Structural Properties of CXCL4L1 and Its Recognition of the CXCR3 N-Terminus.

Chemokine CXCL4L1, a homologue of CXCL4, is a more potent antiangiogenic ligand. Its structural property is correlated with the downstream receptor binding. The two chemokines execute their functions by binding the receptors of CXCR3A and CXCR3B. The receptors differ by an extra 51-residue extension in the CXCR3B N-terminus. To understand the binding specificity, a GB1 protein scaffold was used to carry different CXCR3 extracellular elements, and artificial CXCL4 and CXCL4L1 monomers were engineered for the binding assay. We first characterized the molten globule property of CXCL4L1. The structural property causes the CXCL4L1 tetramer to dissociate into monomers in low concentrations, but native CXCL4 adopts a stable tetramer structure in solution. In the titration experiments, the combination of the CXCR3A N-terminus and receptor extracellular loop 2 provided moderate and comparable binding affinities to CXCL4 and CXCL4L1, while sulfation on the CXCR3A N-terminal tyrosine residues provided binding specificity. However, the CXCR3B N-terminal extension did not show significant enhancement in the binding of CXCL4 or CXCL4L1. This result indicates that the tendency to form a chemokine monomer and the binding affinity together contribute the high antiangiogenic activity of CXCL4L1.

High Level Expression and Purification of Cecropin-like Antimicrobial Peptides in Escherichia coli.

Cecropins are a family of antimicrobial peptides (AMPs) that are widely found in the innate immune system of Cecropia moths. Cecropins exhibit a broad spectrum of antimicrobial and anticancer activities. The structures of Cecropins are composed of 34-39 amino acids with an N-terminal amphipathic α-helix, an AGP hinge and a hydrophobic C-terminal α-helix. KR12AGPWR6 was designed based on the Cecropin-like structural feature. In addition to its antimicrobial activities, KR12AGPWR6 also possesses enhanced salt resistance, antiendotoxin and anticancer properties. Herein, we have developed a strategy to produce recombinant KR12AGPWR6 through a salt-sensitive, pH and temperature dependent intein self-cleavage system. The His6-Intein-KR12AGPWR6 was expressed by E. coli and KR12AGPWR6 was released by the self-cleavage of intein under optimized ionic strength, pH and temperature conditions. The molecular weight and structural feature of the recombinant KR12AGPWR6 was determined by MALDI-TOF mass, CD, and NMR spectroscopy. The recombinant KR12AGPWR6 exhibited similar antimicrobial activities compared to the chemically synthesized KR12AGPWR6. Our results provide a potential strategy to obtain large quantities of AMPs and this method is feasible and easy to scale up for commercial production.

Type IIb Heat Labile Enterotoxin B Subunit as a Mucosal Adjuvant to Enhance Protective Immunity against H5N1 Avian Influenza Viruses.

Human infections with highly pathogenic avian influenza H5N1 viruses persist as a major global health concern. Vaccination remains the primary protective strategy against H5N1 and other novel avian influenza virus infections. We investigated the use of E. coli type IIb heat labile enterotoxin B subunit (LTIIb-B5) as a mucosal adjuvant for intranasal immunizations with recombinant HA proteins against H5N1 avian influenza viruses. Use of LTIIb-B5 adjuvant elicited more potent IgG, IgA, and neutralizing antibody titers in both sera and bronchoalveolar lavage fluids, thus increasing protection against lethal virus challenges. LTIIb-B5 mucosal adjuvanticity was found to trigger stronger Th17 cellular response in spleen lymphocytes and cervical lymph nodes. Studies of anti-IL-17A monoclonal antibody depletion and IL-17A knockout mice also suggest the contribution from Th17 cellular response to anti-H5N1 protective immunity. Our results indicate a link between improved protection against H5N1 live virus challenges and increased Th17 response due to the use of LTIIb-B5 mucosal adjuvant with HA subunit proteins.

Anti-apoptotic BCL-2 regulation by changes in dynamics of its long unstructured loop.

BCL-2, a key protein in inhibiting apoptosis, has a 65-residue-long highly flexible loop domain (FLD) located on the opposite side of its ligand-binding groove. In vivo phosphorylation of the FLD enhances the affinity of BCL-2 for pro-apoptotic ligands, and consequently anti-apoptotic activity. However, it remains unknown as to how the faraway, unstructured FLD modulates the affinity. Here we investigate the protein-ligand interactions by fluorescence techniques and monitor protein dynamics by DEER and NMR spectroscopy tools. We show that phosphomimetic mutations on the FLD lead to a reduction in structural flexibility, hence promoting ligand access to the groove. The bound pro-apoptotic ligands can be displaced by the BCL-2-selective inhibitor ABT-199 efficiently, and thus released to trigger apoptosis. We show that changes in structural flexibility on an unstructured loop can activate an allosteric protein that is otherwise structurally inactive.

N-terminal Backbone Pairing Shifts in CCL5-12AAA14 Dimer Interface: Structural Significance of the FAY Sequence.

CC-type chemokine ligand 5 (CCL5) has been known to regulate immune responses by mediating the chemotaxis of leukocytes. Depending on the environment, CCL5 forms different orders of oligomers to interact with targets and create functional diversity. A recent CCL5 trimer structure revealed that the N-terminal conversed F12-A13-Y14 (12FAY14) sequence is involved in CCL5 aggregation. The CCL5-12AAA14 mutant with two mutations had a deficiency in the formation of high-order oligomers. In the study, we clarify the respective roles of F12 and Y14 through NMR analysis and structural determination of the CCL5-12AAA14 mutant where F12 is involved in the dimer assembly and Y14 is involved in aggregation. The CCL5-12AAA14 structure contains a unique dimer packing. The backbone pairing shifts for one-residue in the N-terminal interface, when compared to the native CCL5 dimer. This difference creates a new structural orientation and leads to the conclusion that F12 confines the native CCL5 dimer configuration. Without F12 anchoring in the position, the interfacial backbone pairing is permitted to slide. Structural plasticity occurs in the N-terminal interaction. This is the first case to report this structural rearrangement through mutagenesis. The study provides a new idea for chemokine engineering and complements the understanding of CCL5 oligomerization and the role of the 12FAY14 sequence.

Integrative Model to Coordinate the Oligomerization and Aggregation Mechanisms of CCL5.

CC-type chemokine ligand 5 (CCL5) is involved in the pathogenesis of many inflammatory conditions. Under physiological conditions, CCL5 oligomerization and aggregation are considered to be responsible for its inflammatory properties. The structural basis of CCL5 oligomerization remains controversial because the current oligomer models contain no consensus interactions. In this study, NMR and biophysical analyses proposed evidence that the CC-type CCL5 dimer acts as the basic unit to constitute the oligomer and that CCL5 oligomerizes alternatively through E66-K25 and E66-R44/K45 interactions. In addition, a newly determined trimer structure, constituted by CCL5 and the E66S mutant, reported an interfacial interaction through the N-terminal 12FAY14 sequence. The interaction contributes to CCL5 aggregation and precipitation but not to oligomerization. In accordance with the observations, an integrative model explains the CCL5 oligomerization and aggregation mechanism in which CCL5 assembly consists of two types of dimer-dimer interactions and one aggregation mechanism. For full-length CCL5, the molecular accumulation triggers oligomerization through the E66-K25 and E66-R44/K45 interactions, and the 12FAY14 interaction acts as a secondary effect to derive aggregation and precipitation. In contrast, the E66-R44/K45 interaction might dominate in CCL5 N-terminal truncations, and the interaction would lead to the filament-like formation in solution.

Salt-sensitive intein for large-scale polypeptide production.

In this chapter, we propose to use salt-sensitive intein as a fusion protein to promote polypeptide expression; the removal of intein from the target sequence requires no enzyme, only a buffer change. The method will be particularly helpful for large-scale polypeptide preparations. Intein is an enzyme that can perform N- and C-terminal self-cleavage. Upon introduction of a mutation to eliminate the N-terminal cleavage activity, the C-terminal cleavage function can still be preserved. This feature was used to develop intein as a fusion protein through conjugation with a given sequence to promote protein expression in a biosynthesis system. Fused intein could later be separated from the target sequence through a C-terminal self-cleavage reaction. Here, a type of salt-sensitive intein is characterized in which ionic strength becomes an effector to control the self-cleavage activity. Low salt concentrations favor the cleavage reaction. Thus, using salt-sensitive intein as a fusion protein simply requires a buffer change to activate the self-cleavage mechanism, which makes it an enzyme-free process. This process has many advantages, including low cost, no extra residue remaining after cleavage, feasibility for preparing proteins starting from a non-Met codon and a special benefit for producing isotope-labeled peptides.

Utilization of HEPES for Enhancing Protein Transfection into Mammalian Cells.

The delivery of active proteins into cells (protein transfection) for biological purposes offers considerable potential for clinical applications. Herein we demonstrate that, with a readily available, inexpensive organic agent, the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) method can be used for simple and efficient protein transfection. By mixing proteins with a pure HEPES solution before they are applied to live cells, proteins with various molecular weights (including antibodies, recombinant proteins, and peptides) were successfully delivered into the cytoplasm of different cell types. The protein transfection efficiency of the HEPES method was not inferior to that of commercially available systems that are both more expensive and time consuming. Studies using endocytotic inhibitors and endosomal markers have revealed that cells internalize HEPES-protein mixtures through endocytosis. Results that HEPES-protein mixtures exhibited a low diffusion coefficient suggest that HEPES might neutralize the charges of proteins and, thus, facilitate their cellular internalization. Upon internalization, the cytosolic antibodies caused the degradation of targeted proteins in TRIM21-expressing cells. In summary, the HEPES method is efficient for protein transfection and has potential for myriad clinical applications.

Understanding the Biomineralization Role of Magnetite-Interacting Components (MICs) From Magnetotactic Bacteria.

Biomineralization is a process that takes place in all domains of life and which usually helps organisms to harden soft tissues by creating inorganic structures that facilitate their biological functions. It was shown that biominerals are under tight biological control via proteins that are involved in nucleation initiation and/or which act as structural skeletons. Magnetotactic bacteria (MTB) use iron biomineralization to create nano-magnetic particles in a specialized organelle, the magnetosome, to align to the geomagnetic field. A specific set of magnetite-associated proteins (MAPs) is involved in regulating magnetite nucleation, size, and shape. These MAPs are all predicted to contain specific 17-22 residue-long sequences involved in magnetite formation. To understand the mechanism of magnetite formation, we focused on three different MAPs, MamC, Mms6 and Mms7, and studied the predicted iron-binding sequences. Using nuclear magnetic resonance (NMR), we differentiated the recognition mode of each MAP based on ion specificity, affinity, and binding residues. The significance of critical residues in each peptide was evaluated by mutation followed by an iron co-precipitation assay. Among the peptides, MamC showed weak ion binding but created the most significant effect in enhancing magnetite particle size, indicating the potency in controlling magnetite particle shape and size. Alternatively, Mms6 and Mms7 had strong binding affinities but less effect in modulating magnetite particle size, representing their major role potentially in initiating nucleation by increasing local metal concentration. Overall, our results explain how different MAPs affect magnetite synthesis, interact with Fe2+ ions and which residues are important for the MAPs functions.

Human CCL5 trimer: expression, purification and initial crystallographic studies.

The chemokine CCL5 is considered to be a potential therapeutic target because of its ability to recruit immune cells to inflammatory sites. CCL5 aggregates under physiological conditions, and high-order oligomer formation is considered to be significant for cell migration, immune-cell activation and HIV cell entry. The structure of the high-order oligomer is unknown and the mechanism by which the oligomer is derived has yet to be established. Here, a CCL5 mutant (CCL5-E66S) which is deficient in oligomer formation was mixed with native CCL5 to prepare a protein trimer. At an optimized ratio the trimeric CCL5 crystallized, and the crystal belonged to the tetragonal space group P41212, with unit-cell parameters a = 56.6, b = 56.6, c = 154.1 Å. The Matthews coefficient (VM) of the crystal is 2.58 Å3 Da-1 (three molecules in the asymmetric unit), with a solvent content of 52.32%. Diffraction data were collected to a resolution of 1.87 Šand the statistics indicated satisfactory data quality. The new structure will reveal the interfaces in the CCL5 oligomer, therefore assisting in understanding the mechanism of CCL5 oligomerization.

The role of CXCR3/LRP1 cross-talk in the invasion of primary brain tumors.

CXCR3 plays important roles in angiogenesis, inflammation, and cancer. However, the precise mechanism of regulation and activity in tumors is not well known. We focused on CXCR3-A conformation and on the mechanisms controlling its activity and trafficking and investigated the role of CXCR3/LRP1 cross talk in tumor cell invasion. Here we report that agonist stimulation induces an anisotropic response with conformational changes of CXCR3-A along its longitudinal axis. CXCR3-A is internalized via clathrin-coated vesicles and recycled by retrograde trafficking. We demonstrate that CXCR3-A interacts with LRP1. Silencing of LRP1 leads to an increase in the magnitude of ligand-induced conformational change with CXCR3-A focalized at the cell membrane, leading to a sustained receptor activity and an increase in tumor cell migration. This was validated in patient-derived glioma cells and patient samples. Our study defines LRP1 as a regulator of CXCR3, which may have important consequences for tumor biology.

Oligomerization State of CXCL4 Chemokines Regulates G Protein-Coupled Receptor Activation.

CXCL4 chemokines have antiangiogenic properties, mediated by different mechanisms, including CXCR3 receptor activation. Chemokines have distinct oligomerization states that are correlated with their biological functions. CXCL4 exists as a stable tetramer under physiological conditions. It is unclear whether the oligomerization state impacts CXCL4-receptor interaction. We found that the CXCL4 tetramer is sensitive to pH and salt concentration. Residues Glu28 and Lys50 were important for tetramer formation, and the first ß-strand and the C-terminal helix are critical for dimerization. By mutating the critical residues responsible for oligomerization, we generated CXCL4 mutants that behave as dimers or monomers under neutral/physiological conditions. The CXCL4 monomer acts as the minimal active unit for interacting CXCR3A, and sulfation of N-terminal tyrosine residues on the receptor is important for binding. Noticeably, CXCL4L1, a CXCL4 variant that differs by three residues in the C-terminal helix, could activate CXCR3A. CXCL4L1 showed a higher tendency to dissociate into monomers, but native CXCL4 did not. This result indicates that monomeric CXCL4 behaves like CXCL4L1. Thus, in this chemokine family, being in the monomeric state seems critical for interaction with CXCR3A.

Computational Prediction of New Intein Split Sites.

Split inteins have emerged as a powerful tool in protein engineering. We describe a reliable in silico method to predict viable split sites for the design of new split inteins. A computational circular permutation (CP) prediction method facilitates the search for internal permissive sites to create artificial circular permutants. In this procedure, the original amino- and carboxyl-termini are connected and new termini are created. The identified new terminal sites are promising candidates for the generation of new split sites with the backbone opening being tolerated by the structural scaffold. Here we show how to integrate the online usage of the CP predictor, CPred, in the search of new split intein sites.

Heparin-Promoted Cellular Uptake of the Cell-Penetrating Glycosaminoglycan Binding Peptide, GBPECP, Depends on a Single Tryptophan.

A 10-residue, glycosaminoglycan-binding peptide, GBPECP, derived from human eosinophil cationic protein has been recently designated as a potent cell-penetrating peptide. A model system containing peptide, glycan, and lipid was monitored by nuclear magnetic resonance (NMR) spectroscopy to determine the cell-penetrating mechanism. Heparin octasaccharide with dodecylphosphocholine (DPC) lipid micelle was titrated into the GBPECP solution. Our data revealed substantial roles for the charged residues Arg5 and Lys7 in recognizing heparin, whereas Arg3 had less effect. The aromatic residue Trp4 acted as an irreplaceable moiety for membrane insertion, as the replacement of Trp4 with Arg4 abolished cell penetration, although it significantly improved the heparin-binding ability. GBPECP bound either heparin or lipid in the presence or absence of the other ligand indicating that the peptide has two alternative binding sites: Trp4 is responsible for lipid insertion, and Arg5 and Lys7 are for GAG binding. We developed a molecular model showing that the two effects synergistically promote the penetration. The loss of either effect would abolish the penetration. GBPECP has been proven to enter cells through macropinocytosis. The GBPECP treatment inhibited A549 lung cancer cell migration and invasion, implying that the cellular microenvironment would be modulated by GBPECP internalization. The intracellular penetration of GBPECP leading to inhibition of epithelial cell migration and invasion depends on the presence of the tryptophan residue in its sequence compared with similar derivative peptides. Therefore, GBPECP shows substantial potential as a novel delivery therapeutic through rapid and effective internalization and interference with cell mobility.

Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer.

The CXCL4 paralog CXCL4L1 is a less studied chemokine that has been suggested to exert an antiangiogenic function. However, CXCL4L1 is also expressed in patient tumors, tumor cell lines, and murine xenografts, prompting a more detailed analysis of its role in cancer pathogenesis. We used genetic and antibody-based approaches to attenuate CXCL4L1 in models of pancreatic ductal adenocarcinoma (PDAC). Mechanisms of expression were assessed in cell coculture experiments, murine, and avian xenotransplants, including through an evaluation of CpG methylation and mutation of critical CpG residues. CXCL4L1 gene expression was increased greatly in primary and metastatic PDAC. We found that myofibroblasts triggered cues in the tumor microenvironment, which led to induction of CXCL4L1 in tumor cells. CXCL4L1 expression was also controlled by epigenetic modifications at critical CpG islands, which were mapped. CXCL4L1 inhibited angiogenesis but also affected tumor development more directly, depending on the tumor cell type. In vivo administration of an mAb against CXCL4L1 demonstrated a blockade in the growth of tumors positive for CXCR3, a critical receptor for CXCL4 ligands. Our findings define a protumorigenic role in PDAC development for endogenous CXCL4L1, which is independent of its antiangiogenic function. Cancer Res; 76(22); 6507-19. ©2016 AACR.

NMR Study Reveals the Receiver Domain of Arabidopsis ETHYLENE RESPONSE1 Ethylene Receptor as an Atypical Type Response Regulator.

The gaseous plant hormone ethylene, recognized by plant ethylene receptors, plays a pivotal role in various aspects of plant growth and development. ETHYLENE RESPONSE1 (ETR1) is an ethylene receptor isolated from Arabidopsis and has a structure characteristic of prokaryotic two-component histidine kinase (HK) and receiver domain (RD), where the RD structurally resembles bacteria response regulators (RRs). The ETR1 HK domain has autophosphorylation activity, and little is known if the HK can transfer the phosphoryl group to the RD for receptor signaling. Unveiling the correlation of the receptor structure and phosphorylation status would advance the studies towards the underlying mechanisms of ETR1 receptor signaling. In this study, using the nuclear magnetic resonance technique, our data suggested that the ETR1-RD is monomeric in solution and the rigid structure of the RD prevents the conserved aspartate residue phosphorylation. Comparing the backbone dynamics with other RRs, we propose that backbone flexibility is critical to the RR phosphorylation. Besides the limited flexibility, ETR1-RD has a unique γ loop conformation of opposite orientation, which makes ETR1-RD unfavorable for phosphorylation. These two features explain why ETR1-RD cannot be phosphorylated and is classified as an atypical type RR. As a control, phosphorylation of the ETR1-RD was also impaired when the sequence was swapped to the fragment of the bacterial typical type RR, CheY. Here, we suggest a molecule insight that the ETR1-RD already exists as an active formation and executes its function through binding with the downstream factors without phosphorylation.

Stress-induced phosphoprotein-1 maintains the stability of JAK2 in cancer cells.

Overexpression of stress-induced phosphoprotein 1 (STIP1) - a co-chaperone of heat shock protein (HSP) 70/HSP90 - and activation of the JAK2-STAT3 pathway occur in several tumors. Combined treatment with a HSP90 inhibitor and a JAK2 inhibitor exert synergistic anti-cancer effects. Here, we show that STIP1 stabilizes JAK2 protein in ovarian and endometrial cancer cells. Knock-down of endogenous STIP1 decreased JAK2 and phospho-STAT3 protein levels. The N-terminal fragment of STIP1 interacts with the N-terminus of JAK2, whereas the C-terminal DP2 domain of STIP1 mediates the interaction with HSP90 and STAT3. A peptide fragment in the DP2 domain of STIP1 (peptide 520) disrupted the interaction between STIP1 and HSP90 and induced cell death through JAK2 suppression. In an animal model, treatment with peptide 520 inhibited tumor growth. In summary, STIP1 modulates the function of the HSP90-JAK2-STAT3 complex. Peptide 520 may have therapeutic potential in the treatment of JAK2-overexpressing tumors.

Structure and function of chicken interleukin-1 beta mutants: uncoupling of receptor binding and in vivo biological activity.

Receptor-binding and subsequent signal-activation of interleukin-1 beta (IL-1ß) are essential to immune and proinflammatory responses. We mutated 12 residues to identify sites important for biological activity and/or receptor binding. Four of these mutants with mutations in loop 9 (T117A, E118K, E118A, E118R) displayed significantly reduced biological activity. Neither T117A nor E118K mutants substantially affected receptor binding, whereas both mutants lack the IL-1ß signaling in vitro but can antagonize wild-type (WT) IL-1ß. Crystal structures of T117A, E118A, and E118K revealed that the secondary structure or surface charge of loop 9 is dramatically altered compared with that of wild-type chicken IL-1ß. Molecular dynamics simulations of IL-1ß bound to its receptor (IL-1RI) and receptor accessory protein (IL-1RAcP) revealed that loop 9 lies in a pocket that is formed at the IL-1RI/IL-1RAcP interface. This pocket is also observed in the human ternary structure. The conformations of above mutants in loop 9 may disrupt structural packing and therefore the stability in a chicken IL-1ß/IL-1RI/IL-1RAcP signaling complex. We identify the hot spots in IL-1ß that are essential to immune responses and elucidate a mechanism by which IL-1ß activity can be inhibited. These findings should aid in the development of new therapeutics that neutralize IL-1 activity.

The First Residue of the PWWP Motif Modulates HATH Domain Binding, Stability, and Protein-Protein Interaction.

Hepatoma-derived growth factor (hHDGF) and HDGF-related proteins (HRPs) contain conserved N-terminal HATH domains with a characteristic structural motif, namely the PWWP motif. The HATH domain has attracted attention because of its ability to bind with heparin/heparan sulfate, DNA, and methylated histone peptide. Depending on the sequence of the PWWP motif, HRP HATHs are classified into P-type (Pro-His-Trp-Pro) and A-type (Ala-His-Trp-Pro) forms. A-type HATH is highly unstable and tends to precipitate in solution. We replaced the Pro residue in P-type HATHHDGF with Ala and evaluated the influence on structure, dynamics, and ligand binding. Nuclear magnetic resonance (NMR) hydrogen/deuterium exchange and circular dichroism (CD) measurements revealed reduced stability. Analysis of NMR backbone (15)N relaxations (R1, R2, and nuclear Overhauser effect) revealed additional backbone dynamics in the interface between the ß-barrel and the C-terminal helix bundle. The ß1-ß2 loop, where the AHWP sequence is located, has great structural flexibility, which aids HATH-HATH interaction through the loop. A-type HATH, therefore, shows a stronger tendency to aggregate when binding with heparin and DNA oligomers. This study defines the role of the first residue of the PWWP motif in modulating HATH domain stability and oligomer formation in binding.

(13)C, (15)N and (1)H resonance assignments of receiver domain of ethylene receptor ETR1.

Ethylene plays versatile functions in regulating plant physiology. Although the high affinity ethylene receptor and its downstream regulators have been identified, the molecular recognition of the receptor interacting domains remains to be established. It has been speculated that the cytoplasmic signaling of the ethylene receptor is a two-component regulatory system involving the conserved receiver domain (RD). Here, we report the NMR chemical shift assignments for RD from Arabidopsis thaliana ethylene receptor ETR1. Nearly complete backbone and side-chain assignments were achieved at pH 6.0 and 25 °C. The assignments and backbone dynamics revealed the secondary structure and showed that ETR1-RD is a monomer in solution. These results will make it possible to monitor downstream binding partners and elucidates our understanding of phosphotransfer in the plant two-component regulatory system in the ethylene signaling pathway.