Yogendra Sharma

Repeated domains of leptospira immunoglobulin-like proteins interact with elastin and tropoelastin.

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17–27HTE) as determined by ELISA (LigBCon4, KD = 0.50 μm; LigBCen7′–8, KD = 0.82 μm; LigBCen9, KD = 1.54 μm; and LigBCen12, KD = 0.73 μm). The interaction of LigBCon4 and 17–27HTE was further confirmed by steady state fluorescence spectroscopy (KD = 0.49 μm) and ITC (KD = 0.54 μm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17–27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.

Neuronal calcium sensor-1 enhancement of InsP3 receptor activity is inhibited by therapeutic levels of lithium

Regulation and dysregulation of intracellular calcium (Ca2+) signaling via the inositol 1,4,5-trisphosphate receptor (InsP3R) has been linked to many cellular processes and pathological conditions. In the present study, addition of neuronal calcium sensor-1 (NCS-1), a high-affinity, low-capacity, calcium-binding protein, to purified InsP3R type 1 (InsP3R1) increased the channel activity in both a calcium-dependent and -independent manner. In intact cells, enhanced expression of NCS-1 resulted in increased intracellular calcium release upon stimulation of the phosphoinositide signaling pathway. To determine whether InsP3R1/NCS-1 interaction could be functionally relevant in bipolar disorders, conditions in which NCS-1 is highly expressed, we tested the effect of lithium, a salt widely used for treatment of bipolar disorders. Lithium inhibited the enhancing effect of NCS-1 on InsP3R1 function, suggesting that InsP3R1/NCS-1 interaction is an essential component of the pathomechanism of bipolar disorder.

A high-molecular-weight outer membrane protein of Xanthomonas oryzae pv. oryzae exhibits similarity to non-fimbrial adhesins of animal pathogenic bacteria and is required for optimum virulence

Transposon insertions in a novel 3.798 kb open reading frame (ORF) of the rice pathogen, Xanthomonas oryzae pv. oryzae (Xoo) cause virulence deficiency and altered colony/lawn morphology. This ORF encodes a protein, XadA, of 1265 amino acids that exhibits significant similarity to non‐fimbrial adhesins of animal pathogenic bacteria such as Yersinia YadA and Moraxella UspA1. An interesting feature is that the YadA similarity region is repeated six times within the XadA sequence and encompasses almost the entire length of the protein. Anti‐XadA antibodies identified a 110 kDa outer membrane protein that was sensitive to protease treatment of whole cells. XadA expression is induced in minimal medium. Homology modelling suggests that XadA adopts a β‐helix conformation‐like pertactin, a non‐fimbrial adhesin of Bordetella pertussis. This work is the first characterization of a non‐fimbrial adhesin‐like molecule in a plant pathogenic bacterium. It extends our knowledge about the repertoire of homologous virulence factors that are deployed by animal and plant pathogenic bacteria to include functions potentially involved in adhesion.

Calcium Binding Properties of γ-Crystallin CALCIUM ION BINDS AT THE GREEK KEY βγ-CRYSTALLIN FOLD

The β- and γ-crystallins are closely related lens proteins that are members of the βγ-crystallin superfamily, which also include many non-lens members. Although β-crystallin is known to be a calcium-binding protein, this property has not been reported in γ-crystallin. We have studied the calcium binding properties of γ-crystallin, and we show that it binds 4 mol eq of calcium with a dissociation constant of 90 μm. It also binds the calcium-mimic spectral probes, terbium and Stains-all. Calcium binding does not significantly influence protein secondary and tertiary structures. We present evidence that the Greek key crystallin fold is the site for calcium ion binding in γ-crystallin. Peptides corresponding to Greek key motif of γ-crystallin (42 residues) and their mutants were synthesized and studied for calcium binding. These peptides adopt β-sheet conformation and form aggregates producing β-sandwich. Our results with peptides show that, in Greek key motif, the amino acid adjacent to the conserved aromatic corner in the “a” strand and three amino acids of the “d” strand participate in calcium binding. We suggest that the βγ superfamily represents a novel class of calcium-binding proteins with the Greek key βγ-crystallin fold as potential calcium-binding sites. These results are of significance in understanding the mechanism of calcium homeostasis in the lens.

Calcium Binds to Leptospiral Immunoglobulin-like Protein, LigB, and Modulates Fibronectin Binding

Pathogenic Leptospira spp. express immunoglobulin-like proteins, LigA and LigB, which serve as adhesins to bind to extracellular matrices and mediate their attachment on host cells. However, nothing is known about the mechanism by which these proteins are involved in pathogenesis. We demonstrate that LigBCen2 binds Ca2+, as evidenced by inductively coupled plasma optical emission spectrometry, energy dispersive spectrometry, 45Ca overlay, and mass spectrometry, although there is no known motif for Ca2+ binding. LigBCen2 binds four Ca2+ as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The dissociation constant, KD, for Ca2+ binding is 7 μm, as measured by isothermal titration calorimetry and calcium competition experiments. The nature of the Ca2+-binding site in LigB is possibly similar to that seen in the βγ-crystallin superfamily, since structurally, both families of proteins possess the Greek key type fold. The conformation of LigBCen2 was significantly influenced by Ca2+ binding as shown by far- and near-UV CD and by fluorescence spectroscopy. In the apo form, the protein appears to be partially unfolded, as seen in the far-UV CD spectrum, and upon Ca2+ binding, the protein acquires significant β-sheet conformation. Ca2+ binding stabilizes the protein as monitored by thermal unfolding by CD (50.7–54.8 °C) and by differential scanning calorimetry (50.0–55.7 °C). Ca2+ significantly assists the binding of LigBCen2 to the N-terminal domain of fibronectin and perturbs the secondary structure, suggesting the involvement of Ca2+ in adhesion. We demonstrate that LigB is a novel bacterial Ca2+-binding protein and suggest that Ca2+ binding plays a pivotal role in the pathogenesis of leptospirosis.

Regulatory and Structural EF-Hand Motifs of Neuronal Calcium Sensor-1 : Mg2+ Modulates Ca2+ Binding, Ca2+-Induced Conformational Changes, and Equilibrium Unfolding Transitions

Neuronal calcium sensor-1 (NCS-1) is a major modulator of Ca(2+) signaling with a known role in neurotransmitter release. NCS-1 has one cryptic (EF1) and three functional (EF2, EF3, and EF4) EF-hand motifs. However, it is not known which are the regulatory (Ca(2+)-specific) and structural (Ca(2+)- or Mg(2+)-binding) EF-hand motifs. To understand the specialized functions of NCS-1, identification of the ionic discrimination of the EF-hand sites is important. In this work, we determined the specificity of Ca(2+) binding using NMR and EF-hand mutants. Ca(2+) titration, as monitored by [(15)N,(1)H] heteronuclear single quantum coherence, suggests that Ca(2+) binds to the EF2 and EF3 almost simultaneously, followed by EF4. Our NMR data suggest that Mg(2+) binds to EF2 and EF3, thereby classifying them as structural sites, whereas EF4 is a Ca(2+)-specific or regulatory site. This was further corroborated using an EF2/EF3-disabled mutant, which binds only Ca(2+) and not Mg(2+). Ca(2+) binding induces conformational rearrangements in the protein by reversing Mg(2+)-induced changes in Trp fluorescence and surface hydrophobicity. In a larger physiological perspective, exchanging or replacing Mg(2+) with Ca(2+) reduces the Ca(2+)-binding affinity of NCS-1 from 90 nM to 440 nM, which would be advantageous to the molecule by facilitating reversibility to the Ca(2+)-free state. Although the equilibrium unfolding transitions of apo-NCS-1 and Mg(2+)-bound NCS-1 are similar, the early unfolding transitions of Ca(2+)-bound NCS-1 are partially influenced in the presence of Mg(2+). This study demonstrates the importance of Mg(2+) as a modulator of calcium homeostasis and active-state behavior of NCS-1.

Fibronectin binds to and induces conformational change in a disordered region of leptospiral immunoglobulin-like protein B

Leptospira interrogans is a pathogenic spirochete that causes disease in both humans and animals. LigB (Leptospiral immunoglobulin-like protein B) contributes to the binding of Leptospira to extracellular matrix proteins such as fibronectin (Fn), fibrinogen, laminin, and collagen. A high affinity Fn-binding region of LigB has been recently localized to LigBCen2, which contains the partial eleventh and full twelfth immunoglobulin-like repeats (LigBCen2R) and 47 amino acids of the non-repeat region (LigBCen2NR) of LigB. In this study, LigBCen2NR was shown to bind to the N-terminal domain (NTD) of Fn (KD = 379 nm) by an enzyme-linked immunosorbent assay and isothermal titration calorimetry. Interestingly, this sequence was not observed to adopt secondary structure by far UV circular dichroism or by differential scanning calorimetry, in agreement with computer-based secondary structure predictions. A low partition coefficient (Kav) measured with gel permeation chromatography, a high hydrodynamic radius (Rh) measured with dynamic light scattering, and the insensitivity of the intrinsic viscosity to guanidine hydrochloride treatment all suggest that LigBCen2NR possesses an extended and disordered structure. Two-dimensional 15N-1H HSQC NMR spectra of intact LigBCen2 in the absence and presence of NTD are consistent with these observations, suggesting the presence of both a β-rich region and an unstructured region in LigBCen2 and that the latter of these selectively interacts with NTD. Upon binding to NTD, LigBCen2NR was observed by CD to adopt a β-strand-rich structure, suggestive of the known β-zipper mode of NTD binding.

The βγ-Crystallin Superfamily Contains a Universal Motif for Binding Calcium,

The betagamma-crystallin superfamily consists of evolutionarily related proteins with domain topology similar to lens beta- and gamma-crystallins, formed from duplicated Greek key motifs. Ca(2+) binding was found in a few betagamma-crystallin members earlier, although its prevalence and diversity as inherent molecular properties among members of the superfamily are not well studied. To increase our understanding of Ca(2+) binding in various betagamma-crystallins, we undertook comprehensive structural and Ca(2+)-binding studies of seven members of the superfamily from bacteria, archaea, and vertebrates, including determination of high-resolution crystal structures of three proteins. Our structural observations show that the determinants of Ca(2+) coordination remain conserved in the form of an N/D-N/D-#-I-S/T-S motif in all domains. However, binding of Ca(2+) elicits varied physicochemical responses, ranging from passive sequestration to active stabilization. The motif in this superfamily is modified in some members like lens crystallins where Ca(2+)-binding abilities are partly or completely compromised. We show that reduction or loss of Ca(2+) binding in members of the superfamily, particularly in vertebrates, is due to the selective presence of unfavorable amino acids (largely Arg) at key Ca(2+)-ligation positions and that engineering of the canonical Ca(2+)-binding residues can confer binding activity on an otherwise inactive domain. Through this work, we demonstrate that betagamma-crystallins with the N/D-N/D-#-I-S/T-S motif form an extensive set of Ca(2+)-binding proteins prevalent in all of the three kingdoms of life.

Calneurons provide a calcium threshold for trans-Golgi network to plasma membrane trafficking

Phosphatidylinositol 4-OH kinase IIIβ (PI-4Kβ) is involved in the regulated local synthesis of phospholipids that are crucial for trans-Golgi network (TGN)-to-plasma membrane trafficking. In this study, we show that the calcium sensor proteins calneuron-1 and calneuron-2 physically associate with PI-4Kβ, inhibit the enzyme profoundly at resting and low calcium levels, and negatively interfere with Golgi-to-plasma membrane trafficking. At high calcium levels this inhibition is released and PI-4Kβ is activated via a preferential association with neuronal calcium sensor-1 (NCS-1). In accord to its supposed function as a filter for subthreshold Golgi calcium transients, neuronal overexpression of calneuron-1 enlarges the size of the TGN caused by a build-up of vesicle proteins and reduces the number of axonal Piccolo-Bassoon transport vesicles, large dense core vesicles that carry a set of essential proteins for the formation of the presynaptic active zone during development. A corresponding protein knockdown has the opposite effect. The opposing roles of calneurons and NCS-1 provide a molecular switch to decode local calcium transients at the Golgi and impose a calcium threshold for PI-4Kβ activity and vesicle trafficking.

Calcium-binding to lens βB2- and βA3-crystallins suggests that all β-crystallins are calcium-binding proteins

Crystallins are the major proteins of a mammalian eye lens. The topologically similar eye lens proteins, β‐ and γ‐crystallins, are the prototype and founding members of the βγ‐crystallin superfamily. βγ‐Crystallins have until recently been regarded as structural proteins. However, the calcium‐binding properties of a few members and the potential role of βγ‐crystallins in fertility are being investigated. Because the calcium‐binding elements of other member proteins, such as spherulin 3a, are not present in βB2‐crystallin and other βγ‐crystallins from fish and mammalian genomes, it was argued that lens βγ‐crystallins should not bind calcium. In order to probe whether β‐crystallins can bind calcium, we selected one basic (βB2) and one acidic (βA3) β‐crystallin for calcium‐binding studies. Using calcium‐binding assays such as 45Ca overlay, terbium binding, Stains‐All and isothermal titration calorimetry, we established that both βB2‐ and βA3‐crystallin bind calcium with moderate affinity. There was no significant change in their conformation upon binding calcium as monitored by fluorescence and circular dichroism spectroscopy. However, 15N‐1H heteronuclear single quantum correlation NMR spectroscopy revealed that amide environment of several residues underwent changes indicating calcium ligation. With the corroboration of calcium‐binding to βB2‐ and βA3‐crystallins, we suggest that all β‐crystallins bind calcium. Our results have important implications for understanding the calcium‐related cataractogenesis and maintenance of ionic homeostasis in the lens.