The pH-dependent structure of calf thymus DNA studied by Raman spectroscopy

The pH-dependent structure of calf thymus DNA is analyzed using Raman spectroscopy. The Raman spectra in the acidic region demonstrate that denaturation occurs in several steps. The binding of H⁺ to adenine and cytosine residues is accompanied by a decrease in the percentage of DNA in the B-conformation and a concurrent increase in a conformation most probably related to the C-form. The denaturation of DNA is observed at pH 3.3 and parallels the protonation of guanine bases. The Raman spectra of calf thymus DNA in the basic region (above pH 10) show that guanine residues are deprotonated at a lower pH value than are thymine residues. In addition, Raman spectra in the basic region detect conformational changes of the phosphate backbone different from those found in the acidic region.     

Charged residues are involved in membrane fusion mediated by a hydrophilic peptide located in vesicular stomatitis virus G protein

Membrane fusion is an essential step of the internalization process of the enveloped animal viruses. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in VSV G protein, comprising the residues 145 to 164, directly involved in membrane interaction and fusion. Unlike fusion peptides from other viruses, this sequence is very hydrophilic, containing six charged residues, but it was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Using a carboxyl-modifying agent, dicyclohexylcarbodiimide (DCCD), and several synthetic mutant peptides, we demonstrated that the negative charges of peptide acidic residues, especially Asp¹⁵³ and Glu¹⁵⁸, participate in the formation of a hydrophobic domain at pH 6.0, which is necessary to the peptide-induced membrane fusion. The formation of the hydrophobic region and the membrane fusion itself were dependent on peptide concentration in a higher than linear fashion, suggesting the involvement of peptide oligomerization. His¹⁴⁸ was also necessary to hydrophobicity and fusion, suggesting that peptide oligomerization occurs through intermolecular electrostatic interactions between the positively-charged His and a negatively-charged acidic residue of two peptide molecules. Oligomerization of hydrophilic peptides creates a hydrophobic region that is essential for the interaction with the membrane that results in fusion.     

Spectroscopic and equilibrium dialysis studies of poly(α-L-glutamic acid)–Cu(II) complexes in the pH range 4–7

The formation of complex between the Cu²⁺ ion and poly(α-L -glutamic acid) [poly(Glu)] in 150 mM NaCl solutions was studied by uv–visible absorption and equilibrium dialysis methods at the mixing ratios of Glu residues to Cu²⁺, R, of 32, 16, and 8 and in the pH range 4–7. The results showed that more than 90% of Cu²⁺ ions bind to the poly(Glu) at pH > 4.9, but the bound Cu(II) begins to dissociate with a decrease in pH. The absorption spectra of bound Cu(II) varied with pH and R in a complicated manner. Three different component spectra were disclosed from the analysis of the pH dependence of the bound spectra. We concluded that poly(Glu)–Cu(II) complexes fall into three classes in the pH range 4–7, with the proportions of these complexes varying with both pH and R. The three complexes predominate either in the helix or extended-coil region, in the helix–coil transition region, or in the helix-aggregate region. The stability constant and binding mode of each Cu(II)–Glu complex were estimated from the dialysis data. With these results, the possible structure of each complex is discussed.     

Rational redesign of neutral endopeptidase binding to merlin and moesin proteins

Neutral endopeptidase (NEP) is a 90‐ to 110‐kDa cell‐surface peptidase that is normally expressed by numerous tissues but whose expression is lost or reduced in a variety of malignancies. The anti‐tumorigenic function of NEP is mediated not only by its catalytic activity but also through direct protein–protein interactions of its cytosolic region with several binding partners, including Lyn kinase, PTEN, and ezrin/radixin/moesin (ERM) proteins. We have previously shown that mutation of the K¹⁹K²⁰K²¹ basic cluster in NEPs' cytosolic region to residues QNI disrupts binding to the ERM proteins. Here we show that the ERM‐related protein merlin (NF2) does not bind NEP or its cytosolic region. Using experimental data, threading, and sequence analysis, we predicted the involvement of moesin residues E¹⁵⁹Q¹⁶⁰ in binding to the NEP cytosolic domain. Mutation of these residues to NL (to mimic the corresponding N¹⁵⁹L¹⁶⁰ residues in the nonbinder merlin) disrupted moesin binding to NEP. Mutation of residues N¹⁵⁹L¹⁶⁰Y¹⁶¹K¹⁶²M¹⁶³ in merlin to the corresponding moesin residues resulted in NEP binding to merlin. This engineered NEP peptide–merlin interaction was diminished by the QNI mutation in NEP, supporting the role of the NEP basic cluster in binding. We thus identified the region of interaction between NEP and moesin, and engineered merlin into a NEP‐binding protein. These data form the basis for further exploration of the details of NEP‐ERM binding and function.     

Functional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its negative regulation by Lon

SulA is induced in Escherichia coli by the SOS response and inhibits cell division through interaction with FtsZ. To determine which region of SulA is essential for the inhibition of cell division, we constructed a series of N-terminal and C-terminal deletions of SulA and a series of alanine substitution mutants. Arginine at position 62, leucine at 67, tryptophan at 77 and lysine at 87, in the central region of SulA, were all essential for the inhibitory activity. Residues 3–27 and the C-terminal 21 residues were dispensable for the activity. The mutant protein lacking N-terminal residues 3–47 was inactive, as was that lacking the C-terminal 34 residues. C-terminal deletions of 8 and 21 residues increased the growth-inhibiting activity in lon ⁺ cells, but not in lon ^? cells. The wild-type and mutant SulA proteins were isolated in a form fused to E. coli maltose-binding protein, and tested in vitro for sensitivity to Lon protease. Lon degraded wild-type SulA and a deletion mutant lacking the N-terminal 93 amino acids, but did not degrade the derivative lacking 21 residues at the C-terminus. Futhermore, the wild-type SulA and the N-terminal deletion mutant formed a stable complex with Lon, while the C-terminal deletion did not. MBP fused to the C-terminal 20 residues of SulA formed a stable complex with, but was not degraded by Lon. When LacZ protein was fused at its C-terminus to 8 or 20 amino acid residues from the C-terminal region of SulA the protein was stable in lon ⁺ cells. These results indicate that the C-terminal 20 residues of SulA permit recognition by, and complex formation with, Lon, and are necessary, but not sufficient, for degradation by Lon.     

Analysis of the DNA binding site of Escherichia coli RecA protein

To investigate the DNA binding site of RecA protein, we constructed 15 recA mutants having alterations in the regions homologous to the other ssDNA binding proteins. The in vivo analyses showed that the mutational change at Arg²⁴³, Lys²⁴⁸, Tyr²⁶⁴, or simultaneously at Lys⁶ and Lys¹⁹, or Lys⁶ and Lys²³ caused severe defects in the recA functions, while other mutational changes did not. Purified RecA-K6A-K23A (Lys⁶ and Lys²³ changed to Ala and Ala, respectively) protein was indistinguishable from the wild-type RecA protein in its binding to DNA. However, the RecA-R243A (Arg²⁴³ changed to Ala) and RecA-Y264A (Tyr²⁶⁴ changed to Ala) proteins were defective in binding to both ss- and ds-DNA. In self-oligomerization property, RecA-R243A was proficient but RecA-Y264A was deficient, suggesting that the RecA-R243A protein had a defect in DNA binding site and the RecA-Y264A protein was defective in its interaction with the adjacent RecA molecule. The region of residues 243–257 including the Arg²⁴³ is highly homologous to the DNA binding motif in the ssDNA binding proteins, while the eukaryotic RecA homologues have a similar structure at the amino-terminal side proximal to the nucleotide binding core. The region of residues 243–257 would be a part of the DNA binding site. The other parts of this site would be the Tyr¹⁰³ and the region of residues 178–183, which were cross-linked to ssDNA. These three regions lie in a line in the crystal structure.     

A calorimetric study of the binding of 2′-deoxyuridine-5′-phosphate and its analogs to thymidylate synthetase

The binding of dUMP, dTMP, UMP, and 5-fluoro-2′-deoxyuridylate (FdUMP) to Lactobacillus casei thymidylate synthetase (TSase) was examined by direct thermal titration. The binding of each ligand was examined in two different buffers, so that proton interactions could be observed. In agreement with an earlier study (N. V. Beaudette, N. Langerman, R. L. Kisliuk, and Y. Gaumont, 1977, Arch. Biochem. Biophys.179, 272–278), dUMP binding is driven predominantly by enthalpy changes at pH 7.4, with 0.77 ± 0.07 mol of protons binding along with the substrate. When the pH is decreased to 5.8, binding affinity increases, and a substantial increase in the entropic contribution to the binding is observed. In contrast to the binding of protons with substrate at pH 7.4, protons are released at pH 5.8. The proton effects suggest a model in which binding occurs through an electrostatic interaction between dianionic nucleotide and protonated enzyme residues. Binding of FdUMP at pH 7.4 involves the uptake of protons, and is also predominantly driven by changes in enthalpy. A good fit to the thermal data is obtained using the single-site binding constant, K = 9.5 × 10⁴m^?1. Our earlier interpretation (Arch. Biochem. Biophys., 1977, 179, 272–278) of the thermal data indicating two sites is in error. Preliminary date are presented which suggest that two-site binding of FdUMP occurs on prolonged incubation during equilibrium dialysis. Binding of the product dTMP shows different behavior. The reaction is entropically driven, suggesting that a significant hydrophobic interaction occurs between the protein and the 5-methyl group of the nucleotide. Only 0.48 ± 0.08 mol of protons are absorbed at pH 7.4. Binding of the nucleotide UMP could not be detected at pH 7.4.     

Residue-specific mobility changes in soluble oligomers of the prion protein define regions involved in aggregation

Prion (PrP) diseases are neurodegenerative diseases characterized by the formation of β-sheet rich, insoluble and protease resistant protein deposits (called PrP^Sc) that occur throughout the brain. Formation of synthetic or in vitro PrP^Sc can occur through on-pathway toxic oligomers. Similarly, toxic and infectious oligomers identified in cell and animal models of prion disease indicate that soluble oligomers are likely intermediates in the formation of insoluble PrP^Sc. Despite the critical role of prion oligomers in disease progression, little is known about their structure. In order, to obtain structural insight into prion oligomers, we generated oligomers by shaking-induced conversion of recombinant, monomeric prion protein PrP^c (spanning residues 90–231). We then obtained two-dimensional solution NMR spectra of the PrP^c monomer, a 40% converted oligomer, and a 94% converted oligomer. Heteronuclear single-quantum correlation (¹H–¹⁵N) studies revealed that, in comparison to monomeric PrP^c, the oligomer has intense amide peak signals in the N-terminal (residues 90–114) and C-terminal regions (residues 226–231). Furthermore, a core region with decreased mobility is revealed from residues ~127 to 225. Within this core oligomer region with decreased mobility, there is a pocket of increased amide peak signal corresponding to the middle of α-helix 2 and the loop between α-helices 2 and 3 in the PrP^c monomer structure. Using high-resolution solution-state NMR, this work reveals detailed and divergent residue-specific changes in soluble oligomeric models of PrP.     

Novel Zn2+-binding Sites in Human Transthyretin: IMPLICATIONS FOR AMYLOIDOGENESIS AND RETINOL-BINDING PROTEIN RECOGNITION*

Human transthyretin (TTR) is a homotetrameric protein involved in several amyloidoses. Zn²⁺ enhances TTR aggregation in vitro, and is a component of ex vivo TTR amyloid fibrils. We report the first crystal structure of human TTR in complex with Zn²⁺ at pH 4.6–7.5. All four structures reveal three tetra-coordinated Zn²⁺-binding sites (ZBS 1–3) per monomer, plus a fourth site (ZBS 4) involving amino acid residues from a symmetry-related tetramer that is not visible in solution by NMR. Zn²⁺ binding perturbs loop E-α-helix-loop F, the region involved in holo-retinol-binding protein (holo-RBP) recognition, mainly at acidic pH; TTR affinity for holo-RBP decreases ∼5-fold in the presence of Zn²⁺. Interestingly, this same region is disrupted in the crystal structure of the amyloidogenic intermediate of TTR formed at acidic pH in the absence of Zn²⁺. HNCO and HNCA experiments performed in solution at pH 7.5 revealed that upon Zn²⁺ binding, although the α-helix persists, there are perturbations in the resonances of the residues that flank this region, suggesting an increase in structural flexibility. While stability of the monomer of TTR decreases in the presence of Zn²⁺, which is consistent with the tertiary structural perturbation provoked by Zn²⁺ binding, tetramer stability is only marginally affected by Zn²⁺. These data highlight structural and functional roles of Zn²⁺ in TTR-related amyloidoses, as well as in holo-RBP recognition and vitamin A homeostasis.     

Modeling by assembly and molecular dynamics simulations of the low Cu2+ occupancy form of the mammalian prion protein octarepeat region: gaining insight into Cu2+-mediated beta-cleavage

The prion protein has garnered considerable interest because of its involvement in prion disease as well as its unresolved cellular function. The octarepeat region in the flexible N-domain is capable of binding copper through multiple coordination modes. Under conditions of low pH and low Cu²⁺ concentration, the four octarepeats (ORs) cooperatively coordinate a single copper ion. Based on the average structure of the PHGG and GWGQ portions of a copper-free OR₂ model from molecular dynamics simulations, the starting structures of the OR₄ complex could be constructed by assembling the repeating structure of PHGG and GWGQ fragments. The resulting model contains a preformed site suitable for Cu²⁺ coordination. Molecular dynamics simulations of Cu²⁺ bound to the assembled OR₄ model (Cu:OR₄) reveal a close association of specific Trp and Gly residues with the Cu²⁺ center. This low Cu²⁺-occupancy form of prion protein is redox-active and can readily initiate cleavage of the OR region, mediated by reactive oxygen species generated by Cu⁺. The OR region is known to be required for β-cleavage, as are the Trp residues within the OR region. The β-cleaved form of the prion protein accumulates in amyloid fibrils. Hence, the close approach of Trp and Gly residues to the Cu²⁺ coordination site in the low Cu²⁺-occupancy form of the OR region may signal an important interaction for the initiation of prion disease.     

Phylogenetic and functional analysis of histidine residues essential for pH-dependent multimerization of von Willebrand factor

von Willebrand factor (VWF) is a multimeric plasma protein that mediates platelet adhesion to sites of vascular injury. The hemostatic function of VWF depends upon the formation of disulfide-linked multimers, which requires the VWF propeptide (D1D2 domains) and adjacent D′D3 domains. VWF multimer assembly occurs in the trans-Golgi at pH ∼6.2 but not at pH 7.4, which suggests that protonation of one or more His residues (pK_a ∼6.0) mediates the pH dependence of multimerization. Alignment of 30 vertebrate VWF sequences identified 13 highly conserved His residues in the D1D2D′D3 domains, and His-to-Ala mutagenesis identified His³⁹⁵ and His⁴⁶⁰ in the D2 domain as critical for VWF multimerization. Replacement of His³⁹⁵ with Lys or Arg prevented multimer assembly, suggesting that reversible protonation of this His residue is essential. In contrast, replacement of His⁴⁶⁰ with Lys or Arg preserved normal multimer assembly, whereas Leu, Met, and Gln did not, indicating that the function of His⁴⁶⁰ depends primarily upon the presence of a positive charge. These results suggest that pH sensing by evolutionarily conserved His residues facilitates the assembly and packaging of VWF multimers upon arrival in the trans-Golgi.     

LexA protein of cyanobacterium Anabaena sp. strain PCC7120 exhibits in vitro pH-dependent and RecA-independent autoproteolytic activity

The LexA protein of the nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120 exhibits a RecA-independent and alkaline pH-dependent autoproteolytic cleavage. The autoproteolytic cleavage of Anabaena LexA occurs at pH 8.5 and above, stimulated by the addition of Ca²⁺ and in the temperature range of 30–57 °C. Mutational analysis of Anabaena LexA protein indicated that the cleavage occurred at the peptide bond between Ala-84 and Gly-85, and optimal cleavage required the presence of Ser-118 and Lys-159, as also observed for LexA protein of Escherichia coli. Cleavage of Anabaena LexA was affected upon deletion of three amino acids, ⁸⁶GLI. These three amino acids are unique to all cyanobacterial LexA proteins predicted to be cleavable. The absence of RecA-dependent cleavage at physiological pH, which has not been reported for other bacterial LexA proteins, is possibly due to the absence of RecA interacting sites on Anabaena LexA protein, corresponding to the residues identified in E. coli LexA, and low cellular levels of RecA in Anabaena. Exposure to SOS-response inducing stresses, such as UV-B and mitomycin C neither affected the expression of LexA in Anabaena nor induced cleavage of LexA in either Anabaena 7120 or E. coli overexpressing Anabaena LexA protein. Though the LexA may be acting as a repressor by binding to the LexA box in the vicinity of the promoter region of specific gene, their derepression may not be via proteolytic cleavage during SOS-inducing stresses, unless the stress induces increase in cytoplasmic pH. This could account for the regulation of several carbon metabolism genes rather than DNA-repair genes under the regulation of LexA in cyanobacteria especially during high light induced oxidative stress.     

Backbone 1HN, 13C, and 15N resonance assignments of the tandem RNA-binding domains of human DGCR8

Double-stranded RNA binding domain (dsRBD) containing proteins are critical components of the microRNA (miRNA) pathway, with key roles in small RNA biogenesis, modification, and regulation. DiGeorge Critical Region 8 (DGCR8) is a 773 amino acid, dsRBD-containing protein that was originally identified in humans as a protein encoded in the region of chromosome 22 that is deleted in patients with DiGeorge syndrome. Now, it is realized that DGCR8 complements the nuclear RNase III Drosha to initiate miRNA biogenesis by promoting efficient recognition and cleavage of primary miRNAs (pri-miRNA). A pair of C-terminal tandem dsRBDs separated by a flexible linker are required for pri-miRNA substrate binding and recognition. The crystal structure of the DGCR8 core region comprising residues 493–720 revealed that each dsRBD adopts the canonical αβββα fold. However, several residues located in important flexible regions including the β1-β2-loop implicated in canonical dsRNA recognition are absent in the crystal structure and no RNA-bound structure of DGCR8 has been reported. Here we report the ¹H^N, ¹³C, and ¹⁵N backbone resonance assignments of the 24 kDa, 214 amino acid human DGCR8^core (residues 493–706) by heteronuclear NMR spectroscopy. Our assignments lay the foundation for a detailed solution state characterization of the dynamical and RNA-binding properties of this protein in solution.     

Structure and dynamics of the M13 coat signal sequence in membranes by multidimensional high-resolution and solid-state NMR spectroscopy

The polypeptide corresponding to the signal sequence of the M13 coat protein and the five N-terminal residues of the mature protein was prepared by solid-phase peptide synthesis with a ¹⁵N isotopic label at the alanine-12 position. Multidimensional solution NMR spectroscopy and molecular modeling calculations indicate that this polypeptide assumes helical conformations between residues 5 and 20, in the presence of sodium dodecylsulfate micelles. This is in good agreement with circular dichroism spectroscopic measurement, which shows an α-helix content of approximately 42%. The α-helix comprises an uninterrupted hydrophobic stretch of ≤12 amino acids, which is generally believed to be too short for a stable transmembrane alignment in a biological bilayer. The monoexponential proton-deuterium exchange kinetics of this hydrophobic helical region is characterized by half-lives of 15–75 minutes (pH 4.2, 323 K). When the polypeptide is reconstituted into phospholipid bilayers, the broad anisotropy of the proton-decoupled ¹⁵N solid-state NMR spectroscopy indicates that the hydrophobic helix is immobilized close to the lipid bilayer surface at the time scale of ¹⁵N solid-state NMR spectroscopy (10⁻⁴ seconds). By contrast, short correlation times, immediate hydrogen-deuterium exchange as well as nuclear Overhauser effect crosspeak analysis suggest that the N and C termini of this polypeptide exhibit a mobile random coil structure. The implications of these structural findings for possible mechanisms of membrane insertion and translocation as well as for membrane protein structure prediction algorithms are discussed. © 1997 Wiley-Liss Inc.     

Human Nucleotide Excision Repair Protein XPA: 1H NMR and CD Solution Studies of a Synthetic Peptide Fragment Corresponding to the Zinc-Binding Domain (101–141)

Abstract

A peptide corresponding to residues 101–141 of the human nucleotide excision repair protein XPA was synthesized with an isoleucine substituted for L138 and its solution structure studied by circular dichroism and homonuclear ¹H NMR spectroscopy. The peptide, (XPA-41), contains a C₄?type zinc-binding motif, C105-(X)₂C108-(X)_l7?C126-(X)₂ C129, which XPA requires for damaged-DNA binding activity. The proton resonances of XPA-41without zinc (apoXPA-41) were assigned using homonuclear TOCSY, NOESY and DQF-COSY data and show the apo-zinc peptide is a random coil. The peptide was folded with the addition of 1.2 equivalents of ZnCl₂ in dilute solution at pH 4.0. Electrospray ionization mass spectroscopy illustrated an increase in the molecular weight of XPA-41 by 65 amu. Circular dichroism spectra of the zinc-folded peptide (zXPA-41) showed the acquisition of elements of secondary structure. Such a conclusion was confirmed with'H NMR data collected at 25°C, pH 6.3. Hα-secondary shifts and NOE patterns indicate that regions V102-C105 and G109-F112 form an anti-parallel β-sheet and residues N128-K137 form a nascent α-helix. Rapid exchange of most amide resonances between S115-C126 prohibited unambiguous assignment of all the proton resonances in this region. However, a 1.19 ppm downfield shift of the H^α resonance of T125 relative to the apo-zinc peptide, together with downfield shifted H^α resonances for the adjacent residues (P124 and L123), suggest a second β-sheet is present in the S115-C126 region. On the basis of structural similarities to GATA-1 (Science 267:438–446), a homology generated structure for zXPA-41 was made, using GATA-1 as the template, which satisfied all the observed NOEs. Using the hybrid homology-NMR based zXPA-41 structure and analogy to GATA-1, models for the role played by the zinc-binding core (101–141) of XPA in DNA damage recognition are proposed.     

Evidence for the residual tertiary structure in the urea-unfolded form of bacteriophage T5 endolysin

Using high-resolution NMR spectroscopy, we studied peculiarities of the unfolding process of the bacteriophage T5 endolysin (EndoT5) by strong denaturants. It was shown that in the absence of zinc ions this protein is mostly unfolded in the solution of 8 M urea or 6 M guanidine hydrochloride. However, in the presence of zinc ions EndoT5 unfolding can be achieved only in acidic solutions (at pH < 4.0), whereas at pH > 4.0 NMR spectra of the metal-bound protein (Zn²⁺–Ca²⁺–EndoT5 or Zn²⁺–EndoT5 complexes) exhibit a few chemical shifts characteristic of the native or native-like proteins. Our data, including the pH–titration curve with the pK of ~5, suggested involvement of the zinc-binding histidines in the stabilization of this protein. Up-field signals that appear in the NMR spectra of apo-EndoT5 in the presence of high concentrations of strong denaturants are probably derived from the amino acid residues included in the formation of structured hydrophobic cluster, which likely corresponds to the 81–93 region of EndoT5 and contains some residual tertiary structure. It is possible also that this hydrophobic fragment serves as a foundation for the formation of structured cluster in the unfolded state.     

Differential hydrogen ion titrations of the histidine residues in Helix pomatia haemocyanin

The properties of the histidine residues in Helix pomatia haemocyanin have been studied by differential hydrogen ion titrations. In oxy-and deoxyhaemocyanin 31 × 10^?5 histidine residues per g protein are titrated in contrast to 35 × 10^?5 residues in apohaemocyanin. The difference corresponds to a stoichiometry of one histidine residue per copper atom bound. Even in apohaemocyanin about 6 × 10^?5 histidine residues per g protein are not titrated in their normal pH region.In the presence of sufficient calcium to displace the dissociation completely out of the titration region the titration curve of apohaemocyanin could he linarized according to the model of Linderstrøm—Lang. In oxy- and deoxyhaemocyanin, however, a distinct deviation from linearity was found under the same conditions. In the absence of calcium the effect of the dissociation adds up to this deviation.The electrostatic interaction factors were determined for the protein at 0.1 M KCl and for the dissociation products: halves and tenths at 1.0 M KCl. The electrostatic interaction factor for the wholes and the halves are much smaller than the values calculated from the Linderstrøm—Lang equation, using the radius of the equivalent sphere either obtained from electron microscopy or from the partial specific volume. This is probably due to solvent penetration. For the tenths at 1.0 M KCl. this effect is small,     

Interaction between cucumber green mottle mosaic virus MP and CP promotes virus systemic infection

The movement protein (MP) and coat protein (CP) of tobamoviruses play critical roles in viral cell-to-cell and long-distance movement, respectively. Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus. The functions of CGMMV MP and CP during viral infection remain largely unclear. Here, we show that CGMMV MP can interact with CP in vivo, and the amino acids at positions 79–128 in MP are vital for the MP–CP interaction. To confirm this finding, we mutated five conserved residues within the residue 79–128 region and six other conserved residues flanking this region, followed by in vivo interaction assays. The results showed that the conserved threonine residue at the position 107 in MP (MP^T107) is important for the MP–CP interaction. Substitution of T107 with alanine (MP^T107A) delayed CGMMV systemic infection in Nicotiana benthamiana plants, but increased CGMMV local accumulation. Substitutions of another 10 conserved residues, not responsible for the MP–CP interaction, with alanine inhibited or abolished CGMMV systemic infection, suggesting that these 10 conserved residues are possibly required for the MP movement function through a CP-independent manner. Moreover, two movement function-associated point mutants (MP^F17A and MP^D97A) failed to cause systemic infection in plants without impacting on the MP–CP interaction. Furthermore, we have found that co-expression of CGMMV MP and CP increased CP accumulation independent of the interaction. MP and CP interaction inhibits the salicylic acid-associated defence response at an early infection stage. Taken together, we propose that the suppression of host antiviral defence through the MP–CP interaction facilitates virus systemic infection.     

Identification of pH-sensing Sites in the Light Harvesting Complex Stress-related 3 Protein Essential for Triggering Non-photochemical Quenching in Chlamydomonas reinhardtii

Light harvesting complex stress-related 3 (LHCSR3) is the protein essential for photoprotective excess energy dissipation (non-photochemical quenching, NPQ) in the model green alga Chlamydomonas reinhardtii. Activation of NPQ requires low pH in the thylakoid lumen, which is induced in excess light conditions and sensed by lumen-exposed acidic residues. In this work we have used site-specific mutagenesis in vivo and in vitro for identification of the residues in LHCSR3 that are responsible for sensing lumen pH. Lumen-exposed protonatable residues, aspartate and glutamate, were mutated to asparagine and glutamine, respectively. By expression in a mutant lacking all LHCSR isoforms, residues Asp¹¹⁷, Glu²²¹, and Glu²²⁴ were shown to be essential for LHCSR3-dependent NPQ induction in C. reinhardtii. Analysis of recombinant proteins carrying the same mutations refolded in vitro with pigments showed that the capacity of responding to low pH by decreasing the fluorescence lifetime, present in the wild-type protein, was lost. Consistent with a role in pH sensing, the mutations led to a substantial reduction in binding the NPQ inhibitor dicyclohexylcarbodiimide.     

The crystal structures of human S100B in the zinc- and calcium-loaded state at three pH values reveal zinc ligand swapping

S100B is a homodimeric zinc-, copper-, and calcium-binding protein of the family of EF-hand S100 proteins. Zn²⁺ binding to S100B increases its affinity towards Ca²⁺ as well as towards target peptides and proteins. Cu²⁺ and Zn²⁺ bind presumably to the same site in S100B. We determined the structures of human Zn²⁺- and Ca²⁺-loaded S100B at pH 6.5, pH 9, and pH 10 by X-ray crystallography at 1.5, 1.4, and 1.65 Å resolution, respectively. Two Zn²⁺ ions are coordinated tetrahedrally at the dimer interface by His and Glu residues from both subunits. The crystal structures revealed that ligand swapping occurs for one of the four ligands in the Zn²⁺-binding sites. Whereas at pH 9, the Zn²⁺ ions are coordinated by His15, His25, His 85′, and His 90′, at pH 6.5 and pH 10, His90′ is replaced by Glu89′. The results document that the Zn²⁺-binding sites are flexible to accommodate other metal ions such as Cu²⁺. Moreover, we characterized the structural changes upon Zn²⁺ binding, which might lead to increased affinity towards Ca²⁺ as well as towards target proteins. We observed that in Zn²⁺-Ca²⁺-loaded S100B the C-termini of helix IV adopt a distinct conformation. Zn²⁺ binding induces a repositioning of residues Phe87 and Phe88, which are involved in target protein binding. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.