Anticooperative Nearest-Neighbor Interactions between Residues in Unfolded Peptides and Proteins

Growing evidence suggests that the conformational distributions of amino acid residues in unfolded peptides and proteins depend on the nature of the nearest neighbors. To explore whether the underlying interactions would lead to a breakdown of the isolated pair hypothesis of the classical random coil model, we further analyzed the conformational propensities that were recently obtained for the two guest residues (x,y) of GxyG tetrapeptides. We constructed a statistical thermodynamics model that allows for cooperative as well as for anticooperative interactions between adjacent residues adopting either a polyproline II or a β-strand conformation. Our analysis reveals that the nearest-neighbor interactions between most of the central residues in the investigated GxyG peptides are anticooperative. Interaction Gibbs energies are rather large at high temperatures (350 K), at which point many proteins undergo thermal unfolding. At room temperature, these interaction energies are less pronounced. We used the obtained interaction parameter in a Zimm-Bragg/Ising-type approach to calculate the temperature dependence of the ultraviolet circular dichroism (CD) of the MAX3 peptide, which is predominantly built by KV repeats. The agreement between simulation and experimental data was found to be satisfactory. Finally, we analyzed the temperature dependence of the CD and ³J(H^NH^α) parameters of the amyloid β_1–9 fragment. The results of this analysis and a more qualitative consideration of the temperature dependence of denatured proteins probed by CD spectroscopy further corroborate the dominance of anticooperative nearest-neighbor interactions. Generally, our results show that unfolded peptides—and most likely also proteins—exhibit some similarity with antiferromagnetic systems.     

Aromatic Amino Acid Residues in Japanese-radish Peroxidase a and Apoenzyme†

The nature of aromatic amino acid residues in Japanese-radish peroxidase a and the apoprotein was investigated by means of spectrophotometry and fluorospectrophotometry. The tyrosine residues in the holoenzyme were masked in the alkali-titration, giving an abnormally high value of 12.6, while they were exposed in the apoenzyme, exhibiting a value of 10.8. The difference spectra in the ultraviolet region between the holo-and apo-enzyme showed characteristic bands of tryptophan and phenylalanine as well as tyrosine. The perturbation of the aromatic amino acid residues by 50% ethyleneglycol was observed in the apoenzyme but not in the holoenzyme. The fluorophotometric experiments also revealed that the aromatic amino acid residues were in different environments in the holo- and apoenzyme. The difference between the conformation of peroxidase and that of the apoprotein was discussed.     

Roles of β-Tubulin Residues Ala428 and Thr429 in Microtubule Formation in Vivo

The C termini of β-tubulin isotypes are regions of high sequence variability that bind to microtubule-associated proteins and motors and undergo various post-translational modifications such as polyglutamylation and polyglycylation. Crystallographic analyses have been unsuccessful in resolving tubulin C termini. Here, we used a stepwise approach to study the role of this region in microtubule assembly. We generated a series of truncation mutants of human βI and βIII tubulin. Transient transfection of HeLa cells with the mutants shows that mutants with deletions of up to 22 residues from βIII and 16 from βI can assemble normally. Interestingly, removal of the next residue (Ala⁴²⁸) results in a complete loss of microtubule formation without affecting dimer formation. C-terminal tail switching of human βI and βIII tubulin suggests that C-terminal tails are functionally equivalent. In short, residues outside of 1–429 of human β-tubulins make no contribution to microtubule assembly. Ala⁴²⁸, in the C-terminal sequence motif N-QQYQDA⁴²⁸, lies at the end of helix H12 of β-tubulin. We hypothesize that this residue is important for maintaining helix H12 structure. Deletion of Ala⁴²⁸ may lead to unwinding of helix H12, resulting in tubulin dimers incapable of assembly. Thr⁴²⁹ plays a more complex role. In the βI isotype of tubulin, Thr⁴²⁹ is not at all necessary for assembly; however, in the βIII isotype, its presence strongly favors assembly. This result is consistent with a likely more complex function of βIII as well as with the observation that evolutionary conservation is total for Ala⁴²⁸ and frequent for Thr⁴²⁹.Microtubules are involved in a great variety of cellular functions. Their constituent protein tubulin is an αβ heterodimer, both α- and β-tubulin existing as multiple isotypes, encoded by different genes and differing in amino acid sequence (1). The differences among the isotypes are highly conserved in evolution. In mammals, the β isotypes are βIa, βIb, βII, βIII, βIVa, βIVb, βV, and βVI. There is evidence that the isotype differences have functional significance. For instance, the βIV isotype is found in all axonemes (2).Structurally, both α- and β-tubulin consist of a globular region of 427 amino acids followed by a C-terminal region of 17–24 amino acids (3–5). The C-terminal region is highly negatively charged, being especially rich in glutamate residues and lacking in basic residues, and is likely to project outward from the rest of the molecule, because of its high negative charge and the electrostatic repulsion among the glutamate residues (3). The three-dimensional structure of the globular domain has been determined by electron and x-ray crystallography (4, 5). However, the C-terminal region has never been localized in the three-dimensional reconstructions except by computer modeling. The probable reasons for this are 1) that, if the C-terminal region projects out from the rest of molecule, it is likely to be very flexible with respect to the rest of the molecule and 2) the C-terminal region undergoes post-translational modification. Both of these can lead to structural heterogeneity and cause the C terminus to be invisible to crystallographic techniques.In this work, we examine the role of the C termini of human β-tubulins to determine the minimal sequence requirement for microtubule incorporation through structure/function analyses. The human βI and βIII tubulin isotypes were utilized based on their high degree of sequence variability clustered at the C terminus (Fig. 1) and the fact that βI is broadly distributed among normal tissues, whereas βIII has a very narrow tissue distribution. These two isotypes share 92% sequence identity, with differences among these isotypes occurring in both the globular domain and the C-terminal region (1).Open in a separate windowFIGURE 1.Sequence alignment of human βIa and βIII tubulin isotypes. Human βIa and βIII tubulin isotypes were aligned with ClustalX 1.83 and processed with BioEdit. Hyphens denote identical residues between sequences.

TABLE 1

The C-terminal amino acid sequences of the human β-tubulin isotypes

Conformational Similarity Indices Between Different Residues in Proteins and α-Helix Propensities

Abstract

Various amino acid similarity matrices have been derived using data on physicochemical properties and molecular evolution. Conformational similarity indices, CS_XX′, between different residues are computed here using the distribution of the main-chain and side-chain torsion angles and the values have been used to cluster amino acids in proteins. A subset of these parameters, CS_AX′ indicates the extent of similarity in the main-chain and side-chain conformations (φ ψ and χ₁) of different residues (X) with Ala (A) and is found to have strong correlation with α-helix propensities. However, no subset of CS_XX′ provides any linear relationship with β-sheet propensities, suggesting that the conformational feature favouring the location of a residue in an a-helix is different from the one favouring the β-sheet. Conformationally similar residues (close CS_AX values) have similar steric framework of the side-chain (linear/branched, aliphatic/aromatic), irrespective of the polarity or hydrophobicity. Cooperative nucleation of helix may be facile for a contiguous stretch of residues with high overall CS_AX values.     

Residues Met^76 and Gin^79 in HLA-G α1 domain involved in KIR2DIA recognition

Human leukocyte antigen-G (HLA-G) has long been speculated as a beneficial factor for a successful pregnancy for its restricted expression on fetal-matemal extravillous cytotrophoblasts and its capability of modulating uterine natural killer cell (uNK) function such as cytotoxicity and cytokine production through NK cell receptors. HLA class I α1 domain is an important killer cell Ig-like receptor (KIR) recognition site and the Met^76 and Gln^79 are unique to HLA-Gin this region. NK cell receptor KIR2DL4 is a specific receptor for HLA-G, yet the recognition site on HLA-G remains unknown. In this study, retroviral transduction was applied to express the wild type HLA-G (HLA-wtG), mutant HLA-G(HLA-mG) on the chronic myelogenous leukemia cell line K562 cells and KIR2DL4 molecule on NK-92 cells,respectively. KIR2DL4-IgG Fc fusion protein was generated to determine the binding specificity between KIR2DL4 and HLA-G. Our results showed that residue Met76, Gln79 mutated to Ala^76.79 in the α1 domain of HLA-G protein could affect the binding affinity between KIR2DL4 and HLA-G, meanwhile, the KIR2DL4 transfected NK-92 cells (NK-92-2DL4) showed a considerably different cytolysis ability against the HLA-wtG and HLA-mG transfected K562 targets.Taken together, our data indicated that residue Met^76 and Gin^79 in HLA-G α1 domain plays a critical role in the recognition of KIR2DL4, which could be an explanation for the isoforms of HLA-G, all containing the α1 domain, with the potential to regulate NK functions.     

Structure–Critical Distribution of Aromatic Residues in the Fibronectin Type III Protein Family

Over a thousand individual Fibronectin type III (FnIII) domain sequences, extracted from more than 60 different FnIII-dependent protein super-structures, were downloaded from curated database resources. Three regions of extreme sequence conservation within the well-characterized FnIII β-sandwich structure were respectively defined by near absolute conservation of a tryptophan (Trp) in β-strand-B, tyrosines (Tyr) in both β-strand-C and β-strand-F, and a leucine (Leu) residue in the unstructured region immediately preceding β-strand-F. Employing these four conserved landmarks, the entire FnIII sequence dataset was vertically registered to align the three conserved regions, and the cumulative distribution of all other amino acid functionality was determined and plotted relative to these landmark residues. Conserved aromatic sites were each found to be flanked by aliphatic residues that assure localization of these sites to the inaccessible hydrophobic interface between major sheet structures. Mapping the location of conserved aromatic sites in numerous PDB structures demonstrated the consistent pair-wise co-localization of the indole side-chain of the conserved strand-B Trp site to within 0.35 nm of the phenolic side-chain of the strand-C Tyr site located 8–14 amino acids distal. Likewise, the side-chain of the strand-F Tyr site co-localized to within 0.45 nm of the aliphatic side-chain of the conserved Leu that uniformly precedes it by six residues. While classic hydropathy-based theories would deem the “burying” of Tyr and Trp side-chains and/or their association with hydrophobic FnIII core residues thermodynamically unnecessary, alternative contributions of conserved Trp and Tyr residues, and particularly the role of the absolutely conserved tyrosine phenolic –OH in native FnIII structure–function are considered. A more global role for conserved FnIII aromaticity is also discussed in light of the aromatic conservation observed in other well-established protein families.     

Host-Specific Enzyme-Substrate Interactions in SPM-1 Metallo-β-Lactamase Are Modulated by Second Sphere Residues

Pseudomonas aeruginosa is one of the most virulent and resistant non-fermenting Gram-negative pathogens in the clinic. Unfortunately, P. aeruginosa has acquired genes encoding metallo-β-lactamases (MβLs), enzymes able to hydrolyze most β-lactam antibiotics. SPM-1 is an MβL produced only by P. aeruginosa, while other MβLs are found in different bacteria. Despite similar active sites, the resistance profile of MβLs towards β-lactams changes from one enzyme to the other. SPM-1 is unique among pathogen-associated MβLs in that it contains “atypical” second sphere residues (S84, G121). Codon randomization on these positions and further selection of resistance-conferring mutants was performed. MICs, periplasmic enzymatic activity, Zn(II) requirements, and protein stability was assessed. Our results indicated that identity of second sphere residues modulates the substrate preferences and the resistance profile of SPM-1 expressed in P. aeruginosa. The second sphere residues found in wild type SPM-1 give rise to a substrate selectivity that is observed only in the periplasmic environment. These residues also allow SPM-1 to confer resistance in P. aeruginosa under Zn(II)-limiting conditions, such as those expected under infection. By optimizing the catalytic efficiency towards β-lactam antibiotics, the enzyme stability and the Zn(II) binding features, molecular evolution meets the specific needs of a pathogenic bacterial host by means of substitutions outside the active site.     

Fibrin Clot Structure and Mechanics Associated with Specific Oxidation of?Methionine Residues in Fibrinogen

Using a combination of structural and mechanical characterization, we examine the effect of fibrinogen oxidation on the formation of fibrin clots. We find that treatment with hypochlorous acid preferentially oxidizes specific methionine residues on the α, β, and γ chains of fibrinogen. Oxidation is associated with the formation of a dense network of thin fibers after activation by thrombin. Additionally, both the linear and nonlinear mechanical properties of oxidized fibrin gels are found to be altered with oxidation. Finally, the structural modifications induced by oxidation are associated with delayed fibrin lysis via plasminogen and tissue plasminogen activator. Based on these results, we speculate that methionine oxidation of specific residues may be related to hindered lateral aggregation of protofibrils in fibrin gels.     

Importance of N-linked Sugar Residues in the Development of Auerbach’s Plexus in the Rat Colon: A Lectin Histochemical Study

The lectin-binding patterns in Auerbach's plexus in the distal portions of the rat colon from 15- to 21-day-old foetuses, newborns, and adults were examined by light and electron microscopy using 16 different lectins (ConA, RCA-1, WGA, PNA, SBA, UEA-1, DBA, LCA, PHA-L, DSA, GS-1, VVA, MPA, BPA, MAA, and PSA). The binding of ConA was shown to increase after day 19 of gestation in parallel with differentiation of Auerbach's plexus, whereas the staining intensity for DSA and RCA-1 increased after day 17 of gestation in accordance with the appearance of the plexus. At the electron microscopical level, DSA binding sites were observed to be localized mainly in the plasma membrane, Golgi apparatus, and nuclear membrane of nerve cells. Positive sites were also observed in the axolemma and in the plasma membrane of nerve cell processes, Schwann cells, and the surrounding smooth muscle cells. PSA, PHA-L, LCA, and WGA showed constant staining during the development after day 15 of gestation. Other lectins, most of which are specific for O-glycosidic mucin-type sugar residues, were essentially negative throughout the developmental stages. Moreover, N-glycanase digestion significantly diminished the positive reactions. N-linked oligosaccharides may thus play important roles in the development and maturation of the Auerbach's plexus, and may be involved in the developmental defect of the plexus, e.g. as occurs in Hirschsprung's disease.     

Residues Met~(76) and Gln~(79) in HLA-G α1 domain involved in KIR2DL4 recognition

INTRODUCTIONThe non-classical HLA class I antigen HLA-G is mainlyexpressed on extravillous cytotrophoblasts that invade de-ciduae in uterine pregnancy. Furthermore, HLA-G canmodulate the function of most immune component cellssuch as NK cells, T cells and…     

On the Role of Basic Residues in Adapting the Reaction Centre–LH1 Complex for Growth at Elevated Temperatures in Purple Bacteria

The purple photosynthetic bacterium Thermochromatium tepidum is a moderate thermophile, with a growth optimum of 48–50 °C. The X-ray crystal structure of the reaction centre from this organism has been determined, and compared with that from mesophilic bacteria such as Blastochloris viridis and Rhodobacter sphaeroides (Nogi T et al. (2000) Proc Natl Acad Sci USA 97: 13561–13566). Structural features that could contribute to the enhanced thermal stability of the Thermochromatium tepidum reaction centre were discussed, including three arginine residues exposed at the periplasmic side of the membrane that are not present in reaction centres from mesophilic organisms, and potentially could increase the affinity of the complex for the surrounding membrane. In the present report these arginine residues, plus a histidine identified from an extensive sequence alignment, were engineered into structurally homologous positions in the Rhodobacter sphaeroides reaction centre, and the effect on the thermal stability of the Rhodobacter sphaeroides complex was examined. We find that these residues do not enhance the thermal stability of the reaction centre, as assessed by absorbance spectroscopy of the bacteriochlorin cofactors in membrane-bound reaction centres. Possible roles of these residues in the Thermochromatium tepidum reaction centre are discussed, and it is proposed that they facilitate stronger binding of the reaction centre to the encircling LH1 antenna complex, through ionic interactions with acidic residues at the C-terminal end of the LH1 α-polypeptide. Such an interaction could enhance the stability of the so-called ‘RC–LH1 core’ complex that is formed between the reaction centre and the LH1 antenna, and which represents the minimal functional photosynthetic unit in all known purple photosynthetic bacteria. Stronger bonding interactions between the two complexes could also contribute to an increase in the rigidity of the photosynthetic membrane in Thermochromatium tepidum, in accord with the general finding that the cytoplasmic membrane from thermophilic eubacteria is less fluid than its counterpart in mesophilic bacteria.     

Residues 377-389 from the δ subunit ofTorpedo californica acetylcholine receptor are located in the cytoplasmic surface

Torpedo californica acetylcholine receptor (AcChR) enriched, sealed vesicles have been specifically labeled on the cytoplasmic surface with pyridoxal 5′-phosphate (Perez-Ramirez, B., and Martinez-Carrion, M., 1989,Biochemistry 28, 5034–5040). After chromatography of the peptide fragments produced by tryptin digestion of labeled AcChR, several fractions containing the phosphopyridoxyl label were obtained. Edman degradation identified one of the fractions, with sequence SRSELMFEKQSER, as corresponding to residues 377–389 in theδ subunit (primary structure). The latter must be a cytoplasmic region of this transmembranous protein, and residueδK385 must reside in a water-soluble exposed domain of the cytosolic side of the membrane. Introduction of phosphopyridoxyl residues allows for their potential use as probes of conformational changes in the cytosolic surface of the receptor molecule.     

Is Use of Both Pulpwood and Logging Residues Instead of Only Logging Residues for Bioenergy Development a Viable Carbon Mitigation Strategy?

This study adopts an integrated life-cycle approach to assess overall carbon saving related with the utilization of wood pellets manufactured using pulpwood and logging residues for electricity generation. Carbon sequestered in wood products and wood present in landfills and avoided carbon emissions due to substitution of grid electricity with the electricity generated using wood pellets are considered part of overall carbon savings. Estimated value of overall carbon saving is compared with the overall carbon saving related to the current use of pulpwood and logging residues. The unit of analysis is a hectare of slash pine (Pinus elliottii) plantation in southern USA. All carbon flows are considered starting from forest management to the decay of wood products in landfills. Exponential decay function is used to ascertain carbon sequestered in wood products and wood present in landfills. Non-biogenic carbon emissions due to burning of wood waste at manufacturing facilities, wood pellets at a power plant, and logging residues on forestlands are also considered. Impacts of harvest age and forest management intensity on overall carbon saving are analyzed as well. The use of pulpwood for bioenergy development reduces carbon sequestered in wood products and wood present in landfills (up to 1.6 metric tons/ha) relative to a baseline when pulpwood is used for paper making and logging residues are used for manufacturing wood pellets. Avoided carbon emissions because of displacement of grid electricity from the electricity generated using wood pellets derived from pulpwood fully compensate the loss of carbon sequestered in wood products and wood present in landfills. The use of both pulpwood and logging residues for bioenergy development is beneficial from carbon perspective. Harvest age is more important in determining overall carbon saving than forest management intensity.     

Plasticity in the Contribution of T Cell Receptor Variable Region Residues to Binding of Peptide–HLA-A2 Complexes

One hypothesis accounting for major histocompatibility complex (MHC) restriction by T cell receptors (TCRs) holds that there are several evolutionary conserved residues in TCR variable regions that contact MHC. While this “germline codon” hypothesis is supported by various lines of evidence, it has been difficult to test. The difficulty stems in part from the fact that TCRs exhibit low affinities for pep/MHC, thus limiting the range of binding energies that can be assigned to these key interactions using mutational analyses. To measure the magnitude of binding energies involved, here we used high-affinity TCRs engineered by mutagenesis of CDR3. The TCRs included a high-affinity, MART-1/HLA-A2-specific single-chain TCR and two other high-affinity TCRs that all contain the same Vα region and recognize the same MHC allele (HLA-A2), with different peptides and Vβ regions. Mutational analysis of residues in CDR1 and CDR2 of the three Vα2 regions showed the importance of the key germline codon residue Y51. However, two other proposed key residues showed significant differences among the TCRs in their relative contributions to binding. With the use of single-position, yeast-display libraries in two of the key residues, MART-1/HLA-A2 selections also revealed strong preferences for wild-type germline codon residues, but several alternative residues could also accommodate binding and, hence, MHC restriction. Thus, although a single residue (Y51) could account for a proportion of the energy associated with positive selection (i.e., MHC restriction), there is significant plasticity in requirements for particular side chains in CDR1 and CDR2 and in their relative binding contributions among different TCRs.     

Substrate Binding and Active Site Residues in RNases E and G: ROLE OF THE 5��-SENSOR*

The paralogous endoribonucleases, RNase E and RNase G, play major roles in intracellular RNA metabolism in Escherichia coli and related organisms. To assay the relative importance of the principal RNA binding sites identified by crystallographic analysis, we introduced mutations into the 5′-sensor, the S1 domain, and the Mg⁺²/Mn⁺² binding sites. The effect of such mutations has been measured by assays of activity on several substrates as well as by an assay of RNA binding. RNase E R169Q and the equivalent mutation in RNase G (R171Q) exhibit the strongest reductions in both activity (the k_cat decrease ∼40- to 100-fold) and RNA binding consistent with a key role for the 5′-sensor. Our analysis also supports a model in which the binding of substrate results in an increase in catalytic efficiency. Although the phosphate sensor plays a key role in vitro, it is unexpectedly dispensable in vivo. A strain expressing only RNase E R169Q as the sole source of RNase E activity is viable, exhibits a modest reduction in doubling time and colony size, and accumulates immature 5 S rRNA. Our results point to the importance of alternative RNA binding sites in RNase E and to alternative pathways of RNA recognition.     

Recognition of Interaction Interface Residues in Low-Resolution Structures of Protein Assemblies Solely from the Positions of Cα Atoms

Background

The number of available structures of large multi-protein assemblies is quite small. Such structures provide phenomenal insights on the organization, mechanism of formation and functional properties of the assembly. Hence detailed analysis of such structures is highly rewarding. However, the common problem in such analyses is the low resolution of these structures. In the recent times a number of attempts that combine low resolution cryo-EM data with higher resolution structures determined using X-ray analysis or NMR or generated using comparative modeling have been reported. Even in such attempts the best result one arrives at is the very course idea about the assembly structure in terms of trace of the Cα atoms which are modeled with modest accuracy.

Methodology/Principal Findings

In this paper first we present an objective approach to identify potentially solvent exposed and buried residues solely from the position of Cα atoms and amino acid sequence using residue type-dependent thresholds for accessible surface areas of Cα. We extend the method further to recognize potential protein-protein interface residues.

Conclusion/ Significance

Our approach to identify buried and exposed residues solely from the positions of Cα atoms resulted in an accuracy of 84%, sensitivity of 83–89% and specificity of 67–94% while recognition of interfacial residues corresponded to an accuracy of 94%, sensitivity of 70–96% and specificity of 58–94%. Interestingly, detailed analysis of cases of mismatch between recognition of interface residues from Cα positions and all-atom models suggested that, recognition of interfacial residues using Cα atoms only correspond better with intuitive notion of what is an interfacial residue. Our method should be useful in the objective analysis of structures of protein assemblies when positions of only Cα positions are available as, for example, in the cases of integration of cryo-EM data and high resolution structures of the components of the assembly.     

Molecular Lesions Associated with Alleles of Decapentaplegic Identify Residues Necessary for Tgf-β/Bmp Cell Signaling in Drosophila Melanogaster

We have identified the molecular lesions associated with six point mutations in the Drosophila TGF-β homologue decapentaplegic (dpp). The sites of these mutations define residues within both the pro and ligand regions that are essential for dpp function in vivo. While all of these mutations affect residues that are highly conserved among TGF-β superfamily members, the phenotypic consequences of the different alleles are quite distinct. Through an analysis of these mutant phenotypes, both in cuticle preparations and with molecular probes, we have assessed the functional significance of specific residues that are conserved among the different members of the superfamily. In addition, we have tested for conditional genetic interactions between the different alleles. We show that two of the alleles are temperature sensitive for the embyronic functions of dpp, such that these alleles are not only embryonic viable as homozygotes but also partially complement other dpp hypomorphs at low temperatures. Our results are discussed with regard to in vitro mutagenesis data on other TGF-β-like molecules, as well as with regard to the regulation of dpp cell signaling in Drosophila.