Arrangement of Annexin A2 tetramer and its impact on the structure and diffusivity of supported lipid bilayers

Annexins are a family of proteins that bind to anionic phospholipid membranes in a Ca2+-dependent manner. Annexin A2 forms heterotetramers (Anx A2t) with the S100A10 (p11) protein dimer. The tetramer is capable of bridging phospholipid membranes and it has been suggested to play a role in Ca2+-dependent exocytosis and cell-cell adhesion of metastatic cells. Here, we employ x-ray reflectivity measurements to resolve the conformation of Anx A2t upon Ca2+-dependent binding to single supported lipid bilayers (SLBs) composed of different mixtures of anionic (POPS) and neutral (POPC) phospholipids. Based on our results we propose that Anx A2t binds in a side-by-side configuration, i.e., both Anx A2 monomers bind to the bilayer with the p11 dimer positioned on top. Furthermore, we observe a strong decrease of lipid mobility upon binding of Anx A2t to SLBs with varying POPS content. X-ray reflectivity measurements indicate that binding of Anx A2t also increases the density of the SLB. Interestingly, in the protein-facing leaflet of the SLB the lipid density is higher than in the substrate-facing leaflet. This asymmetric densification of the lipid bilayer by Anx A2t and Ca2+ might have important implications for the biochemical mechanism of Anx A2t-induced endo- and exocytosis.Comment: 27 pages, 7 figures; supplementary material available upon request from the author

Annexins-Coordinators of Cholesterol Homeostasis in Endocytic Pathways

The spatiotemporal regulation of calcium (Ca2+) storage in late endosomes (LE) and lysosomes (Lys) is increasingly recognized to influence a variety of membrane trafficking events, including endocytosis, exocytosis, and autophagy. Alterations in Ca2+ homeostasis within the LE/Lys compartment are implicated in human diseases, ranging from lysosomal storage diseases (LSDs) to neurodegeneration and cancer, and they correlate with changes in the membrane binding behaviour of Ca2+-binding proteins. This also includes Annexins (AnxA), which is a family of Ca2+-binding proteins participating in membrane traffic and tethering, microdomain organization, cytoskeleton interactions, Ca2+ signalling, and LE/Lys positioning. Although our knowledge regarding the way Annexins contribute to LE/Lys functions is still incomplete, recruitment of Annexins to LE/Lys is greatly influenced by the availability of Annexin bindings sites, including acidic phospholipids, such as phosphatidylserine (PS) and phosphatidic acid (PA), cholesterol, and phosphatidylinositol (4,5)-bisphosphate (PIP2). Moreover, the cytosolic portion of LE/Lys membrane proteins may also, directly or indirectly, determine the recruitment of Annexins to LE. Strikingly, within LE/Lys, AnxA1, A2, A6, and A8 differentially contribute to cholesterol transport along the endocytic route, in particular, cholesterol transfer between LE and other compartments, positioning Annexins at the centre of major pathways mediating cellular cholesterol homeostasis. Underlying mechanisms include the formation of membrane contact sites (MCS) and intraluminal vesicles (ILV), as well as the modulation of LE-cholesterol transporter activity. In this review, we will summarize the current understanding how Annexins contribute to influence LE/Lys membrane transport and associated functions

Identification of Regions Responsible for the Open Conformation of S100A10 Using Chimaeric S100A11/S100A10 Proteins

S100A11 is a dimeric, EF-hand calcium-binding protein. Calcium binding to S100A11 results in a large conformational change that uncovers a broad hydrophobic surface used to interact with phospholipid-binding proteins (annexins A1 and A2), and facilitate membrane vesiculation events. In contrast to other S100 proteins, S100A10 is unable to bind calcium due to deletion and substitution of calcium-ligating residues. Despite this, calcium-free S100A10 assumes an “open” conformation that is very similar to S100A11 in its calcium-bound state (Ca2+-S100A11). To understand how S100A10 is able to adopt an open conformation in the absence of calcium, seven chimeric proteins were constructed where regions from calcium-binding sites I and II, and helices II-IV in S100A11 were replaced with the corresponding regions of S100A10. The chimeric proteins having substitutions in calcium-binding site II displayed increased hydrophobic surface exposure as assessed by ANS fluorescence and phenyl Sepharose binding in the absence of calcium. This response is similar to that observed for Ca2+-S100A11 and calcium-free S100A10. Further, this substitution resulted in calcium-insensitive binding to annexin A2 for one chimeric protein. The results indicate that residues within site II are important in stabilizing the open conformation of S100A10 and presentation of its target-binding site. In contrast, S100A11 chimeric proteins with helical substitutions displayed poorer hydrophobic surface exposure and consequently, unobservable annexin A2 binding. This work represents a first attempt to systematically understand the molecular basis for the calcium-insensitive open conformation of S100A10

Mechanisms of regulated lung surfactant secretion

Scope and Method of Study:This project was to study the molecular mechanisms of lung surfactant secretion from three aspects. The first part was to investigate the interaction between SNARE proteins and annexin A2. GST-tagged protein pull-down assay was the major technique utilized. The physical interaction of recombinant GST-tagged SNARE proteins with annexin A2 were tested. To confirm the interaction between annexin A2 and SNAP-23, additional methods were used, including co-immunoprecipitation, mammalian two-hybrid assays. Immunocytochemistry was used to study the co-localization of annexin A2 and SNAP-23. Deletion and site-directed mutagenesis were used to identify the binding sites for annexin A2 on SNAP-23. Furthermore, an in vitro bio-membrane fusion assay was utilized to study the functional interaction between annexin A2 and SNAP-23. The second part was to identify the v-SNARE protein involved in regulated surfactant secretion. Various VAMP genes were amplified from alveolar type II cells by using RT-PCR. The expression of VAMP proteins were detected with Western blotting. Immunohistochemistry and immunocytochemistry were utilized to study the localization of VAMP in lung and type II cells. In the third part, the proteomic profile of lamellar bodies was analyzed. Lamellar bodies were isolated from rat lungs and the proteins were separated with 1-D and 2-D SDS-PAGE. The proteins were applied to MALDI-TOF MS, and identified by searching the peptide spectra against the Mass Spectrometry protein sequence Database (MSDB) using the Mascot web based search engine.Findings and Conclusions:I.1. SNAP-23 specifically binds with annexin A2 in a Ca2+-dependent manner.2. The cysteine rich domain of SNAP-23 is required for its binding with annexin A2.3. SNAP-23 is required in annexin A2 tetramer mediated membrane fusion between isolated lamellar bodies and the plasma membrane.II.1. VAMP-2 and -8 are expressed in type II cells and lamellar bodies.2. VAMP-2 is localized on lamellar bodies and VAMP-8 is partially localized on lamellar bodies.III.1. Forty-four proteins in lamellar bodies were identified.2. Proteins involved in membrane trafficking and Ca2+-binding may play roles in lamellar body biogenesis and fusion with the plasma membrane

The role of annexin II in vesicle traffic

The annexins are a family of proteins that bind acidic phospholipids in the presence of Ca2+. The association of these proteins with the membranes of secretory granules and endosomes indicates these proteins may play a role in membrane trafficking. One member of the family, annexin II, can exist either as a monomer, heterodimer or heterotetramer in conjunction with the S100 protein p11. The ability of annexin II tetramer to bind both membranes and actin in a Ca2+-dependent manner has led to the hypothesis that annexin II may mediate between vesicle and/or plasma membranes and the cortical cytoskeleton. However, despite intensive biochemical characterisation in vitro, the function of this protein in vivo remains a mystery. In this study annexin II function in living cells was analysed in several different ways using green fluorescent protein (GFP) in full length annexin II-GFP chimeras and chimeras consisting of fragments of annexin II fused to GFP. Transfection of different cell lines with these annexin II-GFP constructs and fluorescence assisted cell sorting (FACS) allowed the generation of multiclonal cell populations expressing annexin II-GFP fusion proteins. These cell populations were analysed for effects on physiological functions - such as secretion (in the RBL cell line) or differentiation (of the PC12 cell line). This line of investigation did not yield evidence to support a role for annexin II in either of these processes. Using novel forms of microscopy the localisation of a full length annexin II- GFP chimera (NAII-GFP) was followed in single cells under physiological conditions. Under conditions of stress NAII-GFP was found to become incorporated into novel actin based structures, reminiscent of Listeria rockets, which propelled pinosomes through the cell interior. This form of vesicle locomotion is dependent on actin polymerisation and may represent a hitherto unrecognised form of vesicle transport

Calcium-Mediated Control of S100 Proteins: Allosteric Communication via an Agitator/Signal Blocking Mechanism.

Allosteric proteins possess dynamically coupled residues for the propagation of input signals to distant target binding sites. The input signals usually correspond to effector is present or effector is not present . Many aspects of allosteric regulation remain incompletely understood. This work focused on S100A11, a dimeric EF-hand protein with two hydrophobic target binding sites. An annexin peptide (Ax) served as the target. Target binding is allosterically controlled by Ca2+ over a distance of ∼26 Å. Ca2+ promotes formation of a [Ca4 S100 Ax2] complex, where the Ax peptides are accommodated between helices III/IV and III\u27/IV\u27. Without Ca2+ these binding sites are closed, precluding interactions with Ax. The allosteric mechanism was probed by microsecond MD simulations in explicit water, complemented by hydrogen exchange mass spectrometry (HDX/MS). Consistent with experimental data, MD runs in the absence of Ca2+ and Ax culminated in target binding site closure. In simulations on [Ca4 S100] the target binding sites remained open. These results capture the essence of allosteric control, revealing how Ca2+ prevents binding site closure. Both HDX/MS and MD data showed that the metalation sites become more dynamic after Ca2+ loss. However, these enhanced dynamics do not represent the primary trigger of the allosteric cascade. Instead, a labile salt bridge acts as an incessantly active agitator that destabilizes the packing of adjacent residues, causing a domino chain of events that culminates in target binding site closure. This agitator represents the starting point of the allosteric signal propagation pathway. Ca2+ binding rigidifies elements along this pathway, thereby blocking signal transmission. This blocking mechanism does not conform to the commonly held view that allosteric communication pathways generally originate at the sites where effectors interact with the protein

Annexins induce curvature on free-edge membranes displaying distinct morphologies

Annexins are a family of proteins characterized by their ability to bind anionic membranes in response to Ca2+-activation. They are involved in a multitude of cellular functions including vesiculation and membrane repair. Here, we investigate the effect of nine annexins (ANXA1-ANXA7, ANXA11, ANXA13) on negatively charged double supported membrane patches with free edges. We find that annexin members can be classified according to the membrane morphology they induce and matching a dendrogam of the annexin family based on full amino acid sequences. ANXA1 and ANXA2 induce membrane folding and blebbing initiated from membrane structural defects inside patches while ANXA6 induces membrane folding originating both from defects and from the membrane edges. ANXA4 and ANXA5 induce cooperative roll-up of the membrane starting from free edges, producing large rolls. In contrast, ANXA3 and ANXA13 roll the membrane in a fragmented manner producing multiple thin rolls. In addition to rolling, ANXA7 and ANXA11 are characterized by their ability to form fluid lenses localized between the membrane leaflets. A shared feature necessary for generating these morphologies is the ability to induce membrane curvature on free edged anionic membranes. Consequently, induction of membrane curvature may be a significant property of the annexin protein family that is important for their function

Hydrogen-Deuterium Exchange Mass Spectrometry and Molecular Dynamics Simulations for Studying Protein Structure and Dynamics

Deciphering properties of proteins are essential for human health and aiding in the development of new pharmaceuticals. This dissertation uses hydrogen-deuterium exchange (HDX) mass spectrometry (MS) and molecular dynamics (MD) simulations to study protein dynamics, for improving the understanding of protein folding/unfolding mechanisms, and ligand binding and allosteric regulation. Chapter 2 uses HDX-MS for probing the conformational dynamics of myoglobin in the presence of N2 bubbles. We propose a dynamic model that reproduces the observed data: “semi-unfolded” “native” “globally unfolded” -\u3e “aggregated”. Chapter 3 focuses on osteoprotegerin (OPG), which hinders bone resorption by inhibiting RANK/RANKL interactions. The dimerization of OPG is regulated by heparan sulfate (HS). Basing on HDX data, a mechanism is proposed for the formation of the RANKL/OPG/HS ternary complex, according to which HS-mediated C-terminal contacts on OPG lower the entropic penalty for RANKL binding. Chapter 4 represents the centerpiece of this thesis. It explores the allosteric regulation of S100A11, a dimeric EF-hand protein with two hydrophobic target binding sites. Both HDX/MS and MD data showed the metalation sites become more dynamic after Ca2+ loss. However, these enhanced dynamics do not represent the trigger of the allosteric cascade. Instead, a labile salt bridge acts as an active “agitator” that destabilizes the packing of adjacent residues, causing a domino chain of events that culminates in target binding site closure. Overall, this thesis highlights how the combination of HDX/MS and computational techniques can provide detailed insights into protein conformational fluctuations and their implications for protein function

Study of the cell biological role of Lowe Syndrome protein OCRL1

Oculocerebrorenal syndrome of Lowe (OCRL) is caused by mutations in a phosphatidylinositol 5-phosphatase, OCRL1, and is believed to lead to an elevation of its preferred substrate, PI(4,5)P2. To date, much of the work on OCRL1 has centred on its role at Golgi and endosomal membranes. However, there is also evidence of plasma membrane activity for OCRL1, where its PI(4,5)P2 substrate is known to be highly abundant. PI(4,5)P2 regulates a wide array of downstream cellular functions such as cytoskeletal dynamics, membrane trafficking and signalling. The tight regulation of PI(4,5)P2 levels and localisation, like other phosphoinositides, provides a framework upon which many of these cellular processes work. In this thesis, effects of OCRL1 loss have been tested through siRNA depletion of OCRL1, focussing where possible on multiple PI(4,5)P2-dependent mechanisms, and also focussing on cells forming polarised epithelia. Firstly, we have visualised the localisation of PI(4,5)P2 in living HeLa cells lacking OCRL1 through immunostaining for Annexin A2, which showed a marked translocation to the plasma membrane. This change in distribution of Annexin A2 suggested that OCRL1 depletion may have an effect on intracellular calcium dynamics as well as PI(4,5)P2 localisation. We also used a GFP-chimera of the well characterised PI(4,5)P2-binding pleckstrin homology domain of PLCδ1. This showed no difference in localisation upon OCRL1 depletion. As OCRL1 is highly enriched at the TGN, we fused the pleckstrin homology domain of PLCδ1 to a mutated pleckstrin homology domain of OSBP known to bind ARF1 at the TGN, to act as a coincidence detector for PI(4,5)P2 at the TGN. This construct also showed no reproducible effect of OCRL1 depletion. Secondly we tested the effect of loss of OCRL1 on cytosolic calcium levels. Using two phospholipase C (PLC) agonists, and a SERCA pump inhibitor, we found no consistent differences in calcium handling upon depletion of OCRL1. Thirdly, we have assessed the potential specialised role that OCRL1 has in polarised epithelial cells, which might relate to the clinical picture in Lowe Syndrome. We found that OCRL1 targets the tight junctions of immortalised lines and primary cells. Through co-immunoprecipitation, we found OCRL1 in complexes with the tight junction scaffold protein ZO-1. Most significantly, we found that depletion of OCRL1 in human polarised epithelial cell lines interfered with epithelial differentiation, reducing cell number and altering morphology, to produce large flat cells. We attribute this phenotype, stronger than any other so far described experimentally, to a defect in tight junction maturation