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.

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.

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.

Pediatric Dental Surgery Under General Anesthesia: Uncooperative Children.

Dental treatment of young pediatric patients can be confounded by lack of cooperation for dental rehabilitation procedures and even examination and/or radiographs. With the recent US Food and Drug Administration warning applied to many anesthetic/sedative agents for children less than 3 years old, a retrospective review of general anesthesia (GA) cases from 1 private pediatric dental practice was studied for age, gender, body mass index, anesthetic duration, airway management used, extent of dental surgical treatment, recovery time, and cardiac/pulmonary complications. For the 2016 calendar year, 351 consecutive GA cases were identified with patients aged 2-13 years. Of these, 336 underwent nasal endotracheal intubation. Forty-six of 351 patients (13%) were younger than 3 years. Median anesthesia duration was approximately 1.7 hours for all age groups. Dental treatment consisting of 8-9 teeth including crowns, fillings, and extractions was most frequently encountered. One hundred sixty-eight patients (48%), however, required care for 10-18 teeth. There were no episodes of significant oxygen desaturation. The overall complication rate was 1.1%, with 2 cases of postextubation croup, 1 case of mild intraoperative bronchospasm, and 1 case of intraoperative bradycardia. Complications did not correlate with children being overweight or obese.

Structure of a 1.5-MDa adhesin that binds its Antarctic bacterium to diatoms and ice.

Bacterial adhesins are modular cell-surface proteins that mediate adherence to other cells, surfaces, and ligands. The Antarctic bacterium Marinomonas primoryensis uses a 1.5-MDa adhesin comprising over 130 domains to position it on ice at the top of the water column for better access to oxygen and nutrients. We have reconstructed this 0.6-µm-long adhesin using a "dissect and build" structural biology approach and have established complementary roles for its five distinct regions. Domains in region I (RI) tether the adhesin to the type I secretion machinery in the periplasm of the bacterium and pass it through the outer membrane. RII comprises ~120 identical immunoglobulin-like ß-sandwich domains that rigidify on binding Ca2+ to project the adhesion regions RIII and RIV into the medium. RIII contains ligand-binding domains that join diatoms and bacteria together in a mixed-species community on the underside of sea ice where incident light is maximal. RIV is the ice-binding domain, and the terminal RV domain contains several "repeats-in-toxin" motifs and a noncleavable signal sequence that target proteins for export via the type I secretion system. Similar structural architecture is present in the adhesins of many pathogenic bacteria and provides a guide to finding and blocking binding domains to weaken infectivity.

Peptide backbone circularization enhances antifreeze protein thermostability.

Antifreeze proteins (AFPs) are a class of ice-binding proteins that promote survival of a variety of cold-adapted organisms by decreasing the freezing temperature of bodily fluids. A growing number of biomedical, agricultural, and commercial products, such as organs, foods, and industrial fluids, have benefited from the ability of AFPs to control ice crystal growth and prevent ice recrystallization at subzero temperatures. One limitation of AFP use in these latter contexts is their tendency to denature and irreversibly lose activity at the elevated temperatures of certain industrial processing or large-scale AFP production. Using the small, thermolabile type III AFP as a model system, we demonstrate that AFP thermostability is dramatically enhanced via split intein-mediated N- and C-terminal end ligation. To engineer this circular protein, computational modeling and molecular dynamics simulations were applied to identify an extein sequence that would fill the 20-Å gap separating the free ends of the AFP, yet impose little impact on the structure and entropic properties of its ice-binding surface. The top candidate was then expressed in bacteria, and the circularized protein was isolated from the intein domains by ice-affinity purification. This circularized AFP induced bipyramidal ice crystals during ice growth in the hysteresis gap and retained 40% of this activity even after incubation at 100°C for 30 min. NMR analysis implicated enhanced thermostability or refolding capacity of this protein compared to the noncyclized wild-type AFP. These studies support protein backbone circularization as a means to expand the thermostability and practical applications of AFPs.

Computational predictions of the embolus-trapping performance of an IVC filter in patient-specific and idealized IVC geometries.

Embolus transport simulations are performed to investigate the dependence of inferior vena cava (IVC) filter embolus-trapping performance on IVC anatomy. Simulations are performed using a resolved two-way coupled computational fluid dynamics/six-degree-of-freedom approach. Three IVC geometries are studied: a straight-tube IVC, a patient-averaged IVC, and a patient-specific IVC reconstructed from medical imaging data. Additionally, two sizes of spherical emboli (3 and 5 mm in diameter) and two IVC orientations (supine and upright) are considered. The embolus-trapping efficiency of the IVC filter is quantified for each combination of IVC geometry, embolus size, and IVC orientation by performing 2560 individual simulations. The predicted embolus-trapping efficiencies of the IVC filter range from 10 to 100%, and IVC anatomy is found to have a significant influence on the efficiency results ([Formula: see text]). In the upright IVC orientation, greater secondary flow in the patient-specific IVC geometry decreases the filter embolus-trapping efficiency by 22-30 percentage points compared with the efficiencies predicted in the idealized straight-tube or patient-averaged IVCs. In a supine orientation, the embolus-trapping efficiency of the filter in the idealized IVCs decreases by 21-90 percentage points compared with the upright orientation. In contrast, the embolus-trapping efficiency is insensitive to IVC orientation in the patient-specific IVC. In summary, simulations predict that anatomical features of the IVC that are often neglected in the idealized models used for benchtop testing, such as iliac vein compression and anteroposterior curvature, generate secondary flow and mixing in the IVC and influence the embolus-trapping efficiency of IVC filters. Accordingly, inter-subject variability studies and additional embolus transport investigations that consider patient-specific IVC anatomy are recommended for future work.

A resolved two-way coupled CFD/6-DOF approach for predicting embolus transport and the embolus-trapping efficiency of IVC filters.

Inferior vena cava (IVC) filters are medical devices designed to provide a mechanical barrier to the passage of emboli from the deep veins of the legs to the heart and lungs. Despite decades of development and clinical use, IVC filters still fail to prevent the passage of all hazardous emboli. The objective of this study is to (1) develop a resolved two-way computational model of embolus transport, (2) provide verification and validation evidence for the model, and (3) demonstrate the ability of the model to predict the embolus-trapping efficiency of an IVC filter. Our model couples computational fluid dynamics simulations of blood flow to six-degree-of-freedom simulations of embolus transport and resolves the interactions between rigid, spherical emboli and the blood flow using an immersed boundary method. Following model development and numerical verification and validation of the computational approach against benchmark data from the literature, embolus transport simulations are performed in an idealized IVC geometry. Centered and tilted filter orientations are considered using a nonlinear finite element-based virtual filter placement procedure. A total of 2048 coupled CFD/6-DOF simulations are performed to predict the embolus-trapping statistics of the filter. The simulations predict that the embolus-trapping efficiency of the IVC filter increases with increasing embolus diameter and increasing embolus-to-blood density ratio. Tilted filter placement is found to decrease the embolus-trapping efficiency compared with centered filter placement. Multiple embolus-trapping locations are predicted for the IVC filter, and the trapping locations are predicted to shift upstream and toward the vessel wall with increasing embolus diameter. Simulations of the injection of successive emboli into the IVC are also performed and reveal that the embolus-trapping efficiency decreases with increasing thrombus load in the IVC filter. In future work, the computational tool could be used to investigate IVC filter design improvements, the effect of patient anatomy on embolus transport and IVC filter embolus-trapping efficiency, and, with further development and validation, optimal filter selection and placement on a patient-specific basis.

The Importance of Hemorheology and Patient Anatomy on the Hemodynamics in the Inferior Vena Cava.

Inferior vena cava (IVC) filters have been used for nearly half a century to prevent pulmonary embolism in at-risk patients. However, complications with IVC filters remain common. In this study, we investigate the importance of considering the hemorheological and morphological effects on IVC hemodynamics by simulating Newtonian and non-Newtonian blood flow in three IVC models with varying levels of geometric idealization. Partial occlusion by an IVC filter and a thrombus is also considered. More than 99% of the infrarenal IVC volume is found to contain flow in the nonlinear region of the shear rate-viscosity curve for blood (less than 100 s-1) in the unoccluded IVCs. Newtonian simulations performed using the asymptotic viscosity for blood over-predict the non-Newtonian Reynolds numbers by more than a factor of two and under-predict the mean wall shear stress (WSS) by 28-54%. Agreement with the non-Newtonian simulations is better using a characteristic viscosity, but local WSS errors are still large (up to 50%) in the partially occluded cases. Secondary flow patterns in the IVC also depend on the viscosity model and IVC morphological complexity. Non-Newtonian simulations required only a marginal increase in computational expense compared with the Newtonian simulations. We recommend that future studies of IVC hemodynamics consider the effects of hemorheology and IVC morphology when accurate predictions of WSS and secondary flow features are desired.

Rational Design of Calpain Inhibitors Based on Calpastatin Peptidomimetics.

Our previously reported structures of calpain bound to its endogenous inhibitor calpastatin have motivated the use of aziridine aldehyde-mediated peptide macrocyclization toward the design of cyclic peptides and peptidomimetics as calpain inhibitors. Inspired by nature's hint that a ß-turn loop within calpastatin forms a broad interaction around calpain's active site cysteine, we have constructed and tested a library of 45 peptidic compounds based on this loop sequence. Four molecules have shown reproducibly low micromolar inhibition of calpain-2. Further systematic sequence changes led to the development of probes that displayed increased potency and specificity of inhibition against calpain over other cysteine proteases. Calculated Ki values were in the low micromolar range, rivaling other peptidomimetic calpain inhibitors and presenting an improved selectivity profile against other therapeutically relevant proteases. Competitive and mixed inhibition against calpain-2 was observed, and an allosteric inhibition site on the enzyme was identified for a noncompetitive inhibitor.

New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.

Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of ChloroIBP was cloned and over-expressed in Escherichia coli to investigate the IBP's biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. In silico structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed ß-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the ß-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the ß-1 and ß-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP.

Modeling repetitive, non-globular proteins.

While ab initio modeling of protein structures is not routine, certain types of proteins are more straightforward to model than others. Proteins with short repetitive sequences typically exhibit repetitive structures. These repetitive sequences can be more amenable to modeling if some information is known about the predominant secondary structure or other key features of the protein sequence. We have successfully built models of a number of repetitive structures with novel folds using knowledge of the consensus sequence within the sequence repeat and an understanding of the likely secondary structures that these may adopt. Our methods for achieving this success are reviewed here.

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.

Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters.

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.

Ca2+-stabilized adhesin helps an Antarctic bacterium reach out and bind ice.

The large size of a 1.5-MDa ice-binding adhesin [MpAFP (Marinomonas primoryensis antifreeze protein)] from an Antarctic Gram-negative bacterium, M. primoryensis, is mainly due to its highly repetitive RII (Region II). MpAFP_RII contains roughly 120 tandem copies of an identical 104-residue repeat. We have previously determined that a single RII repeat folds as a Ca2+-dependent immunoglobulin-like domain. Here, we solved the crystal structure of RII tetra-tandemer (four tandem RII repeats) to a resolution of 1.8 Å. The RII tetra-tandemer reveals an extended (~190-Å × ~25-Å), rod-like structure with four RII-repeats aligned in series with each other. The inter-repeat regions of the RII tetra-tandemer are strengthened by Ca2+ bound to acidic residues. SAXS (small-angle X-ray scattering) profiles indicate the RII tetra-tandemer is significantly rigidified upon Ca2+ binding, and that the protein's solution structure is in excellent agreement with its crystal structure. We hypothesize that >600 Ca2+ help rigidify the chain of ~120 104-residue repeats to form a ~0.6 µm rod-like structure in order to project the ice-binding domain of MpAFP away from the bacterial cell surface. The proposed extender role of RII can help the strictly aerobic, motile bacterium bind ice in the upper reaches of the Antarctic lake where oxygen and nutrients are most abundant. Ca2+-induced rigidity of tandem Ig-like repeats in large adhesins might be a general mechanism used by bacteria to bind to their substrates and help colonize specific niches.

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.

Crystal structure of calpain-3 penta-EF-hand (PEF) domain - a homodimerized PEF family member with calcium bound at the fifth EF-hand.

Calpains are Ca(2+) dependent intracellular cysteine proteases that cleave a wide range of protein substrates to help implement Ca(2+) signaling in the cell. The major isoforms of this enzyme family, calpain-1 and calpain-2, are heterodimers of a large and a small subunit, with the main dimer interface being formed through their C-terminal penta-EF hand (PEF) domains. Calpain-3, or p94, is a skeletal muscle-specific isoform that is genetically linked to limb-girdle muscular dystrophy. Biophysical and modeling studies with the PEF domain of calpain-3 support the suggestion that full-length calpain-3 exists as a homodimer. Here, we report the crystallization of calpain-3's PEF domain and its crystal structure in the presence of Ca(2+) , which provides evidence for the homodimer architecture of calpain-3 and supports the molecular model that places a protease core at either end of the elongated dimer. Unlike other calpain PEF domain structures, the calpain-3 PEF domain contains a Ca(2+) bound at the EF5-hand used for homodimer association. Three of the four Ca(2+) -binding EF-hands of the PEF domains are concentrated near the protease core, and have the potential to radically change the local charge within the dimer during Ca(2+) signaling. Examination of the homodimer interface shows that there would be steric clashes if the calpain-3 large subunit were to try to pair with a calpain small subunit. Database Structural data are available in the Protein Data Bank database under accession number 4OKH.