Comparison of dynamics of wildtype and V94M human UDP-galactose 4-epimerase—A computational perspective on severe epimerase-deficiency galactosemia

UDP-galactose 4′-epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose, an important step in galactose catabolism. Type III galactosemia, an inherited metabolic disease, is associated with mutations in human GALE. The V94M mutation has been associated with a very severe form of type III galactosemia. While a variety of structural and biochemical studies have been reported that elucidate differences between the wildtype and this mutant form of human GALE, little is known about the dynamics of the protein and how mutations influence structure and function. We performed molecular dynamics simulations on the wildtype and V94M enzyme in different states of substrate and cofactor binding. In the mutant, the average distance between the substrate and both a key catalytic residue (Tyr157) and the enzyme-bound NAD⁺ cofactor and the active site dynamics are altered making substrate binding slightly less stable. However, overall stability or dynamics of the protein is not altered. This is consistent with experimental findings that the impact is largely on the turnover number (k_cat), with less substantial effects on K_m. Active site fluctuations were found to be correlated in enzyme with substrate bound to just one of the subunits in the homodimer suggesting inter-subunit communication. Greater active site loop mobility in human GALE compared to the equivalent loop in Escherichia coli GALE explains why the former can catalyze the interconversion of UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine while the bacterial enzyme cannot. This work illuminates molecular mechanisms of disease and may inform the design of small molecule therapies for type III galactosemia.     

Protein kinase C-lambda knockout in embryonic stem cells and adipocytes impairs insulin-stimulated glucose transport

Atypical protein kinase C (aPKC) isoforms have been suggested to mediate insulin effects on glucose transport in adipocytes and other cells. To more rigorously test this hypothesis, we generated mouse embryonic stem (ES) cells and ES-derived adipocytes in which both aPKC-lambda alleles were knocked out by recombinant methods. Insulin activated PKC-lambda and stimulated glucose transport in wild-type (WT) PKC-lambda(+/+), but not in knockout PKC-lambda(-/-), ES cells. However, insulin-stimulated glucose transport was rescued by expression of WT PKC-lambda in PKC-lambda(-/-) ES cells. Surprisingly, insulin-induced increases in both PKC-lambda activity and glucose transport were dependent on activation of proline-rich tyrosine protein kinase 2, the ERK pathway, and phospholipase D (PLD) but were independent of phosphatidylinositol 3-kinase (PI3K) in PKC-lambda(+/+) ES cells. Interestingly, this dependency was completely reversed after differentiation of ES cells to adipocytes, i.e. insulin effects on PKC-lambda and glucose transport were dependent on PI3K, rather than proline-rich tyrosine protein kinase 2/ERK/PLD. As in ES cells, insulin effects on glucose transport were absent in PKC-lambda(-/-) adipocytes but were rescued by expression of WT PKC-lambda in these adipocytes. Our findings suggest that insulin activates aPKCs and glucose transport in ES cells by a newly recognized PI3K-independent ERK/PLD-dependent pathway and provide a compelling line of evidence suggesting that aPKCs are required for insulin-stimulated glucose transport, regardless of whether aPKCs are activated by PI3K-dependent or PI3K-independent mechanisms.     

EM-fold: de novo atomic-detail protein structure determination from medium-resolution density maps

Electron density maps of membrane proteins or large macromolecular complexes are frequently only determined at medium resolution between 4?? and 10??, either by cryo-electron microscopy or X-ray crystallography. In these density maps, the general arrangement of secondary structure elements (SSEs) is revealed, whereas their directionality and connectivity remain elusive. We demonstrate that the topology of proteins with up to 250 amino acids can be determined from such density maps when combined with a computational protein folding protocol. Furthermore, we accurately reconstruct atomic detail in loop regions and amino acid side chains not visible in the experimental data. The EM-Fold algorithm assembles the SSEs de novo before atomic detail is added using Rosetta. In a benchmark of 27 proteins, the protocol consistently and reproducibly achieves models with root mean square deviation values <3??.     

Deregulation of focal adhesion formation and cytoskeletal tension due to loss of A-type lamins

The nuclear lamina mechanically integrates the nucleus with the cytoskeleton and extracellular environment and regulates gene expression. These functions are exerted through direct and indirect interactions with the lamina's major constituent proteins, the A-type lamins, which are encoded by the LMNA gene. Using quantitative stable isotope labeling-based shotgun proteomics we have analyzed the proteome of human dermal fibroblasts in which we have depleted A-type lamins by means of a sustained siRNA-mediated LMNA knockdown. Gene ontology analysis revealed that the largest fraction of differentially produced proteins was involved in actin cytoskeleton organization, in particular proteins involved in focal adhesion dynamics, such as actin-related protein 2 and 3 (ACTR2/3), subunits of the ARP2/3 complex, and fascin actin-bundling protein 1 (FSCN1). Functional validation using quantitative immunofluorescence showed a significant reduction in the size of focal adhesion points in A-type lamin depleted cells, which correlated with a reduction in early cell adhesion capacity and an increased cell motility. At the same time, loss of A-type lamins led to more pronounced stress fibers and higher traction forces. This phenotype could not be mimicked or reversed by experimental modulation of the STAT3-IL6 pathway, but it was partly recapitulated by chemical inhibition of the ARP2/3 complex. Thus, our data suggest that the loss of A-type lamins perturbs the balance between focal adhesions and cytoskeletal tension. This imbalance may contribute to mechanosensing defects observed in certain laminopathies.     

Cryo-Electron Microscopy Structure of an Adenovirus-Integrin Complex Indicates Conformational Changes in both Penton Base and Integrin

A structure of adenovirus type 12 (HAdV12) complexed with a soluble form of integrin αvβ5 was determined by cryo-electron microscopy (cryoEM) image reconstruction. Subnanometer resolution (8 Å) was achieved for the icosahedral capsid with moderate resolution (27 Å) for integrin density above each penton base. Modeling with αvβ3 and α_IIbβ3 crystal structures indicates that a maximum of four integrins fit over the pentameric penton base. The close spacing (∼60 Å) of the RGD protrusions on penton base precludes integrin binding in the same orientation to neighboring RGD sites. Flexible penton-base RGD loops and incoherent averaging of bound integrin molecules explain the moderate resolution observed for the integrin density. A model with four integrins bound to a penton base suggests that integrin might extend one RGD-loop in the direction that could induce a conformational change in the penton base involving clockwise untwisting of the pentamer. A global conformational change in penton base could be one step on the way to the release of Ad vertex proteins during cell entry. Comparison of the cryoEM structure with bent and extended models for the integrin ectodomain reveals that integrin adopts an extended conformation when bound to the Ad penton base, a multivalent viral ligand. These findings shed further light on the structural basis of integrin binding to biologically relevant ligands, as well as on the molecular events leading to HAdV cell entry.A growing number of viruses have been identified as using one of the 24 types of integrin heterodimers as a receptor for cell entry (32). Integrins are cell surface molecules involved in the regulation of adhesion, migration, growth, and differentiation (11). The large multidomained extracellular segments of α and β integrin subunits bind a variety of ligands, including viral ligands, while the smaller intracellular domains interact with cytoskeletal proteins (Fig. ​(Fig.1A).1A). These extracellular and intracellular interactions facilitate bidirectional signaling, with the initiating events occurring either outside of the cell (outside-in signaling) or within the cell (inside-out signaling) (24). Integrin clustering has been established as having an important role in outside-in signaling (9, 19, 20, 44). Clustering results in the formation of focal adhesions, which are organized intracellular complexes, that facilitate downstream signaling cascades within the cell (24).Open in a separate windowFIG. 1.Integrin domains and conformations. (A) Structural domains of integrin αv and β chains, including the extracellular domains, transmembrane-spanning regions, and small cytoplasmic domains, shown in extended schematic forms. The domains are represented as 10Å-resolution density maps based on crystallographic coordinates. The membrane is represented by a gray bar. (Modified from Stewart and Nemerow (32) and reprinted with permission from Elsevier.) (B) Models for soluble αvβ5 integrin with Fos/Jun dimerization domains. Each chain has a six residue glycine-rich linker between the ectodomain and the Fos or Jun dimerization domain. The model of a bent integrin conformation (left) was built as a composite of αvβ3 integrin crystal structures, PDB-IDs 1L5G and 1U8C (42, 43), and the crystal structure of c-Fos/c-Jun bound to DNA, PDB-ID 1FOS (6). The model of an extended integrin conformation (right) is similar to the extended model docked into the HAdV12/αvβ5 cryo structure (Fig. ​(Fig.8B8B).Studies of adenovirus (Ad) interactions with αv integrins provided some of the first evidence of the virus-induced signaling events (13, 14). The Ad penton base capsid protein, which sits at the 12 vertices of the icosahedral capsid, has five prominent Arg-Gly-Asp (RGD) containing loops that are flexible and protrude from the viral surface (31, 48). Receptor-mediated endocytosis of Ad is stimulated by interaction of the RGD-containing penton base with αvβ3 and αvβ5 integrins (34). This interaction leads to receptor clustering, followed by tyrosine phosphorylation/activation of focal adhesion kinase, as well as activation of p130CAS, phosphatidylinositol 3-OH-kinase, and the Rho family of small GTPases, and subsequent actin polymerization and Ad internalization (32). Integrin signaling events also lead to production of proinflammatory cytokines (23) and may result in increased survival of certain host cells through subsequent signaling to protein kinase B (AKT) (25).Multiple studies indicate that after interaction with an RGD-containing ligand a straightening of the integrin extracellular domains occurs, leading to the “extension” or “switchblade” model for integrin activation (16, 45). In the extension model the headpiece domains, which are closest to the RGD interaction site, have a “closed” conformation in the low-affinity, unliganded state. This state is characterized by the close proximity of the α and β subunits at the “knees” or midpoints of the extracellular segments. In contrast, the high-affinity, ligand-bound state in the extension model is distinguished by an “open” headpiece conformation with separation at the knees of the extracellular segments. The location of the RGD binding site between the α-subunit β-propellor and the β-subunit I domain was first visualized in the crystal structure of the αvβ3 extracellular segment with a bound RGD peptide (43). In this structure the RGD site is folded back toward the membrane, and the integrin is in a closed conformation. The closed conformation has also been observed in crystal structures of the αvβ3 ectodomain without an RGD peptide (41) and the α_IIbβ3 ectodomain (47).The open integrin conformation has been characterized as having a large separation of up to ∼70 Å between the knees of α and β subunits (16). Four slightly different open headpiece conformations were observed in crystal structures of the α_IIbβ3 headpiece with bound fibrinogen-mimetic therapeutics (38). These structures show that the change from a closed to an open headpiece conformation is accompanied by a piston-like motion of helix α7 in the β-chain I domain and a large swing of the β-chain hybrid domain of up to 69°, as well as extension and separation of the two integrin chains. Comparison of the available αvβ3 and α_IIbβ3 crystal structures is providing information on the interdomain angle variation and flexibility between domains (47).One aspect of the extension model is that separation of the C-terminal, intracellular portions of the α and β subunits leads to inside-out activation. This concept is supported by nuclear magnetic resonance structures of the cytoplasmic tails of α_IIbβ3 showing that the membrane-proximal helices engage in a weak interaction that can be disrupted by constitutively activating mutations or by talin, a protein found in high concentrations in focal adhesions (33). The concept that the integrin α and β subunits must also separate during outside-in signaling is supported by a study involving a disulfide-bonded mutant of α_IIbβ3 integrin (46). When the α and β subunits are linked in the vicinity of the transmembrane helices the mutant α_IIbβ3 is still able to bind ligand, mediate adhesion, and undergo antibody-induced clustering. However, the disulfide-bonded mutant exhibits defects in focal adhesion formation and focal adhesion kinase activation. Reduction of the disulfide bond or single cysteine mutants rescues signaling.A competing model for integrin activation, called the “deadbolt” model, proposes only small conformational changes in the integrin β-chain I domain upon RGD binding (2). This model is based on crystal structures of the αvβ3 ectodomain with or without an RGD peptide (41, 43). Both of these αvβ3 structures reveal a bent integrin conformation with a closed headpiece conformation. However, the RGD peptide was soaked into a preformed crystal of αvβ3 and crystal contacts may have prevented conformational changes.There are relatively few and only moderate resolution structures of virus-integrin complexes. A moderate resolution cryoEM structure has been determined for the Picornavirus echovirus 1 (EV1) in complex with the I domain of the α2 integrin subunit (39). Docking of crystal structures of EV1 and the α2 I domain into the cryoEM density indicates that the I domain binds within a canyon on the surface of EV1 and that five integrins could potentially bind at one vertex of the icosahedral capsid. Confocal fluorescence microscopy experiments indicated that EV1 causes integrin clustering on human osteosarcoma cells stably transfected with α2 integrin. However, it could not be determined whether the bound integrins were in the inactive (bent) or active (extended) conformation.Moderate resolution (∼21 Å) cryoEM structures of Ad type 2 (HAdV2) and HAdV12 in complex with a soluble form of αvβ5 integrin revealed a ring of integrin density over each penton base capsid protein (5). Better-defined integrin density was observed in the HAdV12/integrin complex, supporting the idea suggested from sequence alignments that the RGD loop of the HAdV12 penton base is shorter and less flexible than that of HAdV2. This study also suggested that the precise spatial arrangement of the five RGD protrusions on the penton base might promote integrin clustering, which may lead to the intracellular signaling events required for virus internalization into a host cell. A similar spacing of RGD-containing integrin-binding sites around the fivefold axis of icosahedral virions has been noted for Ad, foot-and-mouth disease virus, and coxsackievirus A9 (32).We present here a significantly higher-resolution cryoEM structure of HAdV12 complexed with soluble αvβ5 that provides insight into the Ad-integrin interaction. The resolution of the icosahedral capsid portion of the Ad-integrin complex was improved to 8 Å, and the capsid shows clearly resolved α-helices, which allows accurate docking of the penton base crystal structure within the cryoEM density. The resolution of the integrin density is more moderate due to flexibility of the RGD-containing surface loop of penton base and incoherent averaging of integrin heterodimers. Nevertheless, modeling studies with available integrin crystal structures have enabled us to distinguish between a bent or extended conformation (Fig. ​(Fig.1B)1B) when αvβ5 binds to the multivalent ligand presented by the Ad penton base. The cryoEM structural analysis also indicates that integrin induces a conformational change in penton base.     

Molecular Basis of Calcium-Sensitizing and Desensitizing Mutations of the Human Cardiac Troponin C Regulatory Domain: A Multi-Scale Simulation Study

Troponin C (TnC) is implicated in the initiation of myocyte contraction via binding of cytosolic and subsequent recognition of the Troponin I switch peptide. Mutations of the cardiac TnC N-terminal regulatory domain have been shown to alter both calcium binding and myofilament force generation. We have performed molecular dynamics simulations of engineered TnC variants that increase or decrease sensitivity, in order to understand the structural basis of their impact on TnC function. We will use the distinction for mutants that are associated with increased affinity and for those mutants with reduced affinity. Our studies demonstrate that for GOF mutants V44Q and L48Q, the structure of the physiologically-active site II binding site in the -free (apo) state closely resembled the -bound (holo) state. In contrast, site II is very labile for LOF mutants E40A and V79Q in the apo form and bears little resemblance with the holo conformation. We hypothesize that these phenomena contribute to the increased association rate, , for the GOF mutants relative to LOF. Furthermore, we observe significant positive and negative positional correlations between helices in the GOF holo mutants that are not found in the LOF mutants. We anticipate these correlations may contribute either directly to affinity or indirectly through TnI association. Our observations based on the structure and dynamics of mutant TnC provide rationale for binding trends observed in GOF and LOF mutants and will guide the development of inotropic drugs that target TnC.     

Cryo-Electron Microscopy Structure of Adenovirus Type 2 Temperature-Sensitive Mutant 1 Reveals Insight into the Cell Entry Defect

The structure of the adenovirus type 2 temperature-sensitive mutant 1 (Ad2ts1) was determined to a resolution of 10 Å by cryo-electron microscopy single-particle reconstruction. Ad2ts1 was prepared at a nonpermissive temperature and contains the precursor forms of the capsid proteins IIIa, VI, and VIII; the core proteins VII, X (mu), and terminal protein (TP); and the L1-52K protein. Cell entry studies have shown that although Ad2ts1 can bind the coxsackievirus and Ad receptor and undergo internalization via αv integrins, this mutant does not escape from the early endosome and is targeted for degradation. Comparison of the Ad2ts1 structure to that of mature Ad indicates that Ad2ts1 has a different core architecture. The Ad2ts1 core is closely associated with the icosahedral capsid, a connection which may be mediated by preproteins IIIa and VI. Density within hexon cavities is assigned to preprotein VI, and membrane disruption assays show that hexon shields the lytic activity of both the mature and precursor forms of protein VI. The internal surface of the penton base in Ad2ts1 appears to be anchored to the core by interactions with preprotein IIIa. Our structural analyses suggest that these connections to the core inhibit the release of the vertex proteins and lead to the cell entry defect of Ad2ts1.Cryo-electron microscopy (cryo-EM) studies of adenovirus (Ad) combined with atomic resolution structures of component proteins (hexon, penton base, fiber, and protease) have led to a detailed structural model for the mature Ad virion (31). While the Ad protein capsid is icosahedral, the core does not follow the overall symmetry of the particle, and thus the core is not well represented in cryo-EM structures (43). The core is composed of the 36-kb double-stranded DNA (dsDNA) genome complexed with four viral proteins (V, VII, mu, and terminal protein [TP]) and the virally encoded cysteine protease. The core of the mature virion may also contain a few copies of the L1-52K protein (7), a possible scaffolding protein that is present in higher copy numbers in assembling virions (18).The capsid contains the major capsid proteins, hexon, penton base, and fiber, together with four minor capsid proteins (IIIa, VI, VIII, and IX). Cryo-EM difference mapping analyses have led to revised assignments for the locations of the minor capsid proteins, with protein IX on the exterior and the other three proteins on the inner capsid surface (9, 38). A scanning transmission EM study indicated that four trimers of protein IX stabilize the group of nine hexons in the center of each facet (11). However, more recent cryo-EM studies indicated that only the N-terminal domain of protein IX forms these trimeric assemblies (37, 38), while the C-terminal domain, which has a long predicted α-helix with strong propensity for coiled coil formation, associates in helical bundles at the facet edges (38). Two cryo-EM studies support the assignment of the tetrameric helical bundle on the capsid exterior to the C-terminal domain of protein IX (10, 23). Curiously, 12 monomers of protein IX per facet assemble into four trimers with their N-terminal domains and three tetramers with their C-terminal domains.The internal location for protein IIIa below the penton base and surrounding peripentonal hexons was confirmed by a study of virions with N-terminally tagged protein IIIa (39). Although the locations for proteins VI and VIII have not been experimentally confirmed, these proteins are more than likely on the internal side of the capsid, as there is no remaining unassigned cryo-EM density on the exterior of the capsid. In addition, proteins VI and VIII are two of the viral proteins that are produced in precursor form and cleaved by the viral protease during maturation of the assembled virion (22). The protease is presumed to be packaged within the interior of the virion, and therefore the assignment of proteins VI and VIII to the interior of the capsid where they would be accessible to the protease is logical. Density within the internal cavity of all 240 hexon trimers in the Ad capsid has been assigned to protein VI on the basis of biochemical and temperature sensitivity studies (38, 51).Ad cell entry begins with attachment of the Ad fiber to either coxsackievirus and Ad receptor (3) or CD46 (12), which serve as the primary attachment receptors for Ad on most cell types (31). Internalization via clathrin-mediated endocytosis is triggered by association of the Ad penton base with αv integrins (49). Escape from the endosome is facilitated by the membrane lytic activity of protein VI, which is released from the virion in the low-pH environment of the early endosome (50). The stepwise dismantling of the Ad virion during cell entry has been described biochemically (15) but has not been fully characterized structurally. After endosomal escape, the partially uncoated Ad virion is transported along microtubules (44) to the nucleus, where the viral genome is inserted into the nucleus via a nuclear pore complex.Propagation of an Ad2 temperature-sensitive mutant (Ad2ts1) at nonpermissive temperatures (>39°C) results in the synthesis of virions that have an uncoating defect (28, 30, 46). Although these Ad2ts1 particles are capable of interacting with coxsackievirus and Ad receptor and undergoing internalization via association with αv integrins, they are unable to escape the early endosome and thus are targeted for degradation in lysosomes (13, 14). The Ad2ts1 genetic defect is a point mutation (P137L) in protease that is linked to a defect in packaging into the virion (33). In wild-type Ad virions, the protease is activated inside nascent virions by the viral DNA as well as an 11-amino-acid peptide from the C-terminal end of protein VI (22). The Ad protease mediates the maturational cleavage of six structural proteins, i.e., IIIa, VI, VII, VIII, mu, and TP, as well as the presumed scaffolding protein L1-52K (26, 47, 48). In Ad2ts1 particles these cleavages do not occur. The presence of the precursor forms of these proteins in Ad2ts1 is associated with greater capsid stability (42, 50).Here we present a cryo-EM structural study of the Ad2ts1 particle that provides insight into the cell entry defect of this temperature-sensitive mutant. Comparison of the Ad2ts1 structure with that of a mature Ad virion indicates that the major differences are in the interior of the virion.     

Lung surfactant secretion by interalveolar Ca2+ signaling

Although clusters of alveoli form the acinus, which is the most distal respiratory unit, it is not known whether interalveolar communication coordinates acinar surfactant secretion. To address this, we applied real-time digital imaging in conjunction with photo-excited Ca2+ uncaging in intact alveoli of the isolated, blood-perfused rat lung. We loaded alveolar cells with the Ca2+ cage o-nitrophenyl EGTA-AM (NP-EGTA-AM) together with the fluorophores, fluo 4, or LysoTracker green (LTG) to determine, respectively, the cytosolic Ca2+ concentration ([Ca2+]cyt) or type 2 cell secretion. To uncage Ca2+ from NP-EGTA, we exposed a region in a selected alveolus to high-intensity UV illumination. As a result, fluo 4 fluorescence increased, whereas LTG fluorescence decreased, in the photo-targeted region, indicating that uncaging both increased [Ca2+]cyt and induced secretion. Concomitantly, [Ca2+]cyt increases conducted from the uncaging site induced type 2 cell secretion in both the selected alveolus as well as in neighboring alveoli, indicating the presence of interalveolar communication. These conducted responses were inhibited by pretreating alveoli with the connexin43 (Cx43)-inhibiting peptides gap 26 and gap 27. However, although the conducted [Ca2+]cyt increase diminished with distance from the uncaging site, type 2 cell secretion rates were similar at all locations. We conclude that Cx43-dependent, interalveolar Ca2+ signals regulate type 2 cell secretion in adjacent alveoli. Such interalveolar communication might facilitate acinar coordination of alveolar function.