High Affinity Calcium Binding Research Articles

Versatility of subtilisin: A review on structure, characteristics, and applications.

Due to its thermostability and high pH compatibility, subtilisin is most known for its role as an additive for detergents in which it is categorized as a serine protease according to MEROPS database. Subtilisin is typically isolated from various bacterial species of the Bacillus genus such as Bacillus subtilis, B. amyloliquefaciens, B. licheniformis, and various other organisms. It is composed of 268-275 amino acid residues and is initially secreted in the precursor form, preprosubtilisin, which is composed of 29-residues signal peptide, 77-residues propeptide, and 275-residues active subtilisin. Subtilisin is known for the presence of high and low affinity calcium binding sites in its structure. Native subtilisin has general properties of thermostability, tolerance to neutral to high pH, broad specificity, and calcium-dependent stability, which contribute to the versatility of subtilisin applicability. Through protein engineering and immobilization technologies, many variants of subtilisin have been generated, which increase the applicability of subtilisin in various industries including detergent, food processing and packaging, synthesis of inhibitory peptides, therapeutic, and waste management applications.

Determination of the Calcium‐Dependent Activation Mechanism for Metacaspase 1 from Schizophyllum commune

Metacaspases are cysteine proteases implicated in initiating programmed cell death in plants and fungi, and have recently been shown to play a key role in the plant immune response. These proteins are distantly related to caspases, the enzymes that induce apoptosis in animals. Notably, metacaspases require calcium binding for activity, whereas caspases are instead activated by upstream signalling pathways that utilize calcium as a signal transducer. Metacaspase activity is greatest in the presence of millimolar levels of calcium. However, the exact mechanism by which calcium activates the metacaspases is still unclear. Our lab has performed an extensive characterization of metacaspase1 from the fungus Schizophyllum commune (ScMCA-Ia) to determine the likely conformational changes needed for metacaspase activation. The available metacaspase crystal structures from yeast and T. brucei demonstrate calcium-binding to a conserved site of four aspartic acid residues outside of the active site. While calcium itself binds to this allosteric site, the calcium-binding Asps are found on flexible loops and are only a few residues away from the active site. Sequence alignments demonstrate that these Asp residues are conserved in ScMCA-Ia and mutagenesis studies that exchange Asp for Ala reveal that this site is crucial for activity on small peptide substrates. We have used differential scanning fluorimetry to determine the calcium-binding constants of ScMC-Ia and associated mutants. Mutating calcium-binding Asp residues to Glu renders the enzymes inactive, however differential scanning fluorimetry indicates that the mutants still bind to calcium with Kd values similar to wildtype, in the µM range. This suggests that calcium-binding to this high-affinity site changes the position of calcium binding residues, altering the positions of the loops bearing these residues, and ultimately changing the orientation of the active site. Even slight changes to the calcium binding site (Asp to Glu) cannot be tolerated. In addition to the micromolar binding site, a second site that binds calcium in the mM range has been inferred from the low millimolar calcium levels needed for optimal metacaspase activity. Kinetics experiments suggest that the low-affinity calcium binding site is important for substrate binding. Together, these findings comprise a complete model for metacaspase activation in which high-affinity calcium binding reorients the active site for catalysis and low-affinity binding alters the substrate binding pocket to enhance substrate binding and catalysis. This mechanistic understanding of metacaspase activation and conformational changes is needed to design metacaspase-specific inhibitors to treat fungal diseases.

Dementia-linked amyloidosis is associated with brain protein deamidation as revealed by proteomic profiling of human brain tissues.

BackgroundAggregation of malformed proteins is a key feature of many neurodegenerative diseases, but the mechanisms that drive proteinopathy in the brain are poorly understood. We aimed to characterize aggregated proteins in human brain tissues affected by dementia.ResultsTo characterize amyloidal plaque purified from post-mortem brain tissue of dementia patient, we applied ultracentrifugation-electrostatic repulsion hydrophilic interaction chromatography (UC-ERLIC) coupled mass spectrometry-based proteomics technologies. Proteomics profiling of both soluble and aggregated amyloidal plaque demonstrated significant enrichment and deamidation of S100A9, ferritin, hemoglobin subunits, creatine kinase and collagen protein among the aggregated brain proteins. Amyloidal plaques were enriched in the deamidated variant of protein S100A9, and structural analysis indicated that both the low- and high-affinity calcium binding motifs of S100A9 were deamidated exclusively in the aggregated fraction, suggesting altered charge state and function of this protein in brain tissues affected by dementia. The multiple deamidated residues of S100A9 predicts introduction of negative charge that alter Ca++ binding, suggesting increased capacity to form pathological aggregates in the brain.ConclusionUC-coupled proteomics revealed that brain amyloidal plaques are enriched in deamidated proteins, and suggested that altered charge state and calcium-binding capacity of S100A9 may enhance protein aggregation and promote neurodegeneration in the human brain.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0200-z) contains supplementary material, which is available to authorized users.

Regulation of calreticulin–major histocompatibility complex (MHC) class I interactions by ATP

The MHC class I peptide loading complex (PLC) facilitates the assembly of MHC class I molecules with peptides, but factors that regulate the stability and dynamics of the assembly complex are largely uncharacterized. Based on initial findings that ATP, in addition to MHC class I-specific peptide, is able to induce MHC class I dissociation from the PLC, we investigated the interaction of ATP with the chaperone calreticulin, an endoplasmic reticulum (ER) luminal, calcium-binding component of the PLC that is known to bind ATP. We combined computational and experimental measurements to identify residues within the globular domain of calreticulin, in proximity to the high-affinity calcium-binding site, that are important for high-affinity ATP binding and for ATPase activity. High-affinity calcium binding by calreticulin is required for optimal nucleotide binding, but both ATP and ADP destabilize enthalpy-driven high-affinity calcium binding to calreticulin. ATP also selectively destabilizes the interaction of calreticulin with cellular substrates, including MHC class I molecules. Calreticulin mutants that affect ATP or high-affinity calcium binding display prolonged associations with monoglucosylated forms of cellular MHC class I, delaying MHC class I dissociation from the PLC and their transit through the secretory pathway. These studies reveal central roles for ATP and calcium binding as regulators of calreticulin-substrate interactions and as key determinants of PLC dynamics.

Recombinant γT305A fibrinogen indicates severely impaired fibrin polymerization due to the aberrant function of hole ‘a’ and calcium binding sites

IntroductionWe examined a 6-month-old girl with inherited fibrinogen abnormality and no history of bleeding or thrombosis. Routine coagulation screening tests showed a markedly low level of plasma fibrinogen determined by functional measurement and also a low level by antigenic measurement (functional/antigenic ratio=0.295), suggesting hypodysfibrinogenemia. Materials and methodsDNA sequence analysis was performed, and γT305A fibrinogen was synthesized in Chinese hamster ovary cells based on the results. We then functionally analyzed and compared with that of nearby recombinant γN308K fibrinogen. ResultsDNA sequence analysis revealed a heterozygous γT305A substitution (mature protein residue number). The γT305A fibrinogen indicated markedly impaired thrombin-catalyzed fibrin polymerization both in the presence or absence of 1mM calcium ion compared with that of γN308K fibrinogen. Protection of plasmin degradation in the presence of calcium ion or Gly-Pro-Arg-Pro peptide (analogue for so-called knob ‘A’) and factor XIIIa-catalyzed fibrinogen crosslinking demonstrated that the calcium binding sites, hole ‘a’ and D:D interaction sites were all markedly impaired, whereas γN308Kwas impaired at the latter two sites. Molecular modeling demonstrated that γT305 is localized at a shorter distance than γN308 from the high affinity calcium binding site and hole ‘a’. ConclusionOur findings suggest that γT305 might be important for construction of the overall structure of the γ module of fibrinogen. Substitution of γT305A leads to both dysfibrinogenemic and hypofibrinogenemic characterization, namely hypodysfibrinogenemia. We have already reported that recombinant γT305A fibrinogen was synthesized normally and secreted slightly, but was significantly reduced.

Identification of a Determinant of High Affinity Calcium Binding in the Selectivity Filter of a Mammalian Calcium Channel

Voltage-gated calcium channels (CaVs) provide the primary source of calcium influx in excitable cells and couple electrical signals to chemical signaling cascades. Due to CaV size and the difficulty of expressing CaVs at quantities sufficient for high-resolution determination, detailed structural information is limited to isolated cytoplasmic domains. However, CaVs are homologous to voltage-gated sodium channels (NaVs) and NaV structure can serve as a template for CaV structure. We determined the structure of the closed conformation of NaVAe1p, a pore only bacterial NaV derived from NaVAe1, an Alkalilimnicola ehrlichei bacterial NaV. This structure reveals the site of a putative calcium ion at the extracellular mouth of the selectivity filter liganded by four serines. At the equivalent site in mammalian calcium channel selectivity filters, there is a conserved aspartate in one of the calcium channel domains. Our functional studies show that this aspartate is a previously unknown determinant of CaV high affinity calcium binding in the mammalian calcium channel CaV1.2. These findings show the extent of similarities between bacterial sodium channels and eukaryotic voltage gated channels and shed new light on the selectivity filter in mammalian calcium channels.

Structure of a Prokaryotic Sodium Channel Pore Reveals Essential Gating Elements and an Outer Ion Binding Site Common to Eukaryotic Channels

Voltage-gated sodium channels (NaVs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial NaV (BacNaV) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of NaVAe1p, a pore only BacNaV derived from NaVAe1, a BacNaV from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNaVs show that two elements defined by the NaVAe1p structure, an S6 activation gate position and the cytoplasmic tail ‘neck’, are central to BacNaV gating. The structure also reveals the selectivity filter ion entry site, termed the ‘outer ion’ site. Comparison with mammalian voltage gated calcium channel (CaV) selectivity filters, together with functional studies shows that this site forms a previously unknown determinant of CaV high affinity calcium binding. Our findings underscore commonalities between BacNaVs and eukaryotic voltage gated channels and provide a framework for understanding gating and ion permeation in this superfamily.

Formation of the High-Affinity Calcium Binding Site in Pro-subtilisin E with the Insertion Sequence IS1 of Pro-Tk-subtilisin

Subtilisin E is activated from its inactive precursor Pro-subtilisin E by autoprocessing and degradation of the propeptide. Subtilisin E has two calcium binding sites, the high-affinity Ca1 site and the low-affinity Ca2 site. The Ca1 site is conserved in various subtilisin-like proteases and is important for stability. This site is not formed in Pro-subtilisin E, because the structural rearrangement of the N-terminal region of the subtilisin domain upon autoprocessing is necessary for the formation of this site. As a result, Pro-subtilisin E is not fully folded. In contrast, Pro-Tk-subtilisin from Thermococcus kodakarensis is fully folded, because it does not require the structural rearrangement upon autoprocessing for the formation of the Ca1 site due to the presence of the insertion sequence IS1 between the propeptide and subtilisin domains. To examine whether the Ca1 site is formed in Pro-subtilisin E by inserting IS1 between the propeptide and subtilisin domains, the Pro-subtilisin E mutant with this insertion, IS1-Pro-subtilisin E, and its active site mutants, IS1-Pro-S221A and IS1-Pro-S221C, were constructed and characterized. The crystal structure of IS1-Pro-S221A revealed that this protein is fully folded and the Ca1 site is formed. In this structure, IS1 serves as a linker that brings the N-terminus of the subtilisin domain near the Ca1 site. IS1-Pro-S221A in a calcium-bound form was more stable than that in a calcium-free form by 13.1 °C. IS1-Pro-S221C was more rapidly autoprocessed than Pro-S221C. These results suggest that IS1 facilitates the formation of the Ca1 site and the complete folding of Pro-subtilisin E and thereby accelerates its autoprocessing.

Structure of a Prokaryotic Sodium Channel Pore Reveals Essential Gating Elements and an Outer Ion Binding Site Common to Eukaryotic Channels

Voltage-gated sodium channels (NaVs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial NaV (BacNaV) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of NaVAe1p, a pore-only BacNaV derived from NaVAe1, a BacNaV from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNaVs show that two elements defined by the NaVAe1p structure, an S6 activation gate position and the cytoplasmic tail “neck”, are central to BacNaV gating. The structure also reveals the selectivity filter ion entry site, termed the “outer ion” site. Comparison with mammalian voltage-gated calcium channel (CaV) selectivity filters, together with functional studies, shows that this site forms a previously unknown determinant of CaV high-affinity calcium binding. Our findings underscore commonalities between BacNaVs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily.

Structural Insights into Fibrinogen Dynamics Using Amide Hydrogen/Deuterium Exchange Mass Spectrometry

We determined the amide hydrogen/deuterium exchange profile of native human fibrinogen under physiologic conditions. After optimization of the quench and proteolysis conditions, more than 1,200 peptides were identified by mass spectrometry, spanning more than 90% of the constituent Aα, Bβ, and γ chain amino acid sequences. The compact central and distal globular regions of fibrinogen were well protected from deuterium exchange, with the exception of the unfolded amino-terminal segments of the Aα and Bβ chains extending from the central region, and the short γ chain "tail" extending from each distal globular region. The triple-helical coiled-coil regions, which bridge the central region to each distal region, were also well protected with the exception of a moderately fast-exchanging area in the middle of each coiled-coil adjacent to the γ chain carbohydrate attachment site. These dynamic regions appear to provide flexibility to the fibrinogen molecule. The γ chain "out loop" contained within each coiled-coil also exchanged rapidly. The αC domain (Aα 392-610) exchanged rapidly, with the exception of a short segment sandwiched between a conserved disulfide linkage in the N-terminal αC subdomain. This latter finding is consistent with a mostly disordered structure for the αC domain in native fibrinogen. Analysis of the dysfibrinogen Bβ 235 Pro/Leu, which exhibits abnormal fibrin structure, revealed enhanced deuterium exchange surrounding the Pro/Leu substitution site as well as in the vicinity of the high affinity calcium binding site and the A knob polymerization pocket within the γC domain. The implication of these changes with respect to fibrin structure is discussed.

Distinct interactions of Na+ and Ca2+ ions with the selectivity filter of the bacterial sodium channel NaVAb

Rapid and selective ion transport is essential for the generation and regulation of electrical signaling pathways in living organisms. In this study, we use molecular dynamics simulations and free energy calculations to investigate how the bacterial sodium channel NaVAb (Arcobacter butzleri) differentiates between Na+ and Ca2+ ions. Multiple nanosecond molecular dynamics simulations revealed distinct binding patterns for these two cations in the selectivity filter and suggested a high affinity calcium binding site formed by backbone atoms of residues Leu-176 and Thr-175 (SCEN) in the sodium channel selectivity filter.

The Glaucoma-associated Olfactomedin Domain of Myocilin Is a Novel Calcium Binding Protein

Myocilin is a protein found in the trabecular meshwork extracellular matrix tissue of the eye that plays a role in regulating intraocular pressure. Both wild-type and certain myocilin variants containing mutations in the olfactomedin (OLF) domain are linked to the optic neuropathy glaucoma. Because calcium ions are important biological cofactors that play numerous roles in extracellular matrix proteins, we examined the calcium binding properties of the myocilin OLF domain (myoc-OLF). Our study reveals an unprecedented high affinity calcium binding site within myoc-OLF. The calcium ion remains bound to wild-type OLF at neutral and acidic pH. A glaucoma-causing OLF variant, myoc-OLF(D380A), is calcium-depleted. Key differences in secondary and tertiary structure between myoc-OLF(D380A) and wild-type myoc-OLF, as well as limited access to chelators, indicate that the calcium binding site is largely buried in the interior of the protein. Analysis of six conserved aspartate or glutamate residues and an additional 18 disease-causing variants revealed two other candidate residues that may be involved in calcium coordination. Our finding expands our knowledge of calcium binding in extracellular matrix proteins; provides new clues into domain structure, function, and pathogenesis for myocilin; and offers insights into highly conserved, biomedically relevant OLF domains.

Rearranging and concatenating a native RTX domain to understand sequence modularity

The use of repetitive peptide sequences forming predictable secondary structures has been a key paradigm in recent efforts to engineer biomolecular recognition. The modularity and predictability of these scaffolds enables precise identification and mutation of the active interface, providing a level of control which non-repetitive scaffolds often lack. However, the majority of these scaffolds are well-folded stable structures. If the structures had a stimulus-responsive character, this would enable the allosteric regulation of their function. The calcium-responsive beta roll-forming repeats in toxin (RTX) domain potentially offer both of these properties. To further develop this scaffold, we synthesized a set of RTX peptides ranging in size from 5 to 17 repeats, with and without C-terminal capping. We found that while the number of repeats can be altered to tune the size of the RTX face, repeat ordering and C-terminal capping are critical for successful folding. Comparing all of the constructs, we also observed that native configuration with nine repeats exhibited the highest affinity for calcium. In addition, we performed a comparison on a set of known RTX-containing proteins and find that C-terminal repeats often possess deviations from the consensus RTX sequence which may be essential for proper folding. We further find that there seems to be a narrow size range in which RTX domains exist. These results demonstrate that the deviations from the consensus RTX sequence that are observed in natural proteins are important for high-affinity calcium binding and folding. Therefore, the RTX scaffolds will be less modular as compared with other, non-responsive scaffolds, and the sequence-dependent interactions between different repeats will need to be retained in these scaffolds as they are developed in future protein-engineering efforts.

Fibrinogen residue γAla341 is necessary for calcium binding and ‘A-a’ interactions

The fibrinogen γ-module has several important sites relating to fibrinogen function, which include the high affinity calcium binding site, hole 'a' that binds with knob 'A', and the D:D interface. Residue γAla341, which is located in the vicinity of these sites, is altered in three variant fibrinogens: fibrinogen Seoul (γAla341Asp), Tolaga Bay (γAla341Val), and Lyon III (γAla341Thr). In order to investigate the impaired polymerisation of fibrinogens γAla341Asp and γAla341Val to understand the role of γAla341 in fibrin polymerisation and fibrinogen synthesis, we have expressed γAla341Asp and γAla341Val in Chinese hamster ovary (CHO) cells, purified these fibrinogens from the culture media and performed biochemical tests to elucidate their function. Expression in CHO cells was similar for these variants. For both variants the kinetics of thrombin-catalysed FpA release was not different from normal fibrinogen, while FpB release was slower than that of normal. Thrombin-catalysed polymerisation of both variants was dependent on the calcium concentration. At physiologic calcium (1 mM) the variants showed impaired polymerisation with a longer lag period and a slower Vmax than normal fibrinogen. Scanning electron micrographs showed the clots were less organised than normal, having thicker and more twisted fibers, and larger pores. Analysis by SDS-PAGE showed that factor XIIIa-catalysed γ and α chain cross-linking was delayed, and plasmin-catalysed lysis was not reduced by the presence of 5 mM calcium or 5 mM GPRP (Gly-Pro-Arg-Pro). Our data indicate that fibrinogen residue γAla341 is important for the proper conformation of the γ-module, maintaining calcium-binding site and 'A-a' interactions.

Dynamic properties of extremophilic subtilisin-like serine-proteases

The investigation of the structural determinants of enzymatic temperature adaptation is a crucial pre-requisite both in terms of fundamental research and industrial applications to develop new biocatalysts active at different temperature ranges. In several cases, the differences related to cold- or warm-adaptation are related to subtle structural and aminoacidic differences at the molecular level, often hard to detect. In this context, we present a comparative study of psychrophilic, mesophilic and thermophilic subtilisin-like serine proteases by all-atom molecular dynamics (MD) simulations in explicit solvent using a multiple-replica approach. Our results strongly enforce the current view on localized flexibility in crucial functional regions for cold-adapted serine proteases and point out a different optimization and usage of salt-bridge interactions and networks in cold- and warm-adapted enzymes. The analyses allow to identify a subset of structural and dynamic features strictly associated to cold adaptation and which change from cold- to heat-active subtilisins. In particular, the thermophilic subtilisin presents a high affinity calcium binding site which is not structurally conserved in the mesophilic and psychrophilic counterparts, which, as it turns out from the MD analyses, at the same position show a stable salt bridge network and no stabilizing intra-molecular interactions, respectively. These aspects, along with differential flexibility in regions close to the active site or substrate binding pocket, can be an indication of evolution at this protein site toward a lower stability moving from high to low temperature conditions.

Fibrinogen residue γAla341 Is Necessary for Calcium Binding and ‘A-a’ interaction.

AbstractAbstract 1154Fibrinogen, a 340-kDa glycoprotein has essential roles in blood coagulation and platelet aggregation. Fibrinogen is a complex molecule consisting of 3 pairs of Aα, Bβ, and γ chains intertwined to form a tri-nodular molecule with 2 terminal D regions and a central E region. The fibrinogen γ-nodule, a part of D region, has several important sites relating to fibrinogen function, which are the high affinity calcium binding site, hole ‘a’ that binds with knob ‘A’, and the D:D interface. Residue γAla341, which is located in the vicinity of those sites and conserved between all available species, is altered in two variant fibrinogens: fibrinogen Seoul (γAla341Asp) (Song et al., Clin Appl Thromb Hemost 2006) and fibrinogen Tolaga Bay (γAla341Val) (Davis et al., Thromb Haemost 2007). Fibrinogen Seoul showed hypodysfibrinogenemia, and fibrinogen Tolaga Bay hypofibrinogenemia. We have expressed these two variant fibrinogens in CHO cells, purified them from the culture media and performed biochemical tests to elucidate their function. Thrombin-catalyzed kinetics of FpA release was not different (p-value, 0.3, n=3) from normal fibrinogen. Average specificity constant, kcat/Km for FpA was 7.4±1.8, 5.7±1.8, and 8.1±1.2 (mean±SD, 106M-1s-1) for normal, γAla341Val, and γAla341Asp, respectively. However, FpB release from both variants was slower than that of normal (p-value, 0.005 in One Way ANOVA; p-value, 0.006 and 0.002 for normal vs γAla341Val, and normal vs γAla341Asp, respectively in multiple comparison). Average specificity constant for FpB was 3.6±1.1, 1.7±0.4, and 1.4±0.3 (mean±SD, 106M-1s-1) for normal, γAla341Val, and γAla341Asp, respectively. We measured fibrin polymerization by turbidity with the final thrombin and fibrinogen concentration being 0.2mg/mL and 0.1U/mL, respectively. At 10mM calcium we saw no turbidity rising with either variant. Both variants showed impaired polymerization with a longer lag period and a slower Vmax than normal fibrinogen at physiologic 1mM calcium. Lag period, which reflects protofibril formation, for normal, γAla341Val, and γAla341Asp was 110±30, 2,500±150, and 1,200±40, respectively (mean±SD, sec). Vmax, which reflects lateral aggregation, was 81±26, 15±3, and 3±1 (mean±SD, 10-5s-1) for normal, γAla341Val, and γAla341Asp. With the FXIIIa cross-linking, measured by SDS-PAGE, we found that γ and α chain cross-linking was delayed in both variants. We tested the calcium binding and the functionality of ‘a’ polymerization site with the plasmin protection assay. Both variants were not protected from plasminolysis in the presence of 1mM calcium or 0.55mM GPRP, indicative of impaired binding of calcium and knob ‘A’. Given these results, both fibrinogen Seoul and Tolaga Bay likely have a conformational change in their calcium and GPRP binding sites resulting in the impaired fibrin polymerization. In conclusion, we think fibrinogen residue γAla341 is important for calcium binding, ‘A-a’ interactions and the conformation of the γ-nodule. Disclosures:No relevant conflicts of interest to declare.

Impact of calcium on N1 influenza neuraminidase dynamics and binding free energy

The highly pathogenic influenza strains H5N1 and H1N1 are currently treated with inhibitors of the viral surface protein neuraminidase (N1). Crystal structures of N1 indicate a conserved, high affinity calcium binding site located near the active site. The specific role of this calcium in the enzyme mechanism is unknown, though it has been shown to be important for enzymatic activity and thermostability. We report molecular dynamics (MD) simulations of calcium-bound and calcium-free N1 complexes with the inhibitor oseltamivir (marketed as the drug Tamiflu), independently using both the AMBER FF99SB and GROMOS96 force fields, to give structural insight into calcium stabilization of key framework residues. Y347, which demonstrates similar sampling patterns in the simulations of both force fields, is implicated as an important N1 residue that can “clamp” the ligand into a favorable binding pose. Free energy perturbation and thermodynamic integration calculations, using two different force fields, support the importance of Y347 and indicate a +3 to +5 kcal/mol change in the binding free energy of oseltamivir in the absence of calcium. With the important role of structure-based drug design for neuraminidase inhibitors and the growing literature on emerging strains and subtypes, inclusion of this calcium for active site stability is particularly crucial for computational efforts such as homology modeling, virtual screening, and free energy methods. Proteins 2010. © 2010 Wiley-Liss, Inc.

Characterization of Conformational Transitions in Dream Protein using Fluorescence Spectroscopy

DREAM (Down Stream Regulatory Antagonist Modulator) belongs to an important class of calcium binding proteins that are involved in transducing the calcium signal into a biological response in neuronal tissue. DREAM is a unique calcium binding protein that directly binds to DNA and regulates the transcription of prodynorphin and c-fos genes in a calcium dependent fashion. Here we report the conformational transitions in DREAM upon calcium binding by monitoring fluorescence properties of tryptophane residue which is located in proximity to the high affinity calcium binding EF-hand 3. Ca2+ association to the protein leads to the hypsochromic shift in the Trp emission spectrum (max = 338nm) compared to the apo form (max = 343 nm) indicating that the calcium induced conformational transition strongly alters fluorescent probe environment. The changes in the Trp emission spectra are strongly influenced by DREAM concentration confirming that alteration of oligomerization state of DREAM can be readily monitored using fluorescence data. The ligand induced changes in the tryptophan environment were further confirmed by the fluorescence quenching studies using iodide and acrydine quenchers. Trp properties were also analyzed using fluorescence lifetime measurements in frequency domain. In both forms, apo- and Ca2+ bond, the tryptophan lifetime can be described using two exponential decay model , with 1 = 1.5 ns and 1=3.5 ns. The Ca2+ association to protein does not significantly alter tryptophan lifetime but increase the fraction of protein with the lifetime of 1.5 ns.

Concerted but Noncooperative Activation of Nucleotide and Actuator Domains of the Ca-ATPase Upon Calcium Binding

Calcium-dependent domain movements of the actuator (A) and nucleotide (N) domains of the SERCA2a isoform of the Ca-ATPase were assessed using constructs containing engineered tetracysteine binding motifs, which were expressed in insect High-Five cells and subsequently labeled with the biarsenical fluorophore 4′,5′-bis(1,3,2-dithoarsolan-2-yl)fluorescein (FlAsH-EDT2). Maximum catalytic function is retained in microsomes isolated from High-Five cells and labeled with FlAsH-EDT2. Distance measurements using the nucleotide analog TNP-ATP, which acts as a fluorescence resonance energy transfer (FRET) acceptor from FlAsH, identify a 2.4 Å increase in the spatial separation between the N- and A-domains induced by high-affinity calcium binding; this structural change is comparable to that observed in crystal structures.

Concerted but Noncooperative Activation of Nucleotide and Actuator Domains of the Ca-ATPase upon Calcium Binding

Calcium-dependent domain movements of the actuator (A) and nucleotide (N) domains of the SERCA2a isoform of the Ca-ATPase were assessed using constructs containing engineered tetracysteine binding motifs, which were expressed in insect High-Five cells and subsequently labeled with the biarsenical fluorophore 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH-EDT(2)). Maximum catalytic function is retained in microsomes isolated from High-Five cells and labeled with FlAsH-EDT(2). Distance measurements using the nucleotide analog 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP), which acts as a fluorescence resonance energy transfer (FRET) acceptor from FlAsH, identify a 2.4 A increase in the spatial separation between the N- and A-domains induced by high-affinity calcium binding; this structural change is comparable to that observed in crystal structures. No significant distance changes occur across the N-domain between FlAsH and TNP-ATP, indicating that calcium activation induces rigid body domain movements rather than intradomain conformational changes. Calcium-dependent decreases in the fluorescence of FlAsH bound, respectively, to either the N- or A-domains indicate coordinated and noncooperative domain movements, where both A- and N-domains display virtually identical calcium dependencies (i.e., K(d) = 4.8 +/- 0.4 microM). We suggest that occupancy of a single high-affinity calcium binding site induces the rearrangement of the A- and N-domains of the Ca-ATPase to form an intermediate state, which facilitates phosphoenzyme formation from ATP upon occupancy of the second high-affinity calcium site.