C2A Domain Research Articles

Structural insight into host plasma membrane association and assembly of HIV-1 matrix protein

Oligomerization of Pr55Gag is a critical step of the late stage of the HIV life cycle. It has been known that the binding of IP6, an abundant endogenous cyclitol molecule at the MA domain, has been linked to the oligomerization of Pr55Gag. However, the exact binding site of IP6 on MA remains unknown and the structural details of this interaction are missing. Here, we present three high-resolution crystal structures of the MA domain in complex with IP6 molecules to reveal its binding mode. Additionally, extensive Differential Scanning Fluorimetry analysis combined with cryo- and ambient-temperature X-ray crystallography and GNM-based transfer entropy calculations identify the key residues that participate in IP6 binding. Our data provide novel insights about the multilayered HIV-1 virion assembly process that involves the interplay of IP6 with PIP2, a phosphoinositide essential for the binding of Pr55Gag to membrane. IP6 and PIP2 have neighboring alternate binding sites within the same highly basic region (residues 18–33). This indicates that IP6 and PIP2 bindings are not mutually exclusive and may play a key role in coordinating virion particles’ membrane localization. Based on our three different IP6-MA complex crystal structures, we propose a new model that involves IP6 coordination of the oligomerization of outer MA and inner CA domain’s 2D layers during assembly and budding.

Protein Conjugation by Electrophilic Alkynylation Using 5-(Alkynyl)dibenzothiophenium Triflates.

5-(Alkynyl)dibenzothiophenium triflates are introduced as new reagents to prepare different protein conjugates through site-selective cysteine alkynylation. The protocol developed allows a highly efficient label of free cysteine-containing proteins with relevant biological roles, such as ubiquitin, the C2A domain of Synaptotagmin-I, or HER2 targeting nanobodies. An electrophilic bis-alkynylating reagent was also designed. The second alkynylating handle thus introduced in the desired protein enables access to protein-thiol, protein-peptide, and protein-protein conjugates, and even diubiquitin dimers can be prepared through this approach. The low excess of reagent needed, mild reaction conditions used, short reaction times, and stability of the S-C(alkyne) bonds at physiological conditions make this approach an interesting addition to the toolbox of classical, site-selective cysteine-conjugation methods.

Co-ordinated control of the Aurora B abscission checkpoint by PKCε complex assembly, midbody recruitment and retention.

A requirement for PKCε in exiting from the Aurora B dependent abscission checkpoint is associated with events at the midbody, however, the recruitment, retention and action of PKCε in this compartment are poorly understood. Here, the prerequisite for 14-3-3 complex assembly in this pathway is directly linked to the phosphorylation of Aurora B S227 at the midbody. However, while essential for PKCε control of Aurora B, 14-3-3 association is shown to be unnecessary for the activity-dependent enrichment of PKCε at the midbody. This localisation is demonstrated to be an autonomous property of the inactive PKCε D532N mutant, consistent with activity-dependent dissociation. The C1A and C1B domains are necessary for this localisation, while the C2 domain and inter-C1 domain (IC1D) are necessary for retention at the midbody. Furthermore, it is shown that while the IC1D mutant retains 14-3-3 complex proficiency, it does not support Aurora B phosphorylation, nor rescues division failure observed with knockdown of endogenous PKCε. It is concluded that the concerted action of multiple independent events facilitates PKCε phosphorylation of Aurora B at the midbody to control exit from the abscission checkpoint.

The C2 domain of calpain 5 contributes to enzyme activation and membrane localization

The enzymatic characteristics of the ubiquitous calpain 5 (CAPN5) remain undescribed despite its high expression in the central nervous system and links to eye development and disease. CAPN5 contains the typical protease core domains but lacks the C terminal penta-EF hand domain of classical calpains, and instead contains a putative C2 domain. This study used the SH-SY5Y neuroblastoma cell line stably transfected with CAPN5-3xFLAG variants to assess the potential roles of the CAPN5 C2 domain in Ca2+ regulated enzyme activity and intracellular localization. Calcium dependent autoproteolysis of CAPN5 was documented and characterized. Mutation of the catalytic Cys81 to Ala or addition of EGTA prevented autolysis. Eighty μM Ca2+ was sufficient to stimulate half-maximal CAPN5 autolysis in cellular lysates. CAPN5 autolysis was inhibited by tri-leucine peptidyl aldehydes, but less effectively by di-Leu aldehydes, consistent with a more open conformation of the protease core relative to classical calpains. In silico modeling revealed a type II topology C2 domain including loops with the potential to bind calcium. Mutation of the acidic amino acid residues predicted to participate in Ca2+ binding, particularly Asp531 and Asp589, resulted in a decrease of CAPN5 membrane association. These residues were also found to be invariant in several genomes. The autolytic fragment of CAPN5 was prevalent in membrane-enriched fractions, but not in cytosolic fractions, suggesting that membrane association facilitates the autoproteolytic activity of CAPN5. Together, these results demonstrate that CAPN5 undergoes Ca2+-activated autoproteolytic processing and suggest that CAPN5 association with membranes enhances CAPN5 autolysis.

Structure Prediction and Molecular Phylogenetic Analysis of Membrane Interactions in Synaptotagmin-Like Proteins

Membrane trafficking and exocytosis are evolutionarily conserved throughout eukaryotes and involve many interactions between proteins and membrane surfaces. In vertebrates, synaptotagmin-like proteins (Slp) play key roles in trafficking of large dense-core secretory vesicles. Slp family proteins promote secretory vesicle docking to the plasma membrane through their N-terminal Slp homology domains, which bind to vesicular Rab proteins, and their tandem C-terminal C2 domains (C2A and C2B), which bind plasma membrane lipids independently of calcium. Our previous experimental and computational studies showed that the C2A domain of Slp-4 (also called granuphilin) binds with high affinity to membrane surfaces containing phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylserine (PS). A conserved PIP2 binding site centered on three lysine residues in the beta3-beta4 region specifically anchors the domain to PIP2 lipids, while many other basic residues surrounding the Lys-cluster enhance interactions with PS. We hypothesize that the positive electrostatic potential of the membrane binding surface is a key determinant of its membrane affinity and function, and therefore the net charge on the protein surface is likely to be evolutionarily conserved. To test this hypothesis, the C2A domain sequences for Slp-4 and Slp-2 in vertebrates were compared to assess the evolution of their respective polybasic surfaces. Using a molecular phylogenetic approach, clades were identified and the net charges of individual and consensus sequences were calculated. Structural homology models were generated using a crystal structure of Slp-4 C2A, and Poisson-Boltzmann calculations were carried out to quantify the positive electrostatic surfaces. This approach may help characterize electrostatic interactions with charged membrane surfaces and identify conserved differences that govern affinity and function of membrane-binding proteins.

Calcium binds and rigidifies the dysferlin C2A domain in a tightly coupled manner.

The membrane protein dysferlin (DYSF) is important for calcium-activated plasma membrane repair, especially in muscle fibre cells. Nearly 600 mutations in the DYSF gene have been identified that are causative for rare genetic forms of muscular dystrophy. The dysferlin protein consists of seven C2 domains (C2A-C2G, 13%-33% identity) used to recruit calcium ions and traffic accessory proteins and vesicles to injured membrane sites needed to reseal a wound. Amongst these, the C2A is the most prominent facilitating the calcium-sensitive interaction with membrane surfaces. In this work, we determined the calcium-free and calcium-bound structures of the dysferlin C2A domain using NMR spectroscopy and X-ray crystallography. We show that binding two calcium ions to this domain reduces the flexibility of the Ca2+-binding loops in the structure. Furthermore, calcium titration and mutagenesis experiments reveal the tight coupling of these calcium-binding sites whereby the elimination of one site abolishes calcium binding to its partner site. We propose that the electrostatic potential distributed by the flexible, negatively charged calcium-binding loops in the dysferlin C2A domain control first contact with calcium that promotes subsequent binding. Based on these results, we hypothesize that dysferlin uses a 'calcium-catching' mechanism to respond to calcium influx during membrane repair.

Timing of formation and cause of coloration of brown nephrite from the Tiantai Deposit, South Altyn Tagh, northwestern China

The world’s largest and economically valuable nephrite deposits are distributed along the Western Kunlun Orogen in northwestern China. During the past 20 years, extensive white and brown nephrite deposits have been discovered in both China and Russia, and several of these deposits have been found in South Altyn Tagh in the eastern part of the Western Kunlun Orogen. However, the timing of formation, genesis, petrographic characteristics, mineralogical composition, and reasons for the white–brown color of the nephrite remain unclear. We analyzed 50 samples of white, brown, and white–brown nephrite from the Tiantai nephrite deposit in Qiemo County with the aim of understanding the processes of formation, age, and diversity of these nephrites. Three main types of nephrite can be distinguished: white, brown, and dark brown. X-ray diffraction and petrographic studies indicate that the brown and dark-brown nephrite samples have fewer mineral inclusions compared with the white nephrites. The nephrite formed during early retrogression through the sequence of diopside skarn → coarse-grained tremolite skarn and coarse-grained tremolite → fine-grained tremolite. The higher bulk Fe content of the white nephrite (0.76 wt% FeO) relative to the brown nephrite (0.39 wt%), and the uniform, very low Fe2O3 contents of both of these color varieties (<0.15 wt%), suggest that the brown coloration is unrelated to Fe content and oxidation state. The white nephrite is characterized by a wide range of SiO2 contents (49.6–56.7 wt%), in contrast to the brown nephrites, which are characterized by nearly constant SiO2 contents (~58 wt%), close to the ideal content for tremolite. On average, the white nephrite is enriched in Al2O3 (3.22 wt%) and water (3.96 wt% loss on ignition) and depleted in CaO (12.35 wt%) relative to the brown nephrite (0.80 wt% Al2O3, 2.23 wt% loss on ignition, 12.95 wt% CaO). These characteristics, combined with greater transparency and higher Al and water contents in the chlorite formula relative to that of tremolite, as well as a near-absence of Ca in the chlorite formula, can explain the lower transparency and associated more intense coloration (i.e., yellow to brown) of the brown nephrite in terms of the lower modal abundance of chlorite. The spatial distribution of selected elements agrees with this hypothesis, whereby the brown-colored zones are dominated by Ca- and Sr-rich (substituting Ca) domains reflecting the highest modal volume of the tremolite. In contrast, the white zones are characterized by a substantially higher volumetric ratio of Al-rich domains, reflecting the higher chlorite content. Thus, the different coloration is not caused by specific elements, but by modal proportions of tremolite and chlorite. The slightly higher average Cr (10.1 ppm) and Ni (6.4 ppm) contents of brown nephrite, relative to white nephrite (8.7 ppm Cr and 5.5 ppm Ni), might also contribute to the color intensity and brown coloration. Ore-forming fluids involved in the formation of the studied nephrite have isotopic compositions of δ18O = 1.5‰ to 9.4‰ (330–430 °C) and δD = −87‰ to −50‰ (330–450 °C), suggesting that the formation of the Mg-skarn deposit was related to metasomatism of dolomite by fluids derived from local granite/granodiorite intrusions. Zircons from three brown–white samples yield concordant SHRIMP ages of 438 ± 14 Ma (2σ, MSWD=10.4), 916 ± 10 (2σ, MSWD = 1.5), 438 ± 9 Ma (2σ, MSWD = 5.3), and 431.1 ± 2.5 Ma (2σ, MSWD = 2.4). The younger ages (ca. 430 Ma) are close to those for placer nephrite from the Yurungkash and Karakash rivers, as well as those for Yecheng, Alamas, and Buya granodiorites, and probably represent the primary formation ages of nephrite within the Hetian Nephrite Belt. Overall, these ages indicate that the primary deposits formed during pre- or post-orogenic stages in the Western Kunlun Orogen. 40Ar/39Ar dating of hydrothermal muscovite and phlogopite intergrown with tremolite in the nephrite yielded ages of 418.8 ± 3.4 Ma (2σ, MSWD = 0.65), 379.5 ± 3.0 Ma (2σ, MSWD = 0.81), and 354.5 ± 3.6 Ma (2σ, MSWD = 0.71), which suggest multiple metasomatic activity. The similar formation ages of nephrites from Altyn Tagh (433 Ma) and from the previously studied areas of West Kunlun (441–378 Ma) and East Kunlun (416 Ma) indicate that these nephrites formed during the closure of the Proto-Tethys and in the accompanying post-collisional extensional environment.

Multivalent lipid targeting by the calcium-independent C2A domain of synaptotagmin-like protein 4/granuphilin

Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector responsible for docking secretory vesicles to the plasma membrane before exocytosis. Slp-4 binds vesicular Rab proteins via an N-terminal Slp homology domain, interacts with plasma membrane SNARE complex proteins via a central linker region, and contains tandem C-terminal C2 domains (C2A and C2B) with affinity for phosphatidylinositol-(4,5)-bisphosphate (PIP2). The Slp-4 C2A domain binds with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine but much weaker when either the background anionic lipids or PIP2 is removed. Through computational and experimental approaches, we show that this high-affinity membrane binding arises from concerted interaction at multiple sites on the C2A domain. In addition to a conserved PIP2-selective lysine cluster, a larger cationic surface surrounding the cluster contributes substantially to the affinity for physiologically relevant lipid compositions. Although the K398A mutation in the lysine cluster blocks PIP2 binding, this mutated protein domain retains the ability to bind physiological membranes in both a liposome-binding assay and MIN6 cells. Molecular dynamics simulations indicate several conformationally flexible loops that contribute to the nonspecific cationic surface. We also identify and characterize a covalently modified variant that arises through reactivity of the PIP2-binding lysine cluster with endogenous bacterial compounds and binds weakly to membranes. Overall, multivalent lipid binding by the Slp-4 C2A domain provides selective recognition and high-affinity docking of large dense core secretory vesicles to the plasma membrane.

Kinase inhibitors allosterically disrupt a regulatory interaction to enhance PKCα membrane translocation

The eukaryotic kinase domain has multiple intrinsically disordered regions whose conformation dictates kinase activity. Small molecule kinase inhibitors (SMKIs) rely on disrupting the active conformations of these disordered regions to inactivate the kinase. While SMKIs are selected for their ability to cause this disruption, the allosteric effects of conformational changes in disordered regions is limited by a lack of dynamic information provided by traditional structural techniques. In this study, we integrated multiscale molecular dynamics simulations with FRET sensors to characterize a novel allosteric mechanism that is selectively triggered by SMKI binding to the protein kinase Cα domain. The indole maleimide inhibitors BimI and sotrastaurin were found to displace the Gly-rich loop (G-loop) that normally shields the ATP-binding site. Displacement of the G-loop interferes with a newly identified, structurally conserved binding pocket for the C1a domain on the N lobe of the kinase domain. This binding pocket, in conjunction with the N-terminal regulatory sequence, masks a diacylglycerol (DAG) binding site on the C1a domain. SMKI-mediated displacement of the G-loop released C1a and exposed the DAG binding site, enhancing protein kinase Cα translocation both to synthetic lipid bilayers and to live cell membranes in the presence of DAG. Inhibitor chemotype determined the extent of the observed allosteric effects on the kinase domain and correlated with the extent of membrane recruitment. Our findings demonstrate the allosteric effects of SMKIs beyond the confines of kinase catalytic conformation and provide an integrated computational-experimental paradigm to investigate parallel mechanisms in other kinases.

Direct Evidence That Mutations within Dysferlin's C2A Domain Inhibit Lipid Clustering.

Mechanical stress on sarcolemma can create small tears in the muscle cell membrane. Within the sarcolemma resides the multidomain dysferlin protein. Mutations in this protein render it unable to repair the sarcolemma and have been linked to muscular dystrophy. A key step in dysferlin-regulated repair is the binding of the C2A domain to the lipid membrane upon increased intracellular calcium. Mutations mapped to this domain cause loss of binding ability of the C2A domain. There is a crucial need to understand the geometry of dysferlin C2A at a membrane interface as well as cell membrane lipid reorientation when compared to that of a mutant. Here, we describe a comparison between the wild-type dysferlin C2A and a mutation to the conserved aspartic acids in the domain binding loops. To identify both the geometry and the cell membrane lipid reorientation, we applied sum frequency generation (SFG) vibrational spectroscopy and coupled it with simulated SFG spectra to observe and quantify the interaction with a model cell membrane composed of phosphotidylserine and phosphotidylcholine. Observed changes in surface pressure demonstrate that calcium-bridged electrostatic interactions govern the initial interaction of the C2A domains docking with a lipid membrane. SFG spectra taken from the amide-I region for the wild type and variant contain features near 1642, 1663, and 1675 cm-1 related to the C2A domain β-sandwich secondary structure, indicating that the domain binds in a specific orientation. Mapping simulated SFG spectra to the experimentally collected spectra indicated that both wild-type and variant domains have nearly the same orientation to the membrane surface. However, examining the ordering of the lipids that make up a model membrane using SFG, we find that the wild type clusters the lipids as seen by the increase in the ratio of the CD3 and CD2 symmetric intensities by 170% for the wild type and by 120% for the variant. This study highlights the capabilities of SFG to probe with great detail biological mutations in proteins at cell membrane interfaces.

Inhibitory Monoclonal Antibodies and Their Recombinant Derivatives Targeting Surface-Exposed Carbonic Anhydrase XII on Cancer Cells

Monoclonal and recombinant antibodies are widely used for the diagnostics and therapy of cancer. They are generated to interact with cell surface proteins which are usually involved in the development and progression of cancer. Carbonic anhydrase XII (CA XII) contributes to the survival of tumors under hypoxic conditions thus is considered a candidate target for antibody-based therapy. In this study, we have generated a novel collection of monoclonal antibodies (MAbs) against the recombinant extracellular domain of CA XII produced in HEK-293 cells. Eighteen out of 24 MAbs were reactive with cellular CA XII on the surface of live kidney and lung cancer cells as determined by flow cytometry. One MAb 14D6 also inhibited the enzymatic activity of recombinant CA XII as measured by the stopped-flow assay. MAb 14D6 showed the migrastatic effect on human lung carcinoma A549 and renal carcinoma A498 cell lines in a ‘wound healing’ assay. It did not reduce the growth of multicellular lung and renal cancer spheroids but reduced the cell viability by the ATP Bioluminescence assay. Epitope mapping revealed the surface-exposed amino acid sequence (35-FGPDGENS-42) close to the catalytic center of CA XII recognized by the MAb 14D6. The variable regions of the heavy and light chains of MAb 14D6 were sequenced and their complementarity-determining regions were defined. The obtained variable sequences were used to generate recombinant antibodies in two formats: single-chain fragment variable (scFv) expressed in E. coli and scFv fused to human IgG1 Fc fragment (scFv-Fc) expressed in Chinese Hamster Ovary (CHO) cells. Both recombinant antibodies maintained the same specificity for CA XII as the parental MAb 14D6. The novel antibodies may represent promising tools for CA XII-related cancer research and immunotherapy.

18F-C2Am: a targeted imaging agent for detecting tumor cell death in vivo using positron emission tomography

IntroductionTrialing novel cancer therapies in the clinic would benefit from imaging agents that can detect early evidence of treatment response. The timing, extent and distribution of cell death in tumors following treatment can give an indication of outcome. We describe here an 18F-labeled derivative of a phosphatidylserine-binding protein, the C2A domain of Synaptotagmin-I (C2Am), for imaging tumor cell death in vivo using PET.MethodsA one-pot, two-step automated synthesis of N-(5-[18F]fluoropentyl)maleimide (60 min synthesis time, > 98% radiochemical purity) has been developed, which was used to label the single cysteine residue in C2Am within 30 min at room temperature. Binding of 18F-C2Am to apoptotic and necrotic tumor cells was assessed in vitro, and also in vivo, by dynamic PET and biodistribution measurements in mice bearing human tumor xenografts treated with a TRAILR2 agonist or with conventional chemotherapy. C2Am detection of tumor cell death was validated by correlation of probe binding with histological markers of cell death in tumor sections obtained immediately after imaging.Results18F-C2Am showed a favorable biodistribution profile, with predominantly renal clearance and minimal retention in spleen, liver, small intestine, bone and kidney, at 2 h following probe administration. 18F-C2Am generated tumor-to-muscle (T/m) ratios of 6.1 ± 2.1 and 10.7 ± 2.4 within 2 h of probe administration in colorectal and breast tumor models, respectively, following treatment with the TRAILR2 agonist. The levels of cell death (CC3 positivity) following treatment were 12.9–58.8% and 11.3–79.7% in the breast and colorectal xenografts, respectively. Overall, a 20% increase in CC3 positivity generated a one unit increase in the post/pre-treatment tumor contrast. Significant correlations were found between tracer uptake post-treatment, at 2 h post-probe administration, and histological markers of cell death (CC3: Pearson R = 0.733, P = 0.0005; TUNEL: Pearson R = 0.532, P = 0.023).ConclusionThe rapid clearance of 18F-C2Am from the blood pool and low kidney retention allowed the spatial distribution of cell death in a tumor to be imaged during the course of therapy, providing a rapid assessment of tumor treatment response. 18F-C2Am has the potential to be used in the clinic to assess early treatment response in tumors.

Functions of Bombyx mori cathepsin L-like in innate immune response and anti-microbial autophagy

Cathepsins belongs to the cysteine protease family, which are activated by an acidic environment. They play essential biological roles in the innate immunity and development of animals. Here, we identified a 62 kDa cathepsin L-like protease from the silkworm Bombyx mori. It contained putative conserved domains, including an I29 inhibitor domain and a peptidase C1A domain. The expression analysis revealed that cathepsin L-like was highly produced in the fat body, and 20-hydroxyecdysone (20 E) induced its expression. After challenge with three different types of heat-killed pathogens (Escherichia coli, Beauveria bassiana, and Bacillus cereus), the mRNA levels of cathepsin L-like significantly increased and displayed variable expression patterns in the immune tissues, suggesting its potential role in the innate immune response. The suppression of cathepsin L-like altered the expression of immune-related genes associated with the Toll and IMD pathway. Besides, autophagy-related genes such as Atg6, Atg8, VAMP2, Vps4, and syntaxin expression were also altered, indicating that cathepsin L-like regulates innate immunity and autophagy. Fluorescence microscopic analysis exhibited that cathepsin L-like was localized in the cytoplasm, and it was activated and dispersed throughout the cytoplasm and nucleus following the induction of anti-microbial autophagy. Altogether, our data suggest that cathepsin L-like may regulate the innate immune response and anti-microbial autophagy in the silkworm, B. mori.

Synaptotagmin-1 membrane binding is driven by the C2B domain and assisted cooperatively by the C2A domain

Synaptotagmin interaction with anionic lipid (phosphatidylserine/phosphatidylinositol) containing membranes, both in the absence and presence of calcium ions (Ca2+), is critical to its central role in orchestrating neurotransmitter release. The molecular surfaces involved, namely the conserved polylysine motif in the C2B domain and Ca2+-binding aliphatic loops on both C2A and C2B domains, are known. Here we use surface force apparatus combined with systematic mutational analysis of the functional surfaces to directly measure Syt1-membrane interaction and fully map the site-binding energetics of Syt1 both in the absence and presence of Ca2+. By correlating energetics data with the molecular rearrangements measured during confinement, we find that both C2 domains cooperate in membrane binding, with the C2B domain functioning as the main energetic driver, and the C2A domain acting as a facilitator.

Post-tetanic potentiation lowers the energy barrier for synaptic vesicle fusion independently of Synaptotagmin-1.

Previously, we showed that modulation of the energy barrier for synaptic vesicle fusion boosts release rates supralinearly (Schotten, 2015). Here we show that mouse hippocampal synapses employ this principle to trigger Ca2+-dependent vesicle release and post-tetanic potentiation (PTP). We assess energy barrier changes by fitting release kinetics in response to hypertonic sucrose. Mimicking activation of the C2A domain of the Ca2+-sensor Synaptotagmin-1 (Syt1), by adding a positive charge (Syt1D232N) or increasing its hydrophobicity (Syt14W), lowers the energy barrier. Removing Syt1 or impairing its release inhibitory function (Syt19Pro) increases spontaneous release without affecting the fusion barrier. Both phorbol esters and tetanic stimulation potentiate synaptic strength, and lower the energy barrier equally well in the presence and absence of Syt1. We propose a model where tetanic stimulation activates Syt1-independent mechanisms that lower the energy barrier and act additively with Syt1-dependent mechanisms to produce PTP by exerting multiplicative effects on release rates.

Mechanisms of PI(4,5)P2 Enrichment in HIV-1 Viral Membranes

Phosphatidylinositol 4,5-bisphosphate (PIP2) is critical for HIV-1 virus assembly. The viral membrane is enriched in PIP2, suggesting that the virus assembles at PIP2-rich microdomains. We showed previously that in model membranes PIP2 can form nanoscopic clusters bridged by multivalent cations. Here, using purified proteins we quantitated the binding of HIV-1 Gag-related proteins to giant unilamellar vesicles containing either clustered or free PIP2. Myristoylated MA strongly preferred binding to clustered PIP2. By contrast, unmyristoylated HIV-1 MA, RSV MA, and a PH domain all preferred to interact with free PIP2. We also found that HIV-1 Gag multimerization promotes PIP2 clustering. Truncated Gag proteins comprising the MA, CA, and SP domains (MACASP) or the MA and CA domains (MACA) induced self-quenching of acyl chain-labeled fluorescent PIP2 in liposomes, implying clustering. However, HIV-1 MA itself did not induce PIP2 clustering. A CA inter-hexamer dimer interface mutation led to a loss of induced PIP2 clustering in MACA, indicating the importance of protein multimerization. Cryo-electron tomography of liposomes with bound MACA showed an amorphous protein layer on the membrane surface. Thus, it appears that while protein–protein interactions are required for PIP2 clustering, formation of a regular lattice is not. Protein-induced PIP2 clustering and multivalent cation-induced PIP2 clustering are additive. Taken together, these results provide the first evidence that HIV-1 Gag can selectively target pre-existing PIP2-enriched domains of the plasma membrane for viral assembly, and that Gag multimerization can further enrich PIP2 at assembly sites. These effects could explain the observed PIP2 enrichment in HIV-1.

Biophysical Characterization of Cancer-Related Carbonic Anhydrase IX.

Upregulation of carbonic anhydrase IX (CA IX) is associated with several aggressive forms of cancer and promotes metastasis. CA IX is normally constitutively expressed at low levels in selective tissues associated with the gastrointestinal tract, but is significantly upregulated upon hypoxia in cancer. CA IX is a multi-domain protein, consisting of a cytoplasmic region, a single-spanning transmembrane helix, an extracellular CA catalytic domain, and a proteoglycan-like (PG) domain. Considering the important role of CA IX in cancer progression and the presence of the unique PG domain, little information about the PG domain is known. Here, we report biophysical characterization studies to further our knowledge of CA IX. We report the 1.5 Å resolution crystal structure of the wild-type catalytic domain of CA IX as well as small angle X-ray scattering and mass spectrometry of the entire extracellular region. We used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to characterize the spontaneous degradation of the CA IX PG domain and confirm that it is only the CA IX catalytic domain that forms crystals. Small angle X-ray scattering analysis of the intact protein indicates that the PG domain is not randomly distributed and adopts a compact distribution of shapes in solution. The observed dynamics of the extracellular domain of CA IX could have physiological relevance, including observed cleavage and shedding of the PG domain.

The crystal structure of Arabidopsis BON1 provides insights into the copine protein family.

The Arabidopsis thaliana BON1 gene product is a member of the evolutionary conserved eukaryotic calcium-dependent membrane-binding protein family. The copine protein is composed of two C2 domains (C2A and C2B) followed by a vWA domain. The BON1 protein is localized on the plasma membrane, and is known to suppress the expression of immune receptor genes and to positively regulate stomatal closure. The first structure of this protein family has been determined to 2.5-Å resolution and shows the structural features of the three conserved domains C2A, C2B and vWA. The structure reveals the third Ca2+ -binding region in C2A domain is longer than classical C2 domains and a novel Ca2+ binding site in the vWA domain. The structure of BON1 bound to Mn2+ is also presented. The binding of the C2 domains to phospholipid (PSF) has been modeled and provides an insight into the lipid-binding mechanism of the copine proteins. Furthermore, the selectivity of the separate C2A and C2B domains and intact BON1 to bind to different phospholipids has been investigated, and we demonstrated that BON1 could mediate aggregation of liposomes in response to Ca2+ . These studies have formed the basis of further investigations into the important role that the copine proteins play in vivo.

Backbone resonance assignments of the catalytic and regulatory domains of Ca2+/calmodulin-dependent protein kinase 1D

The CaMK subfamily of Ser/Thr kinases are regulated by calmodulin interactions with their C-terminal regions. They are exemplified by Ca2+/calmodulin dependent protein kinase 1δ which is known as CaMK1D, CaMKIδ or CKLiK. CaMK1D mediates intracellular signalling downstream of Ca2+ influx and thereby exhibits amplifications of Ca2+signals and polymorphisms that have been implicated in breast cancer and diabetes. Here we report the backbone 1H, 13C, 15N assignments of the 38 kDa human CaMK1D protein in its free state, including both the canonical bi-lobed kinase fold as well as the autoinhibitory and calmodulin binding domains.

Surface display of spring viremia of carp virus glycoprotein on Lactococcus lactis and its protection efficacy in common carp (Cyprinus carpio L.)

Spring viremia of carp virus (SVCV) causes devastating disease in aquaculture, resulting in significant economic impact. To develop an effective means against SVCV infection, a Lactococcus lactis (L.lactis) based subunit vaccine (pNZ-UGA) was developed based on surface displaying of SVCV glycoprotein using anchoring motif of the cA (C terminus of the peptidoglyvsn-binding) domains of AcmA, a major autolysin from L.lactis. The surface expression of SVCV glycoprotein was verified by indirect immunofluorescence assay. The efficacy of the constructed vaccine was further evaluated in common carp. The results showed that the higher levels of specific IgM could be detected in fish vaccinated with pNZ-UGA, compared with that in PBS and L.lactis groups. Immune-related genes including TNF-α, IL-6b, IL-1β, Cxcr 1, Cxca, IFNg2b, I-IFN, and IgM expression in pNZ-UGA group were strongly up-regulated, revealing that robust innate immune response was induced. Notably, the lowest cumulative mortality (13.46%) was observed in fish vaccinated with pNZ-UGA vaccine after SVCV challenge, whereas the cumulative mortality were 100.00% and 92.31% in PBS and L.lactis groups, respectively. This study suggests the potential use of the recombinant L.lactis with surface displaying antigen proteins as effective vaccines against SVCV and other fish virus infection.