Crystal structure of an insect antifreeze protein reveals ordered waters on the ice-binding surface.

Antifreeze proteins (AFPs) are characterized by their ability to adsorb to the surface of ice crystals and prevent any further crystal growth. AFPs have independently evolved for this purpose in a variety of organisms that encounter the threat of freezing, including many species of polar fish, insects, plants and microorganisms. Despite their diverse origins and structures, it has been suggested that all AFPs can organize ice-like water patterns on one side of the protein (the ice-binding site) that helps bind the AFP to ice. Here, to test this hypothesis, we have solved the crystal structure at 2.05 Šresolution of an AFP from the longhorn beetle, Rhagium mordax with five molecules in the unit cell. This AFP is hyperactive, and its crystal structure resembles that of the R. inquisitor ortholog in having a ß-solenoid fold with a wide, flat ice-binding surface formed by four parallel rows of mainly Thr residues. The key difference between these structures is that the R. inquisitor AFP crystallized with its ice-binding site (IBS) making protein-protein contacts that limited the surface water patterns. Whereas the R. mordax AFP crystallized with the IBSs exposed to solvent enabling two layers of unrestricted ordered surface waters to be seen. These crystal waters make close matches to ice lattice waters on the basal and primary prism planes.

Carrot 'antifreeze' protein has an irregular ice-binding site that confers weak freezing point depression but strong inhibition of ice recrystallization.

Ice-binding proteins (IBPs) are found in many biological kingdoms where they protect organisms from freezing damage as antifreeze agents or inhibitors of ice recrystallization. Here, the crystal structure of recombinant IBP from carrot (Daucus carota) has been solved to a resolution of 2.3 Å. As predicted, the protein is a structural homologue of a plant polygalacturonase-inhibiting protein forming a curved solenoid structure with a leucine-rich repeat motif. Unexpectedly, close examination of its surface did not reveal any large regions of flat, regularly spaced hydrophobic residues that characterize the ice-binding sites (IBSs) of potent antifreeze proteins from freeze-resistant fish and insects. An IBS was defined by site-directed mutagenesis of residues on the convex surface of the carrot solenoid. This imperfect site is reminiscent of the irregular IBS of grass 'antifreeze' protein. Like the grass protein, the carrot IBP has weak freezing point depression activity but is extremely active at nanomolar concentrations in inhibiting ice recrystallization. Ice crystals formed in the presence of both plant proteins grow slowly and evenly in all directions. We suggest that this slow, controlled ice growth is desirable for freeze tolerance. The fact that two plant IBPs have evolved very different protein structures to affect ice in a similar manner suggests this pattern of weak freezing point depression and strong ice recrystallization inhibition helps their host to tolerate freezing rather than to resist it.

Structures of human calpain-3 protease core with and without bound inhibitor reveal mechanisms of calpain activation.

Limb-girdle muscular dystrophy type 2a arises from mutations in the Ca2+-activated intracellular cysteine protease calpain-3. This calpain isoform is abundant in skeletal muscle and differs from the main isoforms, calpain-1 and -2, in being a homodimer and having two short insertion sequences. The first of these, IS1, interrupts the protease core and must be cleaved for activation and substrate binding. Here, to learn how calpain-3 can be regulated and inhibited, we determined the structures of the calpain-3 protease core with IS1 present or proteolytically excised. To prevent intramolecular IS1 autoproteolysis, we converted the active-site Cys to Ala. Small-angle X-ray scattering (SAXS) analysis of the C129A mutant suggested that IS1 is disordered and mobile enough to occupy several locations. Surprisingly, this was also true for the apo version of this mutant. We therefore concluded that IS1 might have a binding partner in the sarcomere and is unstructured in its absence. After autoproteolytic IS1 removal from the active Cys129 calpain-3 protease core, we could solve its crystal structures with and without the cysteine protease inhibitors E-64 and leupeptin covalently bound to the active-site cysteine. In each structure, the active state of the protease core was assembled by the cooperative binding of two Ca2+ ions to the equivalent sites used in calpain-1 and -2. These structures of the calpain-3 active site with residual IS1 and with bound E-64 and leupeptin may help guide the design of calpain-3-specific inhibitors.

Insertion sequence 1 from calpain-3 is functional in calpain-2 as an internal propeptide.

Calpains are intracellular, calcium-activated cysteine proteases. Calpain-3 is abundant in skeletal muscle, where its mutation-induced loss of function causes limb-girdle muscular dystrophy type 2A. Unlike the small subunit-containing calpain-1 and -2, the calpain-3 isoform homodimerizes through pairing of its C-terminal penta-EF-hand domain. It also has two unique insertion sequences (ISs) not found in the other calpains: IS1 within calpain-3's protease core and IS2 just prior to the penta-EF-hand domain. Production of either native or recombinant full-length calpain-3 to characterize the function of these ISs is challenging. Therefore, here we used recombinant rat calpain-2 as a stable surrogate and inserted IS1 into its equivalent position in the protease core. As it does in calpain-3, IS1 occupied the catalytic cleft and restricted the enzyme's access to substrate and inhibitors. Following activation by Ca2+, IS1 was rapidly cleaved by intramolecular autolysis, permitting the enzyme to freely accept substrate and inhibitors. The surrogate remained functional until extensive intermolecular autoproteolysis inactivated the enzyme, as is typical of calpain-2. Although the small-molecule inhibitors E-64 and leupeptin limited intermolecular autolysis of the surrogate, they did not block the initial intramolecular cleavage of IS1, establishing its role as a propeptide. Surprisingly, the large-molecule calpain inhibitor, calpastatin, completely blocked enzyme activity, even with IS1 intact. We suggest that calpastatin is large enough to oust IS1 from the catalytic cleft and take its place. We propose an explanation for why calpastatin can inhibit calpain-2 bearing the IS1 insertion but cannot inhibit WT calpain-3.

Flies expand the repertoire of protein structures that bind ice.

An antifreeze protein (AFP) with no known homologs has been identified in Lake Ontario midges (Chironomidae). The midge AFP is expressed as a family of isoforms at low levels in adults, which emerge from fresh water in spring before the threat of freezing temperatures has passed. The 9.1-kDa major isoform derived from a preproprotein precursor is glycosylated and has a 10-residue tandem repeating sequence xxCxGxYCxG, with regularly spaced cysteines, glycines, and tyrosines comprising one-half its 79 residues. Modeling and molecular dynamics predict a tightly wound left-handed solenoid fold in which the cysteines form a disulfide core to brace each of the eight 10-residue coils. The solenoid is reinforced by intrachain hydrogen bonds, side-chain salt bridges, and a row of seven stacked tyrosines on the hydrophobic side that forms the putative ice-binding site. A disulfide core is also a feature of the similar-sized beetle AFP that is a ß-helix with seven 12-residue coils and a comparable circular dichroism spectrum. The midge and beetle AFPs are not homologous and their ice-binding sites are radically different, with the latter comprising two parallel arrays of outward-pointing threonines. However, their structural similarities is an amazing example of convergent evolution in different orders of insects to cope with change to a colder climate and provide confirmation about the physical features needed for a protein to bind ice.

Allosteric inhibitors of calpains: Reevaluating inhibition by PD150606 and LSEAL.

BACKGROUND: The mercaptoacrylate calpain inhibitor, PD150606, has been shown by X-ray crystallography to bind to a hydrophobic groove in the enzyme's penta-EF-hand domains far away from the catalytic cleft and has been previously described as an uncompetitive inhibitor of calpains. The penta-peptide LSEAL has been reported to be an inhibitor of calpain and was predicted to bind in the same hydrophobic groove. The X-ray crystal structure of calpain-2 bound to its endogenous calpain inhibitor, calpastatin, shows that calpastatin also binds to the hydrophobic grooves in the two penta-EF-hand domains, but its inhibitory domain binds to the protease core domains and blocks the active site cleft directly. METHODS: The mechanisms of inhibition by PD150606 and LSEAL were investigated using steady-state kinetics of cleavage of a fluorogenic substrate by calpain-2 and the protease core of calpain1, as well as by examining the inhibition of casein hydrolysis by calpain and the autoproteolysis of calpain. RESULTS: PD150606 inhibits both full-length calpain-2 and the protease core of calpain-1 with an apparent noncompetitive kinetic model. The penta-peptide LSEAL failed to inhibit either whole calpain or its protease core in vitro. CONCLUSIONS: PD150606 cannot inhibit cleavage by calpain-2 of small substrates via binding to the penta-EF-hand domain. GENERAL SIGNIFICANCE: PD150606 is often described as a calpain-specific inhibitor due to its ability to target the penta-EF-hand domains of calpain, but we show that it must be acting at a site on the protease core domain instead.

A hybrid approach for simulating fluid loading effects on structures using experimental modal analysis and the boundary element method.

Many structural acoustics problems involve a vibrating structure in a heavy fluid. However, obtaining fluid-loaded natural frequencies and damping experimentally can be difficult and expensive. This paper presents a hybrid experimental-numerical approach to determine the heavy-fluid-loaded resonance frequencies and damping of a structure from in-air measurements. The approach combines in-air experimentally obtained mode shapes with simulated in-water acoustic resistance and reactance matrices computed using boundary element (BE) analysis. The procedure relies on accurate estimates of the mass-normalized, in vacuo mode shapes using singular value decomposition and rational fraction polynomial fitting, which are then used as basis modes for the in-water BE analysis. The method is validated on a 4.445 cm (1.75 in.) thick nickel-aluminum-bronze rectangular plate by comparing natural frequencies and damping obtained using the hybrid approach to equivalent data obtained from actual in-water measurements. Good agreement is shown for the fluid-loaded natural frequencies and one-third octave loss factors. Finally, the limitations of the hybrid approach are examined.

Kar3Vik1 mechanochemistry is inhibited by mutation or deletion of the C terminus of the Vik1 subunit.

Force production by kinesins has been linked to structural rearrangements of the N and C termini of their motor domain upon nucleotide binding. In recent crystal structures, the Kar3-associated protein Vik1 shows unexpected homology to these conformational states even though it lacks a nucleotide-binding site. This conservation infers a degree of commonality in the function of the N- and C-terminal regions during the mechanochemical cycle of all kinesins and kinesin-related proteins. We tested this inference by examining the functional effects on Kar3Vik1 of mutating or deleting residues in Vik1 that are involved in stabilizing the C terminus against the core and N terminus of the Vik1 motor homology domain (MHD). Point mutations at two moderately conserved residues near the Vik1 C terminus impaired microtubule gliding and microtubule-stimulated ATP turnover by Kar3Vik1. Deletion of the seven C-terminal residues inhibited Kar3Vik1 motility much more drastically. Interestingly, none of the point mutants seemed to perturb the ability of Kar3Vik1 to bind microtubules, whereas the C-terminal truncation mutant did. Molecular dynamics simulations of these C-terminal mutants showed distinct root mean square fluctuations in the N-terminal region of the Vik1 MHD that connects it to Kar3. Here, the degree of motion in the N-terminal portion of Vik1 highly correlated with that in the C terminus. These observations suggest that the N and C termini of the Vik1 MHD form a discrete folding motif that is part of a communication pathway to the nucleotide-binding site of Kar3.

Calcium-bound structure of calpain and its mechanism of inhibition by calpastatin.

Calpains are non-lysosomal calcium-dependent cysteine proteinases that selectively cleave proteins in response to calcium signals and thereby control cellular functions such as cytoskeletal remodelling, cell cycle progression, gene expression and apoptotic cell death. In mammals, the two best-characterized members of the calpain family, calpain 1 and calpain 2 (micro-calpain and m-calpain, respectively), are ubiquitously expressed. The activity of calpains is tightly controlled by the endogenous inhibitor calpastatin, which is an intrinsically unstructured protein capable of reversibly binding and inhibiting four molecules of calpain, but only in the presence of calcium. To date, the mechanism of inhibition by calpastatin and the basis for its absolute specificity have remained speculative. It was not clear how this unstructured protein inhibits calpains without being cleaved itself, nor was it known how calcium induced changes that facilitated the binding of calpastatin to calpain. Here we report the 2.4-A-resolution crystal structure of the calcium-bound calpain 2 heterodimer bound by one of the four inhibitory domains of calpastatin. Calpastatin is seen to inhibit calpain by occupying both sides of the active site cleft. Although the inhibitor passes through the active site cleft it escapes cleavage in a novel manner by looping out and around the active site cysteine. The inhibitory domain of calpastatin recognizes multiple lower affinity sites present only in the calcium-bound form of the enzyme, resulting in an interaction that is tight, specific and calcium dependent. This crystal structure, and that of a related complex, also reveal the conformational changes that calpain undergoes on binding calcium, which include opening of the active site cleft and movement of the domains relative to each other to produce a more compact enzyme.

A computational method for predicting inferior vena cava filter performance on a patient-specific basis.

A computational methodology for simulating virtual inferior vena cava (IVC) filter placement and IVC hemodynamics was developed and demonstrated in two patient-specific IVC geometries: a left-sided IVC and an IVC with a retroaortic left renal vein. An inverse analysis was performed to obtain the approximate in vivo stress state for each patient vein using nonlinear finite element analysis (FEA). Contact modeling was then used to simulate IVC filter placement. Contact area, contact normal force, and maximum vein displacements were higher in the retroaortic IVC than in the left-sided IVC (144 mm(2), 0.47 N, and 1.49 mm versus 68 mm(2), 0.22 N, and 1.01 mm, respectively). Hemodynamics were simulated using computational fluid dynamics (CFD), with four cases for each patient-specific vein: (1) IVC only, (2) IVC with a placed filter, (3) IVC with a placed filter and model embolus, all at resting flow conditions, and (4) IVC with a placed filter and model embolus at exercise flow conditions. Significant hemodynamic differences were observed between the two patient IVCs, with the development of a right-sided jet, larger flow recirculation regions, and lower maximum flow velocities in the left-sided IVC. These results support further investigation of IVC filter placement and hemodynamics on a patient-specific basis.

Anchored clathrate waters bind antifreeze proteins to ice.

The mechanism by which antifreeze proteins (AFPs) irreversibly bind to ice has not yet been resolved. The ice-binding site of an AFP is relatively hydrophobic, but also contains many potential hydrogen bond donors/acceptors. The extent to which hydrogen bonding and the hydrophobic effect contribute to ice binding has been debated for over 30 years. Here we have elucidated the ice-binding mechanism through solving the first crystal structure of an Antarctic bacterial AFP. This 34-kDa domain, the largest AFP structure determined to date, folds as a Ca(2+)-bound parallel beta-helix with an extensive array of ice-like surface waters that are anchored via hydrogen bonds directly to the polypeptide backbone and adjacent side chains. These bound waters make an excellent three-dimensional match to both the primary prism and basal planes of ice and in effect provide an extensive X-ray crystallographic picture of the AFPice interaction. This unobstructed view, free from crystal-packing artefacts, shows the contributions of both the hydrophobic effect and hydrogen bonding during AFP adsorption to ice. We term this mode of binding the "anchored clathrate" mechanism of AFP action.

Genetic model of selective COX2 inhibition reveals novel heterodimer signaling.

Selective inhibitors of cyclooxygenase-2 (COX2) have attracted widespread media attention because of evidence of an elevated risk of cardiovascular complications in placebo-controlled trials, resulting in the market withdrawal of some members of this class. These drugs block the cyclooxygenase activity of prostaglandin H synthase-2 (PGHS2), but do not affect the associated peroxidase function. They were developed with the rationale of conserving the anti-inflammatory and analgesic actions of traditional nonsteroidal anti-inflammatory drugs (tNSAIDs) while sparing the ability of PGHS1-derived prostaglandins to afford gastric cytoprotection. PGHS1 and PGHS2 coexist in the vasculature and in macrophages, and are upregulated together in inflammatory tissues such as rheumatoid synovia and atherosclerotic plaque. They are each believed to function as homodimers. Here, we developed a new genetic mouse model of selective COX2 inhibition using a gene-targeted point mutation, resulting in a Y385F substitution. Structural modeling and biochemical assays showed the ability of PGHS1 and PGHS2 to heterodimerize and form prostaglandins. The heterodimerization of PGHS1-PGHS2 may explain how the ductus arteriosus closes normally at birth in mice expressing PGHS2 Y385F, but not in PGHS2-null mice.

Structure-function relationships in calpains.

Calpains are a family of complex multi-domain intracellular enzymes that share a calcium-dependent cysteine protease core. These are not degradative enzymes, but instead carry out limited cleavage of target proteins in response to calcium signalling. Selective cutting of cytoskeletal proteins to facilitate cell migration is one such function. The two most abundant and extensively studied members of this family in mammals, calpains 1 and 2, are heterodimers of an isoform-specific 80 kDa large subunit and a common 28 kDa small subunit. Structures of calpain-2, both Ca2+-free and bound to calpastatin in the activated Ca2+-bound state, have provided a wealth of information about the enzyme's structure-function relationships and activation. The main association between the subunits is the pairing of their C-terminal penta-EF-hand domains through extensive intimate hydrophobic contacts. A lesser contact is made between the N-terminal anchor helix of the large subunit and the penta-EF-hand domain of the small subunit. Up to ten Ca2+ ions are co-operatively bound during activation. The anchor helix is released and individual domains change their positions relative to each other to properly align the active site. Because calpains 1 and 2 require ~30 and ~350 µM Ca2+ ions for half-maximal activation respectively, it has long been argued that autoproteolysis, subunit dissociation, post-translational modifications or auxiliary proteins are needed to activate the enzymes in the cell, where Ca2+ levels are in the nanomolar range. In the absence of robust support for these mechanisms, it is possible that under normal conditions calpains are transiently activated by high Ca2+ concentrations in the microenvironment of a Ca2+ influx, and then return to an inactive state ready for reactivation.

Public infrastructure disparities and the microbiological and chemical safety of drinking and surface water supplies in a community bordering a landfill.

The historically African-American Rogers-Eubanks community straddles unincorporated boundaries of two municipalities in Orange County, North Carolina, and predates a regional landfill sited along its border in 1972. Community members from the Rogers-Eubanks Neighborhood Association (RENA), concerned about deterioration of private wells and septic systems and a lack of public drinking water and sewer services, implemented a community-driven research partnership with university scientists and community-based organizations to investigate water and sewer infrastructure disparities and the safety of drinking and surface water supplies. RENA drafted memoranda of agreement with partners and trained community monitors to collect data (inventory households, map water and sewer infrastructure, administer household water and sewer infrastructure surveys, and collect drinking and surface water samples). Respondents to the surveys reported pervasive signs of well vulnerability (100%) and septic system failure (68%). Each 100-m increase in distance from the landfill was associated with a 600 most probable number/100 mL decrease in enterococci concentrations in surface water (95% confidence interval = -1106, -93). Pervasive private household water and sewer infrastructure failures and poor water quality were identified in this community bordering a regional landfill, providing evidence of a need for improved water and sanitation services.

Condensation dynamics in a quantum-quenched Bose gas.

By quenching the strength of interactions in a partially condensed Bose gas, we create a "supersaturated" vapor which has more thermal atoms than it can contain in equilibrium. Subsequently, the number of condensed atoms (N(0)) grows even though the temperature (T) rises and the total atom number decays. We show that the nonequilibrium evolution of the system is isoenergetic and, for small initial N(0), observe a clear separation between T and N(0) dynamics, thus explicitly demonstrating the theoretically expected "two-step" picture of condensate growth. For increasing initial N(0) values, we observe a crossover to classical relaxation dynamics. The size of the observed quench-induced effects can be explained using a simple equation of state for an interacting harmonically trapped atomic gas.

Novel dimeric ß-helical model of an ice nucleation protein with bridged active sites.

BACKGROUND: Ice nucleation proteins (INPs) allow water to freeze at high subzero temperatures. Due to their large size (>120 kDa), membrane association, and tendency to aggregate, an experimentally-determined tertiary structure of an INP has yet to be reported. How they function at the molecular level therefore remains unknown. RESULTS: Here we have predicted a novel ß-helical fold for the INP produced by the bacterium Pseudomonas borealis. The protein uses internal serine and glutamine ladders for stabilization and is predicted to dimerize via the burying of a solvent-exposed tyrosine ladder to make an intimate hydrophobic contact along the dimerization interface. The manner in which PbINP dimerizes also allows for its multimerization, which could explain the aggregation-dependence of INP activity. Both sides of the PbINP structure have tandem arrays of amino acids that can organize waters into the ice-like clathrate structures seen on antifreeze proteins. CONCLUSIONS: Dimerization dramatically increases the 'ice-active' surface area of the protein by doubling its width, increasing its length, and presenting identical ice-forming surfaces on both sides of the protein. We suggest that this allows sufficient anchored clathrate waters to align on the INP surface to nucleate freezing. As PbINP is highly similar to all known bacterial INPs, we predict its fold and mechanism of action will apply to these other INPs.

Effects of interactions on the critical temperature of a trapped Bose gas.

We perform high-precision measurements of the condensation temperature of a harmonically trapped atomic Bose gas with widely tunable interactions. For weak interactions we observe a negative shift of the critical temperature in excellent agreement with mean-field theory. However for sufficiently strong interactions we clearly observe an additional positive shift, characteristic of beyond-mean-field critical correlations. We also discuss nonequilibrium effects on the apparent critical temperature for both very weak and very strong interactions.

Condensed fraction of an atomic Bose gas induced by critical correlations.

We study the condensed fraction of a harmonically trapped atomic Bose gas at the critical point predicted by mean-field theory. The nonzero condensed fraction f(0) is induced by critical correlations which increase the transition temperature T(c) above T(c) (MF). Unlike the T(c) shift in a trapped gas, f(0) is sensitive only to the critical behavior in the quasiuniform part of the cloud near the trap center. To leading order in the interaction parameter a/λ(0), where a is the s-wave scattering length and λ(0) the thermal wavelength, we expect a universal scaling f(0) proportionally (a/λ(0))(4). We experimentally verify this scaling using a Feshbach resonance to tune a/λ(0). Further, using the local density approximation, we compare our measurements with the universal result obtained from Monte Carlo simulations for a uniform system, and find excellent quantitative agreement.

Can a Bose gas be saturated?

We scrutinize the concept of saturation of the thermal component in a partially condensed trapped Bose gas. Using a 39K gas with tunable interactions, we demonstrate strong deviation from Einstein's textbook concept of a saturated vapor. However, the saturation picture can be recovered by extrapolation to the strictly noninteracting limit. We provide evidence for the universality of our observations through additional measurements with a different atomic species, 87Rb.

Relation between malodor, ambient hydrogen sulfide, and health in a community bordering a landfill.

BACKGROUND: Municipal solid waste landfills are sources of air pollution that may affect the health and quality of life of neighboring communities. OBJECTIVES: To investigate health and quality of life concerns of neighbors related to landfill air pollution. METHODS: Landfill neighbors were enrolled and kept twice-daily diaries for 14d about odor intensity, alteration of daily activities, mood states, and irritant and other physical symptoms between January and November 2009. Concurrently, hydrogen sulfide (H(2)S) air measurements were recorded every 15-min. Relationships between H(2)S, odor, and health outcomes were evaluated using conditional fixed effects regression models. RESULTS: Twenty-three participants enrolled and completed 878 twice-daily diary entries. H(2)S measurements were recorded over a period of 80d and 1-h average H(2)S=0.22ppb (SD=0.27; range: 0-2.30ppb). Landfill odor increased 0.63 points (on 5-point Likert-type scale) for every 1ppb increase in hourly average H(2)S when the wind was blowing from the landfill towards the community (95% confidence interval (CI): 0.29, 0.91). Odor was strongly associated with reports of alteration of daily activities (odds ratio (OR)=9.0; 95% CI: 3.5, 23.5), negative mood states (OR=5.2; 95% CI: 2.8, 9.6), mucosal irritation (OR=3.7; 95% CI=2.0, 7.1) and upper respiratory symptoms (OR=3.9; 95% CI: 2.2, 7.0), but not positive mood states (OR=0.6; 95% CI: 0.2, 1.5) and gastrointestinal (GI) symptoms (OR=1.0; 95% CI: 0.4, 2.6). CONCLUSIONS: Results suggest air pollutants from a regional landfill negatively impact the health and quality of life of neighbors.