Structural insights into the role of N-terminal integrity in PhoSL for core-fucosylated N-glycan recognition.

PhoSL (Pholiota squarrosa Lectin) has an exceptional binding affinity for biomolecules with core-fucosylated N-glycans. This modification involves the addition of fucose to the inner N-acetylglucosamine within the N-glycan structure and is known to influence many physiological processes. Nevertheless, the molecular interactions underlying high-affinity binding of native PhoSL to core-fucosylated N-glycans remain largely unknown. In this study, we devised a strategy to produce PhoSL with the essential structural characteristics of the native protein (n-PhoSL). To do so, a fusion protein was expressed in E. coli and purified. Then, enzymatic cleavage and incubation with glutathione were utilized to recapitulate the native primary structure and disulfide bonding pattern. Subsequently, we identified the residues crucial for n-PhoSL binding to core-fucosylated chitobiose (N2F) via NMR spectroscopy. Additionally, crystal structures were solved for both apo n-PhoSL and its N2F complex. These analyses suggested a pivotal role of the N-terminal amine in maintaining the integrity of the binding pocket and actively contributing to core-fucose recognition. In support of this idea, the inclusion of additional residues at the N-terminus considerably reduced binding affinity and PhoSL cytotoxicity toward breast cancer cells. Taken together, these findings can facilitate the utilization of PhoSL in basic research, diagnostics and therapeutic strategies.

In-depth study of DNA binding of Cys2His2 finger domains in testis zinc-finger protein.

Previously, we identified that both fingers 1 and 2 in the three Cys2His2 zinc-finger domains (TZD) of testis zinc-finger protein specifically bind to its cognate DNA; however, finger 3 is non-sequence-specific. To gain insights into the interaction mechanism, here we further investigated the DNA-binding characteristics of TZD bound to non-specific DNAs and its finger segments bound to cognate DNA. TZD in non-specific DNA binding showed smaller chemical shift perturbations, as expected. However, the direction of shift perturbation, change of DNA imino-proton NMR signal, and dynamics on the 15N backbone atom significantly differed between specific and non-specific binding. Using these unique characteristics, we confirmed that the three single-finger segments (TZD1, TZD2 and TZD3) and the two-finger segment (TZD23) non-specifically bind to the cognate DNA. In comparison, the other two-finger segment (TZD12) binding to the cognate DNA features simultaneous non-specific and semi-specific binding, both slowly exchanged in terms of NMR timescale. The process of TZD binding to the cognate DNA is likely stepwise: initially TZD non-specifically binds to DNA, then fingers 1 and 2 insert cooperatively into the major groove of DNA by semi-specific binding, and finally finger 3 non-specifically binds to DNA, which promotes the specific binding on fingers 1 and 2 and stabilizes the formation of a specific TZD-DNA complex.

Solution structure and base specificity of cytotoxic RC-RNase 2 from Rana catesbeiana.

Cytotoxic ribonucleases found in the oocytes and early embryos of frogs with antitumor activity are well-documented. RC-RNase 2, a cytotoxic ribonuclease isolated from oocytes of bullfrog Rana catesbeiana, consists of 105 residues linked with 4 disulfide bridges and belongs to the bovine pancreatic ribonuclease (RNase A) superfamily. Among the RC-RNases, the base preference for RNase 2 is UpG but CpG for RC-RNase 4; while RC-RNase possesses the base specificity of both UpG and CpG. Interestingly, RC-RNase 2 or 4 has much lower catalytic activity but only three-fold less cytotoxicity than RC-RNase. Here, we report the NMR solution structure of rRC-RNase 2, comprising three alpha-helices and two sets of antiparallel beta-sheets. The differences of side-chain conformations of subsite residues among RNase A, RC-RNase, RC-RNase 4 and rRNase 2 are related to their distinct catalytic activities and base preferences. Furthermore, the substrate-related residues in the base specificity among native RC-RNases are derived using the chemical shift perturbation on ligand binding.

Backbone resonance assignments of the 54 kDa dimeric C-terminal domain of murine STING in complex with DMXAA.

The mammalian ER protein STING (stimulators of interferon genes) is an important innate immunity protein for linking detection of novel secondary messengers c-di-GMP, c-di-AMP, cGAMP or cytosolic dsDNA to the activation of TANK kinase 1 and its downstream interferon regulator factor 3. Recently quite a few of crystal structures representing different states of the C-terminal domain (CTD) of human and murine STING (hSTING and mSTING) in complex with c-di-GMP, cGAMP or DMXAA have been reported. However, the C-terminal 42 residues of STING-CTD, which may be important in mediating the downstream reactions, is invisible or absent in all reported X-ray structures. In addition, X-ray crystal structures may be subject to crystal packing force. Hence an alternate method of determining the structure and function of STING in a near physiological condition is essential. We now report the near complete backbone resonance assignments of the 54 kDa dimeric mSTING-CTD in complex with DMXAA, which is the first step in determining its complex structure and understanding why DMXAA, which is a very efficient agent for curing mouse cancer, is totally ineffective in human.

Iron-inducible nuclear translocation of a Myb3 transcription factor in the protozoan parasite Trichomonas vaginalis.

In Trichomonas vaginalis, a novel nuclear localization signal spanning the folded R2R3 DNA-binding domain of a Myb2 protein was previously identified. To study whether a similar signal is used for nuclear translocation by other Myb proteins, nuclear translocation of Myb3 was examined in this report. When overexpressed, hemagglutinin-tagged Myb3 was localized to nuclei of transfected cells, with a cellular distribution similar to that of endogenous Myb3. Fusion to a bacterial tetracycline repressor, R2R3, of Myb3 that spans amino acids (aa) 48 to 156 was insufficient for nuclear translocation of the fusion protein, unless its C terminus was extended to aa 167. The conserved isoleucine in helix 2 of R2R3, which is important for Myb2's structural integrity in maintaining DNA-binding activity and nuclear translocation, was also vital for the former activity of Myb3, but less crucial for the latter. Sequential nuclear influx and efflux of Myb3, which require further extension of the nuclear localization signal to aa 180, were immediately induced after iron repletion. Sequence elements that regulate nuclear translocation with cytoplasmic retention, nuclear influx, and nuclear efflux were identified within the C-terminal tail. These results suggest that the R2R3 DNA-binding domain also serves as a common module for the nuclear translocation of both Myb2 and Myb3, but there are intrinsic differences between the two nuclear localization signals.

Effects of single nucleotide polymorphisms on human N-acetyltransferase 2 structure and dynamics by molecular dynamics simulation.

BACKGROUND: Arylamine N-acetyltransferase 2 (NAT2) is an important catalytic enzyme that metabolizes the carcinogenic arylamines, hydrazine drugs and chemicals. This enzyme is highly polymorphic in different human populations. Several polymorphisms of NAT2, including the single amino acid substitutions R64Q, I114T, D122N, L137F, Q145P, R197Q, and G286E, are classified as slow acetylators, whereas the wild-type NAT2 is classified as a fast acetylator. The slow acetylators are often associated with drug toxicity and efficacy as well as cancer susceptibility. The biological functions of these 7 mutations have previously been characterized, but the structural basis behind the reduced catalytic activity and reduced protein level is not clear. METHODOLOGY/PRINCIPAL FINDINGS: We performed multiple molecular dynamics simulations of these mutants as well as NAT2 to investigate the structural and dynamical effects throughout the protein structure, specifically the catalytic triad, cofactor binding site, and the substrate binding pocket. None of these mutations induced unfolding; instead, their effects were confined to the inter-domain, domain 3 and 17-residue insert region, where the flexibility was significantly reduced relative to the wild-type. Structural effects of these mutations propagate through space and cause a change in catalytic triad conformation, cofactor binding site, substrate binding pocket size/shape and electrostatic potential. CONCLUSIONS/SIGNIFICANCE: Our results showed that the dynamical properties of all the mutant structures, especially in inter-domain, domain 3 and 17-residue insert region were affected in the same manner. Similarly, the electrostatic potential of all the mutants were altered and also the functionally important regions such as catalytic triad, cofactor binding site, and substrate binding pocket adopted different orientation and/or conformation relative to the wild-type that may affect the functions of the mutants. Overall, our study may provide the structural basis for reduced catalytic activity and protein level, as was experimentally observed for these polymorphisms.

Human RegIV protein adopts a typical C-type lectin fold but binds mannan with two calcium-independent sites.

Human RegIV protein, which contains a sequence motif homologous to calcium-dependent (C-type) lectin-like domain, is highly expressed in mucosa cells of the gastrointestinal tract during pathogen infection and carcinogenesis and may be applied in both diagnosis and treatment of gastric and colon cancers. Here, we provide evidence that, unlike other C-type lectins, human RegIV binds to polysaccharides, mannan, and heparin in the absence of calcium. To elucidate the structural basis for carbohydrate recognition by NMR, we generated the mutant with Pro91 replaced by Ser (hRegIV-P91S) and showed that the structural property and carbohydrate binding ability of hRegIV-P91S are almost identical with those of wild-type protein. The solution structure of hRegIV-P91S was determined, showing that it adopts a typical fold of C-type lectin. Based on the chemical shift perturbations of amide resonances, two calcium-independent mannan-binding sites were proposed. One site is similar to the calcium-independent sugar-binding site on human RegIII and Langerin. Interestingly, the other site is adjacent to the conserved calcium-dependent site at position Ca-2 of typical C-type lectins. Moreover, model-free analysis of (15)N relaxation parameters and simplified Carr-Purcell-Meiboom-Gill relaxation dispersion experiments showed that a slow microsecond-to-millisecond time-scale backbone motion is involved in mannan binding by this site, suggesting a potential role for specific carbohydrate recognition. Our findings shed light on the sugar-binding mode of Reg family proteins, and we postulate that Reg family proteins evolved to bind sugar without calcium to keep the carbohydrate recognition activity under low-pH environments in the gastrointestinal tract.

Assignment of disulfide-linked peptides using automatic a1 ion recognition.

We present a novel approach for the assignment of peptides containing disulfide linkages. Dimethyl labeling is introduced to generate labeled peptides which exhibit enhanced a1 ion signals during MS/MS fragmentation. For disulfide-linked peptides, multiple a1 ions can be observed due to multiple N-termini. This distinct feature allows sieving out the disulfide-linked peptides; meanwhile, the N-terminal amino acids can be identified. With such information, the number of possible peptide combinations involved in a disulfide bond dramatically narrows down. Furthermore, we developed a computational algorithm to perform target a1 ion screening followed by molecular weight matching of cysteine-containing peptides with specific amino acids at the N-termini. Once the protein sequence and the peak list from a LC-MS/MS survey scan of labeled peptides are imported, the identities of disulfide-linked peptides can be readily obtained. The presented approach is simple and straightforward, offering a valuable tool for protein structural characterization.

The flexible and clustered lysine residues of human ribonuclease 7 are critical for membrane permeability and antimicrobial activity.

The ubiquitous ribonucleases (RNases) play important roles in RNA metabolism, angiogenesis, neurotoxicity, and antitumor or antimicrobial activity. Only the antimicrobial RNases possess high positively charged residues, although their mechanisms of action remain unclear. Here, we report on the role of cationic residues of human RNase7 (hRNase7) in its antimicrobial activity. It exerted antimicrobial activity against bacteria and yeast, even at 4 degrees C. The bacterial membrane became permeable to the DNA-binding dye SYTOX(R) Green in only a few minutes after bactericidal RNase treatment. NMR studies showed that the 22 positively charged residues (Lys(18) and Arg(4)) are distributed into three clusters on the surface of hRNase7. The first cluster, K(1),K(3),K(111),K(112), was located at the flexible coil near the N terminus, whereas the other two, K(32),K(35) and K(96),R(97),K(100), were located on rigid secondary structures. Mutagenesis studies showed that the flexible cluster K(1),K(3),K(111),K(112), rather than the catalytic residues His(15), Lys(38), and His(123) or other clusters such as K(32),K(35) and K(96),R(97),K(100), is critical for the bactericidal activity. We suggest that the hRNase7 binds to bacterial membrane and renders the membrane permeable through the flexible and clustered Lys residues K(1),K(3),K(111),K(112). The conformation of hRNase7 can be adapted for pore formation or disruption of bacterial membrane even at 4 degrees C.

Human pancreatitis-associated protein forms fibrillar aggregates with a native-like conformation.

Human pancreatitis-associated protein was identified in pathognomonic lesions of Alzheimer disease, a disease characterized by the presence of filamentous protein aggregates. Here, we showed that at physiological pH, human pancreatitis-associated protein forms non-Congo Red-binding, proteinase K-resistant fibrillar aggregates with diameters from 6 up to as large as 68 nm. Interestingly, circular dichroism and Fourier transform infrared spectra showed that, unlike typical amyloid fibrils, which have a cross-beta-sheet structure, these aggregates have a very similar secondary structure to that of the native protein, which is composed of two alpha-helices and eight beta-strands, as determined by NMR techniques. Surface structure analysis showed that the positively charged and negatively charged residues were clustered on opposite sides, and strong electrostatic interactions between molecules were therefore very likely, which was confirmed by cross-linking experiments. In addition, several hydrophobic residues were found to constitute a continuous hydrophobic surface. These results and protein aggregation prediction using the TANGO algorithm led us to synthesize peptide Thr(84) to Ser(116), which, very interestingly, was found to form amyloid-like fibrils with a cross-beta structure. Thus, our data suggested that human pancreatitis-associated protein fibrillization is initiated by protein aggregation primarily because of electrostatic interactions, and the loop from residues 84 to 116 may play an important role in the formation of fibrillar aggregates with a native-like conformation.

Roles of N-terminal pyroglutamate in maintaining structural integrity and pKa values of catalytic histidine residues in bullfrog ribonuclease 3.

Many proteins and bioactive peptides contain an N-terminal pyroglutamate residue (Pyr1). This residue reduces the susceptibility of the protein to aminopeptidases and often has important functional roles. The antitumor ribonuclease RC-RNase 3 (RNase 3) from oocytes of Rana catesbeiana (bullfrog) is one such protein. We have produced recombinant RNase 3 containing the N-terminal Pyr1 (pRNase 3) and found it to be indistinguishable from the native RNase 3 by mass spectrometry and a variety of other biochemical and immunological criteria. We demonstrated by NMR analysis that the Pyr1 of pRNase 3 forms hydrogen bonds with Lys9 and Ile96 and stabilizes the N-terminal alpha-helix in a rigid conformation. In contrast, the N-terminal alpha-helix becomes flexible and the pKa values of the catalytic residues His10 and His97 altered when Pyr1 formation is blocked by an extra methionine at the N terminus in the recombinant mqRNase 3. Thus, our results provide a mechanistic explanation on the essential role of Pyr1 in maintaining the structural integrity, especially at the N-terminal alpha-helix, and in providing the proper environment for the ionization of His10 and His97 residues for catalysis and cytotoxicity against HeLa cells.

The structural integrity exerted by N-terminal pyroglutamate is crucial for the cytotoxicity of frog ribonuclease from Rana pipiens.

Onconase, a cytotoxic ribonuclease from Rana pipiens, possesses pyroglutamate (Pyr) at the N-terminus and has a substrate preference for uridine-guanine (UG). To identify residues responsible for onconase's cytotoxicity, we cloned the rpr gene from genomic DNA and expressed it in Escherichia coli BL21(DE3). The recombinant onconase with Met at the N-terminus had reduced thermostability, catalytic activity and antigenicity. Therefore, we developed two methods to produce onconase without Met. One relied on the endogeneous E.coli methionine aminopeptidase and the other relied on the cleavage of a pelB signal peptide. The Pyr1 substitutional variants maintained similar secondary structures to wild-type onconase, but with less thermostability and specific catalytic activity for the innate substrate UG. However, the non-specific catalytic activity for total RNAs varied depending on the relaxation of base specificity. Pyr1 promoted the structural integrity by forming a hydrogen bond network through Lys9 in alpha1 and Val96 in beta6, and participated in catalytic activity by hydrogen bonds to Lys9 and P(1) catalytic phosphate. Residues Thr35 and Asp67 determined B(1) base specificity, and Glu91 determined B(2) base specificity. The cytotoxicity of onconase is largely determined by structural integrity and specific catalytic activity for UG through Pyr1, rather than non-specific activity for total RNAs.

Solution structure of the cytotoxic RNase 4 from oocytes of bullfrog Rana catesbeiana.

Cytotoxic ribonucleases with antitumor activity are mainly found in the oocytes and early embryos of frogs. Native RC-RNase 4 (RNase 4), consisting of 106 residues linked with four disulfide bridges, is a cytotoxic ribonuclease isolated from oocytes of bullfrog Rana catesbeiana. RNase 4 belongs to the bovine pancreatic ribonuclease (RNase A) superfamily. Recombinant RC-RNase 4 (rRNase 4), which contains an additional Met residue and glutamine instead of pyroglutamate at the N terminus, was found to possess less catalytic and cytotoxic activities than RNase 4. Equilibrium thermal and guanidine-HCl denaturation CD measurements revealed that RNase 4 is more thermally and chemically stable than rRNase 4. However, CD and NMR data showed that there is no gross conformational change between native and recombinant RNase 4. The NMR solution structure of rRNase 4 was determined to comprise three alpha-helices and two sets of antiparallel beta-sheets. Superimposition of each structure with the mean structure yielded an average root mean square deviation (RMSD) of 0.72(+/-0.14)A for the backbone atoms, and 1.42(+/-0.19)A for the heavy atoms in residues 3-105. A comparison of the 3D structure of rRNase 4 with the structurally and functionally related cytotoxic ribonuclease, onconase (ONC), showed that the two H-bonds in the N-terminal pyroglutamate of ONC were not present at the corresponding glutamine residue of rRNase 4. We suggest that the loss of these two H-bonds is one of the key factors responsible for the reductions of the conformational stability, catalytic and cytotoxic activities in rRNase 4. Furthermore, the differences of side-chain conformations of subsite residues among RNase A, ONC and rRNase 4 are related to their distinct catalytic activities and base preferences.

Structural characterizations of fusion peptide analogs of influenza virus hemagglutinin. Implication of the necessity of a helix-hinge-helix motif in fusion activity.

Infection by enveloped viruses initially involves membrane fusion between viral and host cell membranes. The fusion peptide plays a crucial role in triggering this reaction. To clarify how the fusion peptide exerts this specific function, we carried out biophysical studies of three fusion peptide analogs of influenza virus hemagglutinin HA2, namely E5, G13L, and L17A. E5 exhibits an activity similar to the native fusion peptide, whereas G13L and L17A, which are two point mutants of the E5 analog, possess much less fusion activity. Our CD data showed that the conformations of these three analogs in SDS micelles are pH-dependent, with higher alpha-helical contents at acidic pH. Tryptophan fluorescence emission experiments indicated that these three analogs insert deeper into lipid bilayers at acidic pH. The three-dimensional structure of the E5 analog in SDS micelles at pH 4.0 revealed that two segments, Leu(2)-Glu(11) and Trp(14)-Ile(18), form amphipathic helical conformations, with Gly(12)-Gly(13) forming a hinge. The hydrophobic residues in the N- and C-terminal helices form a hydrophobic cluster. At neutral pH, however, the C-terminal helix of Trp(14)-Ile(18) reduces dramatically, and the hydrophobic core observed at acidic pH is severely disrupted. We suggest that the disruption of the C-terminal helix renders the E5 analog fusion-inactive at neutral pH. Furthermore, the decrease of the hinge and the reduction of fusion activity in G13L reveal the importance of the hinge in fusion activity. Also, the decrease in the C-terminal helix and the reduction of fusion activity in L17A demonstrates the importance of the C-terminal helix in fusion activity. Based on these biophysical studies, we propose a model that illustrates the structural change of the HA2 fusion peptide analog and explains how the analog interacts with the lipid bilayer at different pH values.

Solution structure of a K(+)-channel blocker from the scorpion Tityus cambridgei.

A new K(+)-channel blocking peptide identified from the scorpion venom of Tityus cambridgei (Tc1) is composed of 23 amino acid residues linked with three disulfide bridges. Tc1 is the shortest known toxin from scorpion venom that recognizes the Shaker B K(+) channels and the voltage-dependent K(+) channels in the brain. Synthetic Tc1 was produced using solid-phase synthesis, and its activity was found to be the same as that of native Tc1. The pairings of three disulfide bridges in the synthetic Tc1 were identified by NMR experiments. The NMR solution structures of Tc1 were determined by simulated annealing and energy-minimization calculations using the X-PLOR program. The results showed that Tc1 contains an alpha-helix and a 3(10)-helix at N-terminal Gly(4)-Lys(10) and a double-stranded beta-sheet at Gly(13)-Ile(16) and Arg(19)-Tyr(23), with a type I' beta-turn at Asn(17)-Gly(18). Superposition of each structure with the best structure yielded an average root mean square deviation of 0.26 +/- 0.05 A for the backbone atoms and of 1.40 +/- 0.23 A for heavy atoms in residues 2 to 23. The three-dimensional structure of Tc1 was compared with two structurally and functionally related scorpion toxins, charybdotoxin (ChTx) and noxiustoxin (NTx). We concluded that the C-terminal structure is the most important region for the blocking activity of voltage-gated (Kv-type) channels for scorpion K(+)-channel blockers. We also found that some of the residues in the larger scorpion K(+)-channel blockers (31 to 40 amino acids) are not involved in K(+)-channel blocking activity.