The crystal structure of the nuclease domain of colicin E7 suggests a mechanism for binding to double-stranded DNA by the H-N-H endonucleases

The bacterial toxin ColE7 contains an H-N-H endonuclease domain (nuclease ColE7) that digests cellular DNA or RNA non-specifically in target cells, leading to cell death. In the host cell, protein Im7 forms a complex with ColE7 to inhibit its nuclease activity. Here, we present the crystal structure of the unbound nuclease ColE7 at a resolution of 2.1A. Structural comparison between the unbound and bound nuclease ColE7 in complex with Im7, suggests that Im7 is not an allosteric inhibitor that induces backbone conformational changes in nuclease ColE7, but rather one that inhibits by blocking the substrate-binding site. There were two nuclease ColE7 molecules in the P1 unit cell in crystals and they appeared as a dimer related to each other by a non-crystallographic dyad symmetry. Gel-filtration and cross-linking experiments confirmed that nuclease ColE7 indeed formed dimers in solution and that the dimeric conformation was more favored in the presence of double-stranded DNA. Structural comparison of nuclease ColE7 with the His-Cys box homing endonuclease I-PpoI further demonstrated that H-N-H motifs in dimeric nuclease ColE7 were oriented in a manner very similar to that of the betabetaalpha-fold of the active sites found in dimeric I-PpoI. A mechanism for the binding of double-stranded DNA by dimeric H-N-H nuclease ColE7 is suggested.     

X‐ray crystal structure of the N‐terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition

The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N‐terminal domain (NTD), the catalytic core domain, and the C‐terminal domain. The NTD includes an HHCC zinc finger‐like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M‐MuLV) IN N‐terminal region (NTR) constructs that both include an N‐terminal extension domain (NED, residues 1–44) and an HHCC zinc‐finger NTD (residues 45–105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1–105 (NTR_1–105) and 8–105 (NTR_8–105) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR_1–105 packs as a dimer and NTR_8–105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti‐parallel β‐strands and an α‐helix, similar to the NED of prototype foamy virus (PFV) IN. These three β‐strands form an extended β‐sheet with another β‐strand in the HHCC Zn²⁺ binding domain, which is a unique structural feature for the M‐MuLV IN. The HHCC Zn²⁺ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M‐MuLV. Proteins 2017; 85:647–656. © 2016 Wiley Periodicals, Inc.     

Structure of the N‐terminal domain of the metalloprotease PrtV from Vibrio cholerae

The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre‐pro‐protein that undergoes several N‐ and C‐terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N‐terminal domain (residues 23–103) that contains two short α‐helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C‐terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.     

Investigating the inter‐subunit/subdomain interactions and motions relevant to disease mutations in the N‐terminal domain of ryanodine receptors by molecular dynamics simulation

The ryanodine receptors (RyR) are essential to calcium signaling in striated muscles, and numerous disease mutations have been identified in two RyR isoforms, RyR1 in skeletal muscle and RyR2 in cardiac muscle. A deep understanding of the activation/regulation mechanisms of RyRs has been hampered by the shortage of high‐resolution structures and dynamic information for this giant tetrameric complex in different functional states. Toward elucidating the molecular mechanisms of disease mutations in RyRs, we performed molecular dynamics simulation of the N‐terminal domain (NTD) which is not only the best‐resolved structural component of RyRs, but also a hotspot of disease mutations. First, we simulated the tetrameric NTD of wild‐type RyR1 and three disease mutants (K155E, R157Q, and R164Q) that perturb the inter‐subunit interfaces. Our simulations identified a dynamic network of salt bridges involving charged residues at the inter‐subunit/subdomain interfaces and disease‐mutation sites. By perturbing this key network, the above three mutations result in greater flexibility with the highest inter‐subunit opening probability for R157Q. Next, we simulated the monomeric NTD of RyR2 in the presence or absence of a central Cl^– anion which is known to stabilize the interfaces between the three NTD subdomains (A, B, and C). We found that the loss of Cl^– restructures the salt‐bridge network near the Cl^–‐binding site, leading to rotations of subdomain A/B relative to subdomain C and enhanced mobility between the subdomains. This finding supports a mechanism for disease mutations in the NTD of RyR2 via perturbation of the Cl^– binding. The rich structural and dynamic information gained from this study will guide future mutational and functional studies of the NTD of RyRs. Proteins 2017; 85:1633–1644. © 2017 Wiley Periodicals, Inc.     

Flexibility in the N‐terminal actin‐binding domain: Clues from in silico mutations and molecular dynamics

Dystrophin is a long, rod‐shaped cytoskeleton protein implicated in muscular dystrophy (MDys). Utrophin is the closest autosomal homolog of dystrophin. Both proteins have N‐terminal actin‐binding domain (N‐ABD), a central rod domain and C‐terminal region. N‐ABD, composed of two calponin homology (CH) subdomains joined by a helical linker, harbors a few disease causing missense mutations. Although the two proteins share considerable homology (>72%) in N‐ABD, recent structural and biochemical studies have shown that there are significant differences (including stability, mode of actin‐binding) and their functions are not completely interchangeable. In this investigation, we have used extensive molecular dynamics simulations to understand the differences and the similarities of these two proteins, along with another actin‐binding protein, fimbrin. In silico mutations were performed to identify two key residues that might be responsible for the dynamical difference between the molecules. Simulation points to the inherent flexibility of the linker region, which adapts different conformations in the wild type dystrophin. Mutations T220V and G130D in dystrophin constrain the flexibility of the central helical region, while in the two known disease‐causing mutants, K18N and L54R, the helicity of the region is compromised. Phylogenetic tree and sequence analysis revealed that dystrophin and utrophin genes have probably originated from the same ancestor. The investigation would provide insight into the functional diversity of two closely related proteins and fimbrin, and contribute to our understanding of the mechanism of MDys. Proteins 2015; 83:696–710. © 2015 Wiley Periodicals, Inc.     

N‐terminal β‐strand swapping in a consensus‐derived alternative scaffold driven by stabilizing hydrophobic interactions

The crystal structure of an N‐terminal β‐strand‐swapped consensus‐derived tenascin FN3 alternative scaffold has been determined. A comparison with the unswapped structure reveals that the side chain of residue F88 orients differently and packs more tightly with the hydrophobic core of the domain. Dimer formation also results in the burial of a hydrophobic patch on the surface of the domain. Thus, it appears that tighter packing of F88 in the hydrophobic core and burial of surface hydrophobicity provide the driving forces for the N‐terminal β‐strand swapping, leading to the formation of a stable compact dimer. Proteins 2014; 82:1527–1533. © 2014 Wiley Periodicals, Inc.     

Solution structure of the N‐terminal domain of DC‐UbP/UBTD2 and its interaction with ubiquitin

DC‐UbP/UBTD2 is a ubiquitin (Ub) domain‐containing protein first identified from dendritic cells, and is implicated in ubiquitination pathway. The solution structure and backbone dynamics of the C‐terminal Ub‐like (UbL) domain were elucidated in our previous work. To further understand the biological function of DC‐UbP, we then solved the solution structure of the N‐terminal domain of DC‐UbP (DC‐UbP_N) and studied its Ub binding properties by NMR techniques. The results show that DC‐UbP_N holds a novel structural fold and acts as a Ub‐binding domain (UBD) but with low affinity. This implies that the DC‐UbP protein, composing of a combination of both UbL and UBD domains, might play an important role in regulating protein ubiquitination and delivery of ubiquitinated substrates in eukaryotic cells.     

Different antigenicities of the N‐terminal region of cellular and scrapie prion proteins

Limited information is available about conformational differences between the abnormal isoform of prion protein (PrP^Sc) and cellular prion protein (PrP^C) under native conditions. To clarify conformational differences between these two isoforms, PrP‐deficient mice were immunized with brain homogenates of normal and scrapie‐infected animals. All mice generated anti‐PrP antibodies. Peptide array analysis of these serum samples revealed a distinctive epitope of PrP^Sc consisting of QGSPGGN (PrP41–47) at the N‐terminus. This study demonstrated a conformational dissimilarity at the N‐terminus between PrP^Sc and PrP^C, a finding that may provide novel information about conformational features of PrP^Sc.     

Effects of the β‐agonist,isoprenaline, on the down‐regulation,functional responsiveness and trafficking of β2‐adrenergic receptors with N‐terminal polymorphisms

The β₂‐AR (β₂‐adrenergic receptor) is an important target for respiratory and CVD (cardiovascular disease) medications. Clinical studies suggest that N‐terminal polymorphisms of β₂‐AR may act as disease modifiers. We hypothesized that polymorphisms at amino acids 16 and 27 result in differential trafficking and down‐regulation of β₂‐AR variants following β‐agonist exposure. The functional consequences of the four possible combinations of these polymorphisms in the human β₂‐AR (designated β₂‐AR‐RE, β₂‐AR‐GE, β₂‐AR‐RQ and β₂‐AR‐GQ) were studied using site‐directed mutagenesis and recombinant expression in HEK‐293 cells (human embryonic kidney cells). Ligand‐binding assays demonstrated that after 24 h exposure to 1 μM isoprenaline, isoforms with Arg¹⁶ (β₂‐AR‐RE and β₂‐AR‐RQ) underwent increased down‐regulation compared with isoforms with Gly¹⁶ (β₂‐AR‐GE and β₂‐AR‐GQ). Consistent with these differences in down‐regulation between isoforms, prolonged isoprenaline treatment resulted in diminished cAMP response to subsequent isoprenaline challenge in β₂‐AR‐RE relative to β₂‐AR‐GE. Confocal microscopy revealed that the receptor isoforms had similar co‐localization with the early endosomal marker EEA1 following isoprenaline treatment, suggesting that they had similar patterns of internalization. None of the isoforms exhibited significant co‐localization with the recycling endosome marker Rab11 in response to isoprenaline treatment. Furthermore, we found that prolonged isoprenaline treatment led to a higher degree of co‐localization of β₂‐AR‐RE with the lysosomal marker LAMP1 (lysosome‐associated membrane protein 1) compared with that of β₂‐AR‐GE. Taken together, these results indicate that a mechanism responsible for differential responses of these receptor isoforms to the β‐agonist involves differences in the efficiency with which agonist‐activated receptors are trafficked to the lysosomes for degradation, or differences in degradation in the lysosomes.     

N‐terminal diproline and charge group effects on the stabilization of helical conformation in alanine‐based short peptides: CD studies with water and methanol as solvent

Protein folding problem remains a formidable challenge as main chain, side chain and solvent interactions remain entangled and have been difficult to resolve. Alanine‐based short peptides are promising models to dissect protein folding initiation and propagation structurally as well as energetically. The effect of N‐terminal diproline and charged side chains is assessed on the stabilization of helical conformation in alanine‐based short peptides using circular dichroism (CD) with water and methanol as solvent. A1 (Ac–Pro–Pro–Ala–Lys–Ala–Lys–Ala–Lys–Ala–NH₂) is designed to assess the effect of N‐terminal homochiral diproline and lysine side chains to induce helical conformation. A2 (Ac–Pro–Pro–Glu–Glu–Ala–Ala–Lys–Lys–Ala–NH₂) and A3 (Ac–d Pro–Pro–Glu–Glu–Ala–Ala–Lys–Lys–Ala–NH₂) with N‐terminal homochiral and heterochiral diproline, respectively, are designed to assess the effect of Glu...Lys (i , i  + 4) salt bridge interactions on the stabilization of helical conformation. The CD spectra of A1 , A2 and A3 in water manifest different amplitudes of the observed polyproline II (PPII) signals, which indicate different conformational distributions of the polypeptide structure. The strong effect of solvent substitution from water to methanol is observed for the peptides, and CD spectra in methanol evidence A2 and A3 as helical folds. Temperature‐dependent CD spectra of A1 and A2 in water depict an isodichroic point reflecting coexistence of two conformations, PPII and β‐strand conformation, which is consistent with the previous studies. The results illuminate the effect of N‐terminal diproline and charged side chains in dictating the preferences for extended‐β, semi‐extended PPII and helical conformation in alanine‐based short peptides. The results of the present study will enhance our understanding on stabilization of helical conformation in short peptides and hence aid in the design of novel peptides with helical structures. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Syntheses and activities of backbone‐side chain cyclic octapeptide ligands with N‐functionalized phosphotyrosine for the N‐terminal SH2‐domain of the protein tyrosine phosphatase SHP‐1

The protein tyrosine phosphatase SHP‐1 plays an important role in many physiological and pathophysiological processes. This phosphatase is activated through binding of ligands to its SH2‐domains, mainly to the N‐terminal one. Based on a theoretical docking model, backbone‐to‐side chain cyclized octapeptides were designed as ligands. Assembly of such modelled structures required the synthesis of N‐functionalized tyrosine derivatives and their incorporation into the sequence. Because of difficulties encountered in the condensation of N‐protected amino acids to the N‐alkylated tyrosine‐peptide we synthesized and used preformed dipeptide building units. As all attempts to obtain phosphorylated dipeptide units failed, the syntheses had to be performed with a free phenolic function. Use of different N‐alkyl or cycloalkyl residues in the N‐functionalized side chains allowed to investigate the effect of ring size, flexibility and hydrophobicity of formed lactam bridges on stimulatory activity. All tested linear and cyclic octapeptides stimulate the phosphatase activity of SHP‐1. Stimulatory activities of cyclic ligands increase with the chain length of the lactam bridges resulting in increased flexibility and better entropic preformation of the binding conformation. The strong activity of some cyclic octapeptides supports the modelled structure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Design,synthesis, and comparative evaluation of 99mTc(CO)3‐labeled N‐terminal and C‐terminal modified asparagine–glycine–arginine peptide constructs

The present study describes modification of asparagine–glycine–arginine (NGR) peptide at N‐terminally and C‐terminally by introduction of a tridentate chelating scaffold via click chemistry reaction. The N‐terminal and C‐terminal modified peptides were radiometalated with [^99mTc(CO)₃]⁺ precursor. The influence of these moieties at the two termini on the targeting properties of NGR peptide was determined by in vitro cell uptake studies and in vivo biodistribution studies. The two radiolabeled constructs did not exhibit any significant variation in uptake in murine melanoma B16F10 cells during in vitro studies. In vivo studies revealed nearly similar tumor uptake of N‐terminally modified peptide construct 5 and C‐terminally construct 6 at 2 h p.i. (1.9 ± 0.1 vs 2.4 ± 0.2% ID/g, respectively). The tumor‐to‐blood (T/B) and tumor‐to‐liver (T/L) ratios of the two radiometalated peptides were also quite similar. The two constructs cleared from all the major organs (heart, lungs, spleen, stomach, and blood) at 4 h p.i. (<1% ID/g). Blocking studies carried out by coinjection of cCNGRC peptide led to approximately 50% reduction in the tumor uptake at 2 h p.i. This work thus illustrates the possibility of convenient modification/radiometalation of NGR peptide at either N‐ or C‐terminus without hampering tumor targeting and pharmacokinetics.     

Spectroscopic investigations on the conformational changes of lysozyme effected by different sizes of N‐acetyl‐l‐cysteine‐capped CdTe quantum dots

The effect of N‐acetyl‐l ‐cysteine‐capped CdTe quantum dots (NAC‐CdTe QDs) with different sizes on lysozyme was investigated by isothermal titration calorimetry (ITC), enzyme activity assays, and multi‐spectroscopic methods. ITC results proved that NAC‐CdTe QDs can spontaneously bind with lysozyme and hydrophobic force plays a major role in stabilizing QDs–lysozyme complex. Multi‐spectroscopic measurements revealed that NAC‐CdTe QDs caused strong quenching of the lysozyme's fluorescence in a size‐dependent quenching manner. Moreover, the changes of secondary structure and microenvironment in lysozyme caused by the NAC‐CdTe QDs were higher with a bigger size. The results of enzyme activity assays showed that the interaction between lysozyme and NAC‐CdTe QDs inhibited the activity of lysozyme and the inhibiting effect was in a size‐dependent manner. Based on these results, we conclude that NAC‐CdTe QDs with larger particle size had a larger impact on the structure and function of lysozyme.     

Characterizing the release of bioactive N‐glycans from dairy products by a novel endo‐β‐N‐acetylglucosaminidase

Endo‐β‐N‐acetylglucosaminidase isolated from B. infantis ATCC 15697 (EndoBI‐1) is a novel enzyme that cleaves N‐N′‐diacetyl chitobiose moieties found in the N‐glycan core of high mannose, hybrid, and complex N‐glycans. These conjugated N‐glycans are recently shown as a new prebiotic source that stimulates the growth of a key infant gut microbe, Bifidobacterium longum subsp. Infantis. The effects of pH (4.45–8.45), temperature (27.5–77.5°C), reaction time (15–475 min), and enzyme/protein ratio (1:3,000–1:333) were evaluated on the release of N‐glycans from bovine colostrum whey by EndoBI‐1. A central composite design was used, including a two‐level factorial design (2⁴) with four center points and eight axial points. In general, low pH values, longer reaction times, higher enzyme/protein ratio, and temperatures around 52°C resulted in the highest yield. The results demonstrated that bovine colostrum whey, considered to be a by/waste product, can be used as a glycan source with a yield of 20 mg N‐glycan/g total protein under optimal conditions for the ranges investigated. Importantly, these processing conditions are suitable to be incorporated into routine dairy processing activities, opening the door for an entirely new class of products (released bioactive glycans and glycan‐free milk). The new enzyme's activity was also compared with a commercially available enzyme, showing that EndoBI‐1 is more active on native proteins than PNGase F and can be efficiently used during pasteurization, streamlining its integration into existing processing strategies. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1331–1339, 2015     

Site‐specific conjugation of the quencher on peptide's N‐terminal for the synthesis of a targeted non‐spreading activatable optical probe

Optical imaging offers high sensitivity and portability at low cost. The design of ‘smart’ or ‘activatable’ probes can decrease the background noise and increase the specificity of the signal. By conjugating a fluorescent dye and a compatible quencher on each side of an enzyme's substrate, the signal remains in its ‘off ’ state until it reaches the area where a specific enzyme is expressed. However, the signal can leak from that area unless the dye is attached to a molecule able to bind to a specific target also presented in that area. The aim of this study was to (i) specifically conjugate the quencher on the α‐amino group of the peptide's N‐terminus, (ii) conjugate the dye on the ε‐amino group of a lysine in C‐terminus, and (iii) conjugate the carboxyl group of the peptide's C‐terminus to an amino group present on an antibody, using carbodiimide chemistry. The use of protecting groups, such as Boc or Fmoc, to allow site‐specific conjugation, presents several drawbacks including ‘on beads labeling’, additional steps required for deprotection and removal from the resin, decreased yield, and dye degradation. A method of preferential labeling of α‐amino N‐terminal group in slightly acidic solution, proposed by Selo et al. (1996) has partially solved the problem. The present study reports improvements of the method allowing to (i) avoid the homo‐bilabeling, (ii) increase the yield of the N‐terminal labeling by two folds, and (iii) decrease the cost by 44‐fold. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.