C-terminal Core Domain Research Articles

Is the small heat shock protein HSPB7 (cvHsp) a genuine actin-binding protein?

It is postulated that the small heat shock proteins directly interact with actin, affect formation and stabilize actin filaments. To verify this suggestion, we have analyzed interaction of recombinant human small heat shock protein HspB7 with skeletal muscle actin. In blot overlay HspB7 binds both G- and F-actin. The sites of interaction are located in the C-terminal large core domain of actin. In the course of ultracentrifugation F-actin and F-actin/tropomyosin complexes were pelleted and trapped HspB7. However, HspB7 pelleting was nonspecific and saturation was not achieved even at very high HspB7 concentration. HspB7 was unable to retard or prevent heat-induced F-actin aggregation. Native gel electrophoresis and chemical crosslinking failed to detect interaction of G-actin with HspB7, although both these methods clearly demonstrated formation of complexes formed by G-actin with DNAse I and cofilin-2. It is concluded that HspB7 is not a genuine actin-binding protein and its effect on actin filaments seems to be determined by interaction of HspB7 with minor regulatory proteins of actin filaments.

Coupling of Integrin α5 to Annexin A2 by Flow Drives Endothelial Activation.

Atherosclerosis preferentially occurs at specific sites of the vasculature where endothelial cells (ECs) are exposed to disturbed blood flow. Translocation of integrin α5 to lipid rafts promotes integrin activation and ligation, which is critical for oscillatory shear stress (OSS)-induced EC activation. However, the underlying mechanism of OSS promoted integrin α5 lipid raft translocation has remained largely unknown. The objective of this study was to specify the mechanotransduction mechanism of OSS-induced integrin α5 translocation and subsequent EC activation. Mass spectrometry studies identified endothelial ANXA2 (annexin A2) as a potential carrier allowing integrin α5β1 to traffic in response to OSS. Interference by siRNA of AnxA2 in ECs greatly decreased OSS-induced integrin α5β1 translocation to lipid rafts, EC activation, and monocyte adhesion. Pharmacological and genetic inhibition of PTP1B (protein tyrosine phosphatase 1B) blunted OSS-induced integrin α5β1 activation, which is dependent on Piezo1-mediated calcium influx in ECs. Furthermore, ANXA2 was identified as a direct substrate of activated PTP1B by mass spectrometry. Using bioluminescence resonance energy transfer assay, PTP1B-dephosphorylated ANXA2 at Y24 was found to lead to conformational freedom of the C-terminal core domain from the N-terminal domain of ANXA2. Immunoprecipitation assays showed that this unmasked ANXA2-C-terminal core domain specifically binds to an integrin α5 nonconserved cytoplasmic domain but not β1. Importantly, ectopic lentiviral overexpression of an ANXA2Y24F mutant increased and shRNA against Ptp1B decreased integrin α5β1 ligation, inflammatory signaling, and progression of plaques at atheroprone sites in apolipoprotein E (ApoE)-/- mice. However, the antiatherosclerotic effect of Ptp1B shRNA was abolished in AnxA2-/-ApoE-/- mice. Our data elucidate a novel endothelial mechanotransduction molecular mechanism linking atheroprone flow and activation of integrin α5β1, thereby identifying a class of potential therapeutic targets for atherosclerosis. Graphic Abstract: An graphic abstract is available for this article.

Markedly Reduced Expression of Annexin A2 Is Associated with Hypofibrinolysis and Provoked and Unprovoked Venous Thromboembolism

Introduction: Recent evidence indicates that plasma hypofibrinolysis, as measured by clot lysis times (CLT) at or above the 90th percentile, can double the risk of venous thromboembolism (VTE). To our knowledge, there are no studies on the role of cell surface fibrinolysis in thrombosis. Annexin A2 (A2) is a calcium dependent, phospholipid binding protein that forms a heterotetramer with S100A10 (A2-S100A10)2 on the surface of endothelial cells (ECs). This complex serves as a co-receptor for plasminogen and tissue plasminogen activator (tPA), and increases the catalytic efficiency of tPA-dependent cell surface plasmin generation by 60-fold. In mouse studies, global knockout of the annexin A2 gene (Anxa2) is associated with fibrin accumulation and impaired clearance of arterial thrombi. In addition, there are several examples of the regulatory role of A2 in fibrinolysis in human diseases such as antiphospholipid antibody syndrome, cerebral venous thrombosis, and sickle cell disease. In the current study, we aimed to explore the potential role of the A2 system and cell surface based fibrinolysis in the development of VTE. Methods: Study subjects included patients 18-65 years old with history of VTE and healthy controls. Subjects were classified as having provoked or unprovoked VTE based on the presence or absence of identifiable environmental risk factors. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected in EDTA tubes, and used as a surrogate for ECs. Assays performed on PBMCs included determination of the rate of tPA-dependent cell surface plasmin generation using a fluorogenic substrate and analysis of CLT in the presence of phospholipid vesicles. Because A2 accounts for approximately 50% of cell surface based plasmin generation on both ECs and PBMCs, we analyzed total A2 protein expression relative to GAPDH in whole cell lysates of PBMCs using semi-quantitative western blotting. Additional assays included quantitative RT-PCR and ANXA2 gene sequencing. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparisons test. Results: Overall, 116 subjects with VTE (76 with provoked and 40 with unprovoked VTE) and 87 healthy controls were studied between September 2010 and May 2019. Plasma based clot lysis assays revealed that the mean CLT for subjects with VTE was significantly longer than that for healthy controls (37.5 versus 30.7 minutes, p=0.0001). Additionally, the mean rate of cell surface plasmin generation was significantly reduced in subjects with VTE as compared with healthy controls (0.33 RFU/min2 versus 0.49 RFU/min2, p=0.0021). Moreover, none of the 60 healthy controls (0%), but 18 of the 107 subjects with thrombosis (17%) had cell surface plasmin generating capacity in the lowest 10th percentile. In protein expression studies, we observed that none of the 21 healthy controls (0%), but 5 of 41 subjects with thrombosis (12%) had A2 expression in the lowest 10th percentile for the group; 4 of 18 (22%) of those with unprovoked VTE and 1 of 23 (4%) of those with provoked VTE fell into the lowest decile for protein expression. For plasmin generating capacity, 8 of 36 (22%) of subjects with unprovoked VTE and 10 of 71 (14%) of those with provoked VTE occupied the lowest decile. Probing of western blots of samples obtained on two separate occasions several months apart with A2 epitope-specific antibodies revealed abnormalities within either the tPA binding N-terminal tail or C-terminal core domain in proximity to the plasminogen binding site. Neither quantitative RT-PCR nor ANXA2 gene sequencing of selected samples revealed abnormalities in either mRNA or genomic DNA that could explain the reduced A2 expression. Conclusion: These data confirm findings previously reported by Lisman that plasma hypofibrinolysis is associated with VTE and may represent an independent risk factor for VTE. Additionally, we demonstrate for the first time that impaired cell surface based fibrinolysis and aberrations in A2 protein expression are associated with both provoked and unprovoked VTE, and may represent a novel risk factor for thrombosis. Possible explanations for reduced A2 expression include dysfunctional translation of mRNA into protein or post-translational proteolysis of the translated protein. Compromise of the A2-based fibrinolytic system may represent a previously unrecognized contributor to thrombophilia in VTE. Disclosures Ruisi: BMY: Equity Ownership; EMD, subsidiary of Merck KGaA: Employment. De Sancho:Apellis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

The domain architecture of PtkA, the first tyrosine kinase from Mycobacterium tuberculosis, differs from the conventional kinase architecture

The discovery that MptpA (low-molecular-weight protein tyrosine phosphatase A) from Mycobacterium tuberculosis (Mtb) has an essential role for Mtb virulence has motivated research of tyrosine-specific phosphorylation in Mtb and other pathogenic bacteria. The phosphatase activity of MptpA is regulated via phosphorylation on Tyr128 and Tyr129 Thus far, only a single tyrosine-specific kinase, protein-tyrosine kinase A (PtkA), encoded by the Rv2232 gene has been identified within the Mtb genome. MptpA undergoes phosphorylation by PtkA. PtkA is an atypical bacterial tyrosine kinase, as its sequence differs from the sequence consensus within this family. The lack of structural information on PtkA hampers the detailed characterization of the MptpA-PtkA interaction. Here, using NMR spectroscopy, we provide a detailed structural characterization of the PtkA architecture and describe its intra- and intermolecular interactions with MptpA. We found that PtkA's domain architecture differs from the conventional kinase architecture and is composed of two domains, the N-terminal highly flexible intrinsically disordered domain (IDDPtkA) and the C-terminal rigid kinase core domain (KCDPtkA). The interaction between the two domains, together with the structural model of the complex proposed in this study, reveal that the IDDPtkA is unstructured and highly dynamic, allowing for a "fly-casting-like" mechanism of transient interactions with the rigid KCDPtkA This interaction modulates the accessibility of the KCDPtkA active site. In general, the structural and functional knowledge of PtkA gained in this study is crucial for understanding the MptpA-PtkA interactions, the catalytic mechanism, and the role of the kinase-phosphatase regulatory system in Mtb virulence.

Regulation of the Equilibrium between Closed and Open Conformations of Annexin A2 by N-Terminal Phosphorylation and S100A4-Binding

Annexin A2 (ANXA2) has a versatile role in membrane-associated functions including membrane aggregation, endo- and exocytosis, and it is regulated by post-translational modifications and protein-protein interactions through the unstructured N-terminal domain (NTD). Our sequence analysis revealed a short motif responsible for clamping the NTD to the C-terminal core domain (CTD). Structural studies indicated that the flexibility of the NTD andCTD are interrelated and oppositely regulated by Tyr24 phosphorylation and Ser26Glu phosphomimicking mutation. The crystal structure of the ANXA2-S100A4 complex showed that asymmetric binding of S100A4 induces dislocation of the NTD from the CTD and, similar to the Ser26Glu mutation, unmasks the concave side of ANXA2. In contrast, pTyr24 anchors the NTD to the CTD and hampers the membrane-bridging function. This inhibition can be restored by S100A4 and S100A10 binding. Based on our results we provide a structural model for regulation of ANXA2-mediated membrane aggregation by NTD phosphorylation and S100 binding.

Interaction of βA3-Crystallin with Deamidated Mutants of αA- and αB-Crystallins.

Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the α- and β-crystallins. Here, we investigated the effects of the deamidation of αA- and αB-crystallins on their interaction with βA3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT αA- and WT αB-crystallins and their deamidated mutants with βA3-crystallin. The deamidated mutants of αA–crystallin (αA N101D and αA N123D) displayed lower adherence propensity for βA3-crystallin relative to the binding affinity shown by WT αA-crystallin. Among αB-crystallin mutants, αB N78D displayed higher adherence propensity whereas αB N146D mutant showed slightly lower binding affinity for βA3-crystallin relative to that shown by WT αB-crystallin. Under the in vivo condition (FLIM-FRET), both αA-deamidated mutants (αA N101D and αA N123D) exhibited strong interaction with βA3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT αA-crystallin (18±4%). Similarly, the αB N78D and αB N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with βA3-crystallin compared to 18±4% FRET efficiency of WT αB-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of αA- and αB-crystallins with βA3-crystallin suggested that interaction sites most likely reside in the αA CTE and αB NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT αA- and WTαB-crystallins, the deamidated mutants showed strong interactionfor βA3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant’s large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of α-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.

Understanding the Physical and Molecular Basis of Stability of Arabidopsis DNA Pol λ under UV-B and High NaCl Stress.

Here, we have investigated the physical and molecular basis of stability of Arabidopsis DNA Pol λ, the sole X family DNA polymerase member in plant genome, under UV-B and salinity stress in connection with the function of the N-terminal BRCT (breast cancer-associated C terminus) domain and Ser-Pro rich region in the regulation of the overall structure of this protein. Tryptophan fluorescence studies, fluorescence quenching and Bis-ANS binding experiments using purified recombinant full length Pol λ and its N-terminal deletion forms have revealed UV-B induced conformational change in BRCT domain deficient Pol λ. On the other hand, the highly conserved C-terminal catalytic core PolX domain maintained its tertiary folds under similar condition. Circular dichroism (CD) and fourier transform infrared (FT-IR) spectral studies have indicated appreciable change in the secondary structural elements in UV-B exposed BRCT domain deficient Pol λ. Increased thermodynamic stability of the C-terminal catalytic core domain suggested destabilizing effect of the N-terminal Ser-Pro rich region on the protein structure. Urea-induced equilibrium unfolding studies have revealed increased stability of Pol λ and its N-terminal deletion mutants at high NaCl concentration. In vivo aggregation studies using transient expression systems in Arabidopsis and tobacco indicated possible aggregation of Pol λ lacking the BRCT domain. Immunoprecipitation assays revealed interaction of Pol λ with the eukaryotic molecular chaperone HSP90, suggesting the possibility of regulation of Pol λ stability by HSP90 in plant cell. Overall, our results have provided one of the first comprehensive information on the biophysical characteristics of Pol λ and indicated the importance of both BRCT and Ser-Pro rich modules in regulating the stability of this protein under genotoxic stress in plants.

Protein interactions at the C‐terminus of essential cell division protein FtsZ modulate Z‐ring dynamics in Escherichia coli

Highly coordinated spatiotemporal regulation of the cell division pathway ensures the production of two identical daughter cells in Escherichia coli. Constriction and septation at midcell require the assembly of a ring-like dynamic protein structure, called the Z-ring, containing FtsZ, a structural homolog of tubulin that exhibits GTP-dependent polymerization. FtsZ monomers contain an N-terminal polymerization domain, an unstructured linker region, and a conserved C-terminal core domain important for protein interactions. Residues near the FtsZ C-terminus have been implicated in direct interactions with proteins that tether the Z-ring to the membrane, including FtsA and ZipA, and proteins that promote disassembly of FtsZ polymers, including MinC and chaperone-protease ClpXP. To test if protein interactions at the FtsZ C-terminus are important for regulating the dynamic exchange of FtsZ subunits into and out of the Z-ring, we monitored the fluorescence recovery of Z-rings containing Gfp-FtsZ in dividing cells after photobleaching. We demonstrate that multiple mutations near the FtsZ C-terminus lead to the formation of Z-rings that have significantly impaired dynamics in vivo. We also deleted several genes encoding proteins that promote the disassembly of FtsZ polymers in vitro, including MinC and ClpXP, and have observed that cells deleted for these genes contain Z-rings that exchange subunits more slowly than wild type cells. These results show that proteins promoting disassembly of FtsZ polymers have a positive effect on enhancing the exchange of subunits within the FtsZ-ring. Moreover, these results also suggest that proteins modulate Z-ring dynamics through direct interactions with the FtsZ C-terminus.

Cooperative Binding of Annexin A2 to Cholesterol- and Phosphatidylinositol-4,5-Bisphosphate-Containing Bilayers

Biological membranes are organized into dynamic microdomains that serve as sites for signal transduction and membrane trafficking. The formation and expansion of these microdomains are driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Annexin A2 (AnxA2) is a peripherally associated membrane protein that can support microdomain formation in a Ca2+-dependent manner and has been implicated in membrane transport processes. Here, we performed a quantitative analysis of the binding of AnxA2 to solid supported membranes containing the annexin binding lipids phosphatidylinositol-4,5-bisphosphate and phosphatidylserine in different compositions. We show that the binding is of high specificity and affinity with dissociation constants ranging between 22.1 and 32.2 nM. We also analyzed binding parameters of a heterotetrameric complex of AnxA2 with its S100A10 protein ligand and show that this complex has a higher affinity for the same membranes with Kd values of 12 to 16.4 nM. Interestingly, binding of the monomeric AnxA2 and the AnxA2-S100A10 complex are characterized by positive cooperativity. This cooperative binding is mediated by the conserved C-terminal annexin core domain of the protein and requires the presence of cholesterol. Together our results reveal for the first time, to our knowledge, that AnxA2 and its derivatives bind cooperatively to membranes containing cholesterol, phosphatidylserine, and/or phosphatidylinositol-4,5-bisphosphate, thus providing a mechanistic model for the lipid clustering activity of AnxA2.

Lipid Segregation and Membrane Budding Induced by the Peripheral Membrane Binding Protein Annexin A2*

The formation of dynamic membrane microdomains is an important phenomenon in many signal transduction and membrane trafficking events. It is driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Here we analyzed the ability of one peripherally associated membrane protein, annexin A2 (AnxA2), to induce the formation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-rich domains in giant unilamellar vesicles (GUVs) of complex lipid composition. AnxA2 is a cytosolic protein that can bind PI(4,5)P2 and other acidic phospholipids in a Ca(2+)-dependent manner and that has been implicated in cellular membrane dynamics in endocytosis and exocytosis. We show that AnxA2 binding to GUVs induces lipid phase separation and the recruitment of PI(4,5)P2, cholesterol and glycosphingolipids into larger clusters. This property is observed for the full-length monomeric protein, a mutant derivative comprising the C-terminal protein core domain and for AnxA2 residing in a heterotetrameric complex with its intracellular binding partner S100A10. All AnxA2 derivatives inducing PI(4,5)P2 clustering are also capable of forming interconnections between PI(4,5)P2-rich microdomains of adjacent GUVs. Furthermore, they can induce membrane indentations rich in PI(4,5)P2 and inward budding of these membrane domains into the lumen of GUVs. This inward vesiculation is specific for AnxA2 and not shared with other PI(4,5)P2-binding proteins such as the pleckstrin homology (PH) domain of phospholipase Cδ1. Together our results indicate that annexins such as AnxA2 can efficiently induce membrane deformations after lipid segregation, a mechanism possibly underlying annexin functions in membrane trafficking.

Auxiliary KChIP4a Suppresses A-type K+ Current through Endoplasmic Reticulum (ER) Retention and Promoting Closed-state Inactivation of Kv4 Channels

In the brain and heart, auxiliary Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 α-subunits to form a native K(+) channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1-4 members, KChIP4a exhibits a unique N terminus that is known to suppress Kv4 function, but the underlying mechanism of Kv4 inhibition remains unknown. Using a combination of confocal imaging, surface biotinylation, and electrophysiological recordings, we identified a novel endoplasmic reticulum (ER) retention motif, consisting of six hydrophobic and aliphatic residues, 12-17 (LIVIVL), within the KChIP4a N-terminal KID, that functions to reduce surface expression of Kv4-KChIP complexes. This ER retention capacity is transferable and depends on its flanking location. In addition, adjacent to the ER retention motif, the residues 19-21 (VKL motif) directly promote closed-state inactivation of Kv4.3, thus leading to an inhibition of channel current. Taken together, our findings demonstrate that KChIP4a suppresses A-type Kv4 current via ER retention and enhancement of Kv4 closed-state inactivation.

Architecture and Assembly of HIV Integrase Multimers in the Absence of DNA Substrates

We have applied small angle x-ray scattering and protein cross-linking coupled with mass spectrometry to determine the architectures of full-length HIV integrase (IN) dimers in solution. By blocking interactions that stabilize either a core-core domain interface or N-terminal domain intermolecular contacts, we show that full-length HIV IN can form two dimer types. One is an expected dimer, characterized by interactions between two catalytic core domains. The other dimer is stabilized by interactions of the N-terminal domain of one monomer with the C-terminal domain and catalytic core domain of the second monomer as well as direct interactions between the two C-terminal domains. This organization is similar to the "reaching dimer" previously described for wild type ASV apoIN and resembles the inner, substrate binding dimer in the crystal structure of the PFV intasome. Results from our small angle x-ray scattering and modeling studies indicate that in the absence of its DNA substrate, the HIV IN tetramer assembles as two stacked reaching dimers that are stabilized by core-core interactions. These models of full-length HIV IN provide new insight into multimer assembly and suggest additional approaches for enzyme inhibition.

Structure of Yeast Sulfhydryl Oxidase Erv1 Reveals Electron Transfer of the Disulfide Relay System in the Mitochondrial Intermembrane Space

The disulfide relay system in the mitochondrial intermembrane space drives the import of proteins with twin CX(9)C or twin CX(3)C motifs by an oxidative folding mechanism. This process requires disulfide bond transfer from oxidized Mia40 to a substrate protein. Reduced Mia40 is reoxidized/regenerated by the FAD-linked sulfhydryl oxidase Erv1 (EC 1.8.3.2). Full-length Erv1 consists of a flexible N-terminal shuttle domain (NTD) and a conserved C-terminal core domain (CTD). Here, we present crystal structures at 2.0 Å resolution of the CTD and at 3.0 Å resolution of a C30S/C133S double mutant of full-length Erv1 (Erv1FL). Similar to previous homologous structures, the CTD exists as a homodimer, with each subunit consisting of a conserved four-helix bundle that accommodates the isoalloxazine ring of FAD and an additional single-turn helix. The structure of Erv1FL enabled us to identify, for the first time, the three-dimensional structure of the Erv1NTD, which is an amphipathic helix flanked by two flexible loops. This structure also represents an intermediate state of electron transfer from the NTD to the CTD of another subunit. Comparative structural analysis revealed that the four-helix bundle of the CTD forms a wide platform for the electron donor NTD. Moreover, computational simulation combined with multiple-sequence alignment suggested that the amphipathic helix close to the shuttle redox enter is critical for the recognition of Mia40, the upstream electron donor. These findings provide structural insights into electron transfer from Mia40 via the shuttle domain of one subunit of Erv1 to the CTD of another Erv1 subunit.

Mechanistic Insights into the Activation of Rad51-Mediated Strand Exchange from the Structure of a Recombination Activator, the Swi5-Sfr1 Complex

Rad51 forms a helical filament on single-stranded DNA and promotes strand exchange between two homologous DNA molecules during homologous recombination. The Swi5-Sfr1 complex interacts directly with Rad51 and stimulates strand exchange. Here we describe structural and functional aspects of the complex. Swi5 and the C-terminal core domain of Sfr1 form an essential activator complex with a parallel coiled-coil heterodimer joined firmly together via two previously uncharacterized leucine-zipper motifs and a bundle. The resultant coiled coil is sharply kinked, generating an elongated crescent-shaped structure suitable for transient binding within the helical groove of the Rad51 filament. The N-terminal region of Sfr1, meanwhile, has an interface for binding of Rad51. Our data suggest that the snug fit resulting from the complementary geometry of the heterodimer activates the Rad51 filament and that the N-terminal domain of Sfr1 plays a role in the efficient recruitment of the Swi5-Sfr1 complex to the Rad51 filaments.

Peptidoglycan Remodeling in Mycobacterium tuberculosis: Comparison of Structures and Catalytic Activities of RipA and RipB

The success of Mycobacterium tuberculosis in sustaining long-term survival within the host macrophages partly relies on its unique cell envelop that also confers low susceptibility to several antibiotics. Remodeling of the septal peptidoglycan (PG) has been linked to the putative PG hydrolases RipA and RipB. The crystal structures of RipB (Rv1478) and the homologous module of RipA (Rv1477) were determined to 1.60 Å and 1.38 Å resolution, respectively. Both proteins contain a C-terminal core domain resembling the NlpC-type PG hydrolases. However, the structure of RipB exhibits striking differences to the structures of this domain in RipA reported here and previously by others. Major structural differences were found in the N-terminal segments of 70 amino acids and in an adjacent loop, which form part of the substrate binding groove. Both RipA and RipB are able to bind PG. RipA, its C-terminal module and RipB cleave defined PG fragments between d-glutamate and meso-diaminopimelate with pH optima of 5 and 6, respectively. The peptidase module of RipA is also able to degrade Bacillus subtilis PG, which displays peptide stems and cross-links identical with those found in mycobacterial murein. RipB did not show comparable hydrolase activity with this substrate. Removal of the N-terminal segments previously suggested to have a role in auto-inhibition did not change the activity of either RipA or RipB. A comparison of the putative active-site clefts in the two enzymes provides structural insights into the basis of the differences in their substrate specificity.

Multimerization-defective variants of dodecameric secretin PulD

The C-terminal core domain of the secretin PulD from Klebsiella oxytoca forms heat-resistant dodecameric complexes within less than 10 min in an Escherichia coli in vitro transcription-translation system containing liposomes, and is toxic when made in the cytoplasm without a signal peptide. Random mutagenesis of DNA encoding this region of PulD revealed that amino acid changes throughout almost its entire length abolished toxicity. Most of the amino acid substitutions engendered by the mutations retarded or abolished assembly of the dodecameric secretin complex in vitro and/or in the periplasm. Only one of the tested multimerization-defective variants could be rescued by co-production and mixed multimer formation with wild-type secretin in vitro. A three amino acid insertion specifically generated in a region of PulD that was not affected by the spontaneous mutations formed functional multimers that, unlike the wild-type protein, were dissociated by heating in SDS.

CD2AP Structure And Progression Of Renal Disease

CD2AP is a scaffolding molecule that was originally cloned as an interaction partner of CD2 in T lymphocytes. In the kidney, CD2AP is strongly expressed in podocytes, a cell type that regulates the filtration barrier. The protein directly interacts with filamentous actin and a variety of cell membrane proteins including the kidney filter protein nephrin. In addition to discrete binding sites for actin and nephrin, CD2AP possesses three SH3 domains and a proline-rich region containing, in turn, binding sites for SH3 domains. CD2AP is implicated in dynamic actin remodeling and membrane trafficking that occurs during receptor endocytosis and cytokinesis. We have initiated structural studies of recombinant CD2AP protein using electron microscopy and single particle image analysis. Negative stain electron microscopy of revealed uniform particles with a size and morphology suggesting a tetrameric organization, subsequently verified with chemical crosslinking. Single particle image analysis was used to generate a three-dimensional map of the CD2AP tetramer at 21 Å resolution. The electron density map reveals an extended structure allowing the identification of specific subdomains. The tetramer is organized around a central core, including density assigned to the C-terminal coiled-coil domain, surrounded by four loosely attached arms radiating out from the center, which we have assigned to the N-terminal SH3 domains. We have further identified CD2AP as a substrate for cytoplasmic cathepsin L, a protease that is induced in early podocyte damage. Cleavage of CD2AP with cathepsin L results in a C-terminal core domain that is structurally competent but releases the CD2AP binding partner dendrin resulting in translocation of dendrin to the nucleus where it promotes apoptosis. Based on our analysis of the cathepsin L cleavage sites within CD2AP we conclude that cytosolic cathepsin L releases the N-terminal arms producing a structurally competent C-terminal core domain.

Structural Insights into KChIP4a Modulation of Kv4.3 Inactivation

Dynamic inactivation in Kv4 A-type K(+) current plays a critical role in regulating neuronal excitability by shaping action potential waveform and duration. Multifunctional auxiliary KChIP1-4 subunits, which share a high homology in their C-terminal core regions, exhibit distinctive modulation of inactivation and surface expression of pore-forming Kv4 subunits. However, the structural differences that underlie the functional diversity of Kv channel-interacting proteins (KChIPs) remain undetermined. Here we have described the crystal structure of KChIP4a at 3.0A resolution, which shows distinct N-terminal alpha-helices that differentiate it from other KChIPs. Biochemical experiments showed that competitive binding of the Kv4.3 N-terminal peptide to the hydrophobic groove of the core of KChIP4a causes the release of the KChIP4a N terminus that suppresses the inactivation of Kv4.3 channels. Electrophysiology experiments confirmed that the first N-terminal alpha-helix peptide (residues 1-34) of KChIP4a, either by itself or fused to N-terminal truncated Kv4.3, can confer slow inactivation. We propose that N-terminal binding of Kv4.3 to the core of KChIP4a mobilizes the KChIP4a N terminus, which serves as the slow inactivation gate.

Annexin A2 Binding to Endosomes and Functions in Endosomal Transport Are Regulated by Tyrosine 23 Phosphorylation

The phospholipid-binding annexin A2 (AnxA2) is known to play a role in the regulation of membrane and actin dynamics, in particular in the endocytic pathway. The protein is present on early endosomes, where it regulates membrane traffic, including the biogenesis of multivesicular transport intermediates destined for late endosomes. AnxA2 membrane association depends on the protein N terminus and membrane cholesterol but does not involve the AnxA2 ligand p11/S100A10. However, the precise mechanisms that control AnxA2 membrane association and function are not clear. In the present study, we have investigated the role of AnxA2 N-terminal phosphorylation in controlling association to endosomal membranes and functions. We found that endosomal AnxA2 was partially tyrosine-phosphorylated and that mutation of Tyr-23 to Ala (AnxA2Y23A), but not of Ser-25 to Ala, impaired AnxA2 endosome association. We then found that the AnxA2Y23A mutant was unable to bind endosomes in vivo, whereas a phospho-mimicking AnxA2 mutant (Y23D) showed efficient endosome binding capacity. Similarly, we found that AnxA2Y23D interacted more efficiently with liposomes in vitro when compared with AnxA2Y23A. To investigate the role of Tyr-23 in vivo, AnxA2 was knocked down with small interfering RNAs, and then cells were recomplemented with RNA interference-resistant forms of the protein. Using this strategy, we could show that AnxA2Y23D, but not AnxA2Y23A, could restore early-to-late endosome transport after AnxA2 depletion. We conclude that phosphorylation of Tyr-23 is essential for proper endosomal association and function of AnxA2, perhaps because it stabilizes membrane-associated protein via a conformational change.

Annexin A2 Interactions with Rab14 in Alveolar Type II Cells

Annexin A2, a calcium-dependent phospholipid-binding protein, is abundantly expressed in alveolar type II cells where it plays a role in lung surfactant secretion. Nevertheless, little is known about the details of its cellular pathways. To identify annexin A2-regulated or associated proteins, we silenced endogenous annexin A2 expression in rat alveolar type II cells by RNA interference and assessed the change of the cellular transcriptome by DNA microarray analysis. The loss of annexin A2 resulted in the change of 61 genes. Thirteen of the selected genes (11 down-regulated and 2 up-regulated genes) were validated by real time quantitative PCR. When the loss of rat annexin A2 was rescued by overexpressing EGFP-tagged human annexin A2, six of seven selected targets returned to their normal expression level, indicating that these genes are indeed annexin A2-associated targets. One of the targets, Rab14, co-immunoprecipitated with annexin A2. Rab14 also co-localized in part with annexin A2 and lamellar bodies in alveolar type II cells. The silencing of Rab14 resulted in a decrease in surfactant secretion, suggesting that Rab14 may play a role in surfactant secretion.