Crystal structure and standardized geometric analysis of InlJ, a listerial virulence factor and leucine-rich repeat protein with a novel cysteine ladder

We report on the crystal structure of the internalin domain of InlJ, a virulence-associated surface protein of Listeria monocytogenes, at 2.7-Å resolution. InlJ is a member of the internalin family of listerial cell surface proteins characterized by a common N-terminal domain. InlJ bears 15 leucine-rich repeats (LRRs), the same number as in InlA, the prototypical internalin family member. The LRRs of InlJ differ from those of other internalins by having 21, rather than 22, residues and by replacing 1 LRR-defining hydrophobic residue with a conserved cysteine. These cysteines stack to form an intramolecular ladder and regular hydrophobic interactions in consecutive repeats. Analyzing the curvature, twist, and lateral bending angles of InlJ and comparing these with several other LRR proteins, we provide a systematic geometric comparison of LRR protein structures (http://bragi2.helmholtz-hzi.de/Angulator/). These indicate that both cysteine and asparagine ladders stabilize the LRR fold, whereas substitutions in some repeat positions are more likely than others to induce changes in LRR geometry.     

Thermodynamics, kinetics, and salt dependence of folding of YopM, a large leucine-rich repeat protein

Small globular proteins have many contacts between residues that are distant in primary sequence. These contacts create a complex network between sequence-distant segments of secondary structure, which may be expected to promote the cooperative folding of globular proteins. Although repeat proteins, which are composed of tandem modular units, lack sequence-distant contacts, several of considerable length have been shown to undergo cooperative two-state folding. To explore the limits of cooperativity in repeat proteins, we have studied the unfolding of YopM, a leucine-rich repeat (LRR) protein of over 400 residues. Despite its large size and modular architecture (15 repeats), YopM equilibrium unfolding is highly cooperative, and shows a very strong dependence on the concentration of urea. In contrast, kinetic studies of YopM folding indicate a mechanism that includes one or more transient intermediates. The urea dependence of the folding and unfolding rates suggests a relatively small transition state ensemble. As with the urea dependence, we have found an extreme dependence of the free energy of unfolding on the concentration of salt. This salt dependence likely results from general screening of a large number of unfavorable columbic interactions in the folded state, rather than from specific cation binding.     

N-Glycosylation of the Drosophila neural protein Chaoptin is essential for its stability, cell surface transport and adhesive activity

Glycosylation of proteins can modulate their function in a striking variety of systems, including immune responses, neuronal activities and development. The Drosophila protein, Chaoptin (Chp), is essential for the development and maintenance of photoreceptor cells. This protein is heavily glycosylated, but the possible role of this glycosylation is not well-understood. Here we show that mutations introduced into about 1/3 of 16 potential N-linked glycosylation sites within Chp impaired its cell adhesive activities when expressed in Drosophila S2 cells. Mutation of 2/3 of the glycosylation sites resulted in a marked decrease in Chp protein abundance. These results suggest that N-linked glycosylation of Chp is essential for its stability and activity.     

Functional Evolution of Ribonuclease Inhibitor: Insights from Birds and Reptiles

Ribonuclease inhibitor (RI) is a conserved protein of the mammalian cytosol. RI binds with high affinity to diverse secretory ribonucleases (RNases) and inhibits their enzymatic activity. Although secretory RNases are found in all vertebrates, the existence of a non-mammalian RI has been uncertain. Here, we report on the identification and characterization of RI homologs from chicken and anole lizard. These proteins bind to RNases from multiple species but exhibit much greater affinity for their cognate RNases than for mammalian RNases. To reveal the basis for this differential affinity, we determined the crystal structure of mouse, bovine, and chicken RI·RNase complexes to a resolution of 2.20, 2.21, and 1.92 Å, respectively. A combination of structural, computational, and bioinformatic analyses enabled the identification of two residues that appear to contribute to the differential affinity for RNases. We also found marked differences in oxidative instability between mammalian and non-mammalian RIs, indicating evolution toward greater oxygen sensitivity in RIs from mammalian species. Taken together, our results illuminate the structural and functional evolution of RI, along with its dynamic role in vertebrate biology.     

Structure of the dimeric autoinhibited conformation of DAPK2, a pro-apoptotic protein kinase

The death-associated protein kinase (DAPK) family has been characterized as a group of pro-apoptotic serine/threonine kinases that share specific structural features in their catalytic kinase domain. Two of the DAPK family members, DAPK1 and DAPK2, are calmodulin-dependent protein kinases that are regulated by oligomerization, calmodulin binding, and autophosphorylation. In this study, we have determined the crystal and solution structures of murine DAPK2 in the presence of the autoinhibitory domain, with and without bound nucleotides in the active site. The crystal structure shows dimers of DAPK2 in a conformation that is not permissible for protein substrate binding. Two different conformations were seen in the active site upon the introduction of nucleotide ligands. The monomeric and dimeric forms of DAPK2 were further analyzed for solution structure, and the results indicate that the dimers of DAPK2 are indeed formed through the association of two apposed catalytic domains, as seen in the crystal structure. The structures can be further used to build a model for DAPK2 autophosphorylation and to compare with closely related kinases, of which especially DAPK1 is an actively studied drug target. Our structures also provide a model for both homodimerization and heterodimerization of the catalytic domain between members of the DAPK family. The fingerprint of the DAPK family, the basic loop, plays a central role in the dimerization of the kinase domain.     

Crystal structure and characterization of coiled-coil domain of the transient receptor potential channel PKD2L1

The cation-permeable channel PKD2L1 forms a homomeric assembly as well as heteromeric associations with both PKD1 and PKD1L3, with the cytoplasmic regulatory domain (CRD) of PKD2L1 often playing a role in assembly and/or function. Our previous work indicated that the isolated PKD2L1 CRD assembles as a trimer in a manner dependent on the presence of a proposed oligomerization domain. Herein we describe the 2.7 Å crystal structure of a segment containing the PKD2L1 oligomerization domain which indicates that trimerization is driven by the β-branched residues at the first and fourth positions of a heptad repeat (commonly referred to as “a” and “d”) and by a conserved R-h-x-x-h-E salt bridge motif that is largely unique to parallel trimeric coiled coils. Further analysis of the PKD2L1 CRD indicates that trimeric association is sufficiently strong that no other species are present in solution in an analytical ultracentrifugation experiment at the lowest measurable concentration of 750 nM. Conversely, mutation of the “a” and “d” residues leads to formation of an exclusively monomeric species, independent of concentration. Although both monomeric and WT CRDs are stable in solution and bind calcium with 0.9 μM affinity, circular dichroism studies reveal that the monomer loses 25% more α-helical content than WT when stripped of this ligand, suggesting that the CRD structure is stabilized by trimerization in the ligand-free state. This stability could play a role in the function of the full-length complex, indicating that trimerization may be important for both homo- and possibly heteromeric assemblies of PKD2L1.     

Determination of cathepsin V activity and intracellular trafficking by N-glycosylation

Cathepsin V (L2), a lysosomal cysteine protease, is a member of cathepsin family, relating to cancer invasion and metastasis. Cathepsin V contains two predicted N-glycosylation sites, but it has not been reported whether cathepsin V is glycosylated or not. In this study, we clarified the role of N-glycosylation of cathepsin V for its functions. We demonstrated that cathepsin V is N-glycosylated at both Asn²²¹ and Asn²⁹² using mass spectrometry and site-directed mutagenesis. N-glycosylation of cathepsin V was important for transportation to lysosome, secretion, and activity in HT1080 cells. These data demonstrated that functions of cathepsin V are controlled by N-glycosylation.     

Human platelet 12-lipoxygenase, new findings about its activity, membrane binding and low-resolution structure

Human platelet 12-lipoxygenase (hp-12LOX, 662 residues + iron nonheme cofactor) and its major metabolite 12S-hydroxyeicosatetraenoic acid have been implicated in cardiovascular and renal diseases, many types of cancer and inflammatory responses. However, drug development is slow due to a lack of structural information. The major hurdle in obtaining a high-resolution X-ray structure is growing crystals, a process that requires the preparation of highly homogenous, reproducible and stable protein samples. To understand the properties of hp-12LOX, we have expressed and studied the behavior, function and low-resolution structure of the hp-12LOX His-tagged recombinant enzyme and its mutants in solution. We have found that it is a dimer easily converted into bigger aggregates, which are soluble/covalent-noncovalent/reversible. The heavier oligomers show a higher activity at pH 8, in contrast to dimers with lower activity showing two maxima at pH 7 and pH 8, indicating the existence of two different conformers. In the seven-point C → S mutant, aggregation is diminished, activity has one broad peak at pH 8 and there is no change in specificity. Truncation of the N_t-β-barrel domain (PLAT, residues 1-116) reduces activity to ∼ 20% of that shown by the whole enzyme, does not affect regio- or stereospecificity and lowers membrane binding by a factor of ∼ 2. “NoPLAT” mutants show strong aggregation into oligomers containing six or more catalytic domains regardless of the status of the seven cysteine residues tested. Time-of-flight mass spectrometry suggests two arachidonic acid molecules bound to one molecule of enzyme. Small angle X-ray scattering studies (16 Å resolution, χ∼ 1) suggest that two hp-12LOX monomers are joined by the catalytic domains, with the PLAT domains floating on the flexible linkers away from the main body of the dimer.     

Mapping the structure of folding cores in TIM barrel proteins by hydrogen exchange mass spectrometry: the roles of motif and sequence for the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus

To test the roles of motif and amino acid sequence in the folding mechanisms of TIM barrel proteins, hydrogen-deuterium exchange was used to explore the structure of the stable folding intermediates for the of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS). Previous studies of the urea denaturation of sIGPS revealed the presence of an intermediate that is highly populated at approximately 4.5 M urea and contains approximately 50% of the secondary structure of the native (N) state. Kinetic studies showed that this apparent equilibrium intermediate is actually comprised of two thermodynamically distinct species, I(a) and I(b). To probe the location of the secondary structure in this pair of stable on-pathway intermediates, the equilibrium unfolding process of sIGPS was monitored by hydrogen-deuterium exchange mass spectrometry. The intact protein and pepsin-digested fragments were studied at various concentrations of urea by electrospray and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, respectively. Intact sIGPS strongly protects at least 54 amide protons from hydrogen-deuterium exchange in the intermediate states, demonstrating the presence of stable folded cores. When the protection patterns and the exchange mechanisms for the peptides are considered with the proposed folding mechanism, the results can be interpreted to define the structural boundaries of I(a) and I(b). Comparison of these results with previous hydrogen-deuterium exchange studies on another TIM barrel protein of low sequence identify, alpha-tryptophan synthase (alphaTS), indicates that the thermodynamic states corresponding to the folding intermediates are better conserved than their structures. Although the TIM barrel motif appears to define the basic features of the folding free energy surface, the structures of the partially folded states that appear during the folding reaction depend on the amino acid sequence. Markedly, the good correlation between the hydrogen-deuterium exchange patterns of sIGPS and alphaTS with the locations of hydrophobic clusters defined by isoleucine, leucine, and valine residues suggests that branch aliphatic side-chains play a critical role in defining the structures of the equilibrium intermediates.     

Heterologous expression and purification of a biologically active legume lectin from Cratylia mollis seeds (CRAMOLL 1)

CRAMOLL 1 is a mannose/glucose isolectin isolated from Cratylia mollis seeds. This lectin has 82% sequence identity with Con A and essentially the same quaternary structure. As with Con A, CRAMOLL 1 seems to undergo complex post-translational processing which makes it difficult to the use of traditional molecular cloning for heterologous expression. Here we report the expression and purification of functional recombinant CRAMOLL 1 (rCRAMOLL 1) in Escherichia coli. This was accomplished by introducing a chemically synthesized DNA encoding the mature CRAMOLL 1 amino acid sequence into a bacterial expression vector under T7 promoter control. Most of the recombinant lectin was found in insoluble aggregates (inclusion bodies), but we were able to recover reasonable amounts of soluble lectin in the active form by decreasing the protein induction temperature. The recombinant lectin was purified to homogeneity with one-step affinity chromatography. The plant CRAMOLL 1 (pCRAMOLL 1) and rCRAMOLL 1 share several physicochemical properties such as molecular mass, charge density and secondary and tertiary structures. However, pCRAMOLL 1 has a lower thermodynamic stability than rCRAMOLL 1 when probed by acidification, high temperature or high hydrostatic pressure, and this is probably caused by the presence of tetramers composed of fragmented monomers, which are formed in the plant cotyledon but absent from the recombinant protein. rCRAMOLL 1 behaves identically to its plant counterpart with respect to its specificity for monosaccharides, and to its agglutinating activities against rabbit erythrocytes and Trypanosoma cruzi epimastigote cells.     

GAPDH is conformationally and functionally altered in association with oxidative stress in mouse models of amyotrophic lateral sclerosis

It is proposed that conformational changes induced in proteins by oxidation can lead to loss of activity or protein aggregation through exposure of hydrophobic residues and alteration in surface hydrophobicity. Because increased oxidative stress and protein aggregation are consistently observed in amyotrophic lateral sclerosis (ALS), we used a 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (BisANS) photolabeling approach to monitor changes in protein unfolding in vivo in skeletal muscle proteins in ALS mice. We find two major proteins, creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), conformationally affected in the ALS G93A mouse model concordant with a 43% and 41% reduction in enzyme activity, respectively. This correlated with changes in conformation and activity that were detected in CK and GAPDH with in vitro oxidation. Interestingly, we found that GAPDH, but not CK, is conformationally and functionally affected in a longer-lived ALS model (H46R/H48Q), exhibiting a 22% reduction in enzyme activity. We proposed a reaction mechanism for BisANS with nucleophilic amino acids such as lysine, serine, threonine, and tyrosine, and BisANS was found to be primarily incorporated to lysine residues in GAPDH. We identified the specific BisANS incorporation sites on GAPDH in nontransgenic (NTg), G93A, and H46R/H48Q mice using liquid chromatography-tandem mass spectrometry analysis. Four BisANS-containing sites (K52, K104, K212, and K248) were found in NTg GAPDH, while three out of four of these sites were lost in either G93A or H46R/H48Q GAPDH. Conversely, eight new sites (K2, K63, K69, K114, K183, K251, S330, and K331) were found on GAPDH for G93A, including one common site (K114) for H46R/H48Q, which is not found on GAPDH from NTg mice. These data show that GAPDH is differentially affected structurally and functionally in vivo in accordance with the degree of oxidative stress associated with these two models of ALS.     

Proteomics in Parkinson's disease: An unbiased approach towards peripheral biomarkers and new therapies

Parkinson's disease is the most common neurodegenerative movement disorder, affecting about 6 million people worldwide with a slow progression of the symptoms. Its prevalence is expected to double in the most populated areas within the next two decades, according to increasing aged population. Consequently, Parkinson's disease is a socio-economic trouble and a major challenge for the public health system. Parkinson's disease treatment is merely symptomatic, as clinical symptoms appear when about 70% of the involved neurons are lost and potential disease-modifying/neuroprotective therapies would have no effect. In turn, the availability of an objective measure that allows early diagnosis would strongly impact on the costs that biotech- and pharma-companies will sustain in order to develop disease-modifying therapies. The establishment of suitable models to investigate the mechanisms of Parkinson's disease progression and, on the other hand, the discovery and validation of selective and specific molecular biomarkers for early and differential diagnosis are indeed two important goals for a better management of the disease. In this review, we focus on cellular and animal models of Parkinson's disease by describing their advantages and limitations as useful tools to identify pathogenetic pathways that deserve further exploitation. In parallel, we discuss how proteomics may provide a potent tool to observe altered pathways in models or altered biomarkers in patients with an unbiased, hypothesis-free approach.