Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium.

Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about the pathogenic determinants of this organism. We have previously identified a cell-wall-anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad-spectrum binding to extracellular matrix proteins. Here, we analysed the draft genome of strain TX0016 for potential microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Genome-based bioinformatics identified 22 predicted cell-wall-anchored E. faecium surface proteins (Fms), of which 15 (including Acm) had characteristics typical of MSCRAMMs, including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one [Fms10; redesignated second collagen adhesin of E. faecium (Scm)] revealed that recombinant Scm(65) (A- and B-domains) and Scm(36) (A-domain) bound to collagen type V efficiently in a concentration-dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism measurements of recombinant Scm(36) and of Acm(37) indicated that these proteins were rich in beta-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; nine of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated EbpC(fm)), detected a 'ladder' pattern of high-molecular-mass protein bands in a Western blot analysis of cell surface extracts from E. faecium, suggesting that EbpC(fm) is polymerized into a pilus structure. Further analysis of the transcripts of the corresponding gene cluster indicated that fms1 (ebpA(fm)), fms5 (ebpB(fm)) and ebpC(fm) are co-transcribed, a result consistent with those for pilus-encoding gene clusters of other Gram-positive bacteria. All 15 genes occurred frequently in 30 clinically derived diverse E. faecium isolates tested. The common occurrence of MSCRAMM- and pilus-encoding genes and the presence of a second collagen-binding protein may have important implications for our understanding of this emerging pathogen.

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds.

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl](1/2) at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys(36)-Cys(49) and two disulfide bonds formed by two pair of consecutive cysteines, Cys(22)-Cys(23) and Cys(56)-Cys(57), a unique disulfide structure of polypeptide that has not been documented previously.

Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins.

Cells use molecular chaperones and proteases to implement the essential quality control mechanism of proteins. The DegP (HtrA) protein, essential for the survival of Escherichia coli cells at elevated temperatures with homologues found in almost all organisms uniquely has both functions. Here we report a mechanism for DegP to activate both functions via formation of large cage-like 12- and 24-mers after binding to substrate proteins. Cryo-electron microscopic and biochemical studies revealed that both oligomers are consistently assembled by blocks of DegP trimers, via pairwise PDZ1-PDZ2 interactions between neighboring trimers. Such interactions simultaneously eliminate the inhibitory effects of the PDZ2 domain. Additionally, both DegP oligomers were also observed in extracts of E. coli cells, strongly implicating their physiological importance.

Structure of the Hsp110:Hsc70 nucleotide exchange machine.

Hsp70s mediate protein folding, translocation, and macromolecular complex remodeling reactions. Their activities are regulated by proteins that exchange ADP for ATP from the nucleotide-binding domain (NBD) of the Hsp70. These nucleotide exchange factors (NEFs) include the Hsp110s, which are themselves members of the Hsp70 family. We report the structure of an Hsp110:Hsc70 nucleotide exchange complex. The complex is characterized by extensive protein:protein interactions and symmetric bridging interactions between the nucleotides bound in each partner protein's NBD. An electropositive pore allows nucleotides to enter and exit the complex. The role of nucleotides in complex formation and dissociation, and the effects of the protein:protein interactions on nucleotide exchange, can be understood in terms of the coupled effects of the nucleotides and protein:protein interactions on the open-closed isomerization of the NBDs. The symmetrical interactions in the complex may model other Hsp70 family heterodimers in which two Hsp70s reciprocally act as NEFs.

All in the family: atypical Hsp70 chaperones are conserved modulators of Hsp70 activity.

Divergent relatives of the Hsp70 protein chaperone such as the Hsp110 and Grp170 families have been recognized for some time, yet their biochemical roles remained elusive. Recent work has revealed that these "atypical" Hsp70s exist in stable complexes with classic Hsp70s where they exert a powerful nucleotide-exchange activity that synergizes with Hsp40/DnaJ-type cochaperones to dramatically accelerate Hsp70 nucleotide cycling. This represents a novel evolutionary transition from an independent protein-folding chaperone to what appears to be a dedicated cochaperone. Contributions of the atypical Hsp70s to established cellular roles for Hsp70 now must be deciphered.

The role of Sse1 in the de novo formation and variant determination of the [PSI+] prion.

Yeast prions are a group of non-Mendelian genetic elements transmitted as altered and self-propagating conformations. Extensive studies in the last decade have provided valuable information on the mechanisms responsible for yeast prion propagation. How yeast prions are formed de novo and what cellular factors are required for determining prion "strains" or variants--a single polypeptide capable of existing in multiple conformations to result in distinct heritable phenotypes--continue to defy our understanding. We report here that Sse1, the yeast ortholog of the mammalian heat-shock protein 110 (Hsp110) and a nucleotide exchange factor for Hsp70 proteins, plays an important role in regulating [PSI+] de novo formation and variant determination. Overproduction of the Sse1 chaperone dramatically enhanced [PSI+] formation whereas deletion of SSE1 severely inhibited it. Only an unstable weak [PSI+] variant was formed in SSE1 disrupted cells whereas [PSI+] variants ranging from very strong to very weak were formed in isogenic wild-type cells under identical conditions. Thus, Sse1 is essential for the generation of multiple [PSI+] variants. Mutational analysis further demonstrated that the physical association of Sse1 with Hsp70 but not the ATP hydrolysis activity of Sse1 is required for the formation of multiple [PSI+] variants. Our findings establish a novel role for Sse1 in [PSI+] de novo formation and variant determination, implying that the mammalian Hsp110 may likewise be involved in the etiology of protein-folding diseases.

Molecular cross talk between misfolded proteins in animal models of Alzheimer's and prion diseases.

The central event in protein misfolding disorders (PMDs) is the accumulation of a misfolded form of a naturally expressed protein. Despite the diversity of clinical symptoms associated with different PMDs, many similarities in their mechanism suggest that distinct pathologies may cross talk at the molecular level. The main goal of this study was to analyze the interaction of the protein misfolding processes implicated in Alzheimer's and prion diseases. For this purpose, we inoculated prions in an Alzheimer's transgenic mouse model that develop typical amyloid plaques and followed the progression of pathological changes over time. Our findings show a dramatic acceleration and exacerbation of both pathologies. The onset of prion disease symptoms in transgenic mice appeared significantly faster with a concomitant increase on the level of misfolded prion protein in the brain. A striking increase in amyloid plaque deposition was observed in prion-infected mice compared with their noninoculated counterparts. Histological and biochemical studies showed the association of the two misfolded proteins in the brain and in vitro experiments showed that protein misfolding can be enhanced by a cross-seeding mechanism. These results suggest a profound interaction between Alzheimer's and prion pathologies, indicating that one protein misfolding process may be an important risk factor for the development of a second one. Our findings may have important implications to understand the origin and progression of PMDs.

Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli.

Phosphatidylcholine (PC) has been widely used in place of naturally occurring phosphatidylethanolamine (PE) in reconstitution of bacterial membrane proteins. However, PC does not support native structure or function for several reconstituted transport proteins. Lactose permease (LacY) of Escherichia coli, when reconstituted in E. coli phospholipids, exhibits energy-dependent uphill and energy-independent downhill transport function and proper conformation of periplasmic domain P7, which is tightly linked to uphill transport function. LacY expressed in cells lacking PE and containing only anionic phospholipids exhibits only downhill transport and lacks native P7 conformation. Reconstitution of LacY in the presence of E. coli-derived PE, but not dioleoyl-PC, results in uphill transport. We now show that LacY exhibits uphill transport and native conformation of P7 when expressed in a mutant of E. coli in which PC completely replaces PE even though the structure is not completely native. E. coli-derived PC and synthetic PC species containing at least one saturated fatty acid also support the native conformation of P7 dependent on the presence of anionic phospholipids. Our results demonstrate that the different effects of PE and PC species on LacY structure and function cannot be explained by differences in the direct interaction of the lipid head groups with specific amino acid residues alone but are due to more complex effects of the physical and chemical properties of the lipid environment on protein structure. This conclusion is supported by the effect of different lipids on the proper folding of domain P7, which indirectly influences uphill transport function.

Upper crustal deformation in continent-continent collision: A case study from the Bernard nappe complex (Valais, Switzerland)

The Penninic nappes in the Swiss Alps formed during continental collision between the Adriatic and European plates in Cenozoic times. Although intensely studied, the finite geometry of the basement-bearing Penninic nappes in western Switzerland has remained a matter of debate for decades (e.g., “Siviez-Mischabel dilemma”) and the paleogeographic origin of various nappes has been disputed. Here, we present new structural data for the central part of the Penninic Bernard nappe complex, which contains pre-Permian basement and Permo-Mesozoic metasedimentary units. Our lithological and structural observations indicate that the discrepancy between the different structural models proposed for the Bernard nappe complex can be explained by a lateral discontinuity. In the west, the presence of a Permian graben caused complex isoclinal folding, whereas in the east, the absence of such a graben resulted mainly in imbricate thrusting. The overall geometry of the Bernard nappe complex is the result of three main deformation phases: (1) detachment of Mesozoic cover sediments along Triassic evaporites (Evolène phase) during the early stages of collision, (2) Eocene top-to-the-N(NW) nappe stacking (Anniviers phase), and (3) subsequent backfolding and backshearing (Mischabel phase). The southward localized backshearing is key to understand the structural position and paleogeographic origin of units, such as the Frilihorn and Cimes Blanches “nappes” and the Antrona ophiolites. Based on these observations, we present a new tectonic model for the entire Penninic region of western Switzerland and discuss this model in terms of continental collision zone processes.

Cross currents in protein misfolding disorders: interactions and therapy.

Protein Misfolding Disorders (PMDs) are a group of diseases characterized by the accumulation of abnormally folded proteins. Despite the wide range of proteins and tissues involved, PMDs share similar molecular and pathogenic mechanisms. Several epidemiological, clinical and experimental reports have described the co-existence of PMDs, suggesting a possible cross-talk between them. A better knowledge of the molecular basis of PMDs could have important implications for understanding the mechanism by which these diseases appear and progress and ultimately to develop novel strategies for treatment. Due to their similar molecular mechanisms, common therapeutic strategies could be applied for the diseases in this group.

Symmetric inverse consistent nonlinear registration driven by mutual information.

A nonlinear viscoelastic image registration algorithm based on the demons paradigm and incorporating inverse consistent constraint (ICC) is implemented. An inverse consistent and symmetric cost function using mutual information (MI) as a similarity measure is employed. The cost function also includes regularization of transformation and inverse consistent error (ICE). The uncertainties in balancing various terms in the cost function are avoided by alternatively minimizing the similarity measure, the regularization of the transformation, and the ICE terms. The diffeomorphism of registration for preventing folding and/or tearing in the deformation is achieved by the composition scheme. The quality of image registration is first demonstrated by constructing brain atlas from 20 adult brains (age range 30-60). It is shown that with this registration technique: (1) the Jacobian determinant is positive for all voxels and (2) the average ICE is around 0.004 voxels with a maximum value below 0.1 voxels. Further, the deformation-based segmentation on Internet Brain Segmentation Repository, a publicly available dataset, has yielded high Dice similarity index (DSI) of 94.7% for the cerebellum and 74.7% for the hippocampus, attesting to the quality of our registration method.

Ar-40/Ar-39 Evidence for Middle Proterozoic (1300-1500 Ma) Slow Cooling of the Southern Black Hills, South Dakota, Midcontinent, North America: Implications for Early Proterozoic P-T Evolution and Posttectonic Magmatism

Ar-40/Ar-39 total gas and plateau dates from muscovite and biotite in the southern Black Hills, South Dakota, provide evidence for a period of Middle Proterozoic slow cooling. Early Proterozoic (1600-1650 Ma) mica dates were obtained from metasedimentary rocks located in a synformal structure between the Harney Peak and Bear Mountain domes and also south of Bear Mountain. Metamorphic rocks from the dome areas and undeformed samples of the similar to 1710 Ma Harney Peak Granite (HPG) yield Middle Proterozoic mica dates (similar to 1270-1500 Ma). Two samples collected between the synform and Bear Mountain dome yield intermediate total gas mica dates of similar to 1550 Ma. We suggest two end-member interpretations to explain the map pattern of cooling ages: (1) subhorizontal slow cooling of an area which exhibits variation in mica Ar retention intervals or (2) mild folding of a Middle Proterozoic (similar to 1500 Ma) similar to 300 degrees C isotherm. According to the second interpretation, the preservation of older dates between the domes may reflect reactivation of a preexisting synformal structure (and downwarping of relatively cold rocks) during a period of approximately east-west contraction and slow uplift during the Middle Proterozoic. The mica data, together with hornblende data from the Black Hills published elsewhere, indicate that the ambient country-rock temperature at the 3-4 kbar depth of emplacement of the HPG was between 350 degrees C and 500 degrees C, suggesting that the average upper crustal geothermal gradient was 25 degrees-40 degrees C/km prior to intrusion. The thermochronologic data suggest HPG emplacement was followed by a similar to 200 m.y. period of stability and tectonic quiescence with little uplift. We propose that crust thickened during the Early Proterozoic was uplifted and erosionally(?) thinned prior to similar to 1710 Ma and that the HPG magma was emplaced into isostatically stable crust of relatively normal thickness. We speculate that uplift and crustal thinning prior to HPG intrusion was the result of differential thinning of the subcrustal lithosphere beneath the Black Hills. If so, this process would have also caused an increase in mantle heat flux across the Moho and triggered vapor-absent melting of biotite to produce the HPG magma. This scenario for posttectonic granite generation is supported, in part, by the fact that in the whole of the Black Hills, the HPG is spatially associated with the deepest exposed Early Proterozoic country rock.

Softening the Lower Crust: Modes of Syn-Transport Transposition Around and Adjacent to a Deep Crustal Granulite Nappe, Parry Sound Domain, Grenville Province, Ontario, Canada

The Parry Sound domain is a granulite nappe-stack transported cratonward during reactivation of the ductile lower and middle crust in the late convergence of the Mesoproterozoic Grenville orogeny. Field observations suggest the following with respect to the ductile sheath: (1) Formation of a carapace of transposed amphibolite facies gneiss derived from and enveloping the western extremity of the Parry Sound domain and separating it from high-strain gneiss of adjacent allochthons. This ductile sheath formed dynamically around the moving granulite nappe through the development of systems of progressively linked shear zones. (2) Transposition initiated by hydration (amphibolization) of granulite facies gneiss by introduction of fluid along cracks accompanying pegmatite emplacement. Shear zones nucleated along pegmatite margins and subsequently linked and rotated. The source of the pegmatites was most likely subjacent migmatitic and pegmatite-rich units or units over which Parry Sound domain was transported. Comparison of gneisses of the ductile sheath with high-strain layered gneiss of adjacent allochthons show the mode of transposition of penetratively layered gneiss depended on whether or not the gneiss protoliths were amphibolite or granulite facies tectonites before initiation of transposition, resulting in, e.g., folding before shearing, no folding before shearing, respectively. Meter-scale truncation along high-strain gradients at the margins of both types of transposition-related shear zones observed within and marginal to Parry Sound domain mimic features at kilometer scales, implying that apparent truncation by transposition originating in a manner similar to the ductile sheath may be a common feature of deep crustal ductile reworking. Citation: Culshaw, N., C. Gerbi, and J. Marsh (2010), Softening the lower crust: Modes of syn-transport transposition around and adjacent to a deep crustal granulite nappe, Parry Sound domain, Grenville Province, Ontario, Canada, Tectonics, 29, TC5013, doi:10.1029/2009TC002537.

GPR Reflection Profiles of Clark and Commonwealth Glaciers, Dry Valleys, Antarctica

Englacial horizons deeper than 100 m are absent within 100 MHz ground-penetrating radar (GPR) surface profiles we recorded on Clark and Commonwealth Glaciers in the Antarctic Dry Valleys region. Both glaciers show continuous bottom horizons to 280 m, with bottom signal-to-noise ratios near 30 dB. Density horizons should fade below 50 m depth because impermeable ice occurred by 36 m. Folding within Commonwealth Glacier could preclude radar strata beneath about 80 m depth, but there is no significant folding within Clark Glacier. Strong sulfate concentrations and contrasts exist in our shallow ice core. However, it appears that high background concentration levels, and possible decreased concentration contrasts with depth placed their corresponding reflection coefficients at the limit of, or below, our system sensitivity by about 77 m depth. Further verification of this conclusion awaits processing of our deep-core chemistry profiles.

Hierarchical self-assembly of nucleotide-appended oligopyrenotides into defined supramolecular objects

Supramolecular DNA assembly blends DNA building blocks with synthetic organic and inorganic molecules giving structural and functional advantages both to the initial self-assembly process and to the final construct. Synthetic molecules can bring a number of additional interactions into DNA nanotechnology. Incorporating extended aromatic molecules as connectors of DNA strands allows folding of these strands through π-π stacking (DNA “foldamers”). In previous work it was shown that short oligopyrenotides (phosphodiester-linked pyrene oligomers) behave as staircase-like foldamers, which cooperatively self-assemble into two-dimensional supramolecular polymers in aqueous medium. Herein, we demonstrate that a 10-mer DNA-sequence modified with 7 pyrene units (see illustration) forms dimensionally-defined supramolecular polymers under thermodynamic conditions in water. We present the self-assembly behavior, morphological studies, and the spectroscopic properties of the investigated DNA-sequences (illustrative AFM picture shown below).