4.50 and 45.0 mg/kg). In general, F₁ hybrids involving a strain with high induced SCEs and a strain with low induced SCEs exhibit mean SCE values that are closer to the value of the lower strain. F₁ s involving two strains with high SCEs or two strains with low SCEs yield SCEs not different from parental strains. The method of diallel cross analysis showed the order of dominance of these strains in SCE induction to be 129/ReJ BALB/c C3H/S DBA/2 C57BL/6J. These results support the involvement of predominantly nonadditive genetic factors as major gene(s) in SCE induction. In addition, involvement of random and independent events in SCE induction is suggested by the distribution of SCEs which follows a Poisson distribution.     

Peptide scanning-based identification of regions of gamma-II crystallin involved in thermal aggregation: evidence of the involvement of structurally analogous,helix-containing loops from the two double Greek key domains of the molecule

Gamma crystallin is one of three structural proteins present in great abundance in the fiber cells of the vertebrate eye lens. The protein displays a tendency to aggregate readily in the course of heating, cooling, being exposed to ultraviolet radiation, or rapid refolding. To investigate the molecular mechanisms underlying such aggregation, we have employed a peptide-scanning approach aimed at identifying regions of bovine gamma-II crystallin that may be involved in intermolecular interactions leading to aggregation, using assays that measure the competitive inhibition of such aggregation by reagents drawn from a group of contiguous (overlapping) peptides derived from the sequence of the protein itself. Our results suggest that two regions, comprising residues 61-74, and 145-159, play key roles in aggregative interactions. Intriguingly, the two regions (each containing a solvent-exposed, single-turn helix in the native structure) are located in structurally analogous positions in the two homologous double Greek key (beta sheet) domains of the protein, suggesting that helix-strand conversions may operate to facilitate intermolecular beta sheet interactions during aggregation.     

Isolation and NH2-terminal amino acid sequences of rat serum carrier proteins for insulin-like growth factors

Three N-glycosylated carrier proteins (CP) for insulin-like growth factors (apparent molecular weights 30-32, 42 and 45 kDa) were isolated from adult rat serum. They share the same amino terminus (up to amino acid 31) and are constituents of the growth hormone-dependent native 150-200 kDa IGF carrier complex. Residues 12-31 display 60 and 50% sequence homology, respectively, to residues 2-21 of fetal rat and to residues 4-22 of a human amniotic fluid IGF carrier protein. No homology exists with the type I or II IGF receptors. Adult rat serum also contains a fourth IGF CP (24 kDa) whose 9 NH2-terminal amino acids are identical to those of the fetal form. Our findings suggest that the three N-glycosylated components originate from the same IGF carrier protein (adult form) and that the 24 kDa protein is a separate (fetal) species.     

Hepatocyte-mediated SCE induction by indirect mutagens: importance of hepatocyte density and cell-to-cell contact

The SCE-inducing effects of the indirectly acting mutagens cyclophosphamide (CP), dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) were analysed in hepatocyte (hpc)/mammalian cell coculture systems with regard to the importance of the hpc density. V79 cells and human lymphocytes served as target cells. For all 3 compounds steadily increasing genetic effects were observed when the hpc density was increased from 3.2 X 10(4) up to 3.2 X 10(6) viable hpc per culture (25-cm2 flask), i.e. the more hpc available for metabolisation, the more genetic effects induced. The frequency distributions of the CP-induced SCE values were clearly different from those obtained with DMN, especially when high hpc densities were used: distribution patterns obtained for the mutagen with stable metabolites (CP) are characterized by the presence of distinct maxima and the absence of cells with SCE control values, whereas distribution patterns for the mutagen with very short-lived metabolites (DMN) can be described by the absence of maxima and the presence of cells with SCE control values. The frequency distributions of the AFB1-induced SCE values were more similar to the CP type than to the DMN type. From these results it is deduced that close contact between metabolising and target cells is necessary for the detection of the genotoxic effect of DMN. For CP and AFB1 a direct contact seems not to be essential, i.e. reactive intermediates may also be transported via the culture medium to the target cells.     

UPSEC: an algorithm for classifying unaligned protein sequences into functional families.

To classify proteins into functional families based on their primary sequences, popular algorithms such as the k-NN-, HMM-, and SVM-based algorithms are often used. For many of these algorithms to perform their tasks, protein sequences need to be properly aligned first. Since the alignment process can be error-prone, protein classification may not be performed very accurately. To improve classification accuracy, we propose an algorithm, called the Unaligned Protein SEquence Classifier (UPSEC), which can perform its tasks without sequence alignment. UPSEC makes use of a probabilistic measure to identify residues that are useful for classification in both positive and negative training samples, and can handle multi-class classification with a single classifier and a single pass through the training data. UPSEC has been tested with real protein data sets. Experimental results show that UPSEC can effectively classify unaligned protein sequences into their corresponding functional families, and the patterns it discovers during the training process can be biologically meaningful.     

Isolation and characterization of a cDNA clone encoding a cognate 70-kDa heat shock protein of the chloroplast envelope.

The translocation of proteins into the endoplasmic reticulum, the mitochondrion, and the chloroplast has recently been shown to involve homologues of the highly conserved 70-kDa heat shock protein (HSP70) family. In this study, we have isolated and sequenced a full-length cDNA clone encoding a cognate 70-kDa heat shock protein of the spinach chloroplast envelope (SCE70). The cDNA insert is 2,535 base pairs long and codes for 653 amino acid residues of a protein with a predicted molecular mass of 71,731 daltons. The deduced amino acid sequence shows a high degree of homology with HSP70 proteins from other organisms. Southern genomic and RNA analyses reveal different hybridization patterns than that observed for a heat-inducible 70-kDa protein gene. The protein synthesized from the SCE70 cDNA insert co-migrates with a 70-kDa polypeptide of the chloroplast envelope following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis and import studies indicate that SCE70 is associated with the chloroplast outer envelope. The import data suggest that SCE70 is targeted to the envelope membrane via a pathway different from other plastidic precursors but similar to that recently reported for outer envelope proteins SOE1 and OM14.     

Structural Insights into the Regulation of Actin Capping Protein by Twinfilin C-terminal Tail

Twinfilin is a conserved actin regulator that interacts with actin capping protein (CP) via C terminus residues (TWtail) that exhibits sequence similarity with the CP interaction (CPI) motif of CARMIL. Here we report the crystal structure of TWtail in complex with CP. Our structure showed that although TWtail and CARMIL CPI bind CP to an overlapping surface via their middle regions, they exhibit different CP-binding modes at both termini. Consequently, TWtail and CARMIL CPI restrict the CP in distinct conformations of open and closed forms, respectively. Interestingly, V-1, which targets CP away from the TWtail binding site, also favors the open-form CP. Consistently, TWtail forms a stable ternary complex with CP and V-1, a striking contrast to CARMIL CPI, which rapidly dissociates V-1 from CP. Our results demonstrate that TWtail is a unique CP-binding motif that regulates CP in a manner distinct from CARMIL CPI.     

Construction and characterization of protein libraries composed of secondary structure modules

Only a minute fraction of all possible protein sequences can exist in the genomes of all life forms. To explore whether physicochemical constraints or a lack of need causes the paucity of different protein folds, we set out to construct protein libraries without any restriction of topology. We generated different libraries (all alpha-helix, all beta-strand, and alpha-helix plus beta-strand) with an average length of 100 amino acid residues, composed of designed secondary structure modules (alpha-helix, beta-strand, and beta-turn) in various proportions, based primarily on the patterning of polar and nonpolar residues. We wished to explore that part of sequence space that is rich in secondary structure. The analysis of randomly chosen clones from each of the libraries showed that, despite the low sequence homology to known protein sequences, a substantial proportion of the library members containing alpha-helix modules were indeed helical, possess a defined oligomerization state, and showed cooperative chemical unfolding behavior. On the other hand, proteins composed of mainly beta-strand modules tended to form amyloid-like fibrils and were among the least soluble proteins ever reported. We found that a large fraction of members in non-beta-strand-containing protein libraries that are distant from natural proteins in sequence space possess unexpectedly favorable properties. These results reinforce the efficacy of applying binary patterning to design proteins with native-like properties despite lack of restriction in topology. Because of the intrinsic tendency of beta-strand modules to aggregate, their presence requires precise topologic arrangement to prevent fibril formation.     

Protein-DNA interactions: amino acid conservation and the effects of mutations on binding specificity

We investigate the conservation of amino acid residue sequences in 21 DNA-binding protein families and study the effects that mutations have on DNA-sequence recognition. The observations are best understood by assigning each protein family to one of three classes: (i) non-specific, where binding is independent of DNA sequence; (ii) highly specific, where binding is specific and all members of the family target the same DNA sequence; and (iii) multi-specific, where binding is also specific, but individual family members target different DNA sequences. Overall, protein residues in contact with the DNA are better conserved than the rest of the protein surface, but there is a complex underlying trend of conservation for individual residue positions. Amino acid residues that interact with the DNA backbone are well conserved across all protein families and provide a core of stabilising contacts for homologous protein-DNA complexes. In contrast, amino acid residues that interact with DNA bases have variable levels of conservation depending on the family classification. In non-specific families, base-contacting residues are well conserved and interactions are always found in the minor groove where there is little discrimination between base types. In highly specific families, base-contacting residues are highly conserved and allow member proteins to recognise the same target sequence. In multi-specific families, base-contacting residues undergo frequent mutations and enable different proteins to recognise distinct target sequences. Finally, we report that interactions with bases in the target sequence often follow (though not always) a universal code of amino acid-base recognition and the effects of amino acid mutations can be most easily understood for these interactions.     

Conformations of disulfide bridges in proteins

The conformational characteristics of disulfide bridges in proteins have been analyzed using a dataset of 22 protein structures, available at a resolution of less than or equal to 2.0 A, containing a total of 72 disulfide crosslinks. The parameters used in the analysis include (phi, psi) values at Cys residues, bridge dihedral angles chi ss, chi i1, chi j1, chi i2, and chi j2, the distances C alpha i-C alpha j and C beta i-C beta j between the C alpha and C beta atoms of Cys(i) and Cys(j). Eight families of bridge conformations with three or more occurrences have been identified on the basis of these stereochemical parameters. The most populated family corresponds to the "left handed spiral" identified earlier by Richardson [1981) Adv. Protein Chem. 34, 167-330). Disulfide bridging across antiparallel extended strands is observed in alpha-lytic protease, crambin, and beta-trypsin and this structure is shown to be very similar to those obtained in small cystine peptides. Solvent accessible surface area calculations show that the overwhelming majority of disulfide bridges are inaccessible to solvent.