Chinpan Chen

Structural and DNA-binding studies on the bovine antimicrobial peptide, indolicidin: evidence for multiple conformations involved in binding to membranes and DNA

Indolicidin, a l3-residue antimicrobial peptide-amide, which is unusually rich in tryptophan and proline, is isolated from the cytoplasmic granules of bovine neutrophils. In this study, the structures of indolicidin in 50% D3-trifluoroethanol and in the absence and presence of SDS and D38-dodecylphosphocholine were determined using NMR spectroscopy. Multiple conformations were found and were shown to be due to different combinations of contact between the two WPW motifs. Although indolicidin is bactericidal and able to permeabilize bacterial membranes, it does not lead to cell wall lysis, showing that there is more than one mechanism of antimicrobial action. The structure of indolicidin in aqueous solution was a globular and amphipathic conformation, differing from the wedge shape adopted in lipid micelles, and these two structures were predicted to have different functions. Indolicidin, which is known to inhibit DNA synthesis and induce filamentation of bacteria, was shown to bind DNA in gel retardation and fluorescence quenching experiments. Further investigations using surface plasmon resonance confirmed the DNA-binding ability and showed the sequence preference of indolicidin. Based on our biophysical studies and previous results, we present a diagram illustrating the DNA-binding mechanism of the antimicrobial action of indolicidin and explaining the roles of the peptide when interacting with lipid bilayers at different concentrations.

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 °C. The bacterial membrane became permeable to the DNA-binding dye SYTOX® Green in only a few minutes after bactericidal RNase treatment. NMR studies showed that the 22 positively charged residues (Lys18 and Arg4) are distributed into three clusters on the surface of hRNase7. The first cluster, K1,K3,K111,K112, was located at the flexible coil near the N terminus, whereas the other two, K32,K35 and K96,R97,K100, were located on rigid secondary structures. Mutagenesis studies showed that the flexible cluster K1,K3,K111,K112, rather than the catalytic residues His15, Lys38, and His123 or other clusters such as K32,K35 and K96,R97,K100, 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 K1,K3,K111,K112. The conformation of hRNase7 can be adapted for pore formation or disruption of bacterial membrane even at 4 °C.

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.

Multinuclear NMR resonance assignments and the secondary structure of Escherichia coli thioesterase/protease I: A member of a new subclass of lipolytic enzymes

Escherichia coli thioesterase/protease I is a 183 amino acid protein with a molecular mass of 20500. This protein belongs to a new subclass of lipolytic enzymes of the serine protease superfamily, but with a new GDSLS consensus motif, of which no structure has yet been determined. The protein forms a tetramer at pH values above 6.5 and exists as a monomer at lower pH values. Both monomer and tetramer are catalytically active. From analysis of a set of heteronuclear multidimensional NMR spectra with uniform and specific amino acid labeled protein samples, we have obtained near-complete resonance assignments of the backbone 1H,13 C and 15N nuclei (BMRB databank accession number 4060). The secondary structure of E. coli thioesterase/protease I was further deduced from the consensus chemical shift indices, backbone short- and medium-range NOEs, and amide proton exchange rates. The protein was found to consist of four β-strands and seven α-helices, arranged in alternate order. The four β-strands were shown to form a parallel β-sheet. The topological arrangement of the β-strands of -1x, +2x, +1x appears to resemble that of the core region of the αβ hydrolase superfamily, typically found in common lipases and esterases. However, substantial differences, such as the number of β-strands and the location of the catalytic triad residues, make it difficult to give a definitive classification of the structure of E. coli thioesterase/protease I at present.

Oligomerization Is Crucial for the Stability and Function of Heme Oxygenase-1 in the Endoplasmic Reticulum

Heme oxygenase-1 (HO-1), a stress-inducible enzyme anchored in the endoplasmic reticulum (ER) by a single transmembrane segment (TMS) located at the C terminus, interacts with NADPH cytochrome P450 reductase and biliverdin reductase to catalyze heme degradation to biliverdin and its metabolite, bilirubin. Previous studies suggested that HO-1 functions as a monomer. Using chemical cross-linking, co-immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments, here we showed that HO-1 forms dimers/oligomers in the ER. However, oligomerization was not observed with a truncated HO-1 lacking the C-terminal TMS (amino acids 266–285), which exhibited cytosolic and nuclear localization, indicating that the TMS is essential for the self-assembly of HO-1 in the ER. To identify the interface involved in the TMS-TMS interaction, residue Trp-270, predicted by molecular modeling as a potential interfacial residue of TMS α-helices, was mutated, and the effects on protein subcellular localization and activity assessed. The results showed that the W270A mutant was present exclusively in the ER and formed oligomers with similar activity to those of the wild type HO-1. Interestingly, the W270N mutant was localized not only in the ER, but also in the cytosol and nucleus, suggesting it is susceptible to proteolytic cleavage. Moreover, the microsomal HO activity of the W270N mutant was significantly lower than that of the wild type. The W270N mutation appears to interfere with the oligomeric state, as revealed by a lower FRET efficiency. Collectively, these data suggest that oligomerization, driven by TMS-TMS interactions, is crucial for the stabilization and function of HO-1 in the ER.

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.

NMR structural analysis of DNA recognition by a novel Myb1 DNA-binding domain in the protozoan parasite Trichomonas vaginalis

The transcription regulator, tvMyb1, is the first Myb family protein identified in Trichomonas vaginalis. Using an electrophoretic mobility shift assay, we defined the amino-acid sequence from Lys35 to Ser141 (tvMyb135–141) as the minimal DNA-binding domain, encompassing two Myb-like DNA-binding motifs (designated as R2 and R3 motifs) and an extension of 10 residues at the C-terminus. NMR solution structures of tvMyb135–141 show that both the R2 and R3 motifs adopt helix-turn-helix conformations while helix 6 in the R3 motif is longer than its counterpart in vertebrate Myb proteins. The extension of helix 6 was then shown to play an important role in protein stability as well as in DNA-binding activity. The structural basis for the tvMyb135–141/DNA interaction was investigated using chemical shift perturbations, residual dipolar couplings, DNA specificity data and data-driven macromolecular docking by HADDOCK. Our data indicate that the orientation between R2 and R3 motifs dramatically changes upon binding to DNA so as to recognize the DNA major groove through a number of key contacts involving residues in helices 3 and 6. The tvMyb135–141/DNA complex model furthers our understanding of DNA recognition by Myb proteins and this approach could be applied in determining the complex structures involving proteins with multiple domains.

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-β-sheet structure, these aggregates have a very similar secondary structure to that of the native protein, which is composed of two α-helices and eight β-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 Thr84 to Ser116, which, very interestingly, was found to form amyloid-like fibrils with a cross-β 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.

Novel c-di-GMP recognition modes of the mouse innate immune adaptor protein STING.

The mammalian ER protein STING (stimulator of interferon genes; also known as MITA, ERIS, MPYS or TMEM173) is an adaptor protein that links the detection of cytosolic dsDNA to the activation of TANK-binding kinase 1 (TBK1) and its downstream transcription factor interferon regulatory factor 3 (IFN3). Recently, STING itself has been found to be the direct receptor of bacterial c-di-GMP, and crystal structures of several human STING C-terminal domain (STING-CTD) dimers in the apo form or in complex with c-di-GMP have been published. Here, a novel set of structures of mouse STING-CTD (mSTING(137-344)) in apo and complex forms determined from crystals obtained under different crystallization conditions are reported. These novel closed-form structures exhibited considerable differences from previously reported open-form human STING-CTD structures. The novel mSTING structures feature extensive interactions between the two monomers, a unique asymmetric c-di-GMP molecule with one guanine base in an unusual syn conformation that is well accommodated in the dimeric interface with many direct specific interactions and two unexpected equivalent secondary peripheral c-di-GMP binding sites. Replacement of the amino acids crucial for specific c-di-GMP binding in mSTING significantly changes the ITC titration profiles and reduces the IFN-β reporter luciferase activity. Taken together, these results reveal a more stable c-di-GMP binding mode of STING proteins that could serve as a template for rational drug design to stimulate interferon production by mammalian cells.

NMR solution structure of KP-TerB, a tellurite-resistance protein from Klebsiella pneumoniae.

Klebsiella pneumoniae (KP), a Gram‐negative bacterium, is a common cause of hospital‐acquired bacterial infections worldwide. Tellurium (Te) compounds, although relatively rare in the environment, have a long history as antimicrobial and therapeutic agents. In bacteria, tellurite (TeO3 −2) resistance is conferred by the ter (Ter) operon (terZABCDEF). Here, on the basis of 2593 restraints derived from NMR analysis, we report the NMR structure of TerB protein (151 amino acids) of KP (KP‐TerB), which is mainly composed of seven α‐helices and a 310 helix, with helices II to V apparently forming a four‐helix bundle. The ensemble of 20 NMR structures was well‐defined, with a RMSD of 0.32 ± 0.06 Å for backbone atoms and 1.11 ± 0.07 Å for heavy atoms, respectively. A unique property of the KP‐TerB structure is that the positively and negatively charged clusters are formed by the N‐terminal positively and C‐terminal negatively charged residues, respectively. To the best of our knowledge, the protein sequence and structures of KP‐TerB are unique.