Zongchao Jia

Sensitivity of secondary structure propensities to sequence differences between α‐ and γ‐synuclein: Implications for fibrillation

The synucleins are a family of intrinsically disordered proteins involved in various human diseases. α‐Synuclein has been extensively characterized due to its role in Parkinsons disease where it forms intracellular aggregates, while γ‐synuclein is overexpressed in a majority of late‐stage breast cancers. Despite fairly strong sequence similarity between the amyloid‐forming regions of α‐ and γ‐synuclein, γ‐synuclein has only a weak propensity to form amyloid fibrils. We hypothesize that the different fibrillation tendencies of α‐ and γ‐synuclein may be related to differences in structural propensities. Here we have measured chemical shifts for γ‐synuclein and compared them to previously published shifts for α‐synuclein. In order to facilitate direct comparison, we have implemented a simple new technique for re‐referencing chemical shifts that we have found to be highly effective for both disordered and folded proteins. In addition, we have developed a new method that combines different chemical shifts into a single residue‐specific secondary structure propensity (SSP) score. We observe significant differences between α‐ and γ‐synuclein secondary structure propensities. Most interestingly, γ‐synuclein has an increased α‐helical propensity in the amyloid‐forming region that is critical for α‐synuclein fibrillation, suggesting that increased structural stability in this region may protect against γ‐synuclein aggregation. This comparison of residue‐specific secondary structure propensities between intrinsically disordered homologs highlights the sensitivity of transient structure to sequence changes, which we suggest may have been exploited as an evolutionary mechanism for fast modulation of protein structure and, hence, function.

Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation.

The combination of thiol protease activity and calmodulin‐like EF‐hands is a feature unique to the calpains. The regulatory mechanisms governing calpain activity are complex, and the nature of the Ca2+‐induced switch between inactive and active forms has remained elusive in the absence of structural information. We describe here the 2.6 Å crystal structure of m‐calpain in the Ca2+‐free form, which illustrates the structural basis for the inactivity of calpain in the absence of Ca2+. It also reveals an unusual thiol protease fold, which is associated with Ca2+‐binding domains through heterodimerization and a C2‐like β‐sandwich domain. Strikingly, the structure shows that the catalytic triad is not assembled, indicating that Ca2+‐binding must induce conformational changes that re‐orient the protease domains to form a functional active site. The α‐helical N‐terminal anchor of the catalytic subunit does not occupy the active site but inhibits its assembly and regulates Ca2+‐sensitivity through association with the regulatory subunit. This Ca2+‐dependent activation mechanism is clearly distinct from those of classical proteases.

Mimicry of ice structure by surface hydroxyls and water of a beta-helix antifreeze protein.

Insect antifreeze proteins (AFP) are much more effective than fish AFPs at depressing solution freezing points by ice-growth inhibition. AFP from the beetle Tenebrio molitor is a small protein (8.4 kDa) composed of tandem 12-residue repeats (TCTxSxxCxxAx). Here we report its 1.4-Å resolution crystal structure, showing that this repetitive sequence translates into an exceptionally regular β-helix. Not only are the 12-amino-acid loops almost identical in the backbone, but also the conserved side chains are positioned in essentially identical orientations, making this AFP perhaps the most regular protein structure yet observed. The protein has almost no hydrophobic core but is stabilized by numerous disulphide and hydrogen bonds. On the conserved side of the protein, threonine-cysteine-threonine motifs are arrayed to form a flat β-sheet, the putative ice-binding surface. The threonine side chains have exactly the same rotameric conformation and the spacing between OH groups is a near-perfect match to the ice lattice. Together with tightly bound co-planar external water, three ranks of oxygen atoms form a two-dimensional array, mimicking an ice section.

β-Helix structure and ice-binding properties of a hyperactive antifreeze protein from an insect.

Insect antifreeze proteins (AFP) are considerably more active at inhibiting ice crystal growth than AFP from fish or plants. Several insect AFPs, also known as thermal hysteresis proteins, have been cloned and expressed. Their maximum activity is 3–4 times that of fish AFPs and they are 10–100 times more effective at micromolar concentrations. Here we report the solution structure of spruce budworm (Choristoneura fumiferana) AFP and characterize its ice-binding properties. The 9-kDa AFP is a β-helix with a triangular cross-section and rectangular sides that form stacked parallel β-sheets; a fold which is distinct from the three known fish AFP structures. The ice-binding side contains 9 of the 14 surface-accessible threonines organized in a regular array of TXT motifs that match the ice lattice on both prism and basal planes. In support of this model, ice crystal morphology and ice-etching experiments are consistent with AFP binding to both of these planes and thus may explain the greater activity of the spruce budworm antifreeze.

Antifreeze proteins: an unusual receptor–ligand interaction

Antifreeze proteins (AFPs) help organisms to survive below 0 degrees C by inhibiting ice growth. Although AFPs are structurally diverse, they typically present a large proportion of their surface area for binding to ice. Whereas earlier proposed binding mechanisms relied almost entirely on a hydrogen bond match between the AFP and ice, it now seems probable that van der Waals and hydrophobic interactions make a significant contribution to the enthalpy of adsorption. These interactions require intimate surface-surface complementarity between the receptor (AFP) and its ligand (ice).

A Ca2+ Switch Aligns the Active Site of Calpain

Ca(2+) signaling by calpains leads to controlled proteolysis during processes ranging from cytoskeleton remodeling in mammals to sex determination in nematodes. Deregulated Ca(2+) levels result in aberrant proteolysis by calpains, which contributes to tissue damage in heart and brain ischemias as well as neurodegeneration in Alzheimers disease. Here we show that activation of the protease core of mu calpain requires cooperative binding of two Ca(2+) atoms at two non-EF-hand sites revealed in the 2.1 A crystal structure. Conservation of the Ca(2+) binding residues defines an ancestral general mechanism of activation for most calpain isoforms, including some that lack EF-hand domains. The protease region is not affected by the endogenous inhibitor, calpastatin, and may contribute to calpain-mediated pathologies when the core is released by autoproteolysis.

Protein tyrosine phosphatases take off.

Protein tyrosine phosphatases (PTPs) are a family of signal transduction enzymes that dephosphorylate phosphotyrosine containing proteins. Structural and kinetic studies provide a molecular understanding of how these enzymes regulate a wide range of intracellular processes.

Efficient antitumor immunity derived from maturation of dendritic cells that had phagocytosed apoptotic/necrotic tumor cells

Dendritic cells (DCs) that acquired antigen from apoptotic tumor cells are able to induce major histocompatibility complex (MHC) class I‐restricted cytotoxic T lymphocytes and antitumor immunity. In the present study, we investigated the efficiency of antitumor immunity derived from DCs that had phagocytosed apoptotic/necrotic BL6‐10 melanoma cells compared with that of DCs pulsed with the tumor mTRP2 peptide. Our data showed that phagocytosis of apoptotic/necrotic tumor cells resulted in maturation of DCs with up‐regulated expression of proinflammatory cytokines [interleukin (IL)‐1β, IL‐6, tumor necrosis factor‐α, interferon‐γ and granulocyte‐macrophage colony‐stimulating factor], chemokines (MIP‐1α, MIP‐1β and MIP‐2), the CC chemokine receptor CCR7 and the cell surface molecules (MHC class II, CD11b, CD40 and CD86), and down‐regulated expression of the CC chemokine receptors CCR2 and CCR5. These mature DCs displayed enhanced migration toward the CC chemokine MIP‐3β in a chemotaxis assay in vitro and to the regional lymph nodes in an animal model in vivo. Our data also showed that vaccination with DCs that had phagocytosed apoptotic/necrotic BL6‐10 cells was able to (i) more strongly stimulate allogeneic T‐cell proliferation in vitro, (ii) induce an in vivo Th1‐type immune response leading to more efficient tumor‐specific cytotoxic CD8+ T‐cell‐mediated immunity and (iii) eradicate lung metastases in all 6 vaccinated mice compared with mice vaccinated with DCs pulsed with the tumor mTRP2 peptide, in which lung metastases were reduced (mean number of 16 per mouse) but not completely eradicated. Therefore, DCs that had phagocytosed apoptotic/necrotic tumor cells appear to offer new strategies in DC cancer vaccines.

Crystal structures of Escherichia coli phytase and its complex with phytate.

Phytases catalyze the hydrolysis of phytate and are able to improve the nutritional quality of phytate-rich diets. Escherichia coli phytase, a member of the histidine acid phosphatase family has the highest specific activity of all phytases characterized. The crystal structure of E. coli phytase has been determined by a two-wavelength anomalous diffraction method using the exceptionally strong anomalous scattering of tungsten. Despite a lack of sequence similarity, the structure closely resembles the overall fold of other histidine acid phosphatases. The structure of E. coli phytase in complex with phytate, the preferred substrate, reveals the binding mode and substrate recognition. The binding is also accompanied by conformational changes which suggest that substrate binding enhances catalysis by increasing the acidity of the general acid.

Extracellular K+ concentration controls cell surface density of IKr in rabbit hearts and of the HERG channel in human cell lines

Although the modulation of ion channel gating by hormones and drugs has been extensively studied, much less is known about how cell surface ion channel expression levels are regulated. Here, we demonstrate that the cell surface density of both the heterologously expressed K+ channel encoded by the human ether-a-go-go-related gene (HERG) and its native counterpart, the rapidly activating delayed rectifier K+ channel (IKr), in rabbit hearts in vivo is precisely controlled by extracellular K+ concentration ([K+]o) within a physiologically relevant range. Reduction of [K+]o led to accelerated internalization and degradation of HERG channels within hours. Confocal analysis revealed colocalization between HERG and ubiquitin during the process of HERG internalization, and overexpression of ubiquitin facilitated HERG degradation under low [K+]o. The HERG channels colocalized with a marker of multivesicular bodies during internalization, and the internalized HERG channels were targeted to lysosomes. Our results provide the first evidence to our knowledge that the cell surface density of a voltage-gated K+ channel, HERG, is regulated by a biological factor, extracellular K+. Because hypokalemia is known to exacerbate long QT syndrome (LQTS) and Torsades de pointes tachyarrhythmias, our findings provide a potential mechanistic link between hypokalemia and LQTS.