High Affinity Calcium Binding Sites Research Articles

Chicken Feather Waste Valorization Into Nutritive Protein Hydrolysate: Role of Novel Thermostable Keratinase From Bacillus pacificus RSA27

Microbial keratinases exhibit a momentous role in converting keratin biowastes into exceedingly valuable protein supplements. This study reports a novel, highly stable keratinase from Bacillus pacificus RSA27 for the production of pure peptides rich in essential amino acids from chicken feathers. Purified keratinase showed a specific activity of 38.73 U/mg, 2.58-fold purification, and molecular weight of 36 kDa. Kinetic studies using a chicken feather as substrate report Km and Vmax values of 5.69 mg/ml and 142.40 μg/ml/min, respectively, suggesting significant enzyme-substrate affinity/biocatalysis. Identification and in silico structural-functional analysis of keratinase discovered the presence of distinct amino acid residues and their positions. Besides, keratinase possesses a high-affinity calcium-binding site (Asp128, Leu162, Asn164, Ile166, and Val168) and a catalytic triad of Asp119, His151, and Ser308, known attributes of serine protease (subtilisin family). Furthermore, a scale-up to 5 L fermenter revealed complete feather hydrolysis (94.5%) within 24 h with high activity (789 U/ml) and total amino acid of 153.97 μmol/ml. Finally, cytotoxicity evaluation of protein hydrolysate resulted in negligible cytotoxic effects (1.02%) on the mammalian hepatoblastoma cell line, signifying its potential biotechnological applications.

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.

Effect of Calcium on the Hydrolysis Activity of Human Butyrylcholinesterase

The aim of this experiment was to study the effects of calcium ion on the hydrolysis of cationic and anionic substrate by human butyrylcholinesterase (HuBChE). The hydrolysis of aspirin, an anionic substrate, by HuBChE was markedly increased in the presence of increasing concentrations of calcium ion (∼20 mM), as shown by the increasing kcat (∼18-fold). Butyrylthiocholine (BTC), a cationic substrate, was biphasically hydrolyzed with substrate activation; a second BTC molecule caused a 3-fold increase in kcat. At both lower and higher concentrations of BTC, its hydrolysis by HuBChE was slightly slowed down by the addition of calcium ion. Other cationic substrates, propranolol derivatives with butyryl and valeryl groups, were R-preferentially hydrolyzed by HuBChE; the rate of hydrolysis of these compounds was nearly the same in the absence and presence of calcium ion. These data indicate differential effects of calcium ion on HuBChE activity with anionic and cationic substrates. Furthermore, during the hydrolysis of aspirin in the presence of calcium ions, we demonstrated the existence of 2 additional binding sites for calcium, with Km values of 1.8 and 5.9 mM. These binding sites exhibited much lower affinities than the EF-hand motif, previously identified as a high-affinity calcium-binding site.

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.

Investigation of Cytoskeletal Proteins from Tetrahymena thermophila

Over time, organisms have taken different paths to complexity. Although only a single eukaryotic cell, Tetrahymena thermophila derives complexity from its remarkably complex cell surface architecture. Proteomic analysis from our earlier experiments has led to the identification of nearly four dozen proteins isolated from the cortical cytoskeleton. A high molecular coiled coil structural protein (Epc1), an EF‐hand calcium binding protein (Tcb2), and a protein kinase (Epk1) have been investigated. These proteins are believed to have crucial regulatory and structural functions in addition to playing a role in calcium‐dependent regulation of ciliary motion. We are investigating the interactions of these proteins during cell division through confocal microcopy, fluorescence spectroscopy, and NMR solution studies. It was evident through the utilization of confocal microscopy that these proteins were found to localize to a narrow layer of the cortical cytoskeleton called the epiplasm, which was consistent with previous purification experiments. Fluorescence spectroscopy was used in order to monitor conformational changes in the C‐terminal domain of Tcb2, and we identified the presence of one high affinity calcium‐binding site with a low micromolar dissociation constant. The shift peaks observed during (1)H‐(15)N HSQC‐NMR monitored calcium titrations of the Tcb2 C‐terminal domain corroborated evidence of calcium‐dependent conformational changes in this domain. NMR spectra also illustrated that in both the presence and absence of calcium this domain is well folded and upon calcium addition undergoes dramatic conformational change. In the future, we will explore protein distributions relative to one another through FRET and double‐labeling experiments.

Kinetic characterization of Ca2+-ATPase (SERCA1) inhibition by tri-n-butyltin(IV) chloride. A docking conformation proposal

Organotin compounds, such as tri-n-butyltin(IV) chloride (TBT), are widespread toxicants which disrupt different functions in living organisms. TBT interacts with lipid membranes and membrane proteins. The inhibition of the calcium ATPase from sarcoplasmic reticulum membranes by TBT was studied. It was found that the ATPase inhibition could not be reverted in a large time scale; moreover, an excess of TBT over enzyme did not fully inhibit the ATPase activity; therefore, it was concluded that TBT irreversibly inhibits the enzyme, and this inhibition is accompanied by a decrease in the effective TBT concentration. The residual ATP hydrolysis activity was measured at different TBT concentrations with time, and the protective effect of different calcium concentrations on the TBT inhibition was also determined. The simplest kinetic mechanism to successfully explain all the observations and the kinetic behavior was found to be a single irreversible step of the inhibitor binding to the enzyme accompanied with a first-order inhibitor inactivation. A fluorescence study of fluorescein-5-isothiocyanate-labeled enzyme revealed that TBT binding to the enzyme entails a conformational change related to the high- to low-affinity calcium-binding state transition (E1 to E2 transition), resembling the conformational change induced by vanadate linked to the formation of E2 V complex from E1 state. A docking study allowed us to propose a binding pocket for TBT in the membrane region of E1 close to the high-affinity calcium-binding sites, as well as to define the interactions with amino acid residues interfering with calcium sites occupancy.

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.

Assembly of the Respiratory Mucin MUC5B: A NEW MODEL FOR A GEL-FORMING MUCIN

Mucins are essential components in mucus gels that form protective barriers at all epithelial surfaces, but much remains unknown about their assembly, intragranular organization, and post-secretion unfurling to form mucus. MUC5B is a major polymeric mucin expressed by respiratory epithelia, and we investigated the molecular mechanisms involved during its assembly. Studies of intact polymeric MUC5B revealed a single high affinity calcium-binding site, distinct from multiple low affinity sites on each MUC5B monomer. Self-diffusion studies with intact MUC5B showed that calcium binding at the protein site catalyzed reversible cross-links between MUC5B chains to form networks. The site of cross-linking was identified in the MUC5B D3-domain as it was specifically blocked by D3 peptide antibodies. Biophysical analysis and single particle EM of recombinant MUC5B N terminus (D1D2D'D3; NT5B) and subdomains (D1, D1-D2, D2-D'-D3, and D3) generated structural models of monomers and disulfide-linked dimers and suggested that MUC5B multimerizes by disulfide linkage between D3-domains to form linear polymer chains. Moreover, these analyses revealed reversible homotypic interactions of NT5B at low pH and in high calcium, between disulfide-linked NT5B dimers, but not monomers. These results enable a model of MUC5B to be derived, which predicts mechanisms of mucin intracellular assembly and storage, which may be common to the other major gel-forming polymeric mucins.

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.

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.

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.

Calreticulin Is a Thermostable Protein with Distinct Structural Responses to Different Divalent Cation Environments

Calreticulin is a soluble calcium-binding chaperone of the endoplasmic reticulum (ER) that is also detected on the cell surface and in the cytosol. Calreticulin contains a single high affinity calcium-binding site within a globular domain and multiple low affinity sites within a C-terminal acidic region. We show that the secondary structure of calreticulin is remarkably thermostable at a given calcium concentration. Rather than corresponding to complete unfolding events, heat-induced structural transitions observed for calreticulin relate to tertiary structural changes that expose hydrophobic residues and reduce protein rigidity. The thermostability and the overall secondary structure content of calreticulin are impacted by the divalent cation environment, with the ER range of calcium concentrations enhancing stability, and calcium-depleting or high calcium environments reducing stability. Furthermore, magnesium competes with calcium for binding to calreticulin and reduces thermostability. The acidic domain of calreticulin is an important mediator of calcium-dependent changes in secondary structure content and thermostability. Together, these studies indicate interactions between the globular and acidic domains of calreticulin that are impacted by divalent cations. These interactions influence the structure and stability of calreticulin, and are likely to determine the multiple functional activities of calreticulin in different subcellular environments.

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.

Apo and Calcium-Bound Crystal Structures of Cytoskeletal Protein Alpha-14 Giardin (Annexin E1) from the Intestinal Protozoan Parasite Giardia lamblia

Alpha-14 giardin (annexin E1), a member of the alpha giardin family of annexins, has been shown to localize to the flagella of the intestinal protozoan parasite Giardia lamblia. Alpha giardins show a common ancestry with the annexins, a family of proteins most of which bind to phospholipids and cellular membranes in a Ca 2+-dependent manner and are implicated in numerous membrane-related processes including cytoskeletal rearrangements and membrane organization. It has been proposed that alpha-14 giardin may play a significant role during the cytoskeletal rearrangement during differentiation of Giardia. To gain a better understanding of alpha-14 giardin's mode of action and its biological role, we have determined the three-dimensional structure of alpha-14 giardin and its phospholipid-binding properties. Here, we report the apo crystal structure of alpha-14 giardin determined in two different crystal forms as well as the Ca 2+-bound crystal structure of alpha-14 giardin, refined to 1.9, 1.6 and 1.65 Å, respectively. Although the overall fold of alpha-14 giardin is similar to that of alpha-11 giardin, multiwavelength anomalous dispersion phasing was required to solve the alpha-14 giardin structure, indicating significant structural differences between these two members of the alpha giardin family. Unlike most annexin structures, which typically possess N-terminal domains, alpha-14 giardin is composed of only a core domain, followed by a C-terminal extension that may serve as a ligand for binding to cytoskeletal protein partners in Giardia. In the Ca 2+-bound structure we detected five bound calcium ions, one of which is a novel, highly coordinated calcium-binding site not previously observed in annexin structures. This novel high-affinity calcium-binding site is composed of seven protein donor groups, a feature rarely observed in crystal structures. In addition, phospholipid-binding assays suggest that alpha-14 giardin exhibits calcium-dependent binding to phospholipids that coordinate cytoskeletal disassembly/assembly during differentiation of the parasite.

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.

A High Affinity Calcium Binding Site of NCS-1 is Responsible for An Activity-Dependent Facilitation of Presynaptic Calcium Current

A High Affinity Calcium Binding Site of NCS-1 is Responsible for An Activity-Dependent Facilitation of Presynaptic Calcium Current